ANALOGS OF MYRISTIC ACID TO MODULATE HIV-I ASSEMBLY

我们发现肉豆蔻酸(Cl4：0)与 肉豆蔻酰辅酶A：蛋白对HIV-1Pr55gag氨基末端甘氨酸残基的影响 N-肉豆蔻酰基转移酶(NMT)是病毒组装所必需的。我们的分析 NMT的作用机制导致肉豆蔻酸的合成 作为酰基辅酶A合成酶和NMT的替代底物的类似物。 6B族杂原子取代物化改变的类似物 特性(包括减少的疏水性)被发现是选择性的 转移到细胞N-肉豆蔻酰基蛋白质的亚群(部分原因是 该酶的酰辅酶A和辅酶A之间明显的协同作用 多肽结合位点)。一旦合并，它们就会产生依赖于模拟的 蛋白质功能的特殊变化。孵化一个这样的类似物 -13-氧杂十四酸与感染的H9细胞导致其 掺入HIV-1Pr55gag和nef，抑制蛋白水解物 GAG的加工，以及对急性和慢性病毒复制的抑制 无细胞毒性的慢性感染H9细胞。 我们小组建议继续使用生化，有机化学品， 用遗传学和生物物理学的方法来定义动力学机制和 酿酒酵母和人NMT在An中的结构/活性关系 尝试更好地理解如何使用Alternate设计新颖的类比 可通过以下方式选择性地针对病毒蛋白的物理特性 细胞NMT，从而扰乱它们的功能(S)。这将涉及到 (1)人NMT基因的分离及其在E. (2)不同脂肪酸类似物的系统合成 探索酶识别问题的结构基序；(3)定义 利用新近开发的一种连续的 (4)利用酿酒酵母nmt1基因ts突变体进行鉴定。 对(A)催化至关重要的结构特征；(B)机理 潜在的催化作用和(C)其与 肉豆蔻基辅酶A生成系统；(5)核磁共振和X-射线研究 NMT与其配体的相互作用(6)H9细胞和E. 检测类似物对Gag多聚蛋白影响的原核表达系统 病毒蛋白酶前体加工和细胞内靶向；(7) 放射性标记类似物在H9中代谢过程的表征 细胞培养和小鼠体内；以及(8)类似物疗效的评估 在动物模型中，关于化合物选择的决定 临床试验可以尽快进行。

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.