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.
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 degreesC were used to 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 degreesC, 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 microM for purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was 2-fold lower for CPA8 compared with HMC1. A single dose of 100 microM 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.
Lodge
J K
JK
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Jackson-Machelski
E
E
Higgins
M
M
McWherter
C A
CA
Sikorski
J A
JA
Devadas
B
B
Gordon
J I
JI
D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000595:Amino Acid Sequence; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000254:growth & development; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010641:Phenotype
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase