Sikorski J A, Devadas B, Zupec M E, Freeman S K, Brown D L, Lu H F, Nagarajan S, Mehta P P, Wade A C, Kishore N S, Bryant M L, Getman D P, McWherter C A, Gordon J I
G.D. Searle Research and Development, Monsanto Company, St. Louis, Missouri 63198, USA.
Biopolymers. 1997;43(1):43-71. doi: 10.1002/(SICI)1097-0282(1997)43:1<43::AID-BIP5>3.0.CO;2-0.
MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.
肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶（NMT）催化一种罕见的细胞脂肪酸——肉豆蔻酸与多种真核蛋白质的N端甘氨酸残基进行共翻译共价连接。肉豆蔻酰部分对于这些蛋白质生物学功能的表达通常至关重要。仅C14:0的附着提供的疏水性 barely 足以使其与膜稳定结合。因此，N-肉豆蔻酰化蛋白质的分配通常由通过额外的共价或非共价修饰起作用的“开关”调节。白色念珠菌是免疫功能低下人群系统性真菌感染的主要原因，它含有一个对其生存至关重要的单一NMT基因。人和白色念珠菌NMT的酰基辅酶A结合位点的功能特性非常相似。然而，它们的肽结合位点存在明显差异。ADP核糖基化因子（Arf）是这种真菌酶的少数细胞蛋白质底物之一。对源自N端Arf序列（GLYASKLS-NH2）的八肽进行丙氨酸扫描诱变表明，Gly1、Ser5和Lys6在结合中起主要作用。ALYASKLS-NH2是一种对肽具有竞争性的抑制剂[Ki（app） = 15.3 ± 6.4 μM]，对肉豆蔻酰辅酶A是非竞争性的。值得注意的是，用11-氨基十一烷酰基取代N端四肽会产生一种竞争性抑制剂（11-氨基十一烷酰基-SKLS-NH2），其效力比起始八肽高约40倍[Ki（app） = 0.40 ± 0.03 μM]。从C端去除Leu-Ser会产生一种竞争性二肽抑制剂（11-氨基十一烷酰基-SK-NH2），其Ki（app）为11.7 ± 0.4 μM，与起始八肽相当。一种含有C端N-环己基乙基赖氨酰胺部分的衍生二肽抑制剂具有更强效（IC50 = 0.11 ± 0.03 μM）且对细胞羧肽酶消化具有抗性的优点。使柔性的氨基十一烷酰链刚性化会产生非常有效的通用NMT抑制剂（IC50 = 40 - 50 nM）。用2-甲基咪唑取代N端胺并向刚性化元件添加具有R立体化学的苄基α-甲基会产生更有效的抑制剂（IC50 = 20 - 50 nM），与人类酶相比，对真菌的选择性高达500倍。该系列化合物中一种相关的效力较低的成员具有抑菌作用。其生长抑制作用与细胞蛋白质N-肉豆蔻酰化的减少有关，这是使用细胞Arf作为报告分子判断的。这些研究表明NMT是一个新的抗真菌靶点。
