Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds.

pubs.acs.org/acsmedchemlett Letter Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds Brandon A. Vara,* Samuel M. Levi, Abdelghani Achab, David A. Candito, Xavier Fradera, Charles A. Lesburg, Shuhei Kawamura, Brian M. Lacey, Jongwon Lim, Joey L. Methot, Zangwei Xu, Haiyan Xu, Dustin M. Smith, Jennifer A. Piesvaux, J. Richard Miller, Mark Bittinger, Sheila H. Ranganath, David J. Bennett, Erin F. DiMauro, and Alexander Pasternak Cite This: ACS Med. Chem. Lett. 2021, 12, 653−661 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure−activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles. KEYWORDS: HPK1, oncology, immunotherapy, immune-oncology, kinase inhibitor I The protein kinase class of enzymes represents one such group of intracellular regulators which have been recently explored as immune modulators for immuno-oncology.9 Hematopoietic progenitor kinase 1, HPK1 (also known as mitogen-activated protein kinase kinase kinase kinase 1, MAP4K1), a member of the serine/threonine kinase family, is predominantly expressed in hematopoietic cells (e.g., T cells, B cells, neutrophils, dendritic cells (DCs), and macrophages) and has been demonstrated to function as a negative regular of T cell receptor (TCR) and B cell signaling.10,11 Under homeostatic cellular surveillance, active HPK1 phosphorylates S376 of the adaptor protein SLP76, which triggers a binding n contrast to traditional chemotherapy, cancer immunotherapy aims to stimulate or activate the innate and adaptive immune systems to fight malignancies and has become the new gold standard of cancer treatment.1 The immune checkpoint blockade, particularly along the programmed cell death protein-1/programmed cell death-ligand 1 (PD-1/PD-L1)2 and cytotoxic T lymphocyte antigen-4 (CTLA-4) axis,3 targets “exhausted” immune cell populations that demonstrate reduced proliferative activity in response to persistent antigen stimulation.4,5 Despite these advances, clinical data show that response rates and response durability6 have room for improvement, particularly in cases where adaptive immune resistance may be playing a pivotal role in immunosuppression.7 To circumvent potential immunoevasive events, attention has increasingly focused on intracellular pathways that could potentiate T cell-mediated killing of tumor cells, specifically by targeting negative regulators of immune responses that normally function to maintain peripheral tolerance.8 © 2021 American Chemical Society Received: February 12, 2021 Accepted: March 15, 2021 Published: March 19, 2021 653 https://doi.org/10.1021/acsmedchemlett.1c00096 ACS Med. Chem. Lett. 2021, 12, 653−661 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett event of the negative regulator complex 14-3-3. This complexation results in destabilization of the TCR through proteosomal degradation of SLP76 and impedes downstream kinase signaling necessary for T cell initiation and proliferation, dampening an innate immune response.12 Ultimately, of therapeutic interest to the field, loss of HPK1 function in HPK1 kinase-dead syngeneic tumor models MC38 and GL261 (glioma) has shown enhancement of T cell signaling and cytokine production as well as tumor growth inhibition in vivo when combined with an anti-PD-L1 agent.13,14 Additionally, HPK1(−/−) bone marrow-derived DCs expressed higher levels of costimulatory molecules and proinflammatory cytokines, demonstrated enhanced antigen presentation capacity, and were found to be superior to their wild-type counterparts in stimulating T cell proliferation.15 These data, along with other emerging studies,16−18 point to HPK1 as a potential therapeutic immuno-oncology target. Herein, we describe the developments of selective small molecule antagonists of HPK1 that elicit a robust and sustained T cell activation profile in human whole blood, providing in vitro pharmacological tools with the aim to ultimately elucidate the potential impact of HPK1 inhibition toward an antitumor immune response. We leveraged a high throughput screening (HTS) campaign of our compound library (250k) merged with structure-based drug design (SBDD) from HPK1 crystallographic19,20 and kinase literature data. Internal screening efforts identified multiple interesting diaminopyrimidine-based leads,21 including examples from Wee1 kinase,22 Syk,23 and ZAP-70 kinase programs24 with nanomolar potency. Preliminary in silico ligand docking corroborated that the diaminopyrimidine class of small molecules could serve as effective competitive binders of the ATP binding site with modular vectors for additional structure−activity relationship (SAR) studies. Routinely screening compounds in a mini (27 kinases) and broad kinase panel (265 kinases) allowed the team to gauge relative and absolute kinase selectivity, which were key determinants when advancing compounds.25 Crystallographic data of HPK1 show representative kinase architecture with a methionine (Met91) gatekeeper, a flexible glycine-rich (G-loop) domain situated above the ATP binding site, and the “hinge” residues consisting of Glu92, Glu93, and Cys94.20 From these data, we hypothesized adding a hinge interaction to Glu92 and extending toward the Met91 gatekeeper may improve potency and kinase selectivity. In silico modeling also suggested a key salt bridge interaction between Asp101 and the basic amine of a tetrahydroisoquinoline (THIQ, from representative Wee1 compounds22) was a critical anchor for potency and was further corroborated by related literature compounds.26,17 Consistent with our design hypothesis, 1, which features a carboxamide moiety in the 5 position to engage the hinge of HPK1 and a methoxy THIQ, was identified as a subnanomolar compound (HPK1 TR FRET IC50 = 0.5 nM, Figure 1). Compound 1 was shown to be cell active in two in vitro activity assays which drove downstream functional understanding of HPK1 target engagement (TE): an ELISA-based HPK1 TE assay v (...truncated)