Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol.

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

Minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR): quantitative measures for selectivity ratios obtained by screening assays.

Development of inhibitor compounds selective against undesirable targets is critical in drug discovery. Selectivity ratios for candidate compounds are evaluated by dividing potencies from two assays assessing the off-target and target. Because all potency measurements have underlying uncertainty, understanding error propagation is essential to interpreting selectivity data. Assay noise introduces ambiguity in the statistical significance of selectivity ratios, particularly at low replicate numbers when compounds are often prioritized for subsequent testing. The ability to differentiate potency results for any pair of compounds in one assay is evaluated using a metric called minimum significant ratio (MSR). Potency results of one compound tested in a pair of assays can be differentiated by the minimum significant selectivity ratio (MSSR). To differentiate selectivity ratios for any pair of compounds, we extend this concept by proposing two new parameters called the minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR). Importantly, these tools can be used after a single selectivity measurement. We describe these methods and illustrate their usefulness using structure-activity relationship data from a Janus kinase inhibitor project, in which these tools informed a cogent retesting strategy and enabled rapid and objective decision making.

Immunohistochemical expression of Pit-1 protein in human pituitary adenomas.

Pit-1, the pituitary-specific transcriptional factor, has been known as a gene that regulates the functional differentiation of the anterior pituitary gland, especially in GH, PRL, and TSH production. Our immunohistochemical studies were performed to elucidate the functional roles of the pit-1 product in human pituitary adenomas. Eighty-six pituitary adenomas consisted of 51 GH-producing adenomas, 18 nonfunctioning (NF) adenomas, 1 1 PRL adenomas, and 6 TSH adenomas. Indirect immunoperoxidase method was performed using antibodies against GH, PRL, ACTH, the α subunit (SU), FSH ß-SU, LH ß-SU, TSH ß-SU, and pit-1 product. Pit-1 product was expressed in 38 patients in the nuclei of the adenoma cells. The frequency of positive pit-1 product was significantly high in the patients with TSH and GH adenomas compared with those who had NF adenomas. These results suggest a role for pit-1 in functional differentiation of the human pituitary adenomas, especially TSH and GH adenomas.