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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.

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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.In the current ongoing quest for new antibiotics, aminoacyltRNA synthetases (aaRSs) have been regarded as promising targets (5,11,14). The natural antibiotic microcin C (McC) (Fig. 1, compound 1a) targets an aaRS and has been envisaged as a lead compound for further development as an antibacterial agent (17). McC consists of a heptapeptide that is covalently linked through a phosphoramidate bond to adenosine, with, in addition, an aminopropyl moiety esterified to the phosphoramidate linker (4). Once inside a sensitive cell, McC is processed by peptide deformylase and several peptidases that remove the N-terminal formyl group and the peptide part, respectively (7). As a result of intracellular processing, the active compound (compound 2), a modified nonhydrolysable aspartyl-adenylate, is released. Processed McC is a potent inhibitor of aspartyl-tRNA synthetase (AspRS) (8).McC penetrates the outer membrane of the Escherichia coli cell mostly through the OmpF porin, but also through other, yet-unidentified transport systems (M. Novikova, A. Metlitskaya, and K. Severinov, unpublished data), and is subsequently transported through the inner membrane by the YejABEF transporter (10). YejABEF is the only complex responsible for McC transport, since yej mutants are highly resistant to McC and its maturation intermediates and chemical analogues. While intact McC inhibits the growth of sensitive E. coli cells at low micromolar concentrations, processed McC does not affect cell growth, even at millimolar concentrations. Thus, the peptide chain enables McC to function through a Trojan-horse mechanism by promoting active uptake via the YejABEF transporter. The recently improved synthetic approach for the production of McC analogues has led us to investigate the uptake properties of the Yej transporter in more detail. The results obtained could be important for further drug development, where peptides function as carrier moieties for drugs that otherwise would not be able to penetrate the bacterial membranes. Here, we used a number of McC analogues truncated either from their C-or N-terminal sides, or otherwise modified, to determine the minimal peptide chain length sufficient for facilitated transport by Yej. MATERIALS AND METHODSChemistry. Reagents and solvents were purchased from commercial suppliers (Acros, Sigma-Aldrich, Bachem, and Novabiochem) and used as provided unless otherwise indicated....

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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.