Deciphering the Selective Inhibition of ITK and RLK Kinases with PRN694: A Promising Therapeutic Strategy for T-Cell Mediated Diseases

The abstract discusses the development of a new inhibitor, PRN694, targeting the enzymes ITK and RLK, which are crucial for signaling in T and NK cells. Mice lacking ITK show impaired T cell receptor responses, and the absence of RLK further aggravates these issues. Due to their importance, ITK and RLK inhibitors are being considered for treating autoimmune diseases, inflammation, and cancer. However, creating specific inhibitors has been difficult due to the similarity these enzymes share with other human kinases.

The researchers focused on the ATP binding site of the kinases, where they designed PRN694 to bind covalently to specific cysteine residues, thus inhibiting the enzymes' activity. The drug was developed using a combination of covalent bonding and structure-based design, leading to a highly selective inhibitor. In vitro tests showed PRN694's high selectivity and potency against ITK and RLK, with low IC50 values, indicating strong binding affinity.

The inhibitor's effectiveness was confirmed in Jurkat T cells, where it suppressed various T cell receptor pathways. The specificity of PRN694 for ITK was demonstrated when its binding site was removed, leading to a loss of inhibitory effect. PRN694 was also the only compound able to block T cell receptor activation in cells expressing both ITK and RLK.

Further in vitro assays showed that PRN694 could prevent T cell and NK cell activation, inhibit T cell proliferation without cytotoxicity, and block the release of pro-inflammatory cytokines and Th17 cell activation. In vivo assays revealed that PRN694 had a durable effect on ITK, with high occupancy percentages and plasma concentrations measured at different time points.

The study also explored PRN694's therapeutic potential in T-cell leukemia and T-cell driven inflammation. It effectively blocked TCR stimulation in T-cell lymphoma and T-PLL cells, suggesting its potential use in treating T-cell leukemias. Additionally, PRN694 significantly inhibited an oxazolone-induced delayed type hypersensitivity reaction in a model of cell-mediated immune-driven inflammation.

In summary, the researchers have characterized PRN694 as a novel covalent ITK/RLK inhibitor with potential therapeutic applications for treating diseases related to T or NK cells, including inflammation, autoimmune conditions, and cancer.