Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

IL2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) are two members of the Tec family of non-receptor tyrosine kinases which are essential mediators of intracellular signaling in both normal and neoplastic T and natural killer (NK)-cells. ITK-deficient mice display alterations in T cell receptor (TCR) responses including proliferation, cytokine production and activation of downstream pathways, and these defects are exacerbated by RLK deficiency. Given their critical roles, inhibitors of ITK and RLK have therapeutic potential in a number of human autoimmune diseases, inflammatory conditions, and malignancies. Specific inhibitors targeting both ITK and RLK have not been reported and until very recently it has been challenging to develop specific inhibitors with clinical potential given the homology to many other human kinases. Targeting cysteine residues within the ATP binding pocket of kinases is a safe and effective way to design and optimize highly potent and selective kinase inhibitors. In this study, we describe preclinical mechanism of action studies of a novel covalent ITK/RLK inhibitor, PRN694, which covalently binds to cysteine moieties 442 of ITK and 350 of RLK and blocks their kinase activities. We undertook the design of highly selective ITK/RLK inhibitors using a combination of tailored covalency and structure-based design. Molecular modeling and a synthetic feasibility assessment led to the prioritization of several small focused compound libraries. The structure-based medicinal chemistry optimization of one such library culminated in the identification of PRN694. The in vitro selectivity and potency of PRN694 against a panel of 250 kinases revealed only small number of kinase targets, and in a microfluidic kinase assay, PRN694 exhibited high selectivity and potency against ITK and RLK with IC50 values of 0.3 nM and 1.3 nM, respectively. This selectivity was validated in Jurkat T cells with mutated ITK or overexpressed RLK. As expected, PRN694 suppressed a variety of downstream TCR pathways including nuclear activation of NFAT1 and cytoplasmic PLCγ1 and IKBα in TCR-activated Jurkat cells expressing wild type ITK. However, when the covalent binding site for PRN694 was removed, using ITK-C442A, this suppressive effect was lost, indicating specific covalent interaction with ITK. Furthermore, we demonstrated that only PRN694 was able to ablate TCR-induced activation in Jurkat cells which stably express RLK in addition to ITK. These data confirmed that PRN694 was a dual inhibitor of ITK and RLK and could block downstream TCR-activation in T-cells that express both complementary TEC-kinases. In vitro cellular assays also confirmed that PRN694 could prevent TCR or Fc receptor (FcR) - induced cellular and molecular activation in primary T or NK cells, inhibit TCR-induced T-cell proliferation without direct cytotoxicity, and block pro-inflammatory cytokine (including IL-2, TNFα, and IFNγ) release as well as activation of Th17 cells. Additionally, in vivo assays demonstrated durable pharmacodynamic effects of PRN694 on ITK. At 1 hour, 6 hour and 14 hour time points, the percentages of ITK occupancy were 98%, 95% and 54% respectively, and the concentrations of PRN694 in the plasma were 1530 ng/ml, 359.3 ng/ml and 14.8 ng/ml respectively. Further, we examined potential therapeutic applications of PRN694 in the treatment of T-cell leukemias or T-cell driven inflammation. Our results demonstrated an effective blockade of TCR stimulation by PRN694 in the HH cutaneous T-cell lymphoma cell line and primary T-cell prolymphocytic leukemia (T-PLL) cells, suggesting a potential clinical application of this compound in the treatment of T-cell leukemias. ITK and RLK play a critical role in T-cell driven inflammation. Therefore, we also tested the effect of PRN694 on an oxazolone induced delayed type hypersensitivity (DTH) reaction using a well-established in vivo model of inflammation driven by cell-mediated immunity, and found that PRN694 treatment significantly inhibited the DTH reaction. In conclusion, we designed and characterized a novel covalent ITK/RLK inhibitor, PRN694, which has direct therapeutic potential for the treatment of T or NK cell related diseases including inflammation, autoimmune diseases, and malignancy. Disclosures Porcu: Actelion: Honoraria; Infinity: Research Funding; Seattle genetics: Research Funding; Celgene: Honoraria; United States Cutaneous Lymphoma Consortium: Membership on an entity9s Board of Directors or advisory committees; Cutaneous Lymphoma Foundation: Membership on an entity9s Board of Directors or advisory committees. Bradshaw: Principia Biopharma: Employment, Equity Ownership. Owens: Principia Biopharma, Inc: Employment, Equity Ownership. Brameld: Principia Biopharma: Employment, Equity Ownership. Funk: Principia Biopharma Inc.: Employment, Equity Ownership. Hill: Principia Biopharma: Employment. Johnson: Principia Biopharma: Research Funding. Dubovsky: Principia Inc.: Research Funding.