Synthesis and structural insights into the binding mode of the albomycin δ1 core and its analogues in complex with their target aminoacyl-tRNA synthetase.

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5'S, 6'R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5'R,6'S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.

The RimL transacetylase provides resistance to translation inhibitor microcin C.

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.

Base substituted 5'-O-(N-isoleucyl)sulfamoyl nucleoside analogues as potential antibacterial agents.

Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of the corresponding prokaryotic and eukaryotic tRNA synthetases in vitro. Inspired by the aryl-tetrazole containing compounds of Cubist Pharmaceuticals and the modified base as found in the natural antibiotic albomycin, the selectivity issue of the sulfamoylated adenosines prompted us to investigate the pharmacophoric importance of the adenine base. We therefore synthesized and evaluated several isoleucyl-sulfamoyl nucleoside analogues with either uracil, cytosine, hypoxanthine, guanine, 1,3-dideaza-adenine (benzimidazole) or 4-nitro-benzimidazole as the heterocyclic base. Based on the structure and antibacterial activity of microcin C, we also prepared their hexapeptidyl conjugates in an effort to improve their uptake potential. We further compared their antibacterial activity with the parent isoleucyl-sulfamoyl adenosine (Ile-SA), both in in vitro and in cellular assays. Surprisingly, the strongest in vitro inhibition was found for the uracil containing analogue 16f. Unfortunately, only very weak growth inhibitory properties were found as of low uptake. The results are discussed in the light of previous literature findings.

N-alkylated aminoacyl sulfamoyladenosines as potential inhibitors of aminoacylation reactions and microcin C analogues containing D-amino acids.

Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.

Microcin C and albomycin analogues with aryl-tetrazole substituents as nucleobase isosters are selective inhibitors of bacterial aminoacyl tRNA synthetases but lack efficient uptake.

In 1998, Cubist Pharmaceuticals patented a series of aminoacyl tRNA synthetase (aaRS) inhibitors based on aminoacyl sulfamoyladenosines (aaSAs), in which the adenine was substituted by aryl-tetrazole moieties linked to the ribose fragment by a two-carbon spacer. Although potent and specific inhibitors of bacterial IleRS, these compounds did not prove successful in vivo due to low cell permeability and strong binding to serum albumin. In this work, we attempted to improve these compounds by combining them with microcin C (McC) or albomycin (i.e., siderophore-drug conjugate (SDC)) transport modules. We found that aryl-tetrazole variants of McC and albomycin still lacked antibacterial activity. However, these compounds were readily processed by E. coli aminopeptidases with the release of toxic aaRS inhibitors. Hence, the lack of activity in whole-cell assays was due to an inability of the new compounds to be taken up by the cells, thus indicating that the nucleotide moieties of McC and albomycin strongly contribute to facilitated transport of these compounds inside the cell.

Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis.

Microcin C (McC) is heptapeptide adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC, and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its adenosine monophosphate complex reveals specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.

Aminoacyl-tRNA synthetase inhibitors as potential antibiotics.

Increasing resistance to antibiotics is a major problem worldwide and provides the stimulus for development of new bacterial inhibitors with preferably different modes of action. In search for new leads, several new bacterial targets are being exploited beside the use of traditional screening methods. Hereto, inhibition of bacterial protein synthesis is a long-standing validated target. Aminoacyl-tRNA synthetases (aaRSs) play an indispensable role in protein synthesis and their structures proved quite conserved in prokaryotes and eukaryotes. However, some divergence has occurred allowing the development of selective aaRS inhibitors. Following an outline on the action mechanism of aaRSs, an overview will be given of already existing aaRS inhibitors, which are largely based on mimics of the aminoacyl-adenylates, the natural reaction intermediates. This is followed by a discussion on more recent developments in the field and the bioavailability problem.

Microcin C: biosynthesis, mode of action, and potential as a lead in antibiotics development.

The natural compound Microcin C (McC) is a Trojan horse inhibitor of aspartyl tRNA synthetases endowed with strong antibacterial properties, in which a heptapeptide moiety is responsible for active transport of the inhibitory metabolite part into the bacterial cell. The intracellularly formed aspartyl AMP analogue carries a chemically more stable phosphoramidate linkage, in comparison to the labile aspartyl-adenylate, and in addition is esterified with a 3-aminopropyl moiety. Therefore, this compound can target aspartyl-tRNA synthetase. The biochemical production and secretion of McC, and the possibilities to develop new classes of antibiotics using the McC Trojan horse concept in combination with sulfamoylated adenosine analogues will be discussed briefly.

Extended targeting potential and improved synthesis of Microcin C analogs as antibacterials.

Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the function of the YejABEF-transporter and then processed by cellular aminopeptidases. Processed McC (2) is a non-hydrolysable aspartyl-adenylate analog that inhibits aspartyl-tRNA synthetase (AspRS). A new synthesis is described that allows for the production of a wide variety of McC analogs in acceptable amounts. Using this synthesis a number of diverse compounds was synthesized with altered target specificity. Further characteristics of the YejABEF transporters were determined using these compounds.

Characterization of peptide chain length and constituency requirements for YejABEF-mediated uptake of microcin C analogues.

Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required for transport.

The mechanism of microcin C resistance provided by the MccF peptidase.

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. Inside a sensitive cell, McC is processed by aminopeptidases, which release a nonhydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. The mccABCDE operon is sufficient for McC production and resistance of the producing cell to McC. An additional gene, mccF, which is adjacent to but not part of the mccABCDE operon, also provides resistance to exogenous McC. MccF is similar to Escherichia coli LdcA, an L,D-carboxypeptidase whose substrate is monomeric murotetrapeptide L-Ala-D-Glu-meso-A(2)pm-D-Ala or its UDP-activated murein precursor. The mechanism by which MccF provides McC resistance remained unknown. Here, we show that MccF detoxifies both intact and processed McC by cleaving an amide bond between the C-terminal aspartate and the nucleotide moiety. MccF also cleaves the same bond in nonhydrolyzable aminoacyl sulfamoyl adenosines containing aspartyl, glutamyl, and, to a lesser extent, seryl aminoacyl moieties but is ineffective against other aminoacyl adenylates.

MccE provides resistance to protein synthesis inhibitor microcin C by acetylating the processed form of the antibiotic.

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. McC is excreted from producing cells by the MccC transporter. The residual McC that remains in the producing cell can be processed by cellular aminopeptidases with the release of a non-hydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. Accumulation of processed McC inside producing cells should therefore lead to translation inhibition and cessation of growth. Here, we show that a product of another gene of the McC biosynthetic cluster, mccE, acetylates processed McC and converts it into a non-toxic compound. MccE also makes Escherichia coli resistant to albomycin, a Trojan horse inhibitor unrelated to McC that, upon processing, gives rise to a serine coupled to a thioxylofuranosyl pyrimidine, an inhibitor of seryl-tRNA synthetase. We speculate that MccE and related cellular acetyltransferases of the Rim family may detoxify various aminoacyl-nucleotides, either exogenous or those generated inside the cell.

Synthetic microcin C analogs targeting different aminoacyl-tRNA synthetases.

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the alpha-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA(Asp) by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.

Maturation of the translation inhibitor microcin C.

Microcin C (McC), an inhibitor of the growth of enteric bacteria, consists of a heptapeptide with a modified AMP residue attached to the backbone of the C-terminal aspartate through an N-acyl phosphamidate bond. Here we identify maturation intermediates produced by cells lacking individual mcc McC biosynthesis genes. We show that the products of the mccD and mccE genes are required for attachment of a 3-aminopropyl group to the phosphate of McC and that this group increases the potency of inhibition of the McC target, aspartyl-tRNA synthetase.

Escherichia coli peptidase A, B, or N can process translation inhibitor microcin C.

The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.