We have found that covalent attachment of myristate (Cl4:0) to the NH2-terminal Gly residue of HIV-1 Pr55gag by myristoylCoA:protein N-myristoyltransferase (NMT) is necessary for viral assembly. Our analysis of the mechanism of action of NMT has led to the synthesis of myristic acid analogs which are alternative substrates for acylCoA synthetase and NMT. Group 6B heteroatom substituted analogs with altered physical chemical properties (including reduced hydrophobicity) were found to be selectively transferred to subsets of cellular N-myristoylproteins (owing in part to an apparent co-operative interaction between the enzyme's acylCoA and peptide binding site). Once incorporated they produce analog-dependent and specific alterations In protein function. Incubation of one such analog - 13-oxatetradecanoic - acid with infected H9 cells results in its incorporation into HIV-1 Pr55gag and nef, inhibition of proteolytic processing of gag, and inhibition of viral replication in acutely and chronically infected H9 cells without cellular toxicity. Our group proposes to continue to use biochemical, organic chemical, genetic and biophysical methods to define the kinetic mechanisms and structure/activity relationships of S. cerevisiae and human NMTs in an attempt to better understand how to design novel analogs with altered physical properties which can be selectively targeted to viral proteins by cellular NMT and thereby disrupt the their function(s). This will involve (1) isolation of human NMT cDNA and expression of the human enzyme In E. coli; (2) systematic synthesis of fatty acid analogs with varying structural motifs to explore issues of enzyme recognition; (3) definition of the enzyme's kinetic mechanism using a recently developed continuous assay; (4) use ts mutants of the S. cerevisiae NMT1 gene to identify structural features critical for (a) catalysis; (b) the mechanisms underlying catalysis and (c) the nature of its functional Interactions with myristoylCoA generating systems; (5) NMR and x-ray studies of the interactions of NMT with its ligands (6) use of H9 cells and an E. coli-expression system to examine the effects of analogs on gag polyprotein precursor processing by viral protease and intracellular targeting; (7) characterization of the metabolic processing of radiolabeled analogs in H9 cell cultures and in mice; and (8) assessment of the efficacy of analogs in animal models so that decisions concerning compound selection for clinical trials can be made as rapidly as possible.
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