Oxidoreductases of the thioredoxin superfamily possess the C-X-X-C motif. The redox potentials vary over a wide range for these proteins. A crucial determinant of the redox potential has been attributed to the variation of the X-X dipeptide. Here, we substitute Lys for Gly at the first X of Escherichia coli thioredoxin to investigate how a positive charge would affect the redox potential. The substitution does not affect the protein's redox potential. The equilibrium constant obtained from pairwise reaction between the mutant and wild-type proteins equals 1.1, indicating that the replacement does not significantly affect the thiol-disulfide redox equilibrium. However, the catalytic efficiency of thioredoxin reductase on the G33K mutant decreases approximately 2.8 times compared to that of the wild type. The mutation mainly affects K(m), with little effect on k(cat). The mutation also inhibits thioredoxin's ability to reduce insulin disulfide by approximately one-half. Whether the mutant protein supports the growth of phages T3/7 and f1 was tested. The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37 degrees C and 3 x 10(4) times at 42 degrees C relative to that of the wild-type strain, suggesting that interaction between phage gene 5 protein and thioredoxin is hindered. The mutation also reduces the EOP of phage f1 by 8-fold at 37 degrees C and 1.5-fold at 42 degrees C. The global structure of the mutant protein does not change when studied by CD and fluorescence spectra. Therefore, G33K does not significantly affect the overall structure or redox potential of thioredoxin, but primarily interferes with its interaction with other proteins. Together with the G33D mutation, the overall results show that a charged residue at the first X has a greater influence on the molecular interaction of the protein than the redox potential.

A search for structurally diversified Tyk2 JH2 ligands from 6 (BMS-986165), a pyridazine carboxamide-derived Tyk2 JH2 ligand as a clinical Tyk2 inhibitor currently in late development for the treatment of psoriasis, began with a survey of six-membered heteroaryl groups in place of the N-methyl triazolyl moiety in 6. The X-ray co-crystal structure of an early lead (12) revealed a potential new binding pocket. Exploration of the new pocket resulted in two frontrunners for a clinical candidate. The potential hydrogen bonding interaction with Thr599 in the pocket was achieved with a tertiary amide moiety, confirmed by the X-ray co-crystal structure of 29. When the diversity search was extended to nicotinamides, a single fluorine atom addition was found to significantly enhance the permeability, which directly led to the discovery of 7 (BMS-986202) as a clinical Tyk2 inhibitor that binds to Tyk2 JH2. The preclinical studies of 7, including efficacy studies in mouse models of IL-23-driven acanthosis, anti-CD40-induced colitis, and spontaneous lupus, will also be presented.

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In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo-[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure−activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

A novel class of 5-cyanopyrimidine-based inhibitors of p38alpha MAP kinase has been investigated. Analogues optimized through SAR iterations display low nanomolar enzymatic and cellular activity. The in vivo efficacy of this class of p38 inhibitors was demonstrated by 3a and 3b (>50% reduction in TNF levels when orally dosed at 5 mg/kg, 5 h prior to LPS administration in an acute murine model of inflammation). For 3a and 3b, the previously identified N-methoxybenzamide moiety (1) was replaced with N-(isoxazol-3-yl)benzamide, thereby providing increased metabolic stability. Cyanopyrimidine 3a demonstrated 100% oral bioavailability in mouse. High p38 kinase selectivity versus over 20 kinases was observed for analogue 3b. Direct hydrogen bonding of the cyano nitrogen of the 5-cyanopyrimidine core to the backbone NH of Met109 was confirmed by X-ray crystallographic analysis of 3a bound to p38alpha.