Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases

ABSTRACT Microcin C (McC) is a potent antibacterial agent produced by some strains of E scherichia 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 (Ͻ10-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. 1a. McC is a heptapeptide with a formylated N-terminal methionine and a C-terminal aspartate whose ␣-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 aminopep-tidases (12). The product of processing, modified aspartyladenylate (compound 2) (15), closely resembles Asp-AMP (compound 3) (Fig. 1c), the natural reaction intermediate of the tRNA Asp aminoacylation reaction catalyzed by AspRS. However, because the bond between the ␣-carboxyl of C-terminal aspartate and the phosphoramidate nitrogen is nonhydrolyzable, compound 2 inhibits AspRS. Unprocessed McC has no effect on tRNA Asp 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.

MATERIALS AND METHODS
General chemistry. 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. 1 H and 13 C nuclear magnetic resonance spectra of the compounds were recorded on a Bruker UltraShield Avance 300-MHz spectrometer. Spectra were recorded in dimethyl sulfoxide-d 6  acyl)-sulfamoyladenosines (compounds 4 to 6) and their intermediates are not described here.

Synthesis of the hexapeptide Fmoc-MRTGNA-OH (compound 10).
The synthesis of hexapeptide 10 was done by standard solid-phase peptide synthesis using Fmoc-protected amino acids and Wang resin (680 mol 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-chainprotecting 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% H 2 O in trifluoroacetic acid. The resulting Fmoc-hexapeptide 10 was purified by reverse-phase HPLC (RP-HPLC) (solvent A, 5% CH 3  Synthesis of MRTGNAX-SA compounds 7 to 9. Fmoc-MRTGNAD-SA (compound 11). Freshly prepared solutions (20 mg/ml in DMF) of Fmoc-MRTGNA-OH (compound 10; 19.3 mg, 22.3 mol, 1.0 eq), HOBt (5.1 mg, 33.4 mol, 1.5 eq), and DIC (4.2 mg, 33.4 mol, 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 mol, 1.5 eq) and compound 4 (19 mg, 33.4 mol, 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% CH 3  Fmoc-MRTGNAL-SA (compound 13) was prepared following the procedure used for the synthesis of compound 11; compound 6 (7.85 mg, 14.0 mol, 1.0 eq) was reacted with Fmoc-MRTGNA-OH (compound 10; 8.15 mg, 9.36 mol, 1.5 eq) to afford comound 13 Bacterial growth inhibition assays were carried out as described in references 15 and 19. A 200-l 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-␤-Dthiogalactopyranoside. The top agar layer was allowed to solidify. Next, 5-l 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%).
Preparation of S30 extracts. S30 extracts were prepared as described in reference 15. Appropriate cells were grown in 50 ml of LB medium containing 500 g/ml ampicillin. After centrifuging at 3,000 ϫ 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 MgCl 2 , 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 MgCl 2 , 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 ϫ g for 30 min at 4°C. tRNA aminoacylation reaction. The tRNA aminoacylation reactions were performed as described in reference 15 with minor modifications. To 1 l of solution containing inhibitor, 3 l of E. coli S30 extract was added. Next, 16 l 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 MgCl 2 , and 40 M 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. While both groups contribute to the antibacterial activity of McC, a natural derivative lacking both groups is still active (12).

Design and synthesis of
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 ap- VOL. 191, 2009 SYNTHETIC MICROCIN C ANALOGS 6275 proach 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 2Ј,3Ј-O-t-butyl-dimethylsilyl protection instead of 2Ј,3Ј-Oisopropylidene. This modification was developed by Ferreras et al. (7) for the preparation of 5Ј-O-(N-salicyl)-sulfamoyladenosine. We found that this method is preferential, as intermediates are far less prone to undergo intramolecular cyclization to N 3 -5Ј-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 · Et 3 N in the presence of DIPEA (Fig. 1d). Prolonged incubation with Et 3 N-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 5Ј-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.
In vivo activity of McC analogs. 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 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 M MRTGNAX-SAs (Fig. 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 M MRTGNAX-SAs, while sensitivity to XSAs was indistinguishable from that of the wild-type control cells (Fig. 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.
In vitro activity of McC analogs. 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 tRNA Leu but not of tRNA Asp or tRNA Glu ) (Fig. 3a). The addition of MRTGNAX-SAs also inhibited cognate (as determined by the last amino acid of the peptide moiety) tRNA aminoacylation (Fig. 3a). XSAs also inhibited tRNA aminoacylation in extracts prepared from cells lacking pepti- dases A, B, and N (Fig. 3b). In contrast, MRTGNAX-SAs had no effect on tRNA aminoacylation in mutant cell extracts (Fig.  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.
In vivo targets of McC analogs. 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 Acidithiobacil-lus 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. 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.

DISCUSSION
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.