Discovery of novel P1 groups for coagulation factor VIIa inhibition using fragment-based screening.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles.

Discovery of 6-(aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamides as potent, selective dipeptidyl peptidase-4 (DPP4) inhibitors.

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species.

Characterization of S-thiolation on secreted proteins from E. coli by mass spectrometry.

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells.

Differential activation of recombinant human acetyl-CoA carboxylases 1 and 2 by citrate.

The role of citrate as a physiological modulator of mammalian acetyl-CoA carboxylases (ACCs) has been well studied; however, the mechanism has not been clearly defined. In the current study, we found that citrate activated recombinant human ACC2 by more than approximately 1000-fold, but activated recombinant human ACC1 only by approximately 4-fold. The data fit best to a model which accounts for cooperative binding of two citrate molecules.

Involvement of DPP-IV catalytic residues in enzyme-saxagliptin complex formation.

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.

Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes.

Acetyl coenzyme A (acetyl-CoA) carboxylase isozyme 1 (ACC1) and acetyl-CoA carboxylase isozyme 2 (ACC2) are critical for de novo fatty acid synthesis and for the regulation of beta-oxidation. Emerging evidence indicates that one or both isozymes might be therapeutic targets for the treatment of obesity, type 2 diabetes, and dyslipidemia. One of the major obstacles in the field is the lack of readily-available source of recombinant human ACC enzymes to support systematic drug discovery efforts.