Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

pubs.acs.org/acsmedchemlett Letter Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1) Bryan K. Chan,* Eileen Seward, Michael Lainchbury, Thomas F. Brewer, Le An, Toby Blench, Matthew W. Cartwright, Grace Ka Yan Chan, Edna F. Choo, Jason Drummond, Richard L. Elliott, Emanuela Gancia, Lewis Gazzard, Baihua Hu, Graham E. Jones, Xifeng Luo, Andrew Madin, Sushant Malhotra, John G. Moffat, Jodie Pang, Laurent Salphati, Christopher J. Sneeringer, Craig E. Stivala, Binqing Wei, Weiru Wang, Ping Wu, and Timothy P. Heffron Cite This: ACS Med. Chem. Lett. 2022, 13, 84−91 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiroazaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells. KEYWORDS: HPK1, immuno-oncology, structure-based design, kinase inhibitor, CYP inhibition I A high-throughput screen using the kinase domain of HPK1 yielded compound 1, a literature Abl inhibitor6 (Abl IC50 < 0.51 nM), as a promising hit with respect to potency for HPK1 (Ki = 0.4 nM) and lipophilic ligand efficiency7 (LLE = 6.3). In addition to activity against Abl, 1 also showed inhibitory activity against LCK (IC50 = 24 nM, 55-fold selectivity over HPK1). Since Abl8 and LCK9 are essential components of cell survival and TCR signaling, respectively, improving selectivity against these two off-targets was a major focus of our optimization effort. To improve selectivity against Abl, we first explored the difference in the ATP binding site gatekeeper residue between HPK1 (Met) and Abl (Thr).10 Due to the shorter Thr side chain in Abl, modeling suggested that Abl would suffer from a greater loss of favorable van der Waals interactions if the size of the C3 substituent of the azaindole motif were reduced (R1 in Table 1). Gratifyingly, conversion of the anisole substituent (1, Abl IC50 < n contrast to traditional anticancer therapies where the goal is to target pathways that drive tumor proliferation or survival, cancer immunotherapies aim to leverage the patient’s own immune system to combat tumors.1 Over the past several years, a number of antibody inhibitors of immune checkpoints have found considerable clinical success by enhancing cancer patients’ immune T-cell activity. The survival benefits engendered by those approved antibody inhibitors have been impressive; however, the response rate to these agents remains modest. Hence, there is tremendous interest in the identification of additional agents to complement the approved immunomodulators.2 Hematopoietic progenitor kinase 1 (HPK1) is a member of the Ste20 serine/threonine family of kinases that serves as a negative regulator of T-cell receptor (TCR) induced T-cell activation.3 Studies using HPK1 kinase-dead knock-in mouse models have demonstrated that HPK1 kinase activity limits TCR signaling and cytokine production.4 In preclinical syngeneic models, loss of HPK1 kinase function was found to suppress tumor growth. Given the compelling evidence, HPK1 has been proposed to be a promising cancer immunotherapy target.5 As such, the goal of our program was to identify a selective small molecule inhibitor of HPK1. © 2021 American Chemical Society Received: August 30, 2021 Accepted: December 1, 2021 Published: December 8, 2021 84 https://doi.org/10.1021/acsmedchemlett.1c00473 ACS Med. Chem. Lett. 2022, 13, 84−91 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Table 1. Optimization of 7-Azaindoles compd R1 R2 cLogP HPK1 Ki (nM)a LLEb Abl selectivityc LCK selectivityd 1 2 3 4 5 6 2-MeOPh Et H Et Et Et H H H Me Et Cl 3.1 2.1 1.5 2.4 2.8 2.6 0.4 2.1 130 0.4 0.2 0.5 6.3 6.6 5.4 7.0 6.9 6.7 <1× 10× 55× 237× 66× 808× 2556× 501× 58× a All apparent Ki values represent arithmetic means of at least two determinations using the HPK1 Lantha binding biochemical assay. bLLE, lipophilic ligand efficiency based on HPK1 Ki. cSelectivity expressed as the ratio of Abl Ki over HPK1 Ki. dSelectivity expressed as the ratio of LCK Ki over HPK1 Ki. 0.51 nM) to the smaller ethyl group (2, Abl IC50 = 22 nM) resulted in considerable improvement in selectivity against Abl while improving the LLE against HPK1. The increase in LLE suggested that the small alkyl substituent was better suited for HPK1 binding. While compound 2 exhibited only modest Abl selectivity, this promising result suggested that optimizing other vectors of the ligand using a structure-based approach may further enhance the selectivity against Abl. An X-ray structure of 3 bound to HPK1 revealed a binding mode that was atypical among kinase structures (Figure 1A). While the 7-azaindole motif engaged the hinge region of the kinase in a canonical manner, the P-loop of the kinase adopted an unusual “folded” conformation. In the “folded P-loop” conformation, the side chain of Tyr28 was positioned to engage the ligand through hydrophobic interactions.11 Furthermore, the main-chain peptide bond N−H of Gly24 in the P-loop is directed toward the ligand and engaged the amide carbonyl of the ligand via a hydrogen bond. In contrast, in the more usual “extended P-loop” conformation (as exemplified by G1858;12 Figure 1B), both structural features (Gly24 N−H and Tyr28 side chain) would not be available to interact with the azaindole ligands. In the “extended” conformation, the N−H of Gly24 forms a hydrogen bond with Val31 within the P-loop β-sheet, while the side chain of Tyr28 is situated in the DFG region of the binding pocket, away from the hinge region.12 The “folded” P-loop conformation is not commonly observed among kinases that have been studied.11 The ability of HPK1 to adopt the folded conformation was attributed to the unusually high number of glycine residues within its P-loop resulting in a high degree of flexibility, a feature that is absent in the LCK sequence. Literature examples suggested that stabilizing or engaging the “folded” P-loop conformation may have the potential to improve selectivity.11,13 Hence, we hypothesized that further engagement of Gly24 and Tyr28 in the folded Ploop conformation of HPK1 may result in improvement in both (...truncated)