ANALOGS OF MYRISTIC ACID TO MODULATE HIV-I ASSEMBLY

14碳饱和脂肪酸肉豆蔻酸酯通过酰胺连接 与许多病毒的NH 2-末端甘氨酸残基结合 HIV-I的P17 gag和F/3 'orf等蛋白。 定点 Mason-Pfizer猴的NH 2-末端Gly的诱变 病毒Pr 78 gag与Ala的结合已经显示出防止豆蔻酰化， 抑制其随后的蛋白水解加工， 胞浆内A型颗粒积聚，这是由于推测的 阻断衣壳转运到质膜， 释放发生。 我们已经分离出了 这种共翻译或早期翻译后蛋白质修饰， 低等（酵母，小麦胚芽）和高等（大鼠和人类）真核生物。 肉豆蔻酰的脂肪酸和肽底物特异性 CoA：蛋白N-肉豆蔻酰转移酶（NMT）已使用 体外酰化测定。 在这些研究中，我们 合成了肉豆蔻酸酯的氧代和硫代取代的类似物 其疏水性显著降低， 表现出与肉豆蔻酰辅酶A相似的动力学性质。 这些类似物 与肉豆蔻酸酯竞争细胞蛋白质的酰化， 对细胞没有明显的毒性。 具体目标是 多中心研究是（i）扩展我们对酰基辅酶A-NMT的评价 通过持续合成饱和和 具有一个或多个杂原子的不饱和脂肪酸，（ii）至 评估这些类似物抑制逆转录病毒的能力， 特别是HIV-I-在培养的细胞系中的复制，和（iii） 通过克隆来检查NMT-配体相互作用的原子细节， NMT，在E.大肠杆菌，并使其结晶， 而没有配体。

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.