Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor

Abstract

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent value for each JAK isozyme ranged from 31.8 to 2.9M for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and>35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

The role of the JAK/STAT signal pathway in rheumatoid arthritis

“…Decernotinib and tofacitinib are both reversible SMIs. This being the case, Elwood and colleagues, 85 contended that the development of irreversible JAK3 SMIs were likely to be useful and highly effective for altering JAK-directed STAT activation. Thus, a newly synthesized irreversible JAK-3 inhibitor, called Compound 2, was shown to be 4300-fold selective towards JAK3 versus JAK1 in enzyme assays and >35-fold selective in human peripheral blood mononuclear cell assays in assessing JAK/STAT activation by IL-7 versus IL-6 or granulocyte/macrophage colony stimulating factor.…”

Janus kinases to jakinibs: from basic insights to clinical practice

“…It will be important to determine whether neutropenia is seen with this compound. Other JAK3-selective jakinibs are also in development [103,104]. Peficitinib is a pan-JAK inhibitor, but it is also reported to have some selectivity for JAK3.…”

Translational and clinical advances in JAK-STAT biology: The present and future of jakinibs

“…PF‐06651600 is another selective JAK3 inhibitor being studied in RA, alopecia areata, CD, and UC (NCT02974868, NCT02958865, NCT02969044, NCT03395184). Other JAK3‐selective jakinibs are also in development …”