Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low levels, and a defensive cytotoxin at higher levels. The high active-site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock, and cancer. Our structural and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a novel specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents towards remote specificity pockets, accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active-site conservation.

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A series of substrate analogue inhibitors of pancreatic phospholipase A2 has been designed and synthesized. The compounds were tested in a novel dual-screening system based on parallel assays with monomeric and micellar substrates. Intermolecular nuclear Overhauser effects between vinylic protons on one inhibitor and identified active site residues on the bovine pancreatic enzyme have been observed in solution NMR studies of the enzyme-inhibitor complex. It can be deduced from both the biochemical results and the NMR data that the mode of interaction between this type of inhibitor and the active site of phospholipase A2 is essentially the same, irrespective of the presence or absence of an aggregated phospholipid surface. A model of the binding between the enzyme and inhibitor which incorporates the two-dimensional NMR data has been developed. The model can account for the activity of modified inhibitor structures and can be extrapolated to an assessment of the mode of binding of the natural substrate itself.

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The use of GRID in the 3-D QSAR analysis of a series of calcium-channel agonists is described. Partial least-squares analysis of GRID maps showing the interaction energy between an alkyl hydroxyl probe and a series of agonists in 3-D space generated a predictive quantitative model of the variation of biological activity. The macroscopic descriptors CLOGP and CMR were included in the analysis, and the importance of appropriate block scaling is highlighted. The discussion highlights the interpretation of the resulting regression maps, and the steric, electrostatic, lipophilic, and hydrogen-bonding preferences of the calcium-channel receptor are identified.