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• W1981931519 abstract "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 °C were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 containednmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 °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 revealedK i (app) values of 1.8 ± 1 and 9 ± 2.4 μm 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 μm 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. 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 °C were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 containednmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 °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 revealedK i (app) values of 1.8 ± 1 and 9 ± 2.4 μm 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 μm 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. Cryptococcus neoformans is a haploid yeast that causes systemic infection in immunocompromised humans. The organism has tropism for the central nervous system where it produces chronic meningitis. The incidence of infection in patients with acquired immune deficiency syndrome is ∼10% (1Mitchell T.G. Perfect J.R. Clin. Microbiol. Rev. 1995; 8: 515-548Crossref PubMed Google Scholar). New ways of treating cryptococcal meningitis are needed given the limitations of currently available fungicidal and fungistatic agents (e.g. Refs. 2Powderly W.G. Finkelstein D.M. Feinberg J. Frame P. He W. van der Horst C. Koletar S.L. Eyster E. Carey J. Waskin H. Hooton T.M. Hyslop N. Spector S.A. Bozzette S.A. N. Engl. J. Med. 1995; 332: 700-705Crossref PubMed Scopus (309) Google Scholar and 3van der Horst C.M. Saag M.S. Cloud G.A. Hamill R.J. Graybill J.R. Sobel J.D. Johnson P.C. Tuazon C.U. Kerkering T. Moskovitz B.L. Powderly W.G. Dismukes W.E. National Institute of Allergy and Infectious Diseases Mycoses Study Group AIDS Clinical Trials Group N. Engl. J. Med. 1997; 337: 15-21Crossref PubMed Scopus (676) Google Scholar). An ideal drug target would be a fungal gene product that is expressed under the conditions of infection and that is essential for the viability of the organism. The metabolic pathway and/or substrate specificities of the target protein should be distinguishable from those of the human host. Finally, it would be desirable if the protein is common to many fungal pathogens and is required for their survival. Studies of C. neoformans pathogenesis have revealed several virulence genes. They encode products involved in capsule formation (CAP59 and CAP64), synthesis of melanin (CNLAC1), and mating (MFα). GPA1functions as a regulator of each of these functions (4Alspaugh J.A. Perfect J.R. Heitman J. Genes Dev. 1997; 11: 3206-3217Crossref PubMed Scopus (339) Google Scholar). None of these genes is essential for viability (4Alspaugh J.A. Perfect J.R. Heitman J. Genes Dev. 1997; 11: 3206-3217Crossref PubMed Scopus (339) Google Scholar, 5Chang Y.C. Kwon-Chung K.J. Mol. Cell. Biol. 1994; 14: 4912-4919Crossref PubMed Scopus (399) Google Scholar, 6Chang Y.C. Penoyer L.A. Kwon-Chung K.J. Infect. Immun. 1996; 64: 1977-1983Crossref PubMed Google Scholar, 7Salas S.D. Bennett J.E. Kwon-Chung K.J. Perfect J.R. Williamson P.R. J. Exp. Med. 1996; 184: 377-386Crossref PubMed Scopus (320) Google Scholar, 8Wickes B.L. Mayorga M.E. Edman U. Edman J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7327-7331Crossref PubMed Scopus (245) Google Scholar). Genetic studies have shown that ADE2(phosphoribosylaminoimidazole carboxylase) is necessary for the growth of C. neoformans in cerebrospinal fluid (9Perfect J.R. Toffaletti D.L. Rude T.H. Infect. Immun. 1993; 61: 4446-4451Crossref PubMed Google Scholar) and that calcineurin is required for survival at 37 °C (10Odom A. Muir S. Lim E. Toffaletti D.L. Perfect J. Heitman J. EMBO J. 1997; 16: 2576-2589Crossref PubMed Scopus (406) Google Scholar). A number of other C. neoformans genes have been isolated, but their necessity for growth and/or infection has either not been evaluated by direct genetic tests or the results of such tests have been ambiguous (e.g. Refs. 11Livi L.L. Edman U. Schneider G.P. Greene P.J. Santi D.V. Gene (Amst .). 1994; 150: 221-226Crossref PubMed Scopus (14) Google Scholar, 12Edman J.C. Kwon-Chung K.J. Mol. Cell. Biol. 1990; 10: 4538-4544Crossref PubMed Scopus (154) Google Scholar, 13Sirawaraporn W. Cao M. Santi D.V. Edman J.C. J. Biol. Chem. 1993; 268: 8888-8892Abstract Full Text PDF PubMed Google Scholar, 14Parker A.R. Moore T.D.E. Edman J.C. Schwab J.M. Davisson V.J. Gene (Amst .). 1994; 145: 135-138Crossref PubMed Scopus (15) Google Scholar, 15Perfect J.R. Rude T.H. Penning L.M. Johnson S.A. Gene (Amst .). 1992; 122: 213-217Crossref PubMed Scopus (19) Google Scholar, 16Cox G.M. Rude T.H. Dykstra C.C. Perfect J.R. J. Med. Vet. Mycol. 1995; 33: 261-266Crossref PubMed Scopus (48) Google Scholar, 17Spitzer E.D. Spitzer S.G. Gene (Amst .). 1995; 161: 113-117Crossref PubMed Scopus (11) Google Scholar, 18Tolkacheva A. McNamara P. Piekarz E. Courchesne W. Infect. Immun. 1994; 62: 2849-2856Crossref PubMed Google Scholar). One reason for the paucity of such tests is that targeted gene disruption is limited by several factors inC. neoformans. Homologous recombination appears to be inefficient (4Alspaugh J.A. Perfect J.R. Heitman J. Genes Dev. 1997; 11: 3206-3217Crossref PubMed Scopus (339) Google Scholar, 5Chang Y.C. Kwon-Chung K.J. Mol. Cell. Biol. 1994; 14: 4912-4919Crossref PubMed Scopus (399) Google Scholar, 6Chang Y.C. Penoyer L.A. Kwon-Chung K.J. Infect. Immun. 1996; 64: 1977-1983Crossref PubMed Google Scholar, 7Salas S.D. Bennett J.E. Kwon-Chung K.J. Perfect J.R. Williamson P.R. J. Exp. Med. 1996; 184: 377-386Crossref PubMed Scopus (320) Google Scholar, 19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar). There are only a few selectable markers available for conducting such tests (12Edman J.C. Kwon-Chung K.J. Mol. Cell. Biol. 1990; 10: 4538-4544Crossref PubMed Scopus (154) Google Scholar, 20Toffaletti D.L. Rude T.H. Johnston S.A. Durack D.T. Perfect J.T. J. Bacteriol. 1993; 175: 1405-1411Crossref PubMed Google Scholar, 21Cox G.M. Toffaletti D.L. Perfect J.R. J. Med. Vet. Mycol. 1996; 34: 385-391Crossref PubMed Scopus (56) Google Scholar). Moreover, in the absence of a conditional lethal allele, disruption of an essential gene in this haploid organism will produce death, precluding further analysis of the function of the gene. The C. neoformans gene encoding myristoyl-CoA:proteinN-myristoyltransferase (EC 2.1.3.97) (NMT) 1The abbreviations used are: NMT, gene encoding myristoyl-CoA:protein N-myristoyltransferase; Arf, ADP ribosylation factor; kb, kilobase pair; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PCR, polymerase chain reaction. fulfills many of the criteria for an anti-fungal target. Nmt is a monomeric enzyme that catalyzes the co-translational transfer of myristate, a 14-carbon saturated fatty acid, from CoA to the N-terminal Gly residue of nascent proteins. Cellular N-myristoylproteins have diverse biological functions (22Bhatnagar R.S. Gordon J.I. Trends Cell Biol. 1997; 7: 14-20Abstract Full Text PDF PubMed Scopus (136) Google Scholar). The enzyme appears to be ubiquitously expressed in eukaryotes, including the two organisms that are the principal causes of systemic fungal infections in immunosuppressed humans, C. neoformans and Candida albicans (23Weston S.A. Camble R. Colls J. Rosenbrock G. Taylor I. Egerton M. Tucker A.D. Tunncliffe A. Mistry A. Mancia F. de la Fortelle E. Irwin J. Bricogne G. Pauptit R.A. Nat. Struct. Biol. 1998; 5: 213-221Crossref PubMed Scopus (103) Google Scholar, 24Lodge J.K. Johnson R.L. Weinberg R.A. Gordon J.I. J. Biol. Chem. 1994; 269: 2996-3009Abstract Full Text PDF PubMed Google Scholar). In vitro studies of purified orthologous Nmts have shown that their acyl-CoA substrate specificities are highly conserved, whereas their peptide substrate specificities are divergent. These differences in peptide recognition have been exploited to develop species-selective peptidomimetic inhibitors (Refs. 25Devadas B. Zupec M.E. Freeman S.K. Brown D.L. Nagarajan S. Sikorski J.A. McWherter C.A. Getman D.P. Gordon J.I. J. Med. Chem. 1995; 38: 1837-1840Crossref PubMed Scopus (56) Google Scholar, 26Devadas B. Freeman S.K. McWherter C.A. Kuneman D.W. Vinjamoori D.V. Sikorski J.A. Bioorg. Med. Chem. Lett. 1996; 6: 1977-1982Crossref Scopus (12) Google Scholar, 27Devadas B. Freeman S.K. Zupec M.E. Lu H.-F. Nagarajan S.R. Kishore N.S. Lodge J.K. Kuneman D.W. McWherter C.A. Vinjamoori D.V. Getman D.P. Gordon J.I. Sikorski J.A. J. Med. Chem. 1997; 40: 2609-2625Crossref PubMed Scopus (67) Google Scholar, 28McWherter C.A. Rocque W.J. Zupec M.E. Freeman S.K. Brown D.L. Devadas D. Getman D.P. Sikorski J.A. Gordon J.I. J. Biol. Chem. 1997; 272: 11874-11880Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 29Nagarajan S.R. Devadas B. Zupec M.E. Freeman S.K. Brown D.L. Lu H.-F. Mehta P.P. Kishore N.S. McWherter C.A. Getman D.P. Gordon J.I. Sikorski J.A. J. Med. Chem. 1997; 40: 1422-1438Crossref PubMed Scopus (40) Google Scholar; reviewed in Ref. 30Sikorski 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. Biopolymers. 1997; 43: 43-71Crossref PubMed Scopus (56) Google Scholar). In addition, genetic tests have established thatNMT is essential for the growth and viability of bothC. neoformans and C. albicans (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar, 31Weinberg R.A. McWherter C.A. Freeman S.K. Wood D.C. Gordon J.I. Lee S.C. Mol. Microbiol. 1995; 16: 241-250Crossref PubMed Scopus (117) Google Scholar). NMT represents a unique example of an essential C. neoformans gene where a conditional lethal allele has been generated by homologous recombination. This allele was based on a mutant Saccharomyces cerevisiae NMT1 allele (nmt1-451D). nmt1-451D contains a single nucleotide substitution that results in replacement of an absolutely conserved Gly, located 5 residues from the C terminus of the enzyme, with an Asp (32Duronio R.J. Rudnick D.A. Johnson R.L. Johnson R.D. Gordon J.I. J. Cell Biol. 1991; 113: 1313-1330Crossref PubMed Scopus (50) Google Scholar). This substitution reduces the affinity of the enzyme for myristoyl-CoA (33Zhang L. Jackson-Machelski E. Gordon J.I. J. Biol. Chem. 1996; 271: 33131-33140Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The analogous mutation in C. neoformans NMT is Gly487 → Asp (nmt487D). A strain containing several copies of nmt487D, including one at the endogenous locus, was produced (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar). It is a myristic acid auxotroph. Withdrawal of myristate produces rapid growth arrest and death within 4 h (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar). Virulence studies using isogenic NMT andnmt487D strains and an immunosuppressed rabbit model of cryptococcal meningitis established that genetic attenuation of Nmt activity allows the host to rid itself of an otherwise fatal infection (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar). In the present study, we generated a strain of C. neoformanswith a single copy of nmt487D at the endogenous gene locus. This strain was used to correlate cellular proteinN-myristoylation with growth and viability. Moreover, strains expressing wild type C. neoformans or human Nmt were employed to show that a species-selective, fully depeptidized inhibitor of the acyltransferase produces fungicidal effects through an Nmt-dependent mechanism. Strains generated during the course of these studies are described in Table I. Two C. neoformansstrains, MO49 (ade2, NMT) and CMD2 (ADE2, nmt487D), have been described previously (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar, 20Toffaletti D.L. Rude T.H. Johnston S.A. Durack D.T. Perfect J.T. J. Bacteriol. 1993; 175: 1405-1411Crossref PubMed Google Scholar). A strain derived from MO49 with an unmapped insertion of ADE2 was named CAP8 in an earlier report (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar) but is now renamed CPA8 (C ryptococcus with prototropy foradenine) to avoid confusion with capsule-deficient mutants (34Jacobson E.S. Ayers D.J. Harrell A.C. Nicholas C.C. J. Bacteriol. 1982; 150: 1292-1296Crossref PubMed Google Scholar). The following media were used: YPD (1% yeast extract, 2% peptone, 2% glucose); YPD supplemented with 1% Brij 58 (Sigma) and 500 μm myristate or palmitate (NuChek Prep); YNB (yeast nitrogen base; Bio 101); and RPMI 1640 (with glutamine and without bicarbonate; Life Technologies, Inc.).Table ICryptococcus neoformans strainsStrainParental strainHow derivedGenotypeRef.MO49H99 (clinical isolate)Mutagenesisade220Toffaletti D.L. Rude T.H. Johnston S.A. Durack D.T. Perfect J.T. J. Bacteriol. 1993; 175: 1405-1411Crossref PubMed Google ScholarCPA8MO49Transformed/pADE2dApaADE219Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google ScholarCMD2MO49Transformed/pCN28–14ADE2,nmt487D (10 copies)19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google ScholarCMD19MO49Transformed/pCN28–14ADE2,nmt487D (1 copy)This workAMC1CMD19Transformed/pCN36–23ADE2,nmt487D (1 copy), GAL-ARFThis workHMC1CMD19Transformed/pHS20–57ADE2,nmt487D (1 copy), hNMT (10 copies)This workHMC2CMD19Transformed/pHS20–57ADE2,nmt487D (1 copy), hNMT (2 copies)This workCMC1CMD19Transformed/pCN25–28ADE2,nmt487D (1 copy), NMT (10 copies)This workCMC2CMD19Transformed/pCN25–28ADE2,nmt487D (1 copy), NMT (2 copies)This work Open table in a new tab pCN36-23 was employed for forced expression of a C. neoformans ADP-ribosylation factor (Arf) in C. neoformans. The plasmid contains three components: (i) the C. neoformans GAL7 promoter from pGUST11 (35Wickes B.L. Edman J.C. Mol. Microbiol. 1995; 16: 1099-1109Crossref PubMed Scopus (58) Google Scholar) containing an NcoI site in place of the NdeI site at the initiator Met codon (engineered by PCR using 5′-TGTTCCGCCGGTGGAAAGAAGCAGG-3′ and 5′-GGGCCATGGTCAAGAGGGGATTGAGC-3′ as primers and pGUST11 as the template DNA); (ii) the open reading frame of the C. neoformans ARF gene (24Lodge J.K. Johnson R.L. Weinberg R.A. Gordon J.I. J. Biol. Chem. 1994; 269: 2996-3009Abstract Full Text PDF PubMed Google Scholar), obtained by PCR of genomic DNA from strain MO49 (the PCR reaction contained 5′-GGGGCCATGGGCCTTTCTGTCTCC-3′ which incorporates an NcoI site at the initiator ATG, and 5′-CCTTGCGGCCGCGTGCGAAGGTATAAATGGAC-3′ which incorporates anNotI site at the 3′ end of the coding sequence); and (iii) an NotI fragment from pTelHyg (21Cox G.M. Toffaletti D.L. Perfect J.R. J. Med. Vet. Mycol. 1996; 34: 385-391Crossref PubMed Scopus (56) Google Scholar) that includes sequences for maintenance in Escherichia coli and a hygromycin resistance cassette. The ARF insert in pCN36-23 was sequenced in its entirety to verify that no unanticipated mutations had been introduced by PCR. pHS20-57 was used to express human Nmt in C. neoformans. This plasmid consists of five components: (i) a 1.4-kb fragment fromC. neoformans NMT that includes transcriptional regulatory elements from its 5′-nontranscribed domain. This fragment spans the region from the HindIII site of pCN25–28 (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar) to the ATG of the coding sequence where an NdeI site was engineered by PCR; (ii) a 1.7-kb NdeI/EcoRI fragment from pBB218 which includes the coding sequence of a human Nmt cDNA (36Duronio R.J. Reed S.I. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4129-4133Crossref PubMed Scopus (111) Google Scholar); (iii) an EcoRI/BglII fragment from the polylinker of pSP70 (Promega); (iv) a 2.4-kbBamHI/EcoRI fragment from pCN25–28 containing sequences 3′ to the stop codon of C. neoformans NMT (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar); and (v) pGEM4Z (Promega) linearized by digestion withHindIII and EcoRI. Strains M049 and CMD19 (Table I) were transformed using a Bio-Rad Biolistic PDS-1000/HE particle delivery system and published protocols (19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar, 20Toffaletti D.L. Rude T.H. Johnston S.A. Durack D.T. Perfect J.T. J. Bacteriol. 1993; 175: 1405-1411Crossref PubMed Google Scholar). The diameter of the gold particles was 0.6 μm. When strain MO49 was transformed with pCN28-14 (nmt487D; 19), cells were selected based on their ability to grow at 24 °C on YNB supplemented with 0.8 g/liter complete synthetic medium without adenine (CSM-ade: Bio 101), 500 μm myristate, 1% Brij 58, and 1 m sorbitol. When strain CMD19 was transformed with pCN36-23 (ARF), cells were selected at 24 °C on YPD containing 200 units/ml hygromycin (Calbiochem). When CMD19 was transformed with either pCN25-28 (C. neoformans NMT) or pHS20-57 (human NMT), cells were selected for their capacity to grow at 35 °C on YPD without supplements. NMT copy number was quantitated by scanning Southern blots of genomic DNA. Fresh colonies were picked from plates into YPD, and the liquid cultures were adjusted to anA 595 = 0.1. Aliquots (250 μl) were inoculated into 5 ml of YPD or YPD containing 500 μm myristate and 1% Brij 58. Cultures were then incubated with shaking at 24 or 37 °C. Aliquots (10 μl) were withdrawn at various time points and diluted 1:10 with phosphate-buffered saline (PBS), and theA 595 of the sample was determined using a Molecular Devices Thermomax microplate reader. All strains were grown at each condition in triplicate, and each A 595measurement was done in triplicate. Five-milliliter cultures of C. neoformans strains were grown overnight at 24 °C in YPD supplemented 500 μm myristate and 1% Brij 58. The cells were then pelleted by centrifugation at 900 × gfor 10 min at room temperature, washed once in PBS, pelleted once more, and then were resuspended in 0.25 ml of a solution containing 2% SDS, 80 mm Tris, pH 6.8, and 2 mm Pefabloc (Boehringer Mannheim). Following addition of 0.5 ml of zirconia/silica beads (0.5 mm diameter, Biospec Products), the cells were vortexed (3 pulses of 1 min, alternating with 1 min of incubation on ice). The resulting lysates were boiled for 5 min and clarified by centrifugation at 12,000 × g for 5 min. Glycerol (final concentration = 5% v/v), 2-mercaptoethanol (2%), and bromphenol blue (0.002%) were added. Equal amounts of lysate were fractionated by electrophoresis through 10% polyacrylamide gels containing 0.1% SDS (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). The separated proteins were then transferred to Immobilon-P (Millipore) transfer membranes (38Lodge J.K. Jackson-Machelski E. Gordon J.I. Microbiology. 1997; 143: 357-366Crossref PubMed Scopus (36) Google Scholar). Protein blots were probed with a previously characterized rabbit anti-C. albicans Nmt sera (final dilution = 1:1000-fold in blocking buffer; Refs. 24Lodge J.K. Johnson R.L. Weinberg R.A. Gordon J.I. J. Biol. Chem. 1994; 269: 2996-3009Abstract Full Text PDF PubMed Google Scholar and38Lodge J.K. Jackson-Machelski E. Gordon J.I. Microbiology. 1997; 143: 357-366Crossref PubMed Scopus (36) Google Scholar). Antigen-antibody complexes were detected using reagents and protocols supplied in the Tropix Western-Light kit. Fifty-milliliter cultures of C. neoformans strains were grown overnight in YPD, 500 μm myristate, 1% Brij 58 at 24 °C to anA 595 ≈2.0–3.0. The cultures were then centrifuged as above, washed twice in 25 ml of PBS, and resuspended in 4–6 ml of YPD. A 1-ml aliquot was added to 10 ml of YPD or YPD, 500 μm myristate, 1% Brij 58. Following a 2-h incubation at 24 or 37 °C, cells were pelleted, washed once in 5 ml of PBS, and resuspended in 0.5 ml of a solution containing 4% SDS and 0.125m Tris-HCl, pH 6.8. Two volumes of 0.5 mm zirconia/silica beads were added, and cells were disrupted by vortexing as described above. Cleared lysates were prepared and denatured, and the cellular proteins were fractionated using a 16-cm long, 0.75-mm thick, 12% polyacrylamide gel containing 0.1% SDS (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Separated proteins were transferred to Immobilon-P membranes. The protein blots were subsequently incubated with a previously characterized rabbit anti-S. cerevisiae Arf1p sera (R40, a generous gift of R. Kahn, Emory University) diluted 1:50,000 in PBS containing 1% gelatin, 0.2% Tween 20, and 0.1% sodium azide. Antigen-antibody complexes were detected using the Tropix Western-Light kit. Synthesis of this compound was accomplished according to the scheme shown in Fig.1 and involved reacting the amine (1) with the 2-methylimidazole carboxylic acid (2) in the presence of dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (see Ref. 39Devadas B. Freeman S.K. McWherter C.A. Kishore N.S. Lodge J.K. Jackson-Machelski E. Gordon J.I. Sikorski J.A. J. Med. Chem. 1998; 41: 996-1000Crossref PubMed Scopus (49) Google Scholar for a description of the seven-step synthesis of compound 1). The resulting product was treated with trifluoroacetic acid at ambient temperature, and the final product (SC-61213) was isolated by reverse phase high pressure liquid chromatography. The 1H NMR and mass spectral data of SC-61213 were consistent with the structure presented in Fig. 1. A stock solution of 40 mm SC-61213 was prepared in sterile deionized water, and aliquots were stored at −20 °C prior to use. The effects of SC-61213 on purified C. neoformans, human, and S. cerevisiae Nmts were defined using a previously described two-step enzyme assay (40Rudnick D.A. Duronio R.J. Gordon J.I. Hooper N.M. Turner A.J. Lipid Modification of Proteins: A Practical Approach. IRL Press at Oxford University Press, Oxford1992: 37-61Google Scholar). The amount of Nmt added to the 110-μl reaction depended upon the species: 50–100 ng of C. neoformans Nmt purified from E. coli(19Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (170) Google Scholar); 30 ng of a homogeneous preparation of E. coli-derived human Nmt (25Devadas B. Zupec M.E. Freeman S.K. Brown D.L. Nagarajan S. Sikorski J.A. McWherter C.A. Getman D.P. Gordon J.I. J. Med. Chem. 1995; 38: 1837-1840Crossref PubMed Scopus (56) Google Scholar); or 3.5 ng of purified recombinant S. cerevisiae Nmt1p (41Bhatnagar R.S. Schall O.F. Jackson-Machelski E. Sikorski J.A. Devadas B. Gokel G.W. Gordon J.I. Biochemistry. 1997; 36: 6700-6708Crossref PubMed Scopus (41) Google Scholar). The final concentration of [3H]myristoyl-CoA was 0.23 μm. An octapeptide representing the N-terminal sequence of a C. neoformans Arf (GLSVSKLL-NH2) was used as a substrate peptide (final concentration = 10 nm to 2 μm). [3H]Myristoyl-GLSVSKLL-NH2was purified from the reaction mixture by reverse phase high pressure liquid chromatography using a C18 10-μm μBondapak column (dimensions = 3.9 × 300 mm; Waters Corp.) and a linear gradient from H2O, 0.1% trifluoroacetic acid, 0.05% triethylamine to 100% acetonitrile, 0.1% trifluoroacetic acid. The amount of labeled myristoylpeptide recovered was quantitated with an in-line scintillation counter (42Kishore N.S. Lu T.B. Knoll L.J. Katoh A. Rudnick D.A. Mehta P.P. Devadas B. Huhn M. Atwood J.L. Adams S.P. Gokel G.W. Gordon J.I. J. Biol. Chem. 1991; 266: 8835-8855Abstract Full Text PDF PubMed Google Scholar). Peptide K m andV max values were determined for each Nmt from double-reciprocal plots. The K m value at each inhibitor concentration and the type of inhibition were determined using double-reciprocal plots (43Segel I.H. Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry. 2nd Ed. John Wiley & Sons, Inc., New York1976Google Scholar). K i values were calculated from Dixon plots (1/v versus [I]; see Ref. 43Segel I.H. Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry. 2nd Ed. John Wiley & Sons, Inc., New York1976Google Scholar). All assays were performed at least twice, each time in triplicate. The minimal inhibitory concentration of SC-61213 was defined by a broth microdilution anti-fungal test performed according to the protocol described by the National Committee for Clinical Laboratory Standards (NCCLS) tentative standard (44National Committee for Clinical Laboratory Standards Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Tentative Standard, Document M 27-T. National Committee for Clinical Laboratory Standards, Villanova, PA1995Google Scholar). Briefly, RPMI 1640, buffered to pH 7.0 with MOPS or to pH 5.4 with MES, was introduced together with varying amounts of SC-61213 into each well of a 96-well microtiter plate (Falcon).C. neoformans strains CPA8 and HMC1 were then added to the wells according to the NCCLS protocol. Microtiter plates were incubated at 35 °C without shaking. Forty-eight and 72 h after inoculation, the cells in each well were resuspended by pipetting, and the A 550 of the resulting suspension was determined using a Molecular Devices Thermomax plate reader. SC-61213 was tested in a series of 2-fold dilutions over the concentration range 100 to 1.6 μm. Each dilution of the compound was tested on two separate occasions, each time in triplicate. Controls included (i) media without any inoculated C. neoformans to confirm that no contaminants were acquired during the course of the incubation; (ii) media with the isogenic C. neoformans strains but with no drug; and (iii) media containing the strains plus a known fungicidal agent (amphotericin B, 0.25–4 μg/ml). To determine the effects of SC-61213 on protein synthesis, cells were grown overnight at" @default.
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• W1981931519 title "Genetic and Biochemical Studies Establish That the Fungicidal Effect of a Fully Depeptidized Inhibitor of Cryptococcus neoformans Myristoyl-CoA:ProteinN-Myristoyltransferase (Nmt) Is Nmt-dependent" @default.
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