Berthold Wroblowski

Extending kinome coverage by analysis of kinase inhibitor broad profiling data

The explored kinome was extended with broad profiling using the DiscoveRx and Millipore assay panels. The analysis of the profiling of 3368 selected inhibitors on 456 kinases in the DiscoveRx format delivered several insights. First, the coverage depended on the threshold of the selectivity parameter. Second, the relation between hit confirmation rates and inhibitor selectivity showed unexpectedly that higher selectivity can increase the likelihood of false positives. Third, comparing the coverage of a focused to that of a random library showed that the design based on a maximum number of scaffolds was superior to a limited number of scaffolds. Therefore, selective compounds can be used in target validation, enable the jumpstarting of new kinase drug discovery projects, and chart new biological space via phenotypic screening.

A Proteometric Analysis of Human Kinome: Insight into Discriminant Conformation-dependent Residues

Because of the success of imatinib, the first type-II kinase inhibitor approved by the FDA in 2001, sustained efforts have been made by the pharmaceutical industry to discover novel compounds stabilizing the inactive conformation of protein kinases. On the seven type-II inhibitors having reached the market, four were released in 2012, suggesting an acceleration of the research of such a class of compounds. Still, they represent less than a third of the protein kinase inhibitors available to patients today. The identification of key residues involved in the binding of this type of ligands in the kinase active site might ease the design of potent and selective type-II inhibitors. In order to identify those discriminant residues, we have developed a proteometric approach combining residue descriptors of protein kinase sequences and biological activities of various type-II kinase inhibitors. We applied Partial Least Squares (PLS) regression to identify 29 key residues that influence the binding of four type-II inhibitors to most proteins of the kinome. The gatekeeper residue was found to be the most relevant, confirming an essential role in ligand binding as well as in protein kinase conformational changes. Using the newly developed proteometric model, we predicted the propensity of each protein kinase to be inhibited by type-II ligands. The model was further validated using an external data set of protein/ligand activity pairs. Other residues present in the kinase domain, and more specifically in the binding site, have been highlighted by this approach, but their role in biological mechanisms is still unknown.

Exploring protein kinase conformation using swarm-enhanced sampling molecular dynamics.

Protein plasticity, while often linked to biological function, also provides opportunities for rational design of selective and potent inhibitors of their function. The application of computational methods to the prediction of concealed protein concavities is challenging, as the motions involved can be significant and occur over long time scales. Here we introduce the swarm-enhanced sampling molecular dynamics (sesMD) method as a tool to improve sampling of conformational landscapes. In this approach, a swarm of replica simulations interact cooperatively via a set of pairwise potentials incorporating attractive and repulsive components. We apply the sesMD approach to explore the conformations of the DFG motif in the protein p38α mitogen-activated protein kinase. In contrast to multiple MD simulations, sesMD trajectories sample a range of DFG conformations, some of which map onto existing crystal structures. Simulated structures intermediate between the DFG-in and DFG-out conformations are predicted to have druggable pockets of interest for structure-based ligand design.

Abstract 1738: JNJ-42756493 is an inhibitor of FGFR-1, 2, 3 and 4 with nanomolar affinity for targeted therapy

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Alterations in FGFR family members including focal amplification of FGF receptor 1 (FGFR1), mutations in FGFR 2, 3 and 4, translocations involving FGFR 2 and FGFR3, as well as amplification or transcriptional upregulation of various ligand family members have been associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in subsets of various disease settings. A number of agents targeting the FGF signaling axis including small-molecule FGFR targeted agents, with diverse kinase inhibitory and pharmacological profiles, are currently in clinical development. JNJ-42756493 (first disclosure of the structure) has a pharmacological profile that is differentiated from other agents in this class currently under investigation. JNJ-42756493 displays single digit nanomolar FGFR (1, 2, 3 4) tyrosine kinase inhibitory activity. JNJ-42756493 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in engineered cell lines and tumor cell lines dependent upon deregulated FGFR expression. JNJ-42756493 demonstrated highly specific tumor inhibitory effects in FGFR1-4 dependent cell lines, in vitro cell lines based xenografts and direct patient derived xenografts, with no discernible activity in models that were not dependent on FGFR signaling. JNJ-42756493 showed favorable drug like properties and displayed a high distribution to lung, liver and kidney tissue. JNJ-42756493 was well tolerated at efficacious doses and resulted in potent dose-dependent antitumor activity accompanied by pharmacodynamic modulation of tumor FGFR and downstream pathway components. Data presented here highlights JNJ-42756493 as a novel, highly potent and selective small-molecule inhibitor of all four known active FGFR kinase family members with potent antitumor activity against FGFR-dependent tumor models. These data, together with emerging observations from our ongoing Phase 1 clinical trial, position JNJ-42756493 as a differentiated FGFR 1, 2, 3 and 4 kinase inhibitor and support its continued clinical development in lung cancer and other malignancies associated with aberrant FGFR signaling. Citation Format: Timothy Perera, Eleanora Jovcheva, Jorge Vialard, Tinne Verhulst, Norbert Esser, Berthold Wroblowski, Ron Gilissen, Eddy Freyne, Peter King, Suso Platero, Olivier Querolle, Laurence Mevellec, Christopher Murray, Lynsey Fazal, Gordon Saxty, George Ward, Matthew Squires, Neil Thompson, David Newell, Patrick Angibaud. JNJ-42756493 is an inhibitor of FGFR-1, 2, 3 and 4 with nanomolar affinity for targeted therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2014-1738

Abstract 4748: Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Fibroblast growth factors (FGFs) and their receptors (FGFR1 through 4) regulate a variety of key cellular processes, including proliferation, migration, survival, and differentiationa. Aberrant activation of FGF/FGFR is strongly implicated in oncogenic signalling in many tumor types. This has stimulated the development of a number of FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We conducted a fragment screening campaign and this resulted in identification of a 6-aminoquinoxalinyl fragment with a binding affinity in the micromolar range. Structure-guided medicinal chemistry led to the identification of a novel quinoxaline-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. Further optimisation resulted in the generation of JNJ-42756493, a compound with favourable drug-like properties that demonstrated strong anti-tumoral activity in a FGFR2-dependent SNU-16 human gastric carcinoma xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathway of the JNJ-42756493 series and illustrates how a fragment-based drug discovery approach has been efficiently used to discover FGFR1-4 inhibitors with nanomolar affinity. aTurner, N. and Grose, R. Nat. Rev. Cancer, 2010, 10, 116-129. Citation Format: Patrick R. Angibaud, Laurence Mevellec, Gordon Saxty, Christophe Adelinet, Rhalid Akkari, Valerio Berdini, Pascal Bonnet, Marine Bourgeois, Xavier Bourdrez, Anne Cleasby, Helene Colombel, Imre Csoka, Werner Embrechts, Eddy Freyne, Ronaldus Gilissen, Eleonora Jovcheva, Peter King, Jean Lacrampe, Delphine Lardeau, Yannick Ligny, Steve Mcclue, Lieven Meerpoel, David R. Newell, Martin Page, Alexandra Papanikos, Elisabeth Pasquier, Isabelle Pilatte, Virginie Poncelet, Olivier Querolle, David C. Rees, Sharna Rich, Bruno Roux, Elodie Sement, Yvan Simonnet, Matthew Squires, Virginie Tronel, Tinne Verhulst, Jorge Vialard, Marc Willems, Steven J. Woodhead, Berthold Wroblowski, Christopher W. Murray, Timothy Perera. Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2014-4748

Protocols for the Design of Kinase-Focused Compound Libraries

Protocols for the design of kinase‐focused compound libraries are presented. Kinase‐focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure‐based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators.

Abstract 3229: Managing stress: Discovery of inhibitors of the atypical kinase eEF2K and the class III PI3K, VPS34

Adaptation to nutrient deprivation in the tumour microenvironment was recently shown to be dependent on the appropriate regulation of protein elongation rate through activation of the atypical kinase, eukaryotic elongation factor 2 kinase (eEF2K) (Leprivier et al., 2013, Cell 153(5):1064-79). We have solved the crystal structure of the kinase domain of eEF2K, and used structure-based design as well as screening approaches to optimize a chemical series into single-digit nM inhibitors of eEF2K, with remarkable selectivity across the protein kinome (only 5-10 kinases out of 400 tested are inhibited to more than 50% at 1 μM). These compounds inhibit the phosphorylation of eEF2 in nutrient-starved or metabolically stressed cells, and increase protein elongation rates through stabilization of the ribosomal elongation complex under stress. Evotec9s Cellular Target Profiling of these compounds in cell lysates, revealed that a subset of the eEF2K inhibitors also bind with low nM affinity to the class III phosphatidylinositol-3-kinase, VPS34, but not to class I or II PI3Ks, and pull down the entire beclin-UVRAG-VPS34 complex. Proteomic and biochemical screening of the compound set enabled deconvolution of potent EF2K versus VPS34 inhibitors. Inhibition of VPS34 results in abrogation of autophagic flux, as indicated by rapid and massive accumulation of p62, and impairs survival in specific subsets of tumor cell lines, consistent with a pro-survival role for autophagy in those models (Cheng et al., 2013, Pharmacol Rev 65(4):1162-97). Interestingly, a whole-genome pooled shRNA screen in a KRAS/PI3KCA mutant colorectal cancer cell line revealed that reduction of beclin levels significantly increased sensitivity to VPS34 inhibition. In contrast, inhibition of eEF2K does not appear to be anti-proliferative across a wide panel of cancer cell lines under standard cell culture conditions. Our work has provided the first potent inhibitors to unravel the functional relevance of eEF2K and VPS34 in adaptation to cellular stress, and to examine the utility of inhibiting these kinases in nutrient-deprived and/or autophagy-addicted tumours. Citation Format: Matthias Versele, Claire Moore, Christopher G. Proud, Cindy Rockx, Inez Van de Weyer, Kurt Van Baelen, Stephanie Blencke, Sebastian K. Wanndinger, Gaston Diels, Didier Berthelot, Marcel Viellevoye, Bruno Schoentjes, Berthold Wroblowski, Lieven Meerpoel, William N. Hait. Managing stress: Discovery of inhibitors of the atypical kinase eEF2K and the class III PI3K, VPS34. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3229. doi:10.1158/1538-7445.AM2014-3229

Abstract 4800: Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia

Inhibition of Flt3 kinase activity is a promising strategy for the treatment of Flt3 mutant acute myeloid leukemia (AML). However, clinical studies with Flt3 kinase inhibitors have shown that mutations of the gate keeper or an activation loop residue in the Flt3 catalytic domain limits the duration of response. Such mutations generally reduce kinase domain binding affinity and residence time of the kinase inhibitor, and consequently its efficacy. We therefore investigated whether covalent, irreversible binding to Flt3 could overcome some of the limitations of classic non-covalent Flt3 inhibitors. Here, we report for the first time on the high-affinity Cys828-covalent binding mode of a resorcylic acid lactone to the isolated Flt3 kinase domain using X-ray crystallography and kinetic binding assays. In a cellular context (Ba/F3-Flt3-ITD), mutation of Cys828 to Ala reduces potency (IC50) from low nM to microM, demonstrating all relevant Flt3 inhibition in cells critically depends on Cys828. Consistently, the molecule is a low nM inhibitor of both Flt3-ITD and Flt3D835Y in vitro and in BaF3 cells, translating to potent nM inhibition of Flt3-ITD driven AML cell line proliferation, with only microM antiproliferative activity in non-Flt3 driven AML and unrelated leukemia cell lines. Finally, in spite of its fast clearance when dosed to MV4-11 (Flt3-ITD AML) xenograft bearing mice, robust anti-tumor activity was observed using a once-daily treatment schedule. These data demonstrate the feasibility of covalent Flt3 inhibition, and suggest it represents an attractive novel therapeutic approach for the treatment of Flt3-driven AML. Citation Format: Matthias Versele, Burkhard Haefner, Berthold Wroblowski, Ian Stansfield, Laurence Mevellec, Ron Gilissen, Lars Neumann, Martin Augustin, Kris Jacobs, Jan Cools, S Barluenga, M Rothlingshofer, G Karthikeyan, Ricardo Attar, Lieven Meerpoel, Nicolas Winssinger. Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4800.

Abstract 3640: Discovery of quinazolinones as fibroblast growth factor receptor (FGFR1-4) kinase inhibitors

Recent data obtained in several tumor types have identified Fibroblast Growth Factor signaling as a key factor in the molecular pathology of a number of cancers1. This has stimulated the development of a number of agents that block this pathway, including FGFR kinase inhibitors with diverse inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We recently reported that a quinoxaline moiety can efficiently bind the hinge region of FGFR kinase catalytic sites. In continuation of our efforts to discover additional FGFR1-4 inhibitors, we have identified a quinazolinone scaffold as a novel FGFR kinase hinge binder. Initial hits were optimized into compounds displaying nanomolar affinity for FGFR1-4, potent activity in FGFR driven cells and efficacy in a Ba/F3-FGFR3 xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathways of these novel quinazolinone-based FGFR1-4 inhibitors. Turner N. and Grose R. Nature Reviews-Cancer 2010, 10, 116-129 Citation Format: Olivier Querolle, Patrick Angibaud, Helene Colombel, Virginie Caron, Isabelle Pilatte, Virginie Poncelet, Norbert Esser, Ron Gilissen, Peter King, Lieven Meerpoel, Tinne Verhulst, Berthold Wroblowski, Jorge Vialard, Chris W. Murray, David C. Rees, Anita Reningovolo, Gordon Saxty. Discovery of quinazolinones as fibroblast growth factor receptor (FGFR1-4) kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3640. doi:10.1158/1538-7445.AM2015-3640

Abstract 3641: Identification of naphthyridines as potent inhibitors of fibroblast growth factor receptor kinase family

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The fibroblast growth factor receptor (FGFR) tyrosine kinase family members, FGFR1, 2, 3 and 4, have roles in a variety of key cellular processes, including proliferation, migration, survival, and differentiation1. Aberrant activation of FGFRs through mutation, amplification, chromosomal translocation, and ligand up-regulation being strongly implicated in oncogenic signalling in many tumour types, has triggered efforts to identify selective FGFR inhibitors. As a result, several potent FGFR kinase inhibitors are currently being evaluated in clinical studies across many tumor types, including non-small cell lung, breast and bladder cancers. We have designed novel 1,5 and 1,7-naphthyridine derivatives that are potent kinase inhibitors of all FGFR family members in enzymatic and cellular systems. Initial hits were further optimized to increase potency and ADME properties leading to identification of a novel 1,5-naphthyridine-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. In vivo screening using an FGFR3-driven xenograft model revealed efficacious compounds that could be explored further as antitumoral agents. This report represents the first disclosure of the structure-activity relationship and synthesis pathway of novel naphthyridine chemical series displaying nanomolar affinity for FGFRs1, 2, 3 and 4. 1 Dieci M. V., Ardenos M., Andre F., Soria J.C. Cancer Discovery. 2013, 3(3) 264-279. Citation Format: Patrick R. Angibaud, Michel Obringer, Julien Marin, Matthieu Jeanty, Norbert Esser, Ron Gilissen, Peter King, Lieven Meerpoel, Olivier Querolle, David C. Rees, Bruno Roux, Gordon Saxty, Tinne Verhulst, Berthold Wroblowski, Christopher C. Murray, Jorge Vialard. Identification of naphthyridines as potent inhibitors of fibroblast growth factor receptor kinase family. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2015-3641