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.

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.