TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

Abstract Background: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in negatively regulating signal transduction cascades in cells of hematopoietic origin. Recent data generated using potent and highly selective HPK1 inhibitors underscores the role of HPK1 in negatively regulating T, B, and dendritic cell activation and thus orchestrating a comprehensive anti-tumor immune response. In the present study, we seek to further understand the biological effects of HPK1 inhibition in primary human T cells and in syngeneic models. Methods: RNA sequencing and multiplex cytokine analyses were utilized to profile both primary human T cells and the MC38 syngeneic model. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models. Results: In vitro, HPK1 small molecule inhibition resulted in enhanced cytokine production in primary human T cells. In vivo, HPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model. Conclusion: Pharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity. Citation Format: David Ciccone, Vad Lazari, Ian Linney, Michael Briggs, Samantha Carreiro, Christine Loh, Peter Tummino, Joshua McElwee, Alan Collis, Neelu Kaila. Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1649.

Introduction: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in regulating signal transduction cascades in cells of hematopoietic lineage. Recent data from HPK1 knockout animals and kinase-inactive knock-in animals underscores the role of HPK1 in negatively regulating lymphocyte activation. This negative-feedback role of HPK1 downstream of lymphocyte activation and function combined with its restricted expression in cells of hematopoietic origin make it an ideal drug target for enhancing anti-tumor immunity. Experimental Procedures: A structure-based drug design approach was used to identify potent and selective inhibitors of HPK1. Various biochemical and biophysical assays, as well as a primary in vitro T cell activation assay, were utilized for multiple rounds of structure-activity relationship (SAR) studies. In vivo target engagement and pharmacodynamic data were generated using an anti-CD3 mouse model. Results: In vitro, HPK1 small molecule inhibition resulted in enhanced IL-2 production in primary mouse T cells and in purified human T cells stimulated with a suboptimal dose of anti-CD3/anti-CD28. Increased selectivity of HPK1 inhibitors relative to T cell-specific kinases and within the MAP4K family was responsible for further enhancing the IL-2 response in activated T cells. In vivo, qd oral dosing of an HPK1 inhibitor completely abrogated phosphorylated SLP-76, induced by administration of anti-CD3. Furthermore, inflammatory cytokine production was enhanced in vivo upon HPK1 inhibition. Conclusion: Pharmacological blockade of HPK1 kinase activity represents a novel and powerful immunomodulatory approach for anti-tumor immunity. Citation Format: David Ciccone, Jennifer Rocnik, Vad Lazari, Ian Linney, Michael Briggs, Alan Collis, Christine Loh, Mark Ashwell, John Montana, Peter Tummino, Neelu Kaila. HPK1, hematopoietic progenitor kinase 1, is a promising therapeutic target for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 942.

Background HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in regulating signal transduction cascades in cells of hematopoietic origin. Recent data from HPK1 knockout animals and kinase-inactive knock-in animals underscores the role of HPK1 in negatively regulating immune cell activation. This negative-feedback role of HPK1 combined with its restricted expression in cells of hematopoietic origin, make it a compelling drug target for enhancing anti-tumor immunity. Methods A structure-based drug design approach was used to identify potent and selective inhibitors of HPK1. Biochemical assays, as well as primary human and mouse immune cell-based activation assays, were utilized for multiple iterations of structure-activity relationship (SAR) studies. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models. Results A highly potent, HPK1 inhibitor was identified, that showed high selectivity against T cell-specific kinases and kinases in the MAP4K family. In vitro, HPK1 small molecule inhibition resulted in enhanced IL-2 production in primary mouse and human T cells, enhanced IL-6 and IgG production in primary human B cells, and enhanced mouse dendritic cell activation and antigen presentation capacity. Furthermore, HPK1 inhibition alleviated the immuno-suppressive effects of PGE2 on naïve human T cells and restored the proliferative capacity of exhausted human T cells. In vivo, HPK1 inhibitionHPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model. Conclusions Pharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity.