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.

Functional comparison of recombinant acidic mammalian chitinase with enzyme from murine bronchoalveolar lavage.

Acidic mammalian chitinase (AMCase) is an enzyme that selectively degrades the biopolymer chitin. Several chitinase enzymes are utilized by mammals to hydrolyze chitin encountered by inhalation and ingestion. AMCase is distinct from other mammalian chitinases as its activity is retained in strongly acidic conditions (pH <2.0). AMCase expression is induced by antigen-induced mouse models of allergic lung inflammation. This protein has also been implicated in the pathogenesis of asthma although its precise role is poorly defined. We describe a novel way to express and purify active murine AMCase. This material retains properties observed in mouse bronchoalveolar lavage (BAL) fluid with regard to pH preference of activity and its inhibition by cyclic peptide inhibitors argifin and argadin. We found that chitinase in BAL from both antigen-challenged and control animals have similar properties in this regard. This strongly supports the notion the same enzyme (AMCase) gives rise to chitinase activity in both challenged and unchallenged animals. We also describe expression of active human AMCase. The methods described in this paper provide a reliable source of recombinant AMCase that can be utilized to expand understanding of AMCase's role in regulating allergic inflammation.

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode.

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.