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Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Assays using active, phosphorylated protein kinases bias hits toward poorly selective inhibitors that bind within the highly conserved adenosine triphosphate (ATP) pocket. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Kinase cascade assays are typically initiated with target kinases in their unphosphorylated inactive forms, which are activated during the assays. Therefore, these assays are capable of identifying inhibitors that preferentially bind to the unphosphorylated form of the enzyme in addition to those that bind to the active form. We applied this cascade assay to the emerging cancer immunotherapy target hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase that negatively regulates T cell receptor signaling. Using this approach, we discovered an allosteric, inactive conformation-selective triazolopyrimidinone HPK1 inhibitor, compound 1. Compound 1 binds to unphosphorylated HPK1 >24-fold more potently than active HPK1, is not competitive with ATP, and is highly selective against kinases critical for T cell signaling. Furthermore, compound 1 does not bind to the isolated HPK1 kinase domain alone but requires other domains. Together, these data indicate that 1 is an allosteric HPK1 inhibitor that attenuates kinase autophosphorylation by binding to a pocket consisting of residues within and outside of the kinase domain. Our study demonstrates that cascade assays can lead to the discovery of highly selective kinase inhibitors. The triazolopyrimidinone described in this study may represent a privileged chemical scaffold for further development of potent and selective HPK1 inhibitors.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010–20. ©2017 AACR.

Activation of the MET/HGF pathway has been linked to tumor initiation, metastasis, angiogenesis and resistance to therapeutic agents. Here we present pharmacological characterization of OMO-1 (formerly JNJ-38877618), a potent, highly selective, orally bioavailable MET kinase inhibitor with nM binding affinity (Kd=1.4 nM) and enzyme inhibitory activity against wt and M1268T mutant MET (2 and 3 nM IC50). MET inhibitory effects were assessed in proliferation, colony formation and motility assays. OMO-1 displayed nM potency against MET Ampl/mutant and therapy resistant models. In vivo, OMO-1 induced complete inhibition of tumor growth in 3 models: the SNU5 MET amp gastric, U87-MG HGF autocrine glioblastoma and Hs746T MET exon 14 skipping mutant gastric cancer. OMO-1 induced regression of large MET amplified EBC-1 SqNSCLC where OMO-1 led to dose- and time-dependent inhibition of MET kinase activation, with the duration of target shut down considerably exceeding plasma exposure times. Combination treatments were well tolerated and improved EGFR targeted therapy. Although single agent OMO-1 had no effect on NSCLC HCC827 EGFR, combination with Erlotinib led to delayed onset of tumor recurrence. The acquired EGFR inhibitor resistant model HCC827-ER1 was determined to be MET amplified. OMO-1 and erlotinib both inhibited tumor growth of this model whilst combination induced tumor regression. In an EGFR inhibitor resistant PDX having MET amplification, single agent OMO-1 caused tumour stasis whereas MetMab/erlotinib only led to tumor growth delay. The potent preclinical activity we have observed, supports ongoing clinical development of OMO-1 in patients with MET pathway-driven tumors. Citation Format: Marion Libouban, Eleonora Jovcehva, Desiree De Lange, Boudewijn Janssens, Tinne Verhulst, Souichi Ogata, Berthold Wroblowski, Laurence Mevellec, Tianbao Lu, Glen Clack, Timothy Perera. OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4791.

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 Röthlingshöfer, 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.

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

Chromosomal rearrangements resulting in oncogenic fusion proteins containing the ROS1 receptor tyrosine kinase have been described in subsets of a variety of human malignancies including non-small-cell lung cancer (NSCLC), cholangiocarcinoma, and glioblastoma multiforme. Promising clinical responses have been observed in patients bearing tumors with ROS1 fusions treated with Xalkori, an ALK/MET kinase inhibitor that also inhibits Ros1. However, resistance has been observed and a ROS1 kinase domain mutation, G2032R, was identified in a ROS1 fusion positive NSCLC patient who developed resistance to Xalkori treatment. Clinical experience with other receptor tyrosine kinase inhibitors suggests that additional resistance mutations are likely to arise, highlighting the need for therapeutic agents that can overcome this type of resistance. We identified a novel chemical series of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. The structure of human Ros1 in a complex with a ligand from this chemical class was confirmed by X-ray crystallography. Here we describe the structure-activity relationships and synthesis route for this chemical series. A representative compound from this series inhibited isolated recombinant Ros1 kinase activity with an IC50 of approximately 30 nM. This compound inhibited less than 6% of kinases in a panel of 400 at 1 μM concentration. Growth of Ba/F3 cells engineered to express Ros1 autophosphorylation in HCC78 NSCLC cells that harbor a SLC34A2-ROS1 fusion were inhibited at similar concentrations as the isolated protein. This activity translated into potent Ba/F3-Ros1 tumor growth inhibition in mice. This compound was also active on Ba/F3 cells containing Ros1 with a mutation in the gatekeeper residue, L2026M, in cell growth assays in vitro and tumor growth in vivo. Furthermore, this unique binding mode provides scope for activity on additional Ros1 mutations that confer resistance to Xalkori, such as the clinically relevant G2032R mutation. The results shown here describe a novel chemical series with a unique binding mode that has potential for activity in Ros1 driven tumors with mutations that confer resistance to Xalkori. Citation Format: Laurence Mevellec, Berthold Wroblowski, Ron Gilissen, Sophie Descamps, Elisabeth Pasquier, Christophe Adelinet, Marine Bourgeois, Guillaume Mercey, Matthieu Jeanty, Thierry Jousseaume, Aurélie Luguern, Javier Astray Gandara, Said Akzinnay, Etienne Daras, Inge Boeckx, Nele Van Slycken, Mariette Bekkers, Jeroen Van De Ven, Tinne Verhulst, Lieven Meerpoel, Jorge Vialard. Discovery of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. [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 4745. doi:10.1158/1538-7445.AM2014-4745

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. A fragment screening campaign was conducted against the tyrosine kinase domain of FGFR1 to detect low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment inhibitors (molecular weight The poster will focus on the description of previously undescribed compounds bearing an imidazo[1,2-a]pyridine core scaffold where selectivity versus other protein kinases, for example FLT3, is obtained using the X-ray crystal structure and structure-based design. In summary we will illustrate how X-ray crystallography and fragment-based drug design (FBDD) can be used to discover compounds with activity in an FGFR driven xenograft model when dosed by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2011-1361