ANALOGS OF MYRISTIC ACID TO MODULATE HIV-I ASSEMBLY

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The 14 carbon saturated fatty acid myristate is linked via an amide bond to the NH2-terminal glycine residues of a number of viral proteins including the P17gag and F/3'orf of HIV-I. Site directed mutagenesis of the NH2-terminal Gly of the Mason-Pfizer Monkey viral Pr78gag to an Ala has been shown to prevent myristoylation, inhibit its subsequent proteolytic processing, and result in accumulation of intracytoplasmic A-type particles due to a presumed blockade in capsid transport to the plasma membrane where budding and release occur. We have isolated the enzyme responsible for this co- or early post-translational protein modification from lower (yeast, wheat germ) and higher (rat and human) eukaryotes. The fatty acid and peptide substrate specificities of Myristoyl CoA:Protein N-Myristoyltransferase (NMT) has been defined using an in vitro acylation assay. During the course of these studies we have synthesized oxy- and thio-substituted analogs of myristate which have remarkable reductions in their hydrophobicity yet exhibit similar kinetic properties to myristoyl CoA. These analogs compete with myristate for the acylation of cellular proteins but are not apparently toxic to cells. The specific aims of this multicenter study are (i) to extend our evaluation of acylCoA-NMT interactions through the continued synthesis of saturated and unsaturated fatty acids with one or more heteroatoms, (ii) to evaluate the ability of these analogs to inhibit retroviral - specifically HIV-I-replication in cultured cell lines, and (iii) to examine the atomic details of NMT-ligand interactions by cloning NMT, expressing the protein in E. coli and to crystallize it with and without its ligands.