Genetic and biochemical studies establish that the fungicidal effect of a fully depeptidized inhibitor of Cryptococcus neoformans myristoyl-CoA:protein N-myristoyltransferase (Nmt) is Nmt-dependent

被引:37
作者
Lodge, JK
Jackson-Machelski, E
Higgins, M
McWherter, CA
Sikorski, JA
Devadas, B
Gordon, JI
机构
[1] Washington Univ, Sch Med, Dept Mol Biol & Pharmacol, St Louis, MO 63110 USA
[2] GD Searle & Co, Dept Med & Struct Chem, St Louis, MO 63198 USA
关键词
D O I
10.1074/jbc.273.20.12482
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cryptococcus neoformans is a fungal pathogen that causes chronic meningitis in 10% of patients with AIDS. Genetic and biochemical studies were conducted to determine whether myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a target for development of a new class of fungicidal drugs. A single copy of a conditional lethal C. neoformans NMT allele was introduced into the fungal genome by homologous recombination, The allele (nmt487D) produces temperature-sensitive myristic acid auxotrophy, This phenotype is due, in part, to under-myristoylation of a cellular ADP ribosylation factor (Arf) and can be rescued by forced expression of human Nmt. Two isogenic strains with identical growth kinetics at 35 degrees C were used 60 test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 contained nmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 degrees C, survival of HMC1 depends upon its human Nmt. ALYASKLS-NH2, an inhibitor derived from an Arf, was fully depeptidized: p-[(2-methyl-1-imidazol-1-yl)butyl]phenyl-acetyl was used to represent the GLYA tetrapeptide, whereas SKLS was replaced with a chiral tyrosinol scaffold. Kinetic studies revealed Ki ((app)) values of 1.8 +/- 1 and 9 +/- 2.4 mu M for purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was a-fold lower for CPAS compared with HMC1, A single dose of 100 mu M produced a 5-fold greater inhibition of protein synthesis in CPA8 versus HMC1. The strain specificity of these responses indicates that the fungicidal effect was Nmt-dependent. These two strains may be useful for screening chemical Libraries for Nmt-based fungicidal compounds with relatively little activity against the human enzyme.
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页码:12482 / 12491
页数:10
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