The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 Å ). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an ;1000-fold reduction in affinity relative to DPP-IV WT , while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at ;14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.Keywords: DPP-IV; X-ray crystal structure; mutant; ITC; proton NMR; short, strong hydrogen bond; serine protease; saxagliptin Dipeptidyl peptidase IV is a serine protease that modulates the biological activity of specific circulating peptide hormones, chemokines, cytokines, and neuropeptides Abbreviations: ANS, 1-anilino-8-naphthalene sulfonate; DPP-IV, dipeptidyl peptidase IV; GLP-1, glucagon-like peptide-1; ITC, isothermal titration calorimetry; pNA, p-nitroaniline; P n -P n9 , amino acid residues of the substrate which numerically indicate the position relatively to the scissile bond, n being the residues toward the N terminus and n9 being the residues toward the C terminus (this nomenclature was first defined by Schechter and Berger [1967]); SEC-MALS, size-exclusion chromatography-multiple angle light scattering; SKIE, solvent kinetic isotope effect; SSHB, short strong hydrogen bond; SIHB, short ionic hydrogen bond; TSE, thermal stability enhancement.Article and publication are at http://www.proteinscience.org/cgi

This paper provides an unbiased comparison of four commercially available programs for loop sampling, Prime, Modeler, ICM, and Sybyl, each of which uses a different modeling protocol. The study assesses the quality of results and examines the relative strengths and weaknesses of each method. The set of loops to be modeled varied in length from 4-12 amino acids. The approaches used for loop modeling can be classified into two methodologies: ab initio loop generation (Modeler and Prime) and database searches (Sybyl and ICM). Comparison of the modeled loops to the native structures was used to determine the accuracy of each method. All of the protocols returned similar results for short loop lengths (four to six residues), but as loop length increased, the quality of the results varied among the programs. Prime generated loops with RMSDs <2.5 Å for loops up to 10 residues, while the other three methods met the 2.5 Å criteria at seven-residue loops. Additionally, the ability of the software to utilize disulfide bonds and X-ray crystal packing influenced the quality of the results. In the final analysis, the top-ranking loop from each program was rarely the loop with the lowest RMSD with respect to the native template, revealing a weakness in all programs to correctly rank the modeled loops.

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RORγt is an important nuclear receptor that regulates the production of several pro-inflammatory cytokines such as IL-17 and IL-22. As a result, RORγt has been identified as a potential target for the treatment of various immunological disorders such as psoriasis, psoriatic arthritis, and inflammatory bowel diseases. Structure and computer-assisted drug design led to the identification of a novel series of tricyclic RORγt inverse agonists with significantly improved in vitro activity in the reporter (Gal4) and human whole blood assays compared to our previous chemotype. Through careful structure activity relationship, several potent and selective RORγt inverse agonists have been identified. Pharmacokinetic studies allowed the identification of the lead molecule 32 with a low peak-to-trough ratio. This molecule showed excellent activity in an IL-2/IL-23-induced mouse pharmacodynamic study and demonstrated biologic-like efficacy in an IL-23-induced preclinical model of psoriasis.

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Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3, which was confirmed by protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an enhancement in selectivity against key off-target kinases.

Novel tricyclic analogues were designed, synthesized, and evaluated as RORγt inverse agonists. Several of these compounds were potent in an IL-17 human whole blood assay and exhibited excellent oral bioavailability in mouse pharmacokinetic studies. This led to the identification of compound 5, which displayed dose-dependent inhibition of IL-17F production in a mouse IL-2/IL-23 stimulated pharmacodynamic model. In addition, compound 5 was studied in mouse acanthosis and imiquimod-induced models of skin inflammation, where it demonstrated robust efficacy comparable to a positive control. As a result of this excellent overall profile, compound 5 (BMS-986251) was selected as a clinically viable developmental candidate.

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