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

Yiming Zhong,Shuai Dong,Ethan Strattan,Li Ren,Jonathan P Butchar,Kelsey Thornton,Anjali Mishra,Pierluigi Porcu,J Michael Bradshaw,Angelina Bisconte,Timothy D Owens,Erik Verner,Ken A Brameld,Jens Oliver Funk,Ronald J Hill,Amy J Johnson,Jason A Dubovsky

doi:10.1074/jbc.m114.614891

Abstract

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Highlights

inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) are unique to effector lymphocytes and critical for immune activation
Potency, and Specificity of PRN694 —We discovered highly selective ITK/RLK inhibitors using a combination of covalent targeting technology and structure-based design
ITK expression was not detectable in the dendritic cells, macrophages, or RBL-2H3 mast cells we used [41]. These results indicate that in natural killer (NK) cells, but not in dendritic, macrophage, or RBL-2H3 mast cells, which do not express ITK and RLK, Fc receptor (FcR)-induced stimulation is curtailed by PRN694

Summary

Background

ITK and RLK are unique to effector lymphocytes and critical for immune activation. Results: A novel selective covalent ITK/RLK inhibitor called PRN694 was discovered, which blocks T-cell and NK cell activation. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. In vivo experiments demonstrate the pharmacokinetics and pharmacodynamics of PRN694 and show that it attenuates a delayed type hypersensitivity (DTH) reaction in a well established murine model system These results indicate promising clinical applicability of this ITK/RLK dual inhibitor for the treatments of T-cell or NK cell malignancies as well as inflammatory and autoimmune diseases, such as psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, and irritable bowel disease

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION