Oritavancin exhibits dual mode of action to inhibit cell-wall biosynthesis in Staphylococcus aureus

Abstract

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the D-Ala-D-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan D-Ala-D-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.

Figure 1

Chemical structures of four glycopeptides (left column) and their des-N-methylleucyl forms (right column). The terminal leucyl residue of the aglycon core of each glycopeptide is highlighted. Minimum inhibitory concentrations for the growth of vancomycin-susceptible Gram-positive bacteria (see Methods) are in red.

Figure 2

Chemical structure of a peptidoglycan fragment of S. aureus. The five-residue stem on the right has no cross-link to its D-Ala-D-Ala terminus.

Figure 3

Schematic representation of peptidoglycan biosynthesis in S. aureus. A new glycan strand is shown forming at the membrane surface. Transglycosylation inserts Lipid II into this strand. The transformation of immature cell wall to mature (cross-linked) cell wall occurs through transpeptidation. The terminal peptide-stem D-alanine is removed upon transpeptidation. Three representative cross-links are illustrated.

Figure 4

50.3-MHz ¹³C{¹⁹F} REDOR dephasing (ΔS/S₀) for complexes of des-N-methylleucyl-[¹⁹F]oritavancin and whole cells of S. aureus grown on media containing D-[1-¹³C]alanine and a racemase inhibitor (left, solid circles) and [1-¹³C]glycine (right, open circles). The calculated dephasing for a single ¹⁹F-¹³C distance to the nearest cross-link accounts for the D-alanyl dephasing (left, solid line). The calculated dephasing assuming the five distances of a single compact helix for the pentaglycyl bridge matches the experimental glycyl dephasing (right, solid line). The glycyl dephasing is also compared to that observed (reference 19) for a similar complex with [¹⁹F]oritavancin (right panel, dotted line).

Figure 5

20.3-MHz ¹⁵N{¹⁹F} REDOR spectra of a complex of des-N-methylleucyl-[¹⁹F]oritavancin and whole cells of S. aureus grown on media containing L-[ε-¹⁵N]lysine. The full-echo spectrum (S₀) is shown at the bottom of the figure, and the REDOR difference (ΔS = S₀ – S, where S and S₀ are the spectra observed with and without ¹⁹F dephasing pulses, respectively) at the top of the figure. The presence of a REDOR difference at 95 ppm indicates the proximity of ¹⁹F to the bridge link (see Figure 2). A REDOR difference for the corresponding complex with [¹⁹F]oritavancin is not observed (reference 19). The spectra were the result of the accumulation of 20,000 scans. Magic-angle spinning was at 5 kHz.

Figure 6

Possible positions of fluorine relative to the bridging pentaglycyl helix in the peptidoglycan complex of des-N-methylleucyl-[¹⁹F]oritavancin. The pentaglycyl bridge is shown in an α-helical conformation with the carbonyl carbons in black, α carbons in gray, nitrogens in blue, and oxygens in red. The positions that are consistent with the REDOR dephasing of Figure 4 (right, open circles) are indicated by small dots whose colors (inset) indicate the root-mean-square deviation between calculated and experimental dephasing. The best match that is also consistent with the ¹⁵N{¹⁹F} dephasing of Figure 5 places the fluorine of des-N-methylleucyl-[¹⁹F]oritavancin away from the helix axis (arrow). The position of fluorine in the [¹⁹F]oritavancin complex with peptidoglycan (red dot) is on the helix axis.

Figure 7

Oritavancin influence on glycine and lysine metabolism. (Top) 125-MHz ¹³C CPMAS comparison of actively dividing whole cells of S. aureus labeled by [¹³C]glycine with (right) and without (left) [¹⁹F]oritavancin treatment. (Bottom) 50-MHz ¹⁵N CPMAS comparison of actively dividing whole cells of S. aureus labeled by L-[ε-¹⁵N]lysine with (right) and without (left) [¹⁹F]oritavancin treatment. The ¹⁵N spectra have been scaled by whole-cell mass.

Figure 8

A comparison of inhibition of cross-linking from the 30.4-MHz ¹⁵N{¹³C} REDOR spectra of whole cells of S. aureus grown on media containing [¹⁵N]glycine and D-[1-¹³C]alanine and a racemase inhibitor (control, black), plus therapeutic doses of penicillin (blue), [¹⁹F]oritavancin (green), or vancomycin (red). Full-echo spectra normalized to the control are shown at the bottom of the figure and REDOR differences at the top. The REDOR difference measures the relative number of cross-links per peptidoglycan pentagycyl bridging segment. Vancomycin has the least effect on cross-linking and penicillin the largest effect.

Figure 9

Complete accounting of whole-cell D-alanine. Experimental deconvolution of the normalized 75.5-MHz carbonyl-carbon spectra of the four S. aureus samples of Figure 8 are shown, with a structure color coding indicated in the top spectrum. Total label incorporation was 1.00 for the control and 0.53, 0.37, and 0.72 for the vancomycin, oritavancin, and penicillin-treated cells, respectively.

Figure 10

Space-filling model of the des-N-methylleucyl-[¹⁹F]oritavancin complex with the peptidoglycan of S. aureus (right), consistent with the results of Figures 4–6. The drug is in dark gray and the peptidoglycan components in light gray. The corresponding complex with [¹⁹F]oritavancin (left) has slightly altered positions of the D-Ala-D-Ala stem terminus and the fluorobiphenyl moiety.

Figure 11

Template model of cell-wall biosynthesis of wild-type S. aureus. Peptidoglycan stems are shown in blue, pentaglycyl bridging segments in red, and cross-links in green. The orientation of the template strand (the last fully extended glycan chain) is recognized by a combined transglycosylase-transpeptidase (not shown). Addition of a nascent peptidoglycan strand at the membrane exoface is accompanied by partial cross-linking to the bridging segments of the template. (Some stems and bridging segments have been omitted from the figure for clarity.) Interference with template recognition and cross-linking by [¹⁹F]oritavancin results when the drug (purple) attaches to the template proximate to the nascent strand (middle site). Vancomycin bound at the same location does not block cross-linking because the required bulky fluorobiphenyl disaccharide substituent is missing. Both drugs block chain extension when bound to Lipid II (bottom site). The [¹⁹F]oritavancin that is bound between template and mature strands (top site) has the orientation shown in Figure 10 (left), and the ¹⁹F (green) to D-Ala ¹³C (yellow) distance of Figure 4 (left).