Design and Synthesis of Novel Imidazole-Substituted Dipeptide Amides as Potent and Selective Inhibitors of Candida albicans MyristoylCoA:Protein N-Myristoyltransferase and Identification of Related Tripeptide Inhibitors with Mechanism-Based Antifungal Activity

Abstract

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these ...

Aspergillus nidulans swoF Encodes an N -Myristoyl Transferase

“…NMTs are a highly conserved class of eukaryotic proteins; however, their substrate specificities have diverged, with fungal NMTs generally having targets different from those of mammalian NMTs (12,13,27,30,38). This target divergence, coupled with the fact that deletion of the single copy of the NMT gene in fungi is lethal (15,30,43), has led to considerable interest in NMTs as possible targets for a new class of antifun- …”

“…CELL gal drugs. This approach has met with success in C. albicans and C. neoformans, for which compounds that specifically inhibit myristoylation are being tested (11,12,26,29,42). To our knowledge, this option has not yet been explored for A. fumigatus, now the most common fungal pathogen found in autopsies of immunocompromised patients in European hospitals (19).…”

Current Advances in Antifungal Targets and Drug Development

“…Earlier known myristic acid analogues were found to be nonselective inhibitors of NMT [92,93]. Subsequent dipeptide and peptidomimetic inhibitors had potent in vitro activity against the enzyme but failed in cell based assays due to transport problems [94][95][96][97]. Further search led to the identification of low molecular weight compounds like ptoluenesulphonamides, aminobenzothiazoles, quinolines [98][99][100].…”

Design, Synthesis and Antifungal Activity of Novel Benzofuran-Triazole Hybrids

“…Therefore, NMT would be a promising target for the development of novel fungicidal drugs. Up to now, various types of NMT inhibitors such as peptidomimetic [10][11][12], myristic acid analogues [13] and different kinds of heterocycles [14][15][16] have been reported. Among them, benzofuran inhibitors showed high selectivity and powerful antifungal activity [16][17][18][19] (Figure 1).…”