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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 α-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 tRNAAsp 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.
Microcins are small (<10-kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (17). Three microcins, B, C, and J, form a subgroup of posttranslationally modified microcins. Members of this subgroup have highly unusual structures and inhibit cellular enzymes that are validated targets for antibacterial drug development (25). Posttranslationally modified microcins are attractive as drug candidates because of their strong antibacterial action and because virtually limitless numbers of their derivatives can be generated by means of mutation, chemical synthesis, or both. Microcin B (McB), a 43-residue peptide with thiazole and indole rings (13), inhibits DNA gyrase (21). Microcin J, a 21-amino-acid peptide, assumes an unusual threaded lasso structure (2, 23, 27) and inhibits bacterial RNA polymerase (1, 18). The structure of the subject of this study, McC (compound 1) is shown in Fig. Fig.1a.1a. McC is a heptapeptide with a formylated N-terminal methionine and a C-terminal aspartate whose α-carboxyl group is covalently linked to adenosine through an N-acyl phosphoramide bond (10, 14). The phosphoramidate of McC is additionally modified by an O-propylamine group (9).
The passage of McC through the inner layer of the Escherichia coli cell wall is carried out by the YejABEF transporter (19). Once inside the cell, McC is specifically processed by one of the several broad-specificity E. coli cytoplasmic aminopeptidases (12). The product of processing, modified aspartyl-adenylate (compound 2) (15), closely resembles Asp-AMP (compound 3) (Fig. (Fig.1c),1c), the natural reaction intermediate of the tRNAAsp aminoacylation reaction catalyzed by AspRS. However, because the bond between the α-carboxyl of C-terminal aspartate and the phosphoramidate nitrogen is nonhydrolyzable, compound 2 inhibits AspRS. Unprocessed McC has no effect on tRNAAsp aminoacylation, while processed McC has no effect on McC-sensitive cells at concentrations at which intact McC strongly inhibits cell growth. Thus, McC is a Trojan horse inhibitor (22): the peptide part allows McC to enter sensitive cells, where it gets processed, liberating the inhibitory part of the drug.
Aminoacyl-tRNA synthetases (aaRSs) carry out the condensation of genetically encoded amino acids with cognate tRNAs. When 1 of the 20 aaRSs present in the cell is inhibited, the corresponding tRNA is not charged. This leads to protein synthesis inhibition and cell growth arrest. In principle, variation of the last amino acid of the McC peptide, the product of the mccA gene, should allow investigators to obtain McC derivatives targeting aaRSs other than AspRS. Unfortunately, the results of systematic structure-activity analyses of the McC peptide revealed that substitutions in the seventh codon of mccA invariably prevented McC production, presumably by interfering with posttranslational modifications of the MccA peptide by the McC maturation enzymes (11). Indeed, in vitro analysis showed that the C-terminal asparagine of MccA is required for the addition of the adenosine moiety by the MccB protein (24).
Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of their corresponding aaRSs (5, 20, 26). However, these compounds show low in vivo activities due to limited membrane permeability and the absence of a transporter for these compounds. Here, we show that through chemical attachment of aminoacyl-sulfamoyl adenosines to the first 6 amino acids of the MccA peptide, potent antibacterial agents can be generated. The new compounds share the Trojan horse mechanism of action with McC but target aaRSs specified by the last amino acid of the peptide moiety.
Reagents and solvents were from commercial suppliers (Acros, Sigma-Aldrich, Bachem, and Novabiochem) and used as provided, unless indicated otherwise. Dimethylformamide (DMF) and tetrahydroforan were analytical grade and were stored over 4-Å molecular sieves. For reactions involving 9-fluoroenylmethoxy carbonyl (Fmoc)-protected amino acids and peptides, DMF for peptide synthesis (low amine content) was used. All other solvents used for reactions were analytical grade and used as provided. Reactions were carried out in oven-dried glassware under a nitrogen atmosphere and stirred at room temperature, unless indicated otherwise.
1H and 13C nuclear magnetic resonance spectra of the compounds were recorded on a Bruker UltraShield Avance 300-MHz spectrometer. Spectra were recorded in dimethyl sulfoxide-d6 or D2O. The chemical shifts are expressed as δ values in parts per million, using the residual solvent peaks (dimethyl sulfoxide, 1H, 2.50 ppm, and 13C, 39.60 ppm; HOD, 1H, 4.79 ppm) as a reference. Coupling constants are given in hertz. High-resolution mass spectra (HR-MS) were recorded on a quadrupole time-of-flight mass spectrometer (Q-Tof-2; Micromass, Manchester, United Kingdom) equipped with a standard electrospray (ESI) interface; samples were infused in 2-propanol-H2O (1:1) at 3 μl/min.
For thin-layer chromatography, precoated aluminum sheets were used (silica gel 60 F254; Merck). The spots were visualized by UV light. Column chromatography was performed on an ICN silica gel (60A 60-200). For size exclusion chromatography, a 2- by 30-cm column of Sephadex LH-20 was used as the solid phase and methanol-H2O (7:3 [vol/vol]) as the eluent. Preparative high-performance liquid chromatography (HPLC) of final compounds was done using a Waters Xbridge prep C18 (19 by 150 mm) column connected to a Waters 1525 binary HPLC pump and a Waters 2487 dual absorbance detector. Eluent compositions are expressed as vol/vol.
Compounds are shown as their major microspecies at pH 7.4, as determined by the program Marvin (MarvinBeans 4.1.13, 2007; ChemAxon).
The experimental procedures for synthesis of the known 5′-O-(N-l-aminoacyl)-sulfamoyladenosines (compounds 4 to 6) and their intermediates are not described here.
The synthesis of hexapeptide 10 was done by standard solid-phase peptide synthesis using Fmoc-protected amino acids and Wang resin (680 μmol of reactive groups per g) as the solid support. The amino acid building blocks were coupled using activation mixtures consisting of appropriately protected amino acid (4.0 eq), N-hydroxybenzotriazole (HOBt; 4.0 eq), diisopropylcarbodiimide (DIC; 4.0 eq), and N,N-diisopropylethylamine (DIPEA; 2.0 eq) relative to the active support (1.0 eq). For Asn, Arg, and Met, building blocks with acid-labile side-chain-protecting groups were used: Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pmc)-OH, and Fmoc-Thr(OtBu)-OH. Cleavage from the solid support and side chain deprotection were done by treatment with 5% thioanisole and 5% H2O in trifluoroacetic acid. The resulting Fmoc-hexapeptide 10 was purified by reverse-phase HPLC (RP-HPLC) (solvent A, 5% CH3CN, 0.1% HCOOH, H2O; solvent B, 5% H2O, 0.1% HCOOH, CH3CN) and characterized by MS. Yield after purification, starting from 200 mg of resin, was 74.7 mg (63%). HRMS for C39H55N10O11S: calculated, 871.37722; found, 871.37438.
Fmoc-MRTGNAD-SA (compound 11). Freshly prepared solutions (20 mg/ml in DMF) of Fmoc- MRTGNA-OH (compound 10; 19.3 mg, 22.3 μmol, 1.0 eq), HOBt (5.1 mg, 33.4 μmol, 1.5 eq), and DIC (4.2 mg, 33.4 μmol, 1.5 eq) were mixed in a 1.5-ml microcentrifuge tube and shaken for 1 h at room temperature. Next, DIPEA (4.3 mg, 33.4 μmol, 1.5 eq) and compound 4 (19 mg, 33.4 μmol, 1.5 eq) were added and the reaction mixture was shaken for an additional 16 h while protected from light. Next, the reaction mixture was purified by preparative RP-HPLC (solvent A, 25 mM triethylammonium bicarbonate, 5% CH3CN, H2O; solvent B, 25 mM triethylammonium bicarbonate, 5% H2O, CH3CN). The peak containing compound 11 was identified by ESI-MS and lyophilized. HRMS for C53H70N17O19S2 [M-H]−: calculated, 1312.44755; found, 1312.44394.
For MRTGNAD-SA (compound 7), compound 11, obtained in the previous step, was dissolved in DMF (1 ml) and triethylamine (Et3N) (1 ml) was added. The reaction mixture was shaken for 18 h at room temperature and purified by RP-HPLC (twice; solvent A, 25 mM triethylammonium bicarbonate, 5% CH3CN, H2O; solvent B, 25 mM triethylammonium bicarbonate, 5% H2O, CH3CN). The peak corresponding to compound 7 was identified by ESI-MS and lyophilized. Using an estimated 260 value of 14.9 × 10−3 liter/mol/cm, the amount obtained was determined to be 350 μg (0.32 μmol; 1.4% combined yield, coupling and deprotection). HRMS for C38H60N17O17S2 [M-H]−: calculated, 1090.37948; found, 1090.37808.
Fmoc-MRTGNAE-SA (compound 12) was prepared following the procedure used for the synthesis of compound 11; compound 5 (8.10 mg, 14.0 μmol, 1.0 eq) was reacted with Fmoc-MRTGNA-OH (compound 10; 8.15 mg, 9.36 μmol, 1.5 eq) to give compound 12. HRMS for C54H72N17O19S2 [M-H]−: calculated, 1326.46320; found, 1326.45901.
MRTGNAE-SA (compound 8) was prepared following the procedure used for the synthesis of compound 7; compound 12 was deprotected to afford 12 μg (0.011 μmol; 0.1% combined yield, coupling and deprotection). HRMS for C39H62N17O17S2 [M-H]−: calculated, 1104.39513; found, 1104.39346.
Fmoc-MRTGNAL-SA (compound 13) was prepared following the procedure used for the synthesis of compound 11; compound 6 (7.85 mg, 14.0 μmol, 1.0 eq) was reacted with Fmoc-MRTGNA-OH (compound 10; 8.15 mg, 9.36 μmol, 1.5 eq) to afford comound 13. HRMS for C55H76N17O17S2 [M-H]−: calculated, 1310.50468; found, 1310.50428.
MRTGNAL-SA (compound 9) was prepared following the procedure used for the synthesis of compound 7; compound 13 was deprotected to give 170 μg (0.156 μmol; 1.6% combined yield, coupling and deprotection). HRMS for C40H66N17O15S2 [M-H]−: calculated, 1088.43660; found, 1088.43701.
pET-based expression plasmids overproducing AspRS and ProRS from Deinococcus radiodurans were provided by D. Soll, Yale University (see also reference 15). Plasmids overproducing GluRS from Acidithiobacillus ferrooxidans and LeuRS from Methanothermobacter thermautotrophicus were generously provided by Michael Ibba (Ohio State University). A plasmid overproducing E. coli LeuRS was created by PCR amplifying the appropriate gene from E. coli genomic DNA and cloning it into a pET21 vector. For microbiological tests, these plasmids were introduced into E. coli BL21(DE3) cells.
Bacterial growth inhibition assays were carried out as described in references 15 and 19. A 200-μl aliquot of overnight culture of appropriate E. coli cells was combined with 5 ml of soft (0.8%) LB agar and poured on the surface of square (10- by 10-cm) petri dish containing solidified 1.5% LB agar. In the case of experiments involving overproduction of AaRSs, both the top and the bottom layers of agar contained appropriate antibiotics and 0.5 mM isopropyl-β-d-thiogalactopyranoside. The top agar layer was allowed to solidify. Next, 5-μl drops of solutions containing various concentrations of compounds to be tested were carefully deposited on the surface of the plate be using a P20 Pipetman. The drops were allowed to dry, and plates were incubated for 6 to 8 h at 37°C. The size of anabiosys halos (growth inhibition zones) centered around the sites where inhibitor drops were deposited were recorded. Each experiment was simultaneously conducted in triplicate (i.e., using three different plates). The results of the measurements were highly reproducible (standard deviations of less than 10%).
S30 extracts were prepared as described in reference 15. Appropriate cells were grown in 50 ml of LB medium containing 500 μg/ml ampicillin. After centrifuging at 3,000 × g for 10 min the supernatant was discarded and the pellet was resuspended in 40 ml of buffer containing 20 mM Tris-HCl or HEPES-KOH (pH 8.0), 10 mM MgCl2, and 100 mM KCl. The cell suspension was centrifuged again as above. This procedure was repeated two times. The pellet was resuspended in 1 ml of 20 mM Tris-HCl or HEPES-KOH (pH 8.0), 10 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol, and kept at 0°C. Subsequently, the cells were sonicated for 10 s and left at 0°C for 10 min. This procedure was repeated five to eight times. The lysate was centrifuged at 15,000 × g for 30 min at 4°C.
The tRNA aminoacylation reactions were performed as described in reference 15 with minor modifications. To 1 μl of solution containing inhibitor, 3 μl of E. coli S30 extract was added. Next, 16 μl of the following aminoacylation mixture was added: 30 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 5 g/liter bulk E. coli tRNA, 3 mM ATP, 30 mM KCl, 8 mM MgCl2, and 40 μM of specified radiolabeled amino acid. The reaction products were precipitated in cold 10% trichloroacetic acid (TCA) on Whatman 3MM papers 5 min after the aminoacylation mixture was added. The aminoacylation reaction was carried out at room temperature. Depending on whether or not processing was needed, variable time intervals were included between the addition of the cell extract and the addition of the aminoacylation mixture. After thorough washing with cold 10% TCA, the papers were washed twice with acetone and dried on a heating plate. Following the addition of scintillation liquid, the amount of radioactivity was determined in a scintillation counter.
Given our failure to generate McC derivatives with targets other than AspRS by means of mutation (9), we decided to obtain McC analogs by total chemical synthesis. In all, three different compounds were prepared. Compound 7 was designed to target AspRS, which is also the target of McC. Two other compounds (8 and 9) were expected to target GluRS and LeuRS, respectively. To simplify synthesis, synthetic compounds differed from natural McC in several ways. First, the N-acyl O-aminopropylphosphoramidate linker of the aspartyl-adenylate moiety of processed McC (compound 2) was replaced by its close analog sulfamate, giving 5′-O-(N-L-aspartyl)-sulfamoyladenosine [DSA; compound 4; XSA abbreviations represent 5′-O-(N-l-aminoacyl)-sulfamoyladenosine, where X can be replaced by any 1-letter amino acid code to indicate the corresponding amino acid derivative] as the expected processing product of the compound expected to retain the target specificity of McC and ESA (compound 5) and LSA (compound 6) as the expected processing products of the other two compounds. DSA inhibits AspRS with an inhibition constant of 8.0 nM (3). ESA and LSA inhibit, respectively, GluRS and LeuRS with inhibition constants of 2.8 nM (4) and 8.4 nM (8). Compounds 7, 8, and 9 lack the O-aminopropyl group present in intact McC, as well as the N-formyl group. While both groups contribute to the antibacterial activity of McC, a natural derivative lacking both groups is still active (12).
As nothing was known in advance about the stability of compounds 7 to 9, the use of strong acid, strong base, and strong nucleophiles during their synthesis was avoided, to limit possible decomposition. We chose to use a convergent approach of coupling of Fmoc-protected hexapeptide 10 (Fmoc-MRTGNA-OH), corresponding to the first 6 amino acids of MccA prepared by standard solid-phase peptide synthesis, with XSAs 4 to 6. This was followed by deprotection of the coupling product to yield MRTGNAX-SAs 7 to 9. The existing synthesis scheme for XSAs was modified to allow for the preparation of larger amounts of the compounds. The main difference between our method and previous syntheses (3, 4, 6) was the use of 2′,3′-O-t-butyl-dimethylsilyl protection instead of 2′,3′-O-isopropylidene. This modification was developed by Ferreras et al. (7) for the preparation of 5′-O-(N-salicyl)-sulfamoyladenosine. We found that this method is preferential, as intermediates are far less prone to undergo intramolecular cyclization to N3-5′- cycloadenosine (18).
Triethylamine salts of XSAs 4 to 6 were coupled to Fmoc-MRTGNA-OH (compound 10) by in situ preactivation of hexapeptide 10 with HOBt in the presence of DIC and subsequent reaction of the activated hexapeptide with XSA · Et3N in the presence of DIPEA (Fig. (Fig.1d).1d). Prolonged incubation with Et3N-DMF at room temperature led to successful deprotection allowing us, following HPLC purification, to obtain synthetic McC analogs in amounts sufficient for biological and biochemical analyses. The identity of the 5′-O-(N-l-aminoacyl)-sulfamoyladenosine intermediates 4 to 6 was confirmed by HR-MS and nuclear magnetic resonance. The identities of the Fmoc-MRTGNA-OH hexapeptide 10 and the synthetic McC analogs 7 to 9 were confirmed by HR-MS.
The antibacterial activities of MRTGNAX-SAs 7 to 9 and corresponding XSAs 4 to 6 were determined by monitoring the appearance of growth inhibition zones (anabiosys halos) on lawns of McC-sensitive E. coli K-12 BW28357 cells (10). As controls, McC and its derivative without the N-terminal formyl and the aminopropyl groups were used; as we have described elsewhere (16), such a derivative is produced by E. coli cells lacking aminopeptidases A, B, and N and harboring an McC-producing plasmid with a disrupted mccD and/or mccE gene). The results, presented in Fig. Fig.2a,2a, can be summarized as follows. First, all MRTGNAX-SAs possessed antibacterial activity. Clear zones of growth inhibition were observed with a 10 μM solution of MRTGNAD-SA, the most active of the synthetic compounds. Growth inhibition zones of comparable size were detected with a 25 μM solution of MRTGNAL-SA and a 50 μM solution of MRTGNAE-SA. MRTGNAD-SA was less active than intact McC but considerably more active than the McC derivative without the N-terminal formyl and the aminopropyl groups. Second, detectable growth inhibition zones were observed with 100 μM and 250 μM solutions of LSA and DSA, respectively. No antibacterial activity of ESA was detected at concentrations of up to 1,000 μM. We therefore conclude that the addition of the MRTGNA heptapeptide increases the antibacterial activity of XSAs by at least an order of magnitude. Moreover, a comparison of MRTGNAD-SA and the McC derivative lacking the formyl and propylamine groups indicates that the sulfamoyl linkage increases antibacterial activity. Additional modifications such as the presence of an N-terminal formyl group or the presence of an aminopropyl group may increase the antibacterial activities of MRTGNAD-SA and other MRTGNAX-SAs even further.
Cells carrying mutations in the yej genes coding for the YejABEF inner membrane ABC transporter are resistant to McC because they are unable to internalize the drug (19). BW28357 cells harboring a deletion in the yejA gene (19) were resistant to up to 100 μM MRTGNAX-SAs (Fig. (Fig.2b).2b). In contrast, the sensitivities of these cells to XSAs were indistinguishable from those of the wild-type control cells (compare Fig. 2a and b). We therefore conclude that the YejABEF transporter is responsible for the uptake of MRTGNAX-SAs. We further conclude that YejABEF is not involved in XSAs transport.
Once inside the cell, McC is deformylated and then processed by the action of one of the three broad-specificity aminopeptidases, A, B, or N (12). BW28357 cells harboring a triple deletion of the pepA, pepB, and pepN genes coding, respectively, for peptidases A, B, and N, are resistant to the drug because they cannot process it (12). These cells were also resistant to up to 100 μM MRTGNAX-SAs, while sensitivity to XSAs was indistinguishable from that of the wild-type control cells (Fig. (Fig.2c).2c). The results therefore suggest that peptidases A, B, and N are required for processing of MRTGNAX-SAs (see below, also). Additional analysis involving McC-sensitive double (pepA pepB, pepA pepN, and pepB pepN) and single (pepA, pepB, and pepN) mutants revealed that they were all sensitive to MRTGNAX-SAs (data not shown). Thus, any one of the three peptidases is sufficient to impart sensitivity to MRTGNAX-SAs.
Our next step was to determine the intracellular targets of MRTGNAX-SAs 7 to 9. To this end, in vitro tRNA aminoacylation reactions in E. coli extracts were carried out using radioactively labeled aspartate, glutamate, and leucine. As expected, each XSA 4 to 6 inhibited aminoacylation of cognate tRNA but had no effect on noncognate tRNA aminoacylation (i.e., LSA abolished aminoacylation of tRNALeu but not of tRNAAsp or tRNAGlu) (Fig. (Fig.3a).3a). The addition of MRTGNAX-SAs also inhibited cognate (as determined by the last amino acid of the peptide moiety) tRNA aminoacylation (Fig. (Fig.3a).3a). XSAs also inhibited tRNA aminoacylation in extracts prepared from cells lacking peptidases A, B, and N (Fig. (Fig.3b).3b). In contrast, MRTGNAX-SAs had no effect on tRNA aminoacylation in mutant cell extracts (Fig. (Fig.3b).3b). We therefore conclude that synthetic McC-like compounds are processed by aminopeptidases and, upon processing, inhibit AaRSs specified by the last amino acid of the peptide moiety.
The results presented so far suggest that synthetic McC analogs enter the cell through the YejABEF transporter, are processed by cytoplasmic aminopeptidases, and then target AaRSs specified by their last amino acids. To prove this conjecture, we determined whether E. coli BL21(DE3) cells overproducing AspRS from Deinococcus radiodurans, GluRS from Acidithiobacillus ferrooxidans, or LeuRS Methanothermobacter thermautotrophicus become resistant to compounds 7, 8, and 9. As control, cells overproducing D. radiodurans ProRS were used. Previously, we showed that overproduction of D. radiodurans AspRS but not ProRS makes E. coli resistant to McC (15). Because the initial plasmid overproducing LeuRS from thermophilic A. ferrooxidans did not lead to changes in sensitivity to any of the compounds tested (data not shown), a plasmid overproducing E. coli LeuRS was created. As can be seen in Fig. Fig.4,4, only overproduction of “cognate” mesophilic AaRSs afforded protection from synthetic McC analogs and McC. Likewise, AspRS and LeuRS overproduction led to increased resistance to DSA and LSA. No protection from ESA could be observed (data not shown), as this compound lacks antibacterial activity (see above). Based on these results we conclude that, in vivo, synthetic McC analogs target AaRSs whose identities are determined by the nature of the last amino acid of an McC analog.
The principal result of our work is the demonstration that McC can be used as a platform to prepare synthetic compounds that target AaRSs other than AspRS, the target of wild-type McC. This is a significant advance, since neither site-specific mutagenesis of the mccA gene coding for the peptide moiety of McC (25) nor bioinformatics searches for mcc gene homologs (25) have led to compounds with altered target specificities.
Our experiments reveal that synthetic McC-like compounds retain the essential Trojan horse features of the original compound. First, the McC-like compounds are at least 10 times more active than the corresponding XSAs, due to the contribution of the MccA hexapeptide MTRGNA. The facilitated transport of McC-like compounds is due to the action of the YejABEF transporter, which is also responsible for McC uptake (19). Finally, the McC-like compounds are processed inside the cytoplasm of sensitive cells by the same broad-specificity aminopeptidases that process McC (12).
The current and previous results (11) show that changes in the McC peptide moiety, including substitutions altering the enzyme-inhibiting part of McC and changes in the linker between the peptide and nucleotide parts of McC, are tolerated with limited effects on activity and may even increase the whole-cell antibacterial activity of McC-like compounds. This modularity of McC is interesting from a drug development point of view. An important question that remains is how much the structure of the toxic part may deviate from the native processed McC (compound 2) structure to retain the uptake advantage. The antibacterial activity of MRTGNAL-SA (compound 9) suggests that these differences can be quite extensive, as this compound differs from compound 2 both at the linker moiety and the aminoacyl side chain (an uncharged isobutyl side chain versus an anionic carboxymethyl). Thus, total chemical synthesis should allow investigators, in principle, to generate McC-like compounds targeting each of the 20 AaRSs in the cell. It would be interesting to see if amides or esters consisting of the MRTGNA peptide and inhibitors of essential bacterial cell components other than AaRSs also lead to antibacterials with improved potencies.
By bypassing the need for specific maturation enzymes acting on the MccA heptapeptide, our results open several avenues for preparation of novel McC-like compounds that act on bacteria other than those targeted by McC. For example, compounds containing peptide moieties shorter than the MccA heptapeptide can be readily prepared and tested. Conceivably, such compounds may enter sensitive cells through transporters other than YejABEF. Since McC-like compounds are processed by ubiquitous broadly specific aminopeptidases and since the ultimate target, an aaRS, is evolutionarily conserved, such compounds may inhibit the growth of bacteria other than E. coli. An alternative strategy is to couple a nonhydrolyzable XSA to a peptide known to specifically enter a particular group of bacteria and thus create a narrow-spectrum inhibitor. Finally, peptide library-based approaches can be used for the generation and screening of McC-like compounds with desired properties. This work is currently under way in our laboratories.
We thank Katrijn Bockstael for help in the solid-phase synthesis of the peptide, Jef Rozenski and Stephanie Vandenwaeyenberg for recording the mass spectra, and Piet Herdewijn and Roger Busson for helpful discussions.
This work was supported by the U54 AI057158 NIH/NIAID North Eastern Biodefense Center grant, a Burroughs Wellcome Career Award, a Russian Academy of Sciences Presidium program in Molecular and Cellular Biology grant, and a Rutgers University Technology Commercialization Fund Grant (to K.S.) and by Russian Foundation for Basic Research grant 06-04-48865 (to A.M.).
Published ahead of print on 14 August 2009.