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. 2021 Mar 19;12(4):653-661.
doi: 10.1021/acsmedchemlett.1c00096. eCollection 2021 Apr 8.

Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds

Brandon A Vara  1 , Samuel M Levi  1 , Abdelghani Achab  1 , David A Candito  1 , Xavier Fradera  1 , Charles A Lesburg  1 , Shuhei Kawamura  1 , Brian M Lacey  1 , Jongwon Lim  1 , Joey L Methot  1 , Zangwei Xu  1 , Haiyan Xu  1 , Dustin M Smith  1 , Jennifer A Piesvaux  1 , J Richard Miller  1 , Mark Bittinger  1 , Sheila H Ranganath  1 , David J Bennett  1 , Erin F DiMauro  1 , Alexander Pasternak  1
Affiliations

Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds

Brandon A Vara et al. ACS Med Chem Lett. .

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.

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Conflict of interest statement

The authors declare the following competing financial interest(s): The authors declare competing financial interests as Merck & Co., Inc., Kenilworth, NJ, USA stakeholders, stockholders and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA employees.

Figures

Figure 1
Figure 1
Profile of the initial lead compound 1. Rat PK: Wistar Han; 0.1 mg/kg IV. Mouse PK: CL57BL/6; 1/10 mg/kg IV/PO. Cl measured in mL/min/kg. MRT: mean resident time; Cl: total clearance; Clu: unbound clearance; %F: oral bioavailability.
Figure 2
Figure 2
Chemical visualization of the diaminopyrimidine core structure as a type-I HPK1 inhibitor and primary regions of SAR exploration. Initial compounds are exemplified, including 1. *Lipophilic ligand efficiency; LLE (experimental) based on HPK1 TR FRET IC50. #Standard error for IC50 and EC50 potency ranges are considered within 3× of the presented value, and data presented are an average of multiple runs, usually n = >3.
Figure 3
Figure 3
(a) Cocrystal structure of 1 with the HPK1 kinase domain, highlighting key secondary structural features and the hydrophobic region that sits below the P-loop, pictured in tan (PDB 7M0K). (b) Cocrystal structure of 1 bound to HPK1 highlighting key hinge binding residues (Glu92 and Cys94) and a salt bridge interaction with the solvent exposed THIQ moiety and Asp101.
Figure 4
Figure 4
Optimized assembly of diaminopyrimidine HPK1 inhibitor scaffolds.
Figure 5
Figure 5
Compounds 1 and 7 and their respective IL-2 secretion (ELISA EC50) dose–response curves (raw data). Calculated EC50 values from separate PMBC donor samples (353 and 374) are presented. A standard threshold set at “100% Effect” (dotted line) vs the control compound S1 presents a qualitative assessment for elevated or sustained IL-2 responses when inhibitor is present. In the examples presented, 7 maintains robust and desirable curve shapes in both PBMC donors, whereas 1 exhibits a sustained curve in only one donor.
Figure 6
Figure 6
Single crystal X-ray of 27 bound to HPK1, detailing the hydrophobic interactions and orientation of the 2-fluoro-6-(trifluoromethyl)aniline ring system (PDB 7M0M).
Figure 7
Figure 7
Complete profile for 27.
See this image and copyright information in PMC

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