Generation of Broad-Spectrum Antifungal Drug Candidates from the Natural Product Compound Aureobasidin A.

The natural product aureobasidin A (AbA) is a potent, well-tolerated antifungal agent with robust efficacy in animals. Although native AbA is active against a number of fungi, it has little activity against Aspergillus fumigatus, an important human pathogen, and attempts to improve the activity against this organism by structural modifications have to date involved chemistries too complex for continued development. This report describes novel chemistry for the modification of AbA. The key step involves functionalization of the phenylalanine residues in the compound by iridium-catalyzed borylation. This is followed by displacement of the pinacol boron moiety to form the corresponding bromide or iodide and substitution by Suzuki biaryl coupling. The approach allows for synthesis of a truly wide range of derivatives and has produced compounds with A. fumigatus minimal inhibitory concentrations (MIC) of <0.5 µg/mL. The approach is readily adaptable to large-scale synthesis and industrial production.

Inhibition of inositol phosphorylceramide synthase by the cyclic peptide aureobasidin A.

By using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent K(i) values of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a K(i) of 1.4 microM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The K(m) values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V(max) for the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a K(i) of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent K(i) of 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.

Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938.

The gene (aba1) encoding the NRPS complex responsible for the synthesis of the cyclic peptide antibiotic Aureobasidin A (AbA) in Aureobasidium pullulans BP-1938, was cloned using a combination of PCR and library screening approaches. The aba1 gene was found to consist of a single, intronless open reading frame (ORF) of 34,980 bp, encoding an 11,659 amino acid protein with a calculated molecular mass of 1,286,254 Da. Putative promoter and translation start elements were identified upstream from the putative ATG in the aba1 gene, and a consensus poly(A) addition signal (AATAAA) was identified 191 bp downstream of the translation termination codon (TGA). As predicted by the structure AbA, the aba1 gene encodes an enzyme composed of nine biosynthetic modules, eight of which contain adenylation domains with recognizable amino acid specificity-conferring code elements, and four of which contain embedded methylation domains. The biosynthetic module located at position one in the aba1 gene lacks recognizable specificity-conferring code elements, consistent with it being responsible for incorporation of the 2-hydroxy-3-methylpentanoic acid residue at that position in AbA. An unusual feature of the aba1 gene sequence is a very high degree of shared identity among eight of the biosynthetic modules, at both the nucleotide and amino acid level. The majority of the modules share better than 70% nucleotide identity with another module in the complex, and modules with the same amino acid adenylation specificity share up to 95% identity. Insertion of a hygromycin B phosphotransferase (HPT) gene cassette in place of the module 4 sequence in aba1 resulted in a cessation of AbA production, thus validating that the isolated gene encodes the AbA biosynthesis complex.

Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1.

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin-type O-glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc-T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc-glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzyme's affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.

Effect of membrane perturbants on the activity and phase distribution of inositol phosphorylceramide synthase; development of a novel assay.

The effect of 26 different membrane-perturbing agents on the activity and phase distribution of inositol phosphorylceramide synthase (IPC synthase) activity in crude Candida albicans membranes was investigated. The nonionic detergents Triton X-100, Nonidet P-40, Brij, Tween, and octylglucoside all inactivated the enzyme. However, at moderate concentrations, the activity of the Triton X-100- and octylglucoside-solubilized material could be partially restored by inclusion of 5 mM phosphatidylinositol (PI) in the solubilization buffer. The apparent molecular mass of IPC synthase activity solubilized in 2% Triton X-100 was between 1.5 x 10(6) and 20 x 10(6) Da, while under identical conditions, octylglucoside-solubilized activity remained associated with large presumably membrane-like structures. Increased detergent concentrations produced more drastic losses of enzymatic activity. The zwitterionic detergents Empigen BB, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), Zwittergent 3-10, and amidosulfobetaine (ASB)-16 all appeared capable of solubilizing IPC synthase. However, these agents also inactivated the enzyme essentially irreversibly. Solubilization with lysophospholipids again resulted in drastic losses of enzymatic activity that were not restored by the inclusion of PI. Lysophosphatidylinositol also appeared to compete, to some extent, with the donor substrate phosphatidylinositol. The sterol-containing agent digitonin completely inactivated IPC synthase. By contrast, sterol-based detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and taurodeoxycholate (tDOC) had little or no effect on the enzyme activity. The IPC synthase activity in C. albicans membranes remained largely intact and sedimentable at CHAPS concentrations (4%) where >90% of the phospholipids and 60% of the total proteins were extracted from the membranes. At 2.5% CHAPS, a concentration where approximately 50% of the protein and 80% of the phospholipids are solubilized, there was no detectable loss of enzyme activity, and it was found that the detergent-treated membranes had significantly improved properties compared to crude, untreated membranes as the source of IPC synthase activity. In contrast to assays utilizing intact membranes or Triton X-100 extracts, assays using CHAPS- or tDOC-washed membranes were found to be reproducible, completely dependent on added acceptor substrate (C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-ceramide), and >95% dependent on added donor substrate (PI). Product formation was linear with respect to both enzyme concentration and time, and transfer efficiency was improved more than 20-fold as compared to assays using crude membranes. Determination of kinetic parameters for the two IPC synthase substrates using CHAPS-washed membranes resulted in K(m) values of 3.3 and 138.0 microM for C(6)-NBD-ceramide and PI, respectively. In addition, the donor substrate, PI, was found to be inhibitory at high concentrations with an apparent K(i) of 588.2 microM.

Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1.

All UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases cloned to date contain a lectin domain at the C-terminus, consisting of three tandem repeat sequences (alpha,beta, and gamma). We previously reported that the alpha repeat of one of the most ubiquitous isozymes, GalNAc-T1, is a functional lectin that recognizes O-linked GalNAc residues on the acceptor polypeptides with multiple acceptor sites; the domain appears not to be involved in the glycosylation of acceptors with a single acceptor site. In this report, we studied the function of the beta and gamma repeats in the GalNAc-T1 lectin domain, by site-directed mutagenesis and analysis of the catalytic properties of mutant enzymes. We found that the beta repeat recognizes GalNAc and is involved in glycosylation of acceptors with multiple glycosylation sites. The gamma repeat, on the other hand, showed no significant GalNAc-binding activity. These results indicate that the lectin domain of GalNAc-T1 has at least two functional repeats, allowing the possibility of multivalent interactions with GalNAc residues on the acceptor polypeptide during glycosylation.

The lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 is involved in O-glycosylation of a polypeptide with multiple acceptor sites.

Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW)(3) repeat sequence at the C terminus that consists of three tandem repeats (alpha, beta, and gamma). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp(444) in the alpha repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp(444) and the corresponding aspartate residues in the beta and the gamma repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1).

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.