High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure−activity relationships of C 2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and “unbound” (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2‘ pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure−activity relationships, and molecular recognition of this series of C 2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2‘ is also discussed.

The bacterial cell wall is a polymer consisting of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, cross-linked via peptides appended to MurNAc. The final steps in the formation of cell wall, also referred to as murein, are catalyzed by high-molecular-weight, class A penicillin-binding proteins (PBPs). These bifunctional enzymes catalyze both glycosyltransfer, to form the carbohydrate backbone of murein, and transpeptidation, to form the interstrand peptide linkages. Using PBP1b from Eschericia coli, an in vitro kinetic characterization of the glycosyltransfer reaction was carried out. Initial studies with unlabeled substrate (Lipid II) revealed that activity is strongly influenced by DMSO, as well as metal and detergent. In addition, a continuous fluoresence assay was developed and used to determine the effect of pH on the reaction. A single basic residue was titrated, with a pK(a) of 7.0. Taken together, these data suggest a mechanism for PBP1b where the glycosyltransfer reaction is catalyzed by the concerted effect of an active site base to deprotonate the glycosyl acceptor and a divalent metal to assist departure of the leaving group of the glycosyl donor.

In developing inhibitors of the LIM kinases, the initial lead molecules combined potent target inhibition with potent cytotoxic activity. However, as subsequent compounds were evaluated, the cytotoxic activity separated from inhibition of LIM kinases. A rapid determination of the cytotoxic mechanism and its molecular target was enabled by integrating data from two robust core technologies. Highcontent assays and gene expression profiling both indicated an effect on microtubule stability. Although the cytotoxic compounds are still kinase inhibitors, and their structures did not predict tubulin as an obvious target, these results provided the impetus to test their effects on microtubule polymerization directly. Unexpectedly, we confirmed tubulin itself as a molecular target of the cytotoxic kinase inhibitor compounds. This general approach to mechanism of action questions could be extended to larger data sets of quantified phenotypic and gene expression data. [Mol Cancer Ther 2008;7(11):3490-8]