Prof. Dr. Daniel Rauh

Medical Chemistry and Chemical Biology
TU Dortmund University


  • A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2
    Palei, S. and Weisner, J. and Vogt, M. and Gontla, R. and Buchmuller, B. and Ehrt, C. and Grabe, T. and Kleinbölting, S. and Müller, M. and Clever, G.H. and Rauh, D. and Summerer, D.
    RSC Medicinal Chemistry (2022)
    Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action. © 2022 RSC.
    view abstract10.1039/d2md00186a
  • Covalent Allosteric Inhibitors of Akt Generated Using a Click Fragment Approach
    van der Westhuizen, L. and Weisner, J. and Taher, A. and Landel, I. and Quambusch, L. and Lindemann, M. and Uhlenbrock, N. and Müller, M.P. and Green, I.R. and Pelly, S.C. and Rauh, D. and van Otterlo, W.A.L.
    ChemMedChem (2022)
    Akt is a protein kinase that has been implicated in the progression of cancerous tumours. A number of covalent allosteric Akt inhibitors are known, and based on these scaffolds, a small library of novel potential covalent allosteric imidazopyridine-based inhibitors was designed. The envisaged compounds were synthesised, with click chemistry enabling a modular approach to a number of the target compounds. The binding modes, potencies and antiproliferative activities of these synthesised compounds were explored, thereby furthering the structure activity relationship knowledge of this class of Akt inhibitors. Three novel covalent inhibitors were identified, exhibiting moderate activity against Akt1 and various cancer cell lines, potentially paving the way for future covalent allosteric inhibitors with improved properties. © 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH
    view abstract10.1002/cmdc.202100776
  • Design and synthesis of Nrf2-derived hydrocarbon stapled peptides for the disruption of protein-DNA-interactions
    Wiedemann, B. and Kamps, D. and Depta, L. and Weisner, J. and Cvetreznik, J. and Tomassi, S. and Gentz, S. and Hoffmann, J.-E. and Müller, M.P. and Koch, O. and Dehmelt, L. and Rauh, D.
    PLoS ONE 17 (2022)
    Misregulation and mutations of the transcription factor Nrf2 are involved in the development of a variety of human diseases. In this study, we employed the technology of stapled peptides to address a protein-DNA-complex and designed a set of Nrf2-based derivatives. Varying the length and position of the hydrocarbon staple, we chose the best peptide for further evaluation in both fixed and living cells. Peptide 4 revealed significant enrichment within the nucleus compared to its linear counterpart 5, indicating potent binding to DNA. Our studies suggest that these molecules offer an interesting strategy to target activated Nrf2 in cancer cells. Copyright: © 2022 Wiedemann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    view abstract10.1371/journal.pone.0267651
  • Optimization of Covalent MKK7 Inhibitors via Crude Nanomole-Scale Libraries
    Gehrtz, P. and Marom, S. and Bührmann, M. and Hardick, J. and Kleinbölting, S. and Shraga, A. and Dubiella, C. and Gabizon, R. and Wiese, J.N. and Müller, M.P. and Cohen, G. and Babaev, I. and Shurrush, K. and Avram, L. and Resnick, E. and Barr, H. and Rauh, D. and London, N.
    Journal of Medicinal Chemistry 65 (2022)
    High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them via the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated via conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter. © 2022 American Chemical Society.
    view abstract10.1021/acs.jmedchem.1c02206
  • Persister state-directed transitioning and vulnerability in melanoma
    Chauvistré, H. and Shannan, B. and Daignault-Mill, S.M. and Ju, R.J. and Picard, D. and Egetemaier, S. and Váraljai, R. and Gibhardt, C.S. and Sechi, A. and Kaschani, F. and Keminer, O. and Stehbens, S.J. and Liu, Q. and Yin, X. and Jeyakumar, K. and Vogel, F.C.E. and Krepler, C. and Rebecca, V.W. and Kubat, L. and Lueong, S.S. and Forster, J. and Horn, S. and Remke, M. and Ehrmann, M. and Paschen, A. and Becker, J.C. and Helfrich, I. and Rauh, D. and Kaiser, M. and Gul, S. and Herlyn, M. and Bogeski, I. and Rodríguez-López, J.N. and Haass, N.K. and Schadendorf, D. and Roesch, A.
    Nature Communications 13 (2022)
    Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(–)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination. © 2022, The Author(s).
    view abstract10.1038/s41467-022-30641-9
  • Cellular model system to dissect the isoform-selectivity of Akt inhibitors
    Quambusch, L. and Depta, L. and Landel, I. and Lubeck, M. and Kirschner, T. and Nabert, J. and Uhlenbrock, N. and Weisner, J. and Kostka, M. and Levy, L.M. and Schultz-Fademrecht, C. and Glanemann, F. and Althoff, K. and Müller, M.P. and Siveke, J.T. and Rauh, D.
    Nature Communications 12 (2021)
    The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms’ distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties. © 2021, The Author(s).
    view abstract10.1038/s41467-021-25512-8
  • Insight into Targeting Exon20 Insertion Mutations of the Epidermal Growth Factor Receptor with Wild Type-Sparing Inhibitors
    Lategahn, J. and Tumbrink, H.L. and Schultz-Fademrecht, C. and Heimsoeth, A. and Werr, L. and Niggenaber, J. and Keul, M. and Parmaksiz, F. and Baumann, M. and Menninger, S. and Zent, E. and Landel, I. and Weisner, J. and Jeyakumar, K. and Heyden, L. and Russ, N. and Müller, F. and Lorenz, C. and Brägelmann, J. and Spille, I. and Grabe, T. and Müller, M.P. and Heuckmann, J.M. and Klebl, B.M. and Nussbaumer, P. and Sos, M.L. and Rauh, D.
    Journal of Medicinal Chemistry (2021)
    Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1H-pyrrolo[2,3-b]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations. © 2022 The Authors. Published by American Chemical Society.
    view abstract10.1021/acs.jmedchem.1c02080
  • Resistance to avapritinib in pdgfra-driven gist is caused by secondary mutations in the pdgfra kinase domain
    Grunewald, S. and Klug, L.R. and Mühlenberg, T. and Lategahn, J. and Falkenhorst, J. and Town, A. and Ehrt, C. and Wardelmann, E. and Hartmann, W. and Schildhaus, H.-U. and Treckmann, J. and Fletcher, J.A. and Jung, S. and Czodrowski, P. and Miller, S. and Schmidt-Kittler, O. and Rauh, D. and Heinrich, M.C. and Bauer, S.
    Cancer Discovery 11 (2021)
    Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapri-tinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance. © 2020 American Association for Cancer Research.
    view abstract10.1158/2159-8290.CD-20-0487
  • Complex Crystal Structures of EGFR with Third-Generation Kinase Inhibitors and Simultaneously Bound Allosteric Ligands
    Niggenaber, J. and Heyden, L. and Grabe, T. and Müller, M.P. and Lategahn, J. and Rauh, D.
    ACS Medicinal Chemistry Letters 11 (2020)
    Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) and currently the gold-standard for the treatment of patients suffering from non-small cell lung cancer (NSCLC) harboring T790M-mutated epidermal growth factor receptor (EGFR). The outcome of the treatment, however, is limited by the emergence of the C797S resistance mutation. Allosteric inhibitors have a different mode of action and were developed to overcome this limitation. However, most of these innovative molecules are not effective as a single agent. Recently, mutated EGFR was successfully addressed with osimertinib combined with the allosteric inhibitor JBJ-04-125-02, but surprisingly, structural insights into their binding mode were lacking. Here, we present the first complex crystal structures of mutant EGFR in complex with third-generation inhibitors such as osimertinib and mavelertinib in the presence of simultaneously bound allosteric inhibitors. These structures highlight the possibility of further combinations targeting EGFR and lay the foundation for hybrid inhibitors as next-generation TKIs. © 2020 American Chemical Society.
    view abstract10.1021/acsmedchemlett.0c00472
  • Conformational selection: Vs. induced fit: Insights into the binding mechanisms of p38α MAP Kinase inhibitors
    Roser, P. and Weisner, J. and Stehle, J. and Rauh, D. and Drescher, M.
    Chemical Communications 56 (2020)
    The conformational dynamics of a kinase's activation loop have been challenging to assess due to the activation loop's intrinsic flexibility. To directly probe the conformational equilibrium of the activation loop of mitogen-activated protein kinase p38α, we present an approach based on site-directed spin labeling, electron paramagnetic resonance (EPR) distance restraints, and multilateration. We demonstrate that the activation loop of apo p38α resides in a highly flexible equilibrium state and we reveal that binding of small molecules significantly alters this equilibrium and the populated sub-states. This journal is © The Royal Society of Chemistry.
    view abstract10.1039/d0cc02539a
  • Inhibition of Tumor VEGFR2 Induces Serine 897 EphA2-Dependent Tumor Cell Invasion and Metastasis in NSCLC
    Volz, C. and Breid, S. and Selenz, C. and Zaplatina, A. and Golfmann, K. and Meder, L. and Dietlein, F. and Borchmann, S. and Chatterjee, S. and Siobal, M. and Schöttle, J. and Florin, A. and Koker, M. and Nill, M. and Ozretić, L. and Uhlenbrock, N. and Smith, S. and Büttner, R. and Miao, H. and Wang, B. and Reinhardt, H.C. and Rauh, D. and Hallek, M. and Acker-Palmer, A. and Heukamp, L.C. and Ullrich, R.T.
    Cell Reports 31 (2020)
    Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in ∼20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients. © 2020 The Author(s) Anti-angiogenic treatment targeting VEGFR2 signaling has shown limited efficacy in lung cancer patients. Volz et al. show that VEGFR2 inhibition leads to a pro-invasive phenotype in VEGFR2-positive non-small cell lung cancer cells, which is mediated by phosphorylation of EphA2-S897. © 2020 The Author(s)
    view abstract10.1016/j.celrep.2020.107568
  • KRasG12C inhibitors in clinical trials: A short historical perspective
    Goebel, L. and Müller, M.P. and Goody, R.S. and Rauh, D.
    RSC Medicinal Chemistry 11 (2020)
    KRas is the most frequently mutated oncogene in human cancer, and even 40 years after the initial discovery of Ras oncogenes in 1982, no approved drug directly targets Ras in Ras-driven cancer. New information and approaches for direct targeting of mutant Ras have fueled hope for the development of direct KRas inhibitors. In this review, we provide a comprehensive historical perspective of the development of promising KRasG12C inhibitors that covalently bind to the mutated cysteine residue in the switch-II pocket and trap the protein in the inactive GDP bound state. After decades of failure, three covalent G12C-specific inhibitors from three independent companies have recently entered clinical trials and therefore represent new hope for patients suffering from KRasG12C driven cancer. This journal is © The Royal Society of Chemistry.
    view abstract10.1039/d0md00096e
  • Spotlight on AKT: Current Therapeutic Challenges
    Landel, I. and Quambusch, L. and Depta, L. and Rauh, D.
    ACS Medicinal Chemistry Letters 11 (2020)
    The protein kinase B (Akt) exemplifies an important switch of cell death and survival within the PI3K/Akt signaling pathway, which renders Akt a valuable target in diseases such as cancer. Herein, we give a short overview of clinical applications involving Akt, outline promising and innovative approaches to investigate the role of this kinase in diseases, and highlight the current challenges that require thorough investigation to set the groundwork for successful therapeutic strategies. © 2020 American Chemical Society.
    view abstract10.1021/acsmedchemlett.9b00548
  • Structure Defines Function: Clinically Relevant Mutations in ErbB Kinases
    Niggenaber, J. and Hardick, J. and Lategahn, J. and Rauh, D.
    Journal of Medicinal Chemistry 63 (2020)
    The ErbB receptor tyrosine kinase family members EGFR (epidermal growth factor receptor) and Her2 are among the prominent mutated oncogenic drivers of non-small cell lung cancer (NSCLC). Their importance in proliferation, apoptosis, and cell death ultimately renders them hot targets in cancer therapy. Small-molecule tyrosine kinase inhibitors seem well suited to be tailor-made therapeutics for EGFR mutant NSCLC; however, drug resistance mutations limit their success. Against this background, the elucidation and visualization of the three-dimensional structure of cancer-related kinases provide valuable insights into their molecular functions. This field has undergone a revolution because X-ray crystal structure determinations aided structure-based drug design approaches and clarified the effect of activating and resistance-conferring mutations. Here, we present an overview of important mutations affecting EGFR and Her2 and highlight their influence on the kinase domain conformations and active site accessibility. © 2019 American Chemical Society.
    view abstract10.1021/acs.jmedchem.9b00964
  • Targeting Her2-insYVMA with Covalent Inhibitors - A Focused Compound Screening and Structure-Based Design Approach
    Lategahn, J. and Hardick, J. and Grabe, T. and Niggenaber, J. and Jeyakumar, K. and Keul, M. and Tumbrink, H.L. and Becker, C. and Hodson, L. and Kirschner, T. and Klövekorn, P. and Ketzer, J. and Baumann, M. and Terheyden, S. and Unger, A. and Weisner, J. and Müller, M.P. and Van Otterlo, W.A.L. and Bauer, S. and Rauh, D.
    Journal of Medicinal Chemistry 63 (2020)
    Mutated or amplified Her2 serves as a driver of non-small cell lung cancer or mediates resistance toward the inhibition of its family member epidermal growth factor receptor with small-molecule inhibitors. To date, small-molecule inhibitors targeting Her2 which can be used in clinical routine are lacking, and therefore, the development of novel inhibitors was undertaken. In this study, the well-established pyrrolopyrimidine scaffold was modified with structural motifs identified from a screening campaign with more than 1600 compounds, which were applied against wild-type Her2 and its mutant variant Her2-A775_G776insYVMA. The resulting inhibitors were designed to covalently target a reactive cysteine in the binding site of Her2 and were further optimized by means of structure-based drug design utilizing a set of obtained complex crystal structures. In addition, the analysis of binding kinetics and absorption, distribution, metabolism, and excretion parameters as well as mass spectrometry experiments and western blot analysis substantiated our approach. ©
    view abstract10.1021/acs.jmedchem.0c00870
  • 2-Azo-, 2-diazocine-thiazols and 2-azo-imidazoles as photoswitchable kinase inhibitors: Limitations and pitfalls of the photoswitchable inhibitor approach
    Schehr, M. and Ianes, C. and Weisner, J. and Heintze, L. and Müller, M.P. and Pichlo, C. and Charl, J. and Brunstein, E. and Ewert, J. and Lehr, M. and Baumann, U. and Rauh, D. and Knippschild, U. and Peifer, C. and Herges, R.
    Photochemical and Photobiological Sciences 18 (2019)
    In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors. © 2019 The Royal Society of Chemistry and Owner Societies.
    view abstract10.1039/c9pp00010k
  • A novel scaffold for EGFR inhibition: Introducing N-(3-(3-phenylureido)quinoxalin-6-yl) acrylamide derivatives
    do Amaral, D.N. and Lategahn, J. and Fokoue, H.H. and da Silva, E.M.B. and Sant’Anna, C.M.R. and Rauh, D. and Barreiro, E.J. and Laufer, S. and Lima, L.M.
    Scientific Reports 9 (2019)
    Clinical data acquired over the last decade on non-small cell lung cancer (NSCLC) treatment with small molecular weight Epidermal Growth Factor Receptor (EGFR) inhibitors have shown significant influence of EGFR point mutations and in-frame deletions on clinical efficacy. Identification of small molecules capable of inhibiting the clinically relevant EGFR mutant forms is desirable, and novel chemical scaffolds might provide knowledge regarding selectivity among EGFR forms and shed light on new strategies to overcome current clinical limitations. Design, synthesis, docking studies and in vitro evaluation of N-(3-(3-phenylureido)quinoxalin-6-yl) acrylamide derivatives (7a-m) against EGFR mutant forms are described. Compounds 7h and 7l were biochemically active in the nanomolar range against EGFRwt and EGFRL858R. Molecular docking and reaction enthalpy calculations have shown the influence of the combination of reversible and covalent binding modes with EGFR on the inhibitory activity. The inhibitory profile of 7h against a panel of patient-derived tumor cell lines was established, demonstrating selective growth inhibition of EGFR related cells at 10 μM among a panel of 30 cell lines derived from colon, melanoma, breast, bladder, kidney, prostate, pancreas and ovary tumors. © 2019, The Author(s).
    view abstract10.1038/s41598-018-36846-7
  • Characterization of Covalent Pyrazolopyrimidine-MKK7 Complexes and a Report on a Unique DFG-in/Leu-in Conformation of Mitogen-Activated Protein Kinase Kinase 7 (MKK7)
    Wolle, P. and Engel, J. and Smith, S. and Goebel, L. and Hennes, E. and Lategahn, J. and Rauh, D.
    Journal of Medicinal Chemistry 62 (2019)
    Pyrazolopyrimidines are well-established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor or Bruton's tyrosine kinase, and we recently described their potential in targeting mitogen-activated protein kinase kinase 7 (MKK7). Herein, we report the structure-activity relationship of pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation. © 2019 American Chemical Society.
    view abstract10.1021/acs.jmedchem.9b00472
  • Co-crystal structure determination and cellular evaluation of 1,4-dihydropyrazolo[4,3-c] [1,2] benzothiazine 5,5-dioxide p38α MAPK inhibitors
    Bartolini, D. and Bührmann, M. and Barreca, M.L. and Manfroni, G. and Cecchetti, V. and Rauh, D. and Galli, F.
    Biochemical and Biophysical Research Communications 511 (2019)
    p38α mitogen-activated protein kinase (MAPK) is an attracting pharmacological target in inflammatory diseases and cancer. Searching for new and more efficient p38-MAPK inhibitors, two recently developed pyrazolobenzothiazine-based (COXP4M12 and COXH11) compounds were investigated in this study using a cellular model of p38 activation. This consisted of HT29 human colorectal adenocarcinoma cells exposed to H 2 O 2 or lipopolysaccharide (LPS). Immunoblot data confirmed the inhibitory effect of COXP4M12 and COXH11 on p38 substrate phosphorylation (MAPK-APK2 and ATF2 transcription factor). Compound cytotoxicity was very low and apparent efficacy of these inhibitors was comparable with that of SB203580, a commercially available type I inhibitor of p38. All these compounds also inhibit upstream kinases that promote p38-MAPK phosphorylation and co-activate the stress-activated protein kinase JNK, while ERK1/2 MAPK phosphorylation was unaffected. Compound-target kinase interaction was investigated by means of co-crystallization experiments that provided further structural and molecular insight on the inhibitory mechanism and optimization strategy of this new class of p38-MAPK inhibitors. © 2019 Elsevier Inc.
    view abstract10.1016/j.bbrc.2019.02.063
  • Covalent-Allosteric Inhibitors to Achieve Akt Isoform-Selectivity
    Quambusch, L. and Landel, I. and Depta, L. and Weisner, J. and Uhlenbrock, N. and Müller, M.P. and Glanemann, F. and Althoff, K. and Siveke, J.T. and Rauh, D.
    Angewandte Chemie - International Edition 58 (2019)
    Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure-guided approach for the design of structurally diverse and pharmacologically beneficial covalent-allosteric modifiers, which enabled an investigation of the isoform-specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform-selective covalent-allosteric Akt inhibitors that emerged from this approach showed a conclusive structure–activity relationship and broke ground in the development of selective probes to delineate the isoform-specific functions of Akt kinases. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/anie.201909857
  • Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S
    Lategahn, J. and Keul, M. and Klövekorn, P. and Tumbrink, H.L. and Niggenaber, J. and Müller, M.P. and Hodson, L. and Flaßhoff, M. and Hardick, J. and Grabe, T. and Engel, J. and Schultz-Fademrecht, C. and Baumann, M. and Ketzer, J. and Mühlenberg, T. and Hiller, W. and Günther, G. and Unger, A. and Müller, H. and Heimsoeth, A. and Golz, C. and Blank-Landeshammer, B. and Kollipara, L. and Zahedi, R.P. and Strohmann, C. and Hengstler, J.G. and Van Otterlo, W.A.L. and Bauer, S. and Rauh, D.
    Chemical Science 10 (2019)
    Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode. This journal is © The Royal Society of Chemistry.
    view abstract10.1039/c9sc03445e
  • Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules
    Goody, R.S. and Müller, M.P. and Rauh, D.
    Cell Chemical Biology 26 (2019)
    In this review we discuss and compare recently introduced molecules that are able to react covalently with an oncogenic mutant of KRas, KRas G12C. Two different classes of compounds in question have been developed, both leading to the mutant being locked in the inactive (guanosine diphosphate [GDP]-bound) state. The first are compounds that interact reversibly with the switch-II pocket (S-IIP) before covalent interaction. The second class interact in a competitive manner with the GDP/guanosine triphosphate (GTP) binding site. The fundamental physico-chemical principles of the two inhibitor classes are evaluated. For GDP/GTP-competing molecules, we show that special attention must be paid to the influence of guanine nucleotide exchange factors (GEFs) and their elevated activity in cells harboring abnormally activated Ras mutants. A new approach is suggested involving compounds that interact with the guanine binding site of the GTPase, but in a manner that is independent of the interaction of the GTPase with its cognate GEF. © 2019 Recent work has shown that two classes of small molecules can target oncogenic KRas G12C covalently, offering a potential approach to mutant-specific cancer therapy. Goody et al. summarize these results and analyze them in detail in terms of the known kinetics of Ras, Sos, and nucleotide interactions. © 2019
    view abstract10.1016/j.chembiol.2019.07.005
  • Preclinical efficacy of covalent-allosteric AKT inhibitor borussertib in combination with trametinib in KRAS-mutant pancreatic and colorectal cancer
    Weisner, J. and Landel, I. and Reintjes, C. and Uhlenbrock, N. and Trajkovic-Arsic, M. and Dienstbier, N. and Hardick, J. and Ladigan, S. and Lindemann, M. and Smith, S. and Quambusch, L. and Scheinpflug, R. and Depta, L. and Gontla, R. and Unger, A. and Muller, H. and Baumann, M. and Schultz-Fademrecht, C. and Gunther, G. and Maghnouj, A. and Muller, M.P. and Pohl, M. and Teschendorf, C. and Wolters, H. and Viebahn, R. and Tannapfel, A. and Uhl, W. and Hengstler, J.G. and Hahn, S.A. and Siveke, J.T. and Rauh, D.
    Cancer Research 79 (2019)
    Aberrations within the PI3K/AKT signaling axis are frequently observed in numerous cancer types, highlighting the relevance of these pathways in cancer physiology and pathology. However, therapeutic interventions employing AKT inhibitors often suffer from limitations associated with target selectivity, efficacy, or dose-limiting effects. Here we present the first crystal structure of autoinhibited AKT1 in complex with the covalent-allosteric inhibitor borussertib, providing critical insights into the structural basis of AKT1 inhibition by this unique class of compounds. Comprehensive biological and preclinical evaluation of borussertib in cancer-related model systems demonstrated a strong antiproliferative activity in cancer cell lines harboring genetic alterations within the PTEN, PI3K, and RAS signaling pathways. Furthermore, borussertib displayed antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models of mutant KRAS pancreatic and colon cancer. Significance: Borussertib, a first-in-class covalent-allosteric AKT inhibitor, displays antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models and provides a starting point for further pharmacokinetic/ dynamic optimization. © 2019 American Association for Cancer Research.
    view abstract10.1158/0008-5472.CAN-18-2861
  • Structural and chemical insights into the covalent-allosteric inhibition of the protein kinase Akt
    Uhlenbrock, N. and Smith, S. and Weisner, J. and Landel, I. and Lindemann, M. and Le, T.A. and Hardick, J. and Gontla, R. and Scheinpflug, R. and Czodrowski, P. and Janning, P. and Depta, L. and Quambusch, L. and Müller, M.P. and Engels, B. and Rauh, D.
    Chemical Science 10 (2019)
    The Ser/Thr kinase Akt (Protein Kinase B/PKB) is a master switch in cellular signal transduction pathways. Its downstream signaling influences cell proliferation, cell growth, and apoptosis, rendering Akt a prominent drug target. The unique activation mechanism of Akt involves a change of the relative orientation of its N-terminal pleckstrin homology (PH) and the kinase domain and makes this kinase suitable for highly specific allosteric modulation. Here we present a unique set of crystal structures of covalent-allosteric interdomain inhibitors in complex with full-length Akt and report the structure-based design, synthesis, biological and pharmacological evaluation of a focused library of these innovative inhibitors. © 2019 The Royal Society of Chemistry.
    view abstract10.1039/c8sc05212c
  • Targeting the MKK7-JNK (Mitogen-Activated Protein Kinase Kinase 7-c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors
    Wolle, P. and Hardick, J. and Cronin, S.J.F. and Engel, J. and Baumann, M. and Lategahn, J. and Penninger, J.M. and Rauh, D.
    Journal of Medicinal Chemistry 62 (2019)
    The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold. © 2019 American Chemical Society.
    view abstract10.1021/acs.jmedchem.9b00102
  • An Unusual Intramolecular Halogen Bond Guides Conformational Selection
    Tesch, R. and Becker, C. and Müller, M.P. and Beck, M.E. and Quambusch, L. and Getlik, M. and Lategahn, J. and Uhlenbrock, N. and Costa, F.N. and Polêto, M.D. and Pinheiro, P.D.S.M. and Rodrigues, D.A. and Sant'Anna, C.M.R. and Ferreira, F.F. and Verli, H. and Fraga, C.A.M. and Rauh, D.
    Angewandte Chemie - International Edition 57 (2018)
    PIK-75 is a phosphoinositide-3-kinase (PI3K) α-isoform-selective inhibitor with high potency. Although published structure–activity relationship data show the importance of the NO2 and the Br substituents in PIK-75, none of the published studies could correctly determine the underlying reason for their importance. In this publication, we report the first X-ray crystal structure of PIK-75 in complex with the kinase GSK-3β. The structure shows an unusual U-shaped conformation of PIK-75 within the active site of GSK-3β that is likely stabilized by an atypical intramolecular Br⋅⋅⋅NO2 halogen bond. NMR and MD simulations show that this conformation presumably also exists in solution and leads to a binding-competent preorganization of the PIK-75 molecule, thus explaining its high potency. We therefore suggest that the site-specific incorporation of halogen bonds could be generally used to design conformationally restricted bioactive substances with increased potencies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/anie.201804917
  • Augmented Reality in Scientific Publications - Taking the Visualization of 3D Structures to the Next Level
    Wolle, P. and Müller, M.P. and Rauh, D.
    ACS Chemical Biology 13 (2018)
    The examination of three-dimensional structural models in scientific publications allows the reader to validate or invalidate conclusions drawn by the authors. However, either due to a (temporary) lack of access to proper visualization software or a lack of proficiency, this information is not necessarily available to every reader. As the digital revolution is quickly progressing, technologies have become widely available that overcome the limitations and offer to all the opportunity to appreciate models not only in 2D, but also in 3D. Additionally, mobile devices such as smartphones and tablets allow access to this information almost anywhere, at any time. Since access to such information has only recently become standard practice, we want to outline straightforward ways to incorporate 3D models in augmented reality into scientific publications, books, posters, and presentations and suggest that this should become general practice. © 2018 American Chemical Society.
    view abstract10.1021/acschembio.8b00153
  • C797S Resistance: The Undruggable EGFR Mutation in Non-Small Cell Lung Cancer?
    Grabe, T. and Lategahn, J. and Rauh, D.
    ACS Medicinal Chemistry Letters 9 (2018)
    The first evidence of osimertinib resistance mediated by the epidermal growth factor receptor (EGFR) mutation C797S was reported three years ago. Since then, no major breakthroughs have been achieved to target the clinically relevant mutant variant that impedes covalent bond formation with irreversible EGFR inhibitors. Although several biochemically active compounds have been described, only a few inhibitors that potently act on the cellular level or in vivo have been introduced so far. Herein, we give an overview of current approaches in the field and highlight the challenges that need to be addressed in future research projects to overcome the C797S-mediated drug resistance. © 2018 American Chemical Society.
    view abstract10.1021/acsmedchemlett.8b00314
  • Chemical modulation of transcription factors
    Wiedemann, B. and Weisner, J. and Rauh, D.
    MedChemComm 9 (2018)
    Transcription factors (TFs) constitute a diverse class of sequence-specific DNA-binding proteins, which are key to the modulation of gene expression. TFs have been associated with human diseases, including cancer, Alzheimer's and other neurodegenerative diseases, which makes this class of proteins attractive targets for chemical biology and medicinal chemistry research. Since TFs lack a common binding site or structural similarity, the development of small molecules to efficiently modulate TF biology in cells and in vivo is a challenging task. This review highlights various strategies that are currently being explored for the identification and development of modulators of Myc, p53, Stat, Nrf2, CREB, ER, AR, HIF, NF-κB, and BET proteins. © The Royal Society of Chemistry.
    view abstract10.1039/c8md00273h
  • Direct monitoring of the conformational equilibria of the activation loop in the mitogen-activated protein kinase p38α
    Roser, P. and Weisner, J. and Simard, J.R. and Rauh, D. and Drescher, M.
    Chemical Communications 54 (2018)
    Conformational transitions in protein kinases are crucial for the biological function of these enzymes. Here, we characterize and assess conformational equilibria of the activation loop and the effect of small molecule inhibitors in the MAP kinase p38α. Our work experimentally revealed the existence of a two-state equilibrium for p38α while the addition of inhibitors shifts the equilibrium between these two states. © 2018 The Royal Society of Chemistry.
    view abstract10.1039/c8cc06128a
  • Donated chemical probes for open science
    Müller, S. and Ackloo, S. and Arrowsmith, C.H. and Bauser, M. and Baryza, J.L. and Blagg, J. and Böttcher, J. and Bountra, C. and Brown, P.J. and Bunnage, M.E. and Carter, A.J. and Damerell, D. and Dötsch, V. and Drewry, D.H. and Edwards, A.M. and Edwards, J. and Elkins, J.M. and Fischer, C. and Frye, S.V. and Gollner, A. and Grimshaw, C.E. and IJzerman, A. and Hanke, T. and Hartung, I.V. and Hitchcock, S. and Howe, T. and Hughes, T.V. and Laufer, S. and Li, V.M. and Liras, S. and Marsden, B.D. and Matsui, H. and Mathias, J. and O'Hagan, R.C. and Owen, D.R. and Pande, V. and Rauh, D. and Rosenberg, S.H. and Roth, B.L. and Schneider, N.S. and Scholten, C. and Singh Saikatendu, K. and Simeonov, A. and Takizawa, M. and Tse, C. and Thompson, P.R. and Treiber, D.K. and Viana, A.Y. and Wells, C.I. and Willson, T.M. and Zuercher, W.J. and Knapp, S. and Mueller-Fahrnow, A.
    eLife 7 (2018)
    Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (<ext-link ext-link-type="uri" xlink:href=""></ext-link><ext-link ext-link-type="uri" xlink:href="">/</ext-link>). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project. © 2018, Müller et al.
    view abstract10.7554/eLife.34311
  • Insights into the Kinetics of the Resistance Formation of Bacteria against Ciprofloxacin Poly(2-methyl-2-oxazoline) Conjugates
    Schmidt, M. and Romanovska, A. and Wolf, Y. and Nguyen, T.-D. and Krupp, A. and Tumbrink, H.L. and Lategahn, J. and Volmer, J. and Rauh, D. and Luetz, S. and Krumm, C. and Tiller, J.C.
    Bioconjugate Chemistry 29 (2018)
    The influence on the resistance formation of polymers attached to antibiotics has rarely been investigated. In this study, ciprofloxacin (CIP) was conjugated to poly(2-methyl-2-oxazoline)s with an ethylene diamine end group (Me-PMOx28-EDA) via two different spacers (CIP modified with α,α′-dichloro-p-xylene - xCIP, CIP modified with chloroacetyl chloride - eCIP). The antibacterial activity of the conjugates against a number of bacterial strains shows a great dependence on the nature of the spacer. The Me-PMOx39-EDA-eCIP, containing a potentially cleavable linker, does not exhibit a molecular weight dependence on antibacterial activity in contrast to Me-PMOx27-EDA-xCIP. The resistance formation of both conjugates against Staphylococcus aureus and Escherichia coli was investigated. Both conjugates showed the potential to significantly delay the formation of resistant bacteria compared to the unmodified CIP. Closer inspection of a possible resistance mechanism by genome sequencing of the topoisomerase IV region of resistant S. aureus revealed that this bacterium mutates at the same position when building up resistance to CIP and to Me-PMOx27-EDA-xCIP. However, the S. aureus cells that became resistant against the polymer conjugate are fully susceptible to CIP. Thus, conjugation of CIP with PMOx seems to alter the resistance mechanism. © 2018 American Chemical Society.
    view abstract10.1021/acs.bioconjchem.8b00361
  • Lessons To Be Learned: The Molecular Basis of Kinase-Targeted Therapies and Drug Resistance in Non-Small Cell Lung Cancer
    Lategahn, J. and Keul, M. and Rauh, D.
    Angewandte Chemie - International Edition 57 (2018)
    The treatment of non-small cell lung cancer (NSCLC) is currently experiencing a revolution. Over the last decade, the knowledge gained about the biochemical features of biomarkers and their predictive abilities has led to the development of targeted small-molecule inhibitors that present an alternative to harsh chemotherapy. The use of these new therapies has improved the quality of life and increased the survival of patients. The occurrence of inevitable drug resistance requires the constant development of precision medicine. The detailed understanding of the target biology and the search for innovative chemical approaches has encouraged investigations in this field. Herein, we review selected aspects of the molecular targets and present an overview of current topics and challenges in the rational development of small molecules to target NSCLC. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/anie.201710398
  • Overcoming EGFR G724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors
    Fassunke, J. and Müller, F. and Keul, M. and Michels, S. and Dammert, M.A. and Schmitt, A. and Plenker, D. and Lategahn, J. and Heydt, C. and Brägelmann, J. and Tumbrink, H.L. and Alber, Y. and Klein, S. and Heimsoeth, A. and Dahmen, I. and Fischer, R.N. and Scheffler, M. and Ihle, M.A. and Priesner, V. and Scheel, A.H. and Wagener, S. and Kron, A. and Frank, K. and Garbert, K. and Persigehl, T. and Püsken, M. and Haneder, S. and Schaaf, B. and Rodermann, E. and Engel-Riedel, W. and Felip, E. and Smit, E.F. and Merkelbach-Bruse, S. and Reinhardt, H.C. and Kast, S.M. and Wolf, J. and Rauh, D. and Büttner, R. and Sos, M.L.
    Nature Communications 9 (2018)
    The emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFRT790M-negative but EGFRG724S-positive subclones and osimertinib resistance. We demonstrate that EGFRG724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFRG724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFRG724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFRG724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFRG724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors. © 2018, The Author(s).
    view abstract10.1038/s41467-018-07078-0
  • RASPELD to Perform High-End Screening in an Academic Environment toward the Development of Cancer Therapeutics
    Wolle, P. and Weisner, J. and Keul, M. and Landel, I. and Lategahn, J. and Rauh, D.
    ChemMedChem 13 (2018)
    The identification of compounds for dissecting biological functions and the development of novel drug molecules are central tasks that often require screening campaigns. However, the required architecture is cost- and time-intensive. Herein we describe the devices and technologies that comprise a Robotics-Assisted Screening Platform for Efficient Ligand Discovery (RASPELD), which we set up in an academic laboratory. RASPELD provides semi-automated high-end screening, and it can be maintained by graduate students. We demonstrate its successful application in biochemical and cellular screens for the identification and validation of bioactive chemical entities as candidate cancer-relevant inhibitors. Specifically, we examined the interaction between a transcription factor, Nrf2, and its key regulator, Keap1. We also examined drug-resistant mutants of the epidermal growth factor receptor (EGFR). Screening campaigns with more than 30 000 compounds were performed in a reasonable period of time. We identified the molecule RSL6586 as a starting point for hit optimization, which is currently ongoing. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/cmdc.201800477
  • Try Me: Promiscuous Inhibitors Still Allow for Selective Targeted Protein Degradation
    Müller, M.P. and Rauh, D.
    Cell Chemical Biology 25 (2018)
    In this issue of Cell Chemical Biology, Bondeson et al. (2018), Burslem et al. (2018), and Huang et al. (2018) systematically characterize proteolysis-targeting chimeras (PROTACs) regarding their specificity and general advantages of targeted proteolysis of cellular proteins and provide interesting insights into possible future developments. In this issue of Cell Chemical Biology, Bondeson et al. (2018), Burslem et al. (2018), and Huang et al. (2018) systematically characterize proteolysis-targeting chimeras (PROTACs) regarding their specificity and general advantages of targeted proteolysis of cellular proteins and provide interesting insights into possible future developments. © 2018 Elsevier Ltd
    view abstract10.1016/j.chembiol.2018.01.004
  • Characterization of Covalent-Reversible EGFR Inhibitors
    Smith, S. and Keul, M. and Engel, J. and Basu, D. and Eppmann, S. and Rauh, D.
    ACS Omega 2 (2017)
    Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRIs) provide a valuable alternative approach to classical covalent inhibitors. This special class of inhibitors can be optimized for an extended drug-target residence time. For CRIs, it was shown that the fast addition of thiols to electron-deficient olefins leads to a covalent bond that can break reversibly under proteolytic conditions. Research groups are just beginning to include CRIs in their arsenal of compound classes, and, with that, the understanding of this interesting set of chemical warheads is growing. However, systems to assess both characteristics of the covalent-reversible bond in a simple experimental setting are sparse. Here, we have developed an efficient methodology to characterize the covalent and reversible properties of CRIs and to investigate their potential in targeting clinically relevant variants of the receptor tyrosine kinase EGFR. © 2017 American Chemical Society.
    view abstract10.1021/acsomega.7b00157
  • Covalent Lipid Pocket Ligands Targeting p38α MAPK Mutants
    Bührmann, M. and Hardick, J. and Weisner, J. and Quambusch, L. and Rauh, D.
    Angewandte Chemie - International Edition 56 (2017)
    A chemical genetic approach is presented to covalently target a unique lipid binding pocket in the protein kinase p38α, whose function is not yet known. Based on a series of cocrystal structures, a library of 2-arylquinazolines that were decorated with electrophiles were designed and synthesized to covalently target tailored p38α mutants containing artificially introduced cysteine residues. Matching protein–ligand pairs were identified by MS analysis and further validated by MS/MS studies and protein crystallography. The covalent ligands that emerged from this approach showed excellent selectivity towards a single p38α mutant and will be applicable as suitable probes in future studies of biological systems to dissect the function of the lipid pocket by means of pharmacological perturbations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/anie.201706345
  • Design, Synthesis, and Biological Evaluation of Novel Type I1/2 p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R-Spine
    Walter, N.M. and Wentsch, H.K. and Bührmann, M. and Bauer, S.M. and Döring, E. and Mayer-Wrangowski, S. and Sievers-Engler, A. and Willemsen-Seegers, N. and Zaman, G. and Buijsman, R. and Lämmerhofer, M. and Rauh, D. and Laufer, S.A.
    Journal of Medicinal Chemistry 60 (2017)
    We recently reported 1a (skepinone-L) as a type I p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, as a type I inhibitor, it is entirely ATP-competitive and shows just a moderate residence time. Thus, the scope was to develop a new class of advanced compounds maintaining the structural binding features of skepinone-L scaffold like inducing a glycine flip at the hinge region and occupying both hydrophobic regions I and II. Extending this scaffold with suitable residues resulted in an interference with the kinase's R-Spine. By synthesizing 69 compounds, we could significantly prolong the target residence time with one example to 3663 s, along with an excellent selectivity score of 0.006 and an outstanding potency of 1.0 nM. This new binding mode was validated by cocrystallization, showing all binding interactions typifying type I1/2 binding. Moreover, microsomal studies showed convenient metabolic stability of the most potent, herein reported representatives. © 2017 American Chemical Society.
    view abstract10.1021/acs.jmedchem.7b00745
  • Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor
    Tomassi, S. and Lategahn, J. and Engel, J. and Keul, M. and Tumbrink, H.L. and Ketzer, J. and Mühlenberg, T. and Baumann, M. and Schultz-Fademrecht, C. and Bauer, S. and Rauh, D.
    Journal of Medicinal Chemistry 60 (2017)
    The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder. © 2017 American Chemical Society.
    view abstract10.1021/acs.jmedchem.6b01626
  • Inhibitors to Overcome Secondary Mutations in the Stem Cell Factor Receptor KIT
    Kaitsiotou, H. and Keul, M. and Hardick, J. and Mühlenberg, T. and Ketzer, J. and Ehrt, C. and Krüll, J. and Medda, F. and Koch, O. and Giordanetto, F. and Bauer, S. and Rauh, D.
    Journal of Medicinal Chemistry 60 (2017)
    In modern cancer therapy, the use of small organic molecules against receptor tyrosine kinases (RTKs) has been shown to be a valuable strategy. The association of cancer cells with dysregulated signaling pathways linked to RTKs represents a key element in targeted cancer therapies. The tyrosine kinase mast/stem cell growth factor receptor KIT is an example of a clinically relevant RTK. KIT is targeted for cancer therapy in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). However, acquired resistance mutations within the catalytic domain decrease the efficacy of this strategy and are the most common cause of failed therapy. Here, we present the structure-based design and synthesis of novel type II kinase inhibitors to overcome these mutations in KIT. Biochemical and cellular studies revealed promising molecules for the inhibition of mutated KIT. © 2017 American Chemical Society.
    view abstract10.1021/acs.jmedchem.7b00841
  • Optimized 4,5-diarylimidazoles as potent/selective inhibitors of Protein Kinase CK1δ and their structural relation to P38α MAPK
    Halekotte, J. and Witt, L. and Ianes, C. and Krüger, M. and Bührmann, M. and Rauh, D. and Pichlo, C. and Brunstein, E. and Luxenburger, A. and Baumann, U. and Knippschild, U. and Bischof, J. and Peifer, C. and Koch, P. and Laufer, S.
    Molecules 22 (2017)
    The involvement of protein kinase CK1δ in the pathogenesis of severe disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, familial advanced sleep phase syndrome, and cancer has dramatically increased interest in the development of effective small molecule inhibitors for both therapeutic application and basic research. Unfortunately the design of CK1 isoform-specific compounds has proved to be highly complicated due to the existence of six evolutionarily conserved human CK1 members that possess similar, different, or even opposite physiological and pathophysiological implications. Consequently only few potent and selective CK1δ inhibitors have been reported so far and structurally divergent approaches are urgently needed in order to establish SAR that might enable complete discrimination of CK1 isoforms and related p38α MAPK. In this study we report on design and characterization of optimized 4,5-diarylimidazoles as highly effective ATP-competitive inhibitors of CK1δ with compounds 11b (IC50 CK1δ = 4 nM, IC50 CK1ϵ = 25 nM), 12a (IC50 CK1δ = 19 nM, IC50 CK1ϵ = 227 nM), and 16b (IC50 CK1δ = 8 nM, IC50 CK1ϵ = 81 nM) being among the most potent CK1δ-targeting agents published to date. Inhibitor compound 11b, displaying potential as a pharmacological tool, has further been profiled over a panel of 321 protein kinases exhibiting high selectivity. Cellular efficacy has been evaluated in human pancreatic cancer cell lines Colo357 (EC50 = 3.5 μM) and Panc89 (EC50 = 1.5 μM). SAR is substantiated by X-ray crystallographic analysis of 16b in CK1δ and 11b in p38α. © 2017 by the authors.
    view abstract10.3390/molecules22040522
  • Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine
    Wentsch, H.K. and Walter, N.M. and Bührmann, M. and Mayer-Wrangowski, S. and Rauh, D. and Zaman, G.J.R. and Willemsen-Seegers, N. and Buijsman, R.C. and Henning, M. and Dauch, D. and Zender, L. and Laufer, S.
    Angewandte Chemie - International Edition 56 (2017)
    Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I 1/2 binders for p38α MAP kinase. Type I 1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/anie.201701185
  • Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34
    Robke, L. and Laraia, L. and Carnero Corrales, M.A. and Konstantinidis, G. and Muroi, M. and Richters, A. and Winzker, M. and Engbring, T. and Tomassi, S. and Watanabe, N. and Osada, H. and Rauh, D. and Waldmann, H. and Wu, Y.-W. and Engel, J.
    Angewandte Chemie - International Edition 56 (2017)
    Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/anie.201703738
  • Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK
    Bührmann, M. and Wiedemann, B.M. and Müller, M.P. and Hardick, J. and Ecke, M. and Rauh, D.
    PLoS ONE 12 (2017)
    In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography. © 2017 Bührmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    view abstract10.1371/journal.pone.0184627
  • Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor
    Engel, J. and Smith, S. and Lategahn, J. and Tumbrink, H.L. and Goebel, L. and Becker, C. and Hennes, E. and Keul, M. and Unger, A. and Müller, H. and Baumann, M. and Schultz-Fademrecht, C. and Günther, G. and Hengstler, J.G. and Rauh, D.
    Journal of Medicinal Chemistry 60 (2017)
    Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile. © 2017 American Chemical Society.
    view abstract10.1021/acs.jmedchem.7b00515
  • Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma
    Brägelmann, J. and Dammert, M.A. and Dietlein, F. and Heuckmann, J.M. and Choidas, A. and Böhm, S. and Richters, A. and Basu, D. and Tischler, V. and Lorenz, C. and Habenberger, P. and Fang, Z. and Ortiz-Cuaran, S. and Leenders, F. and Eickhoff, J. and Koch, U. and Getlik, M. and Termathe, M. and Sallouh, M. and Greff, Z. and Varga, Z. and Balke-Want, H. and French, C.A. and Peifer, M. and Reinhardt, H.C. and Örfi, L. and Kéri, G. and Ansén, S. and Heukamp, L.C. and Büttner, R. and Rauh, D. and Klebl, B.M. and Thomas, R.K. and Sos, M.L.
    Cell Reports 20 (2017)
    Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients. © 2017 The Authors
    view abstract10.1016/j.celrep.2017.08.082
  • Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically Resistant L858R/T790M/C797S EGFR Mutant: Targeting of Both Hydrophobic Regions and the Phosphate Binding Site
    Günther, M. and Lategahn, J. and Juchum, M. and Döring, E. and Keul, M. and Engel, J. and Tumbrink, H.L. and Rauh, D. and Laufer, S.
    Journal of Medicinal Chemistry 60 (2017)
    Inhibition of the epidermal growth factor receptor represents one of the most promising strategies in the treatment of lung cancer. Acquired resistance compromises the clinical efficacy of EGFR inhibitors during long-term treatment. The recently discovered EGFR-C797S mutation causes resistance against third-generation EGFR inhibitors. Here we present a rational approach based on extending the inhibition profile of a p38 MAP kinase inhibitor toward mutant EGFR inhibition. We used a privileged scaffold with proven cellular potency as well as in vivo efficacy and low toxicity. Guided by molecular modeling, we synthesized and studied the structure-activity relationship of 40 compounds against clinically relevant EGFR mutants. We successfully improved the cellular EGFR inhibition down to the low nanomolar range with covalently binding inhibitors against a gefitinib resistant T790M mutant cell line. We identified additional noncovalent interactions, which allowed us to develop metabolically stable inhibitors with high activities against the osimertinib resistant L858R/T790M/C797S mutant. © 2017 American Chemical Society.
    view abstract10.1021/acs.jmedchem.7b00316
  • A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant
    Radi, M. and Schneider, R. and Fallacara, A.L. and Botta, L. and Crespan, E. and Tintori, C. and Maga, G. and Kissova, M. and Calgani, A. and Richters, A. and Musumeci, F. and Rauh, D. and Schenone, S.
    Bioorganic and Medicinal Chemistry Letters 26 (2016)
    The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor. © 2016 Elsevier Ltd
    view abstract10.1016/j.bmcl.2016.06.051
  • Hope and Disappointment: Covalent Inhibitors to Overcome Drug Resistance in Non-Small Cell Lung Cancer
    Engel, J. and Lategahn, J. and Rauh, D.
    ACS Medicinal Chemistry Letters 7 (2016)
    In the last five years, the detailed understanding of how to overcome T790M drug resistance in non-small cell lung cancer (NSCLC) has culminated in the development of a third-generation of covalent EGFR inhibitors with excellent clinical outcomes. However, the emergence of a newly discovered acquired drug resistance challenges the concept of small molecule targeted cancer therapy in NSCLC. © 2015 American Chemical Society.
    view abstract10.1021/acsmedchemlett.5b00475
  • Insight into the Inhibition of Drug-Resistant Mutants of the Receptor Tyrosine Kinase EGFR
    Engel, J. and Becker, C. and Lategahn, J. and Keul, M. and Ketzer, J. and Mühlenberg, T. and Kollipara, L. and Schultz-Fademrecht, C. and Zahedi, R.P. and Bauer, S. and Rauh, D.
    Angewandte Chemie - International Edition 55 (2016)
    Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/anie.201605011
  • Monitoring Conformational Changes in the Receptor Tyrosine Kinase EGFR
    Becker, C. and Öcal, S. and Nguyen, H.D. and Phan, T. and Keul, M. and Simard, J.R. and Rauh, D.
    ChemBioChem 17 (2016)
    The receptor tyrosine kinase EGFR is regulated by complex conformational changes, and this conformational control is disturbed in certain types of cancer. Many ligands are known to bind EGFR in its active conformation, thereby preventing ATP from binding. Only a few ligands are known to stabilize EGFR in its inactive conformation, thus providing novel strategies for perturbing EGFR activity. We report a direct binding assay that enables the identification of novel ligands that bind to and stabilize the inactive conformation of EGFR. Too active? Stabilize the inactive conformation. EGFR is a target for tumor therapy, with many small molecules known to bind its kinase domain in the active conformation. A binding assay was developed to search for ligands stabilizing its inactive conformation, thus providing a tool for new approaches to target its dynamic regulatory mechanisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/cbic.201600115
  • A Synergistic Interaction between Chk1- and MK2 Inhibitors in KRAS-Mutant Cancer
    Dietlein, F. and Kalb, B. and Jokic, M. and Noll, E.M. and Strong, A. and Tharun, L. and Ozretić, L. and Künstlinger, H. and Kambartel, K. and Randerath, W.J. and Jüngst, C. and Schmitt, A. and Torgovnick, A. and Richters, A. and Rauh, D. and Siedek, F. and Persigehl, T. and Mauch, C. and Bartkova, J. and Bradley, A. and Sprick, M.R. and Trumpp, A. and Rad, R. and Saur, D. and Bartek, J. and Wolf, J. and Büttner, R. and Thomas, R.K. and Reinhardt, H.C.
    Cell 162 (2015)
    Summary KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers. © 2015 Elsevier Inc.
    view abstract10.1016/j.cell.2015.05.053
  • Combining X-ray crystallography and molecular modeling toward the optimization of pyrazolo[3,4-d ]pyrimidines as potent c-Src inhibitors active in vivo against neuroblastoma
    Tintori, C. and Fallacara, A.L. and Radi, M. and Zamperini, C. and Dreassi, E. and Crespan, E. and Maga, G. and Schenone, S. and Musumeci, F. and Brullo, C. and Richters, A. and Gasparrini, F. and Angelucci, A. and Festuccia, C. and Delle Monache, S. and Rauh, D. and Botta, M.
    Journal of Medicinal Chemistry 58 (2015)
    c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells. © 2014 American Chemical Society.
    view abstract10.1021/jm5013159
  • Covalent-Allosteric Kinase Inhibitors
    Weisner, J. and Gontla, R. and Van der westhuizen, L. and Oeck, S. and Ketzer, J. and Janning, P. and Richters, A. and Mühlenberg, T. and Fang, Z. and Taher, A. and Jendrossek, V. and Pelly, S.C. and Bauer, S. and Van otterlo, W.A.L. and Rauh, D.
    Angewandte Chemie - International Edition 54 (2015)
    Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/anie.201502142
  • Discovery of inter-domain stabilizers-a novel assay system for allosteric Akt inhibitors
    Fang, Z. and Simard, J.R. and Plenker, D. and Nguyen, H.D. and Phan, T. and Wolle, P. and Baumeister, S. and Rauh, D.
    ACS Chemical Biology 10 (2015)
    In addition to the catalytically active kinase domain, most kinases feature regulatory domains that govern their activity. Modulating and interfering with these interdomain interactions presents a major opportunity for understanding biological systems and developing novel therapeutics. Therefore, small molecule inhibitors that target these interactions through an allosteric mode of action have high intrinsic selectivity, as these interactions are often unique to a single kinase or kinase family. Here we report the development of iFLiK (interface-Fluorescent Labels in Kinases), a fluorescence-based assay that can monitor such interdomain interactions. Using iFLiK, we have demonstrated selective detection of allosteric Akt inhibitors that induce an inactive closed conformation unique to Akt. This methodology easily distinguished small molecule allosteric inhibitors from classic ATP-competitive inhibitors. Screening an in-house compound library with iFLiK, we were able to identify novel compounds with a scaffold that has not been previously described for allosteric Akt inhibitors. © 2014 American Chemical Society.
    view abstract10.1021/cb500355c
  • Identification and further development of potent TBK1 inhibitors
    Richters, A. and Basu, D. and Engel, J. and Ercanoglu, M.S. and Balke-Want, H. and Tesch, R. and Thomas, R.K. and Rauh, D.
    ACS Chemical Biology 10 (2015)
    The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules. © 2014 American Chemical Society.
    view abstract10.1021/cb500908d
  • Intermittent high-dose treatment with erlotinib enhances therapeutic efficacy in EGFR-mutant lung cancer
    Schöttle, J. and Chatterjee, S. and Volz, C. and Siobal, M. and Florin, A. and Rokitta, D. and Hinze, Y. and Dietlein, F. and Plenker, D. and König, K. and Albus, K. and Heuckmann, J.M. and Rauh, D. and Franz, T. and Neumaier, B. and Fuhr, U. and Heukamp, L.C. and Ullrich, R.T.
    Oncotarget 6 (2015)
    Treatment with EGFR kinase inhibitors improves progression-free survival of patients with EGFR-mutant lung cancer. However, all patients with initial response will eventually acquire resistance and die from tumor recurrence. We found that intermittent high-dose treatment with erlotinib induced apoptosis more potently and improved tumor shrinkage significantly than the established low doses. In mice carrying EGFR-mutant xenografts intermittent high-dose treatment (200 mg/kg every other day) was tolerable and prolonged progression-free survival and reduced the frequency of acquired resistance. Intermittent EGFR-targeted high-dose schedules induce more profound as well as sustained target inhibition and may afford enhanced therapeutic efficacy.
    view abstract10.18632/oncotarget.6276
  • Monitoring ligand-induced conformational changes for the identification of estrogen receptor agonists and antagonists
    Mayer-Wrangowski, S.C. and Rauh, D.
    Angewandte Chemie - International Edition 54 (2015)
    Nuclear receptors are transcription factors that are important targets for current drug discovery efforts as they play a role in many pathological processes. Their activity can be regulated by small molecules like hormones and drugs that can have agonistic or antagonistic functions. These ligands bind to the receptor and account for diverse conformational changes that are crucial determinants for the receptor activity. Here, we set out to develop FLiN (fluorescent labels in nuclear receptors), a direct binding assay that detects conformational changes in the estrogen receptor. The assay is based on the introduction of a cysteine residue and subsequent specific labeling of the receptor with a thiol-reactive fluorophore. Changes in the receptor conformation upon ligand binding lead to differences in the microenvironment of the fluorophore and alter its emission spectrum. The FLiN assay distinguishes between different binding modes and is suitable for high-throughput screening. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/anie.201410148
  • Neuritogenic Militarinone-Inspired 4-Hydroxypyridones Target the Stress Pathway Kinase MAP4K4
    Schröder, P. and Förster, T. and Kleine, S. and Becker, C. and Richters, A. and Ziegler, S. and Rauh, D. and Kumar, K. and Waldmann, H.
    Angewandte Chemie - International Edition 54 (2015)
    Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4-hydroxy-2-pyridone core structure induce pronounced neurite outgrowth, but their protein target has not been identified. Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The most potent 4-hydroxy-2-pyridone is a selective ATP-competitive inhibitor of MAP4K4 but not of the other stress pathway related kinases, as proven by biochemical analysis and by a crystal structure of the inhibitor in complex with MAP4K4. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases. No stress: Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/anie.201501515
  • Special issue focused on two areas pertinent to chemical biology: Post-translational modifications and new frontiers on kinases
    Rauh, D.
    ACS Chemical Biology 10 (2015)
    view abstract10.1021/acschembio.5b00010
  • Structure-based design and synthesis of covalent-reversible inhibitors to overcome drug resistance in EGFR
    Basu, D. and Richters, A. and Rauh, D.
    Bioorganic and Medicinal Chemistry 23 (2015)
    The clinical success of covalent kinase inhibitors in the treatment of EGFR-dependent non-small cell lung cancer (NSCLC) has rejuvenated the appreciation of reactive small molecules. Acquired drug resistance against first-line EGFR inhibitors remains the major bottleneck in NSCLC and is currently addressed by the application of fine-tuned covalent drugs. Here we report the design, synthesis and biochemical evaluation of a novel class of EGFR inhibitors with a covalent yet reversible warhead. A series of WZ4002 analogs, derived from anilinopyrimidine and 3-substituted-2-cyanoacrylamide scaffolds, exhibit strong and selective inhibitory activity against clinically relevant EGFRL858R and EGFRL858R/T790M. © 2015 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.bmc.2015.04.038
  • Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach
    Engel, J. and Richters, A. and Getlik, M. and Tomassi, S. and Keul, M. and Termathe, M. and Lategahn, J. and Becker, C. and Mayer-Wrangowski, S. and Grütter, C. and Uhlenbrock, N. and Krüll, J. and Schaumann, N. and Eppmann, S. and Kibies, P. and Hoffgaard, F. and Heil, J. and Menninger, S. and Ortiz-Cuaran, S. and Heuckmann, J.M. and Tinnefeld, V. and Zahedi, R.P. and Sos, M.L. and Schultz-Fademrecht, C. and Thomas, R.K. and Kast, S.M. and Rauh, D.
    Journal of Medicinal Chemistry 58 (2015)
    Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797. © 2015 American Chemical Society.
    view abstract10.1021/acs.jmedchem.5b01082
  • A special thematic compilation/special issue crossover with biochemistry, journal of medicinal chemistry, and ACS medicinal chemistry letters focused on kinases
    Rauh, D.
    ACS Chemical Biology 9 (2014)
    view abstract10.1021/cb500150d
  • Cell-autonomous and non-cell-autonomous mechanisms of transformation by amplified FGFR1 in lung cancer
    Malchers, F. and Dietlein, F. and Schöttle, J. and Lu, X. and Nogova, L. and Albus, K. and Fernandez-Cuesta, L. and Heuckmann, J.M. and Gautschi, O. and Diebold, J. and Plenker, D. and Gardizi, M. and Scheffler, M. and Bos, M. and Seidel, D. and Leenders, F. and Richters, A. and Peifer, M. and Florin, A. and Mainkar, P.S. and Karre, N. and Chandrasekhar, S. and George, J. and Silling, S. and Rauh, D. and Zander, T. and Ullrich, R.T. and Christian Reinhardt, H. and Ringeisen, F. and Büttner, R. and Heukamp, L.C. and Wolf, J. and Thomas, R.K.
    Cancer Discovery 4 (2014)
    The 8p12 locus (containing the FGFR1 tyrosine kinase gene) is frequently amplified in squamous cell lung cancer. However, it is currently unknown which of the 8p12- amplified tumors are also sensitive to fibroblast growth factor receptor (FGFR) inhibition. We found that, in contrast with other recurrent amplifications, the 8p12 region included multiple centers of amplification, suggesting marked genomic heterogeneity. FGFR1 -amplified tumor cells were dependent on FGFR ligands in vitro and in vivo. Furthermore, ectopic expression of FGFR1 was oncogenic, which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of FGFR1 - amplified tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting that patients with FGFR1- amplified and MYC-overexpressing tumors may benefit from FGFR inhibitor therapy. Thus, both cell-autonomous and non-cell-autonomous mechanisms of transformation modulate FGFR dependency in FGFR1 -amplified lung cancer, which may have implications for patient selection for treatment with FGFR inhibitors. © 2014 American Association for Cancer Research.
    view abstract10.1158/2159-8290.CD-13-0323
  • Correlating structure and ligand affinity in drug discovery: A cautionary tale involving second shell residues
    Tziridis, A. and Rauh, D. and Neumann, P. and Kolenko, P. and Menzel, A. and Bräuer, U. and Ursel, C. and Steinmetzer, P. and Stürzebecher, J. and Schweinitz, A. and Steinmetzer, T. and Stubbs, M.T.
    Biological Chemistry 395 (2014)
    A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.
    view abstract10.1515/hsz-2014-0158
  • FLiK and FLiP: Direct Binding Assays for the Identification of Stabilizers of Inactive Kinase and Phosphatase Conformations
    Rauh, D. and Simard, J.R.
    Concepts and Case Studies in Chemical Biology (2014)
    Protein function is often modulated by the events of phosphorylation and dephosphorylation. Over the last two decades, targeting these events with small organic molecules is a primary focus of chemical biology and medicinal chemistry research. However, the conserved nature of both the nucleotide binding sites (kinases) and phosphate binding sites (phosphatases) contributes to reduced inhibitor selectivity and limited efficacy. To address this problem, a series of fluorescent-based high-throughput screening technologies were developed that can discriminate for binders and inhibitors, which stabilize inactive enzyme conformations by binding within less conserved allosteric pockets. © 2014 Wiley-VCH Verlag & Co. KGaA. All rights reserved.
    view abstract10.1002/9783527687503.ch2
  • FLiK: A direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations
    Simard, J.R. and Rauh, D.
    Methods in Enzymology 548 (2014)
    Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the Kd of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput. © 2014 Elsevier Inc. All rights reserved.
    view abstract10.1016/B978-0-12-397918-6.00006-9
  • Identification of type II and III DDR2 inhibitors
    Richters, A. and Nguyen, H.D. and Phan, T. and Simard, J.R. and Grütter, C. and Engel, J. and Rauh, D.
    Journal of Medicinal Chemistry 57 (2014)
    Discoidin domain-containing receptors (DDRs) exhibit a unique mechanism of action among the receptor tyrosine kinases (RTKs) because their catalytic activity is induced by extracellular collagen binding. Moreover, they are essential components in the assimilation of extracellular signals. Recently, DDRs were reported to be significantly linked to tumor progression in breast cancer by facilitating the processes of invasion, migration, and metastasis. Here, we report the successful development of a fluorescence-based, direct binding assay for the detection of type II and III DFG-out binders for DDR2. Using sequence alignments and homology modeling, we designed a DDR2 construct appropriate for fluorescent labeling. Successful assay development was validated by sensitive detection of a reference DFG-out binder. Subsequent downscaling led to convenient application to high-throughput screening formats. Screening of a representative compound library identified high-affinity DDR2 ligands validated by orthogonal activity-based assays, and a subset of identified compounds was further investigated with respect to DDR1 inhibition. © 2014 American Chemical Society.
    view abstract10.1021/jm500167q
  • An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms
    Rauh, D. and Fischer, F. and Gertz, M. and Lakshminarasimhan, M. and Bergbrede, T. and Aladini, F. and Kambach, C. and Becker, C. F. W. and Zerweck, J. and Schutkowski, M. and Steegborn, C.
    Nature Communications 4 (2013)
    Sirtuin enzymes regulate metabolism and aging processes through deacetylation of acetyl-lysines in target proteins. More than 6,800 mammalian acetylation sites are known, but few targets have been assigned to most sirtuin isoforms, hampering our understanding of sirtuin function. Here we describe a peptide microarray system displaying 6,802 human acetylation sites for the parallel characterisation of their modification by deacetylases. Deacetylation data for all seven human sirtuins obtained with this system reveal isoform-specific substrate preferences and deacetylation substrate candidates for all sirtuin isoforms, including Sirt4. We confirm malate dehydrogenase protein as a Sirt3 substrate and show that peroxiredoxin 1 and high-mobility group B1 protein are deacetylated by Sirt5 and Sirt1, respectively, at the identified sites, rendering them likely new in vivo substrates. Our microarray platform enables parallel studies on physiological acetylation sites and the deacetylation data presented provide an exciting resource for the identification of novel substrates for all human sirtuins.
    view abstract10.1038/ncomms3327
  • BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis
    Sreeramulu, S. and Mostizky, Y. and Sunitha, S. and Shani, E. and Nahum, H. and Salomon, D. and Hayun, L.B. and Gruetter, C. and Rauh, D. and Ori, N. and Sessa, G.
    Plant Journal 74 (2013)
    Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor-like cytoplasmic kinase sub-family (RLCK-XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T-DNA insertion lines for eight BSK genes (BSK1-BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24-epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR-responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3-like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3-like kinases. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.
    view abstract10.1111/tpj.12175
  • Chemical biology - Current and next challenges
    Rauh, D.
    ACS Chemical Biology 8 (2013)
    view abstract10.1021/cb400013t
  • De Novo design of protein kinase inhibitors by in silico identification of hinge region-binding fragments
    Urich, R. and Wishart, G. and Kiczun, M. and Richters, A. and Tidten-Luksch, N. and Rauh, D. and Sherborne, B. and Wyatt, P.G. and Brenk, R.
    ACS Chemical Biology 8 (2013)
    Protein kinases constitute an attractive family of enzyme targets with high relevance to cell and disease biology. Small molecule inhibitors are powerful tools to dissect and elucidate the function of kinases in chemical biology research and to serve as potential starting points for drug discovery. However, the discovery and development of novel inhibitors remains challenging. Here, we describe a structure-based de novo design approach that generates novel, hinge-binding fragments that are synthetically feasible and can be elaborated to small molecule libraries. Starting from commercially available compounds, core fragments were extracted, filtered for pharmacophoric properties compatible with hinge-region binding, and docked into a panel of protein kinases. Fragments with a high consensus score were subsequently short-listed for synthesis. Application of this strategy led to a number of core fragments with no previously reported activity against kinases. Small libraries around the core fragments were synthesized, and representative compounds were tested against a large panel of protein kinases and subjected to co-crystallization experiments. Each of the tested compounds was active against at least one kinase, but not all kinases in the panel were inhibited. A number of compounds showed high ligand efficiencies for therapeutically relevant kinases; among them were MAPKAP-K3, SRPK1, SGK1, TAK1, and GCK for which only few inhibitors are reported in the literature. © 2013 American Chemical Society.
    view abstract10.1021/cb300729y
  • Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low ATP competitiveness and outstanding whole blood activity
    Fischer, S. and Wentsch, H.K. and Mayer-Wrangowski, S.C. and Zimmermann, M. and Bauer, S.M. and Storch, K. and Niess, R. and Koeberle, S.C. and Grütter, C. and Boeckler, F.M. and Rauh, D. and Laufer, S.A.
    Journal of Medicinal Chemistry 56 (2013)
    p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy. © 2012 American Chemical Society.
    view abstract10.1021/jm301539x
  • Highly enantioselective catalytic synthesis of neurite growth-promoting secoyohimbanes
    Antonchick, A.P. and López-Tosco, S. and Parga, J. and Sievers, S. and Schürmann, M. and Preut, H. and Höing, S. and Schöler, H.R. and Sterneckert, J. and Rauh, D. and Waldmann, H.
    Chemistry and Biology 20 (2013)
    Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation. © 2013 Elsevier Ltd.
    view abstract10.1016/j.chembiol.2013.03.011
  • Metabolically stable dibenzo[ b, e ]oxepin-11(6 H)-ones as highly selective p38 MAP kinase inhibitors: Optimizing anti-cytokine activity in human whole blood
    Baur, B. and Storch, K. and Martz, K.E. and Goettert, M.I. and Richters, A. and Rauh, D. and Laufer, S.A.
    Journal of Medicinal Chemistry 56 (2013)
    Five series of metabolically stable disubstituted dibenzo[b,e]oxepin-11(6H) -ones were synthesized and tested in a p38α enzyme assay for their inhibition of tumor necrosis factor-α (TNF-α) release in human whole blood. Compared to the monosubstituted dibenzo[b,e]oxepin-11(6H)-one derivatives, it has been shown that the additional introduction of hydrophilic residues at position 9 leads to a substantial improvement of the inhibitory potency and metabolic stability. Using protein X-ray crystallography, the binding mode of the disubstituted dibenzoxepinones and the induction of a glyince flip in the hinge region were confirmed. The most potent compound of this series, 32e, shows an outstanding biological activity on isolated p38α, with an IC50 value of 1.6 nM, extraordinary selectivity (by a factor &gt;1000, Kinase WholePanelProfiler), and low ATP competitiveness. The ability to inhibit the release of TNF-α from human whole blood was optimized down to an IC50 value of 125 nM. With the promising dibenzoxepinone inhibitor 3i, a pharmacokinetic study in mice was conducted. © 2013 American Chemical Society.
    view abstract10.1021/jm401276h
  • Natural-product-derived fragments for fragment-based ligand discovery
    Over, B. and Wetzel, S. and Grütter, C. and Nakai, Y. and Renner, S. and Rauh, D. and Waldmann, H.
    Nature Chemistry 5 (2013)
    Fragment-based ligand and drug discovery predominantly employs sp 2 -rich compounds covering well-explored regions of chemical space. Despite the ease with which such fragments can be coupled, this focus on flat compounds is widely cited as contributing to the attrition rate of the drug discovery process. In contrast, biologically validated natural products are rich in stereogenic centres and populate areas of chemical space not occupied by average synthetic molecules. Here, we have analysed more than 180,000 natural product structures to arrive at 2,000 clusters of natural-product-derived fragments with high structural diversity, which resemble natural scaffolds and are rich in sp 3 -configured centres. The structures of the cluster centres differ from previously explored fragment libraries, but for nearly half of the clusters representative members are commercially available. We validate their usefulness for the discovery of novel ligand and inhibitor types by means of protein X-ray crystallography and the identification of novel stabilizers of inactive conformations of p38α MAP kinase and of inhibitors of several phosphatases. © 2013 Macmillan Publishers Limited. All rights reserved.
    view abstract10.1038/nchem.1506
  • Overcoming compound fluorescence in the FLiK screening assay with red-shifted fluorophores
    Schneider, R. and Gohla, A. and Simard, J.R. and Yadav, D.B. and Fang, Z. and Van Otterlo, W.A.L. and Rauh, D.
    Journal of the American Chemical Society 135 (2013)
    In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple π-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays. © 2013 American Chemical Society.
    view abstract10.1021/ja403074j
  • Selective detection of allosteric phosphatase inhibitors
    Schneider, R. and Beumer, C. and Simard, J.R. and Grütter, C. and Rauh, D.
    Journal of the American Chemical Society 135 (2013)
    Normal cellular function, such as signal transduction, is largely controlled by the reversible phosphorylation of cellular proteins catalyzed by two major classes of enzymes, kinases and phosphatases. A misbalance in this complex and dynamic interplay leads to a variety of severe diseases, such as cancer, inflammation, or autoimmune diseases. This makes kinases as well as phosphatases equally attractive targets for therapeutic manipulation by small molecules. While the development of kinase inhibitors has resulted in several blockbuster drugs, such as imatinib, with remarkable success in the clinic and sales of many billions of U.S. dollars per year, not a single phosphatase inhibitor has yet been approved for clinical use. Similar to the kinase world, substrate-competitive phosphatase inhibitors have been developed but were not suitable for further development into clinical candidates due to their charge and limited selectivity. Research efforts, therefore, have shifted to the exploitation of allosteric sites that can regulate phosphatase activity and may enable the discovery of novel modulators of phosphatase activity with much improved pharmacological properties. However, assay systems, which enable the straightforward discovery of these inhibitor types, are missing. Here, we present a novel binding assay capable of detecting ligands of an allosteric pocket of the protein tyrosine phosphatase 1B. This assay is suitable for high-throughput screening and selectively detects ligands which bind to this unique site with a clear discrimination from substrate-competitive ligands. © 2013 American Chemical Society.
    view abstract10.1021/ja4030484
  • Strategies for the selective regulation of kinases with allosteric modulators: Exploiting exclusive structural features
    Fang, Z. and Grütter, C. and Rauh, D.
    ACS Chemical Biology 8 (2013)
    The modulation of kinase function has become an important goal in modern drug discovery and chemical biology research. In cancer-targeted therapies, kinase inhibitors have been experiencing an upsurge, which can be measured by the increasing number of kinase inhibitors approved by the FDA in recent years. However, lack of efficacy, limited selectivity, and the emergence of acquired drug resistance still represent major bottlenecks in the clinic and challenge inhibitor development. Most known kinase inhibitors target the active kinase and are ATP competitive. A second class of small organic molecules, which address remote sites of the kinase and stabilize enzymatically inactive conformations, is rapidly moving to the forefront of kinase inhibitor research. Such allosteric modulators bind to sites that are less conserved across the kinome and only accessible upon conformational changes. These molecules are therefore thought to provide various advantages such as higher selectivity and extended drug target residence times. This review highlights various strategies that have been developed to utilizing exclusive structural features of kinases and thereby modulating their activity allosterically. © 2012 American Chemical Society.
    view abstract10.1021/cb300663j
  • Structural characterization of the RLCK family member BSK8: A pseudokinase with an unprecedented architecture
    Grütter, C. and Sreeramulu, S. and Sessa, G. and Rauh, D.
    Journal of Molecular Biology 425 (2013)
    Brassinosteroid signaling kinases (BSKs) are plant-specific receptor-like cytoplasmic protein kinases involved in the brassinosteroid signaling pathway. Unlike common protein kinases, they possess a naturally occurring alanine residue at the "gatekeeper" position, as well as other sequence variations. How BSKs activate downstream proteins such as BSU1, as well as the structural consequences of their unusual sequential features, was unclear. We crystallized the catalytic domain of BSK8 and solved its structure by multiple-wavelength anomalous dispersion phasing methods to a resolution of 1.5 Å. In addition, a co-crystal structure of BSK8 with 5-adenylyl imidodiphosphate (AMP-PNP) revealed unusual conformational arrangements of the nucleotide phosphate groups and catalytic key motifs, typically not observed for active protein kinases. Sequential analysis and comparisons with known pseudokinase structures suggest that BSKs represent constitutively inactive protein kinases that regulate brassinosteroid signal transfer through an allosteric mechanism. © 2013. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.jmb.2013.07.034
  • Targeting gain of function and resistance mutations in Abl and KIT by hybrid compound design
    Richters, A. and Ketzer, J. and Getlik, M. and Grütter, C. and Schneider, R. and Heuckmann, J.M. and Heynck, S. and Sos, M.L. and Gupta, A. and Unger, A. and Schultz-Fademrecht, C. and Thomas, R.K. and Bauer, S. and Rauh, D.
    Journal of Medicinal Chemistry 56 (2013)
    Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies. © 2013 American Chemical Society.
    view abstract10.1021/jm4004076
  • A chemical genetic approach for covalent inhibition of analogue-sensitive Aurora kinase
    Koch, A. and Rode, H.B. and Richters, A. and Rauh, D. and Hauf, S.
    ACS Chemical Biology 7 (2012)
    The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular processes. However, due to the covalent inhibitors' possible off-target reactivities, it is required that the overall shape of the inhibitor as well as the intrinsic reactivity of the electrophile are precisely tuned to favor the reaction with only the desired cysteine. Here we report on the design and biological characterization of covalent anilinoquinazolines as potent inhibitors of genetically engineered Aurora kinase in fission yeast. © 2012 American Chemical Society.
    view abstract10.1021/cb200465c
  • A framework for identification of actionable cancer genome dependencies in small cell lung cancer
    Sos, M.L. and Dietlein, F. and Peifer, M. and Schoẗtle, J. and Balke-Want, H. and Mul̈ler, C. and Koker, M. and Richters, A. and Heynck, S. and Malchers, F. and Heuckmann, J.M. and Seidel, D. and Eyers, P.A. and Ullrich, R.T. and Antonchick, A.P. and Vintonyak, V.V. and Schneider, P.M. and Ninomiya, T. and Waldmann, H. and Buẗtner, R. and Rauh, D. and Heukamp, L.C. and Thomas, R.K.
    Proceedings of the National Academy of Sciences of the United States of America 109 (2012)
    Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.
    view abstract10.1073/pnas.1207310109
  • Change or be changed: Reflections of the workshop 'Future in Medicinal Chemistry'
    Brenk, R. and Rauh, D.
    Bioorganic and Medicinal Chemistry 20 (2012)
    view abstract10.1016/j.bmc.2012.04.038
  • Design, Synthesis, and biological evaluation of novel disubstituted dibenzosuberones as highly potent and selective inhibitors of p38 mitogen activated protein kinase
    Koeberle, S.C. and Fischer, S. and Schollmeyer, D. and Schattel, V. and Grütter, C. and Rauh, D. and Laufer, S.A.
    Journal of Medicinal Chemistry 55 (2012)
    Synthesis, biological testing, structure-activity relationships (SARs), and selectivity of novel disubstituted dibenzosuberone derivatives as p38 MAP kinase inhibitors are described. Hydrophilic moieties were introduced at the 7-, 8-, and 9-position of the 2-phenylamino-dibenzosuberones, improving physicochemical properties as well as potency. Extremely potent inhibitors were obtained, with half-maximal inhibitory concentration (IC 50) values in the low nM range in a whole blood assay measuring the inhibition of cytokine release. The high potency of the target compounds together with the outstanding selectivity of this novel class of compounds toward p38 mitogen activated protein (MAP) kinase as compared to other kinases indicate them to be most applicable as tools in pharmacological research and eventually they may foster a new generation of anti-inflammatory drugs. © 2012 American Chemical Society.
    view abstract10.1021/jm300327h
  • Detailed study of N,N '-(diisopropylphenyl)-terrylene-3,4:11,12-bis(dicarboximide) as electron acceptor for solar cells application
    Gorenflot, J. and Sperlich, A. and Baumann, A. and Rauh, D. and Vasilev, A. and Li, C. and Baumgarten, M. and Deibel, C. and Dyakonov, V.
    Synthetic Metals 161 (2012)
    We report on terrylene-3,4:11,12-bis(dicarboximide) (TDI) as electron acceptor for bulk-heterojunction solar cells using poly(3-hexyl thiophene) (P3HT) as complementary donor component. Enhanced absorption was observed in the blend compared to pure P3HT. As shown by the very efficient photoluminescence (PL) quenching, the generated excitons are collected at the interface between the donor and acceptor, where they separate into charges which we detect by photoinduced absorption and electron-spin resonance (ESR). Time-of-flight (TOF) photoconductivity measurements reveal a good electron mobility of 10(-3) cm(2)V(-1)s(-1) in the blend. Nevertheless, the photocurrent in solar cells was found to be surprisingly low. Supported by the external quantum efficiency (EQE) spectrum as well as morphological studies by way of X-ray diffraction and atomic force microscopy, we explain our observation by the formation of a TDI hole blocking layer at the anode interface, which prevents the efficiently generated charges to be extracted. (C) 2011 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.synthmet.2011.09.041
  • Direct binding assay for the detection of type IV allosteric inhibitors of Abl
    Schneider, R. and Becker, C. and Simard, J.R. and Getlik, M. and Bohlke, N. and Janning, P. and Rauh, D.
    Journal of the American Chemical Society 134 (2012)
    Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site. © 2012 American Chemical Society.
    view abstract10.1021/ja303858w
  • Epidermal growth factor receptor (EGFR) signaling and covalent EGFR inhibition in lung cancer
    Heuckmann, J.M. and Rauh, D. and Thomas, R.K.
    Journal of Clinical Oncology 30 (2012)
    view abstract10.1200/JCO.2012.43.1825
  • Fluorescence labels in kinases: A high-throughput kinase binding assay for the identification of DFG-out binding ligands
    Simard, J.R. and Rauh, D.
    Methods in Molecular Biology 800 (2012)
    Despite the hundreds of kinase inhibitors currently in discovery and pre-clinical phases, the number of kinase inhibitors which have been approved and are on the market remains low by comparison. This discrepancy reflects the challenges which accompany the development of kinase inhibitors which are relatively specific and less toxic. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibiting kinase activity, but the highly conserved nature of the ATP-binding site contributes to poor inhibitor selectivity, issues which have particularly hampered the development of novel kinase inhibitors. We developed a high-throughput screening technology that can discriminate for inhibitors which stabilize the inactive "DFG-out" kinase conformation by binding within an allosteric pocket adjacent to the ATP-binding site. Here, we describe how to use this approach to measure the K d of ligands, as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries at high throughput. © 2012 Springer Science+Business Media, LLC.
    view abstract10.1007/978-1-61779-349-3_8
  • Fluorophore labeled kinase detects ligands that bind within the MAPK insert of p38α kinase
    Getlik, M. and Simard, J.R. and Termathe, M. and Grütter, C. and Rabiller, M. and van Otterlo, W.A.L. and Rauh, D.
    PLoS ONE 7 (2012)
    The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. © 2012 Getlik et al.
    view abstract10.1371/journal.pone.0039713
  • Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer
    Peifer, M. and Fernández-Cuesta, L. and Sos, M.L. and George, J. and Seidel, D. and Kasper, L.H. and Plenker, D. and Leenders, F. and Sun, R. and Zander, T. and Menon, R. and Koker, M. and Dahmen, I. and Müller, C. and Di Cerbo, V. and Schildhaus, H.-U. and Altmüller, J. and Baessmann, I. and Becker, C. and De Wilde, B. and Vandesompele, J. and Böhm, D. and Ansén, S. and Gabler, F. and Wilkening, I. and Heynck, S. and Heuckmann, J.M. and Lu, X. and Carter, S.L. and Cibulskis, K. and Banerji, S. and Getz, G. and Park, K.-S. and Rauh, D. and Grütter, C. and Fischer, M. and Pasqualucci, L. and Wright, G. and Wainer, Z. and Russell, P. and Petersen, I. and Chen, Y. and Stoelben, E. and Ludwig, C. and Schnabel, P. and Hoffmann, H. and Muley, T. and Brockmann, M. and Engel-Riedel, W. and Muscarella, L.A. and Fazio, V.M. and Groen, H. and Timens, W. and Sietsma, H. and Thunnissen, E. and Smit, E. and Heideman, D.A.M. and Snijders, P.J.F. and Cappuzzo, F. and Ligorio, C. and Damiani, S. and Field, J. and Solberg, S. and Brustugun, O.T. and Lund-Iversen, M. and Sänger, J. and Clement, J.H. and Soltermann, A. and Moch, H. and Weder, W. and Solomon, B. and Soria, J.-C. and Validire, P. and Besse, B. and Brambilla, E. and Brambilla, C. and Lantuejoul, S. and Lorimier, P. and Schneider, P.M. and Hallek, M. and Pao, W. and Meyerson, M. and Sage, J. and Shendure, J. and Schneider, R. and Büttner, R. and Wolf, J. and Nürnberg, P. and Perner, S. and Heukamp, L.C. and Brindle, P.K. and Haas, S. and Thomas, R.K.
    Nature Genetics 44 (2012)
    Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background. © 2012 Nature America, Inc. All rights reserved.
    view abstract10.1038/ng.2396
  • Molecular medicine: Symbiosis of chemistry and medicine - Targeted against cancer
    Mayer-Wrangowski, S. and Rauh, D.
    BioSpektrum 18 (2012)
    Selective kinase inhibitors have become an important class of anti-cancer agents. However, clinical success of these agents is mostly limited to a small subset of patients, who are often defined by specific genomic lesions within their tumor cells. Recent progress in cancer genetics and medicinal chemistry allows for the first time effective personalization of tumor therapy with so far unreached benefit for some patient populations. © Springer-Verlag 2012.
    view abstract10.1007/s12268-012-0195-7
  • Skepinone-L is a selective p38 mitogen-activated protein kinase inhibitor
    Koeberle, S.C. and Romir, J. and Fischer, S. and Koeberle, A. and Schattel, V. and Albrecht, W. and Grütter, C. and Werz, O. and Rauh, D. and Stehle, T. and Laufer, S.A.
    Nature Chemical Biology 8 (2012)
    Until now, a lack of inhibitors with high potency and selectivity in vivo has hampered investigation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We describe the design of skepinone-L, which is, to our knowledge, the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, skepinone-L is a valuable probe for chemical biology research, and it may foster the development of a unique class of kinase inhibitors. © 2012 Nature America, Inc. All rights reserved.
    view abstract10.1038/nchembio.761
  • Structure-based design, synthesis and biological evaluation of N-pyrazole, N′-thiazole urea inhibitors of MAP kinase p38α
    Getlik, M. and Grütter, C. and Simard, J.R. and Nguyen, H.D. and Robubi, A. and Aust, B. and Van Otterlo, W.A.L. and Rauh, D.
    European Journal of Medicinal Chemistry 48 (2012)
    In this paper, we present the structure-based design, synthesis and biological activity of N-pyrazole, N′-thiazole-ureas as potent inhibitors of p38α mitogen-activated protein kinase (p38α MAPK). Guided by complex crystal structures, we employed the initially identified N-aryl, N′-thiazole urea scaffold and introduced key structural elements that allowed the formation of novel hydrogen bonding interactions within the allosteric site of p38α, resulting in potent type III inhibitors. [4-(3-tert-Butyl-5-{[(1,3-thiazol-2-ylamino)carbonyl]amino}-1H-pyrazol-1-yl) -phenyl]acetic acid 18c was found to be the most potent compound within this series and inhibited p38α activity with an IC 50 of 135 ± 21 nM. Its closest analog, ethyl [4-(3-tert-butyl-5-{[(1,3-thiazol-2-ylamino) carbonyl]amino}-1H-pyrazol-1-yl)phenyl]acetate 18b, effectively inhibited p38α mediated phosphorylation of the mitogen activated protein kinase activated protein kinase 2 (MK2) in HeLa cells. © 2011 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.ejmech.2011.11.019
  • Targeting GSK3 from Ustilago maydis: Type-II kinase inhibitors as potential antifungals
    Grütter, C. and Simard, J.R. and Mayer-Wrangowski, S.C. and Schreier, P.H. and Pérez-Martín, J. and Richters, A. and Getlik, M. and Gutbrod, O. and Braun, C.A. and Beck, M.E. and Rauh, D.
    ACS Chemical Biology 7 (2012)
    Protein kinases are key enzymes in the complex regulation of cellular processes in almost all living organisms. For this reason, protein kinases represent attractive targets to stop the growth of eukaryotic pathogens such as protozoa and fungi. However, using kinase inhibitors to fight against these organisms bears several challenges since most of them are unselective and will also affect crucial host kinases. Here we present the X-ray structure of glycogen synthase kinase 3 from the fungal plant pathogen Ustilago maydis (UmGSK3) and its inhibition by type-II kinase inhibitors. Despite the high sequence homology between the human and the fungal variant of this vital kinase, we found substantial differences in the conformational plasticity of their active sites. Compounds that induced such conformational changes could be used to selectively inhibit the fungal kinase. This study serves as an example of how species-specific selectivity of inhibitors can be achieved by identifying and addressing the inactive state of a protein kinase. In addition to this, our study gives interesting insights into the molecular plasticity of UmGSK3 by revealing a previously unknown inactive conformation of this important kinase family. © 2012 American Chemical Society.
    view abstract10.1021/cb300128b
  • Targeting the hinge glycine flip and the activation loop: Novel approach to potent p38α inhibitors
    Martz, K.E. and Dorn, A. and Baur, B. and Schattel, V. and Goettert, M.I. and Mayer-Wrangowski, S.C. and Rauh, D. and Laufer, S.A.
    Journal of Medicinal Chemistry 55 (2012)
    The p38 MAP kinase is a key player in signaling pathways regulating the biosynthesis of inflammatory cytokines. Small molecule p38 inhibitors suppress the production of these cytokines. Therefore p38 is a promising drug target for novel anti-inflammatory drugs. In this study, we report novel dibenzepinones, dibenzoxepines, and benzosuberones as p38α MAP kinase inhibitors. Previously reported dibenzepinones and dibenzoxepines were chemically modified by introduction of functional groups or removal of a phenyl ring. This should result in targeting of the hydrophobic region I, the "deep pocket", and the hinge glycine flip of the kinase. Potent inhibitors with IC50 values in the single digit nanomolar range (up to 3 nM) were identified. Instead of targeting the "deep pocket" in the DFG-out conformation, interactions with the DFG-motif in the in-conformation could be observed by protein X-ray crystallography. © 2012 American Chemical Society.
    view abstract10.1021/jm300951u
  • ALK mutations conferring differential resistance to structurally diverse ALK inhibitors
    Heuckmann, J.M. and Hölzel, M. and Sos, M.L. and Heynck, S. and Balke-Want, H. and Koker, M. and Peifer, M. and Weiss, J. and Lovly, C.M. and Grütter, C. and Rauh, D. and Pao, W. and Thomas, R.K.
    Clinical Cancer Research 17 (2011)
    Purpose: EML4-ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4-ALK-positive tumors. Experimental Design: By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684. Results: Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4-ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066. Conclusions: Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4-ALK fusions and ALK mutations. ©2011 AACR.
    view abstract10.1158/1078-0432.CCR-11-1648
  • Enantioselective synthesis of the spirotryprostatin A scaffold
    Antonchick, A.P. and Schuster, H. and Bruss, H. and Schürmann, M. and Preut, H. and Rauh, D. and Waldmann, H.
    Tetrahedron 67 (2011)
    The pentacyclic spirotryprostatin scaffold embodies an oxindole with an all-carbon quaternary stereocenter. The scaffold can efficiently be accessed in a one-pot reaction sequence consisting of a highly enantioselective 1,3-dipolar cycloaddition, N-acylation of the resulting stereochemically complex 3,3′-pyrrolidinyl-spirooxindole core with Fmoc-proline and spontaneous ring closure upon N-deprotection. © 2011 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.tet.2011.04.056
  • Identification and further development of thiazolidinones spiro-fused to indolin-2-ones as potent and selective inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase B
    Vintonyak, V.V. and Warburg, K. and Over, B. and Hübel, K. and Rauh, D. and Waldmann, H.
    Tetrahedron 67 (2011)
    Tuberculosis continues to be a major cause of morbidity and mortality throughout the world. Protein tyrosine phosphatases from Mycobacterium tuberculosis are attractive targets for developing novel strategies in battling tuberculosis due to their role in the intracellular survival of M. tuberculosis in various infection models. Here, we report on the identification and further development of thiazolidinones spiro-fused to indolin-2-ones as a new class of potent and selective inhibitors of M. tuberculosis protein tyrosine phosphatase B. Detailed structure-activity relationship (SAR) studies revealed that a nitro-substituted 2-oxoindole core together with a dihalogenated anilide and a halogenated N-benzyl moiety are essential for strong inhibitory activity against MptpB (M. tuberculosis protein tyrosine phosphatase B). Small structural modification of the identified compounds led to significant improvement of compound solubility and cell permeability retaining inhibitory activity in the micromolar range. The configuration of the spiro-center was found to be crucial for the inhibitory activity and the separation of the racemate revealed the R-(-)-enantiomers as the biologically active component. The reported MptpB inhibitors show excellent selectivity against a selected panel of protein tyrosine phosphatases, including MptpA (M. tuberculosis protein tyrosine phosphatase A), PTP1B (protein tyrosine phosphatase 1B), SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase), PTPN2, h-PTPβ (human protein tyrosine phosphatase β), and VHR (Vaccinia virus VH1-related dual-specific protein phosphatase) and further highlight the identified thiazolidinones spiro-fused to indolin-2-ones as a promising class of new compounds that might prove useful for chemical biology research to dissect MptpB function and eventually foster the development of next generation antibiotics. © 2011 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.tet.2011.04.026
  • Identification of Ustilago maydis Aurora kinase as a novel antifungal target
    Tückmantel, S. and Greul, J.N. and Janning, P. and Brockmeyer, A. and Grütter, C. and Simard, J.R. and Gutbrod, O. and Beck, M.E. and Tietjen, K. and Rauh, D. and Schreier, P.H.
    ACS Chemical Biology 6 (2011)
    Infestation of crops by pathogenic fungi has continued to have a major impact by reducing yield and quality, emphasizing the need to identify new targets and develop new agents to improve methods of crop protection. Here we present Aurora kinase from the phytopathogenic fungus Ustilago maydis as a novel target for N-substituted diaminopyrimidines, a class of small-molecule kinase inhibitors. We show that Aurora kinase is essential in U. maydis and that diaminopyrimidines inhibit its activity in vitro. Furthermore, we observed an overall good correlation between in vitro inhibition of Aurora kinase and growth inhibition of diverse fungi in vivo. In vitro inhibition assays with Ustilago and human Aurora kinases indicate that some compounds of the N-substituted diaminopyrimidine class show specificity for the Ustilago enzyme, thus revealing their potential as selective fungicides. © 2011 American Chemical Society.
    view abstract10.1021/cb200112y
  • Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer
    Hammerman, P.S. and Sos, M.L. and Ramos, A.H. and Xu, C. and Dutt, A. and Zhou, W. and Brace, L.E. and Woods, B.A. and Lin, W. and Zhang, J. and Deng, X. and Lim, S.M. and Heynck, S. and Peifer, M. and Simard, J.R. and Lawrence, M.S. and Onofrio, R.C. and Salvesen, H.B. and Seidel, D. and Zander, T. and Heuckmann, J.M. and Soltermann, A. and Moch, H. and Koker, M. and Leenders, F. and Gabler, F. and Querings, S. and Ansén, S. and Brambilla, E. and Brambilla, C. and Lorimier, P. and Brustugun, O.T. and Helland, A. and Petersen, I. and Clement, J.H. and Groen, H. and Timens, W. and Sietsma, H. and Stoelben, E. and Wolf, J. and Beer, D.G. and Tsao, M.S. and Hanna, M. and Hatton, C. and Eck, M.J. and Janne, P.A. and Johnson, B.E. and Winckler, W. and Greulich, H. and Bass, A.J. and Cho, J. and Rauh, D. and Gray, N.S. and Wong, K.-K. and Haura, E.B. and Thomas, R.K. and Meyerson, M.
    Cancer Discovery 1 (2011)
    Although genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations that drive squamous cell cancer (SCC) of the lung. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of lung SCCs and cell lines. Lung SCC cell lines harboring DDR2 mutations were selectively killed by knockdown of DDR2 by RNA interference or by treatment with the multitargeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation that was blocked by dasatinib. A patient with lung SCC that responded to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. Because dasatinib is already approved for use, these findings could be used to rapidly generate clinical trials. siGNificaNce: DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs. © 2011 American Association for Cancer Research.
    view abstract10.1158/2159-8274.CD-11-0005
  • Synthesis and biological evaluation of 7-substituted-1-(3-bromophenylamino) isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase and epidermal growth factor receptor
    Rode, H.B. and Sos, M.L. and Grütter, C. and Heynck, S. and Simard, J.R. and Rauh, D.
    Bioorganic and Medicinal Chemistry 19 (2011)
    Here we present the synthesis and biological activity of a series of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase (MLCK) and the epidermal growth factor receptor kinase (EGFR). The inhibitory effect of these molecules was found to be dependent on the nature of the substituents at the 7-position of the isoquinoline scaffold. © 2010 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.bmc.2010.11.007
  • Using small molecules to target protein phosphatases
    Vintonyak, V.V. and Waldmann, H. and Rauh, D.
    Bioorganic and Medicinal Chemistry 19 (2011)
    The site specific functionalization of phosphate groups with amino acid side chains of substrate proteins represents one of the most important regulatory mechanisms of biological systems. Phosphorylation and dephosphorylation are reversibly catalyzed by protein kinases and protein phosphatases, and the aberrant regulation of these enzymes is associated with the onset and progression of various disease states such as cancer, diabetes, neurodegenerative and autoimmune disorders, making these proteins attractive targets for drug discovery. Here we report on strategies currently explored for the identification and development of various inhibitors directed against clinically relevant phosphatases. While over the last years, inhibition of phosphorylation has evolved into a key strategy in targeted therapies, the development of clinically relevant phosphatase inhibitors still faces major bottlenecks and is often plagued by limited selectivity and unfavorable pharmacokinetics. The reader will gain a better understanding of the importance of the field and its current limitations. © 2011 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.bmc.2011.02.047
  • A scaffold-tree-merging strategy for prospective bioactivity annotation of γ-pyrones
    Wetzel, S. and Wilk, W. and Chammaa, S. and Sperl, B. and Roth, A.G. and Yektaoglu, A. and Renner, S. and Berg, T. and Arenz, C. and Giannis, A. and Oprea, T.I. and Rauh, D. and Kaiser, M. and Waldmann, H.
    Angewandte Chemie - International Edition 49 (2010)
    Tactical target setting: The merging of natural product and non-natural product based hierarchical scaffold trees annotated with bioactivity (see schematic illustration) together with brachiation along structural lines of biological relevance provides a novel strategy for the prospective identification of protein targets for compound collections inspired by natural product structures. (Figure Presented) © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/anie.200906555
  • Analysis of compound synergy in high-throughput cellular screens by population-based lifetime modeling
    Peifer, M. and Weiss, J. and Sos, M.L. and Koker, M. and Heynck, S. and Netzer, C. and Fischer, S. and Rode, H. and Rauh, D. and Rahnenführer, J. and Thomas, R.K.
    PLoS ONE 5 (2010)
    Despite the successful introduction of potent anti-cancer therapeutics, most of these drugs lead to only modest tumorshrinkage or transient responses, followed by re-growth of tumors. Combining different compounds has resulted in enhanced tumor control and prolonged survival. However, methods querying the efficacy of such combinations have been hampered by limited scalability, analytical resolution, statistical feasibility, or a combination thereof. We have developed a theoretical framework modeling cellular viability as a stochastic lifetime process to determine synergistic compound combinations from high-throughput cellular screens. We apply our method to data derived from chemical perturbations of 65 cancer cell lines with two inhibitors. Our analysis revealed synergy for the combination of both compounds in subsets of cell lines. By contrast, in cell lines in which inhibition of one of both targets was sufficient to induce cell death, no synergy was detected, compatible with the topology of the oncogenically activated signaling network. In summary, we provide a tool for the measurement of synergy strength for combination perturbation experiments that might help define pathway topologies and direct clinical trials. ©2010 Peifer et al.
    view abstract10.1371/journal.pone.0008919
  • Characterization of irreversible kinase inhibitors by directly detecting covalent bond formation: A tool for dissecting kinase drug resistance
    Klüter, S. and Simard, J.R. and Rode, H.B. and Grütter, C. and Pawar, V. and Raaijmakers, H.C.A. and Barf, T.A. and Rabiller, M. and Van Otterlo, W.A.L. and Rauh, D.
    ChemBioChem 11 (2010)
    Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants. Dissecting the mechanisms of kinase drug resistance: We describe a straightforward assay system, which allowed real-time detection of irreversible kinase inhibition without requiring ATP or time-dependent IC50 measurements. This assay system provided an effective tool for dissecting drug-resistance mechanisms resulting from point mutations at the gatekeeper position. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/cbic.201000352
  • Chemogenomic profiling provides insights into the limited activity of irreversible EGFR inhibitors in tumor cells expressing the T790M EGFR resistance mutation
    Sos, M.L. and Rode, H.B. and Heynck, S. and Peifer, M. and Fischer, F. and Klüter, S. and Pawar, V.G. and Reuter, C. and Heuckmann, J.M. and Weiss, J. and Ruddigkeit, L. and Rabiller, M. and Koker, M. and Simard, J.R. and Getlik, M. and Yuza, Y. and Chen, T.-H. and Greulich, H. and Thomas, R.K. and Rauh, D.
    Cancer Research 70 (2010)
    Reversible epidermal growth factor receptor (EGFR) inhibitors are the first class of small molecules to improve progression-free survival of patients with EGFR-mutated lung cancers. Second-generation EGFR inhibitors introduced to overcome acquired resistance by the T790M resistance mutation of EGFR have thus far shown limited clinical activity in patients with T790M-mutant tumors. In this study, we systematically analyzed the determinants of the activity and selectivity of the second-generation EGFR inhibitors. A focused library of irreversible as well as structurally corresponding reversible EGFR-inhibitors was synthesized for chemogenomic profiling involving over 79 genetically defined NSCLC and 19 EGFR-dependent cell lines. Overall, our results show that the growth-inhibitory potency of all irreversible inhibitors against the EGFR T790M resistance mutation was limited by reduced target inhibition, linked to decreased binding velocity to the mutant kinase. Combined treatment of T790M-mutant tumor cells with BIBW-2992 and the phosphoinositide-3-kinase/ mammalian target of rapamycin inhibitor PI-103 led to synergistic induction of apoptosis. Our findings offer a mechanistic explanation for the limited efficacy of irreversible EGFR inhibitors in EGFR T790M gatekeeper-mutant tumors, and they prompt combination treatment strategies involving inhibitors that target signaling downstream of the EGFR. ©2010 American Association for Cancer Research.
    view abstract10.1158/0008-5472.CAN-09-3106
  • Displacement assay for the detection of stabilizers of inactive kinase conformations
    Klüter, S. and Grütter, C. and Naqvi, T. and Rabiller, M. and Simard, J.R. and Pawar, V. and Getlik, M. and Rauh, D.
    Journal of Medicinal Chemistry 53 (2010)
    Targeting protein kinases with small molecules outside the highly conserved ATP pocket to stabilize inactive kinase conformations is becoming a more desirable approach in kinase inhibitor research, since these molecules have advanced pharmacological properties compared to compounds exclusively targeting the ATP pocket. Traditional screening approaches for kinase inhibitors are often based on enzyme activity, but they may miss inhibitors that stabilize inactive kinase conformations by enriching the active state of the kinase. Here we present the development of a kinase binding assay employing a pyrazolourea type III inhibitor and enzyme fragment complementation (EFC) technology that is suitable to screen stabilizers of enzymatically inactive kinases. To validate this assay system, we report the binding characteristics of a series of kinase inhibitors to inactive p38R and JNK2. Additionally, we present protein X-ray crystallography studies to examine the binding modes of potent quinoline-based DFG-out binders in p38α. © 2009 American Chemical Society.
    view abstract10.1021/jm901297e
  • Fluorophore labeling of the glycine-rich loop as a method of identifying inhibitors that bind to active and inactive kinase conformations
    Simard, J.R. and Getlik, M. and Grütter, C. and Schneider, R. and Wulfert, S. and Rauh, D.
    Journal of the American Chemical Society 132 (2010)
    Targeting protein kinases with small organic molecules is a promising strategy to regulate unwanted kinase activity in both chemical biology and medicinal chemistry research. Traditionally, kinase inhibitors are identified in activity-based screening assays using enzymatlcally active kinase preparations to measure the perturbation of substrate phosphorylation, often resulting in the enrichment of classical ATP competitive (Type I) inhibitors. However, addressing enzymatically incompetent kinase conformations offers new opportunities for targeted therapies and is moving to the forefront of kinase inhibitor research. Here we report the development of a new FLIK (Fluorescent Labels in Kinases) binding assay to detect small molecules that induce changes in the conformation of the glycine-rich loop. Due to cross-talk between the glyclne-rlch loop and the activation loop in kinases, this alternative labeling approach can also detect ligands that stabilize inactive kinase conformations, including slow-binding Type II and Type III kinase inhibitors. Protein X-ray crystallography validated the assay results and identified a novel DFG-out binding mode for a quinazollne-based inhibitor In p38a kinase. We also detected the hlgh-afflnlty binding of a clinically relevant and specific VEGFR2 inhibitor, and we provide structural details of its binding mode In p38a, in which it stabilizes the DFG-out conformation. Last, we demonstrate the power of this new FLIK labeling strategy to detect the binding of Type ligands that induce conformational changes in the glyclnerich loop as a means of gaining affinity for the target kinase. This approach may be a useful alternative to develop direct binding assays for kinases that do not adopt the DFG-out conformation while also avoiding the use of expensive kits, detection reagents, or radioactivity frequently employed with activity-based assays. © 2010 American Chemical Society.
    view abstract10.1021/ja908083e
  • Highly enantioselective synthesis and cellular evaluation of spirooxindoles inspired by natural products
    Antonchick, A.P. and Gerding-Reimers, C. and Catarinella, M. and Schürmann, M. and Preut, H. and Ziegler, S. and Rauh, D. and Waldmann, H.
    Nature Chemistry 2 (2010)
    In biology-oriented synthesis the underlying scaffold classes of natural products selected in evolution are used to define biologically relevant starting points in chemical structure space for the synthesis of compound collections with focused structural diversity. Here we describe a highly enantioselective synthesis of natural-product-inspired 3,3′ -pyrrolidinyl spirooxindoles-which contain an all-carbon quaternary centre and three tertiary stereocentres. This synthesis takes place by means of an asymmetric Lewis acid-catalysed 1,3-dipolar cycloaddition of an azomethine ylide to a substituted 3-methylene-2-oxindole using 1-3 mol% of a chiral catalyst formed from a N,P-ferrocenyl ligand and CuPF6 (CH3 CN)4. Cellular evaluation has identified a molecule that arrests mitosis, induces multiple microtubule organizing centres and multipolar spindles, causes chromosome congression defects during mitosis and inhibits tubulin regrowth in cells. Our findings support the concept that compound collections based on natural-product-inspired scaffolds constructed with complex stereochemistry will be a rich source of compounds with diverse bioactivity. © 2010 Macmillan Publishers Limited. All rights reserved.
    view abstract10.1038/nchem.730
  • Identification of Thiazolidinones Spiro-Fused to IndoIin-2-ones as Potent and Selective Inhibitors of the Mycobacterium tuberculosis Protein Tyrosine Phosphatase B
    Vintonyak, V.V. and Warburg, K. and Kruse, H. and Grimme, S. and Hübet, K. and Rauh, D. and Waldmann, H.
    Angewandte Chemie - International Edition 49 (2010)
    (Figure Presented) The best of 40000: Detailed structureactivity- relationship studies revealed key structural elements of indolin-2-on-3- spirothiazolidinones (see example) and their appropriate configuration for strong inhibitory activity against the pathophysiologically relevant title protein. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/anie.201002138
  • Proteus in the world of proteins: Conformational changes in protein kinases
    Rabiller, M. and Getlik, M. and Klüter, S. and Richters, A. and Tückmantel, S. and Simard, J.R. and Rauh, D.
    Archiv der Pharmazie 343 (2010)
    The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by introducing variations to a highly conserved theme. The activity of each kinase domain is controlled by layers of regulatory mechanisms involving different combinations of post-translational modifications, intramolecular contacts, and intermolecular interactions. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has encouraged extensive investigations of their biological function and three-dimensional structures, yielding a more detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes. In the present review, we discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of specific inhibitors that lock protein kinases in inactive conformations. In addition, we highlight recent developments to monitor ligand-induced structural changes in protein kinases and for screening and identifying inhibitors that stabilize enzymatically incompetent kinase conformations. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/ardp.201000028
  • RAF kinase inhibitors in cancer treatment: Like a bull in a China shop?
    Robubi, A. and Waldmann, H. and Rauh, D.
    ChemBioChem 11 (2010)
    Opposite effects: RAF kinase inhibitors are currently under intensive investigation for the treatment of various cancers. Recently, several groups have reported independently that at least some of these compounds actually activate RAF kinases (dimer interface shown) in certain cellular settings. These findings will have enormous consequences in patient selection and for further target selection and drug development. (Figure Presented) © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/cbic.201000348
  • Small-molecule inhibition of APT1 affects Ras localization and signaling
    Dekker, F.J. and Rocks, O. and Vartak, N. and Menninger, S. and Hedberg, C. and Balamurugan, R. and Wetzel, S. and Renner, S. and Gerauer, M. and Schölermann, B. and Rusch, M. and Kramer, J.W. and Rauh, D. and Coates, G.W. and Brunsveld, L. and Bastiaens, P.I.H. and Waldmann, H.
    Nature Chemical Biology 6 (2010)
    Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localizationĝ€"and thereby unregulated signalingĝ€"caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.
    view abstract10.1038/nchembio.362
  • Stabilizing inactive kinase conformations
    Rauh, D.
    Nachrichten aus der Chemie 58 (2010)
    view abstract10.1002/nadc.201069050
  • Synthesis and biological evaluation of 4-anilinoquinolines as potent inhibitors of epidermal growth factor receptor
    Pawar, V.G. and Sos, M.L. and Rode, H.B. and Rabiller, M. and Heynck, S. and Van Otterlo, W.A.L. and Thomas, R.K. and Rauh, D.
    Journal of Medicinal Chemistry 53 (2010)
    The mutant receptor tyrosine kinase EGFR is a validated and therapeutically amenable target for genotypically selected lung cancer patients. Here we present the synthesis and biological evaluation of a series of 6- and 7-substituted 4-anilinoquinolines as potent type I inhibitors of clinically relevant mutant variants of EGFR. Quinolines 3a and 3e were found to be highly active kinase inhibitors in biochemical assays and were further investigated for their biological effect on EGFR-dependent Ba/F3 cells and non-small cell lung cancer (NSCLC) cell lines. © 2010 American Chemical Society.
    view abstract10.1021/jm901877j
  • drug design

  • proteins

« back