Reversible Janus kinase (JAK) inhibitors such as Tofacitinib(Changelian, et al., 2003;Flanagan, et al., 2010) and Decernotinib(Farmer, et al., 2015;Mahajan, et al., 2015) block cytokine signaling and are efficacious in treating autoimmune diseases (Kremer, et al., 2009;Fleischmann, et al., 2015;Fleischmann, et al., 2015;Krueger, et al., 2016;Sandborn, et al., 2012). However therapeutic doses are limited due to inhibition of other JAK/STAT pathways associated with hematopoiesis, lipid biogenesis, infection and immune responses(Kahn C, 2012). A selective JAK3 inhibitor may have a better therapeutic index, however, 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 Km for each JAK isozyme ranged from 31.8 μM for JAK1 to 2.9 μM for JAK3. To confirm compound activity in cells, we developed a novel enzyme complimentation 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 Km 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 (IL-2 vs. IL-6) or 140-fold (IL-2 vs. EPO or GMCSF) selective in cellular reporter assays and >35-fold selective in human PBMC assays (IL-7 vs. 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.
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