Mechanism of action and limited cross-resistance of new lipopeptide MX-2401 - PubMed (original) (raw)

. 2011 Jun;55(6):2743-54.

doi: 10.1128/AAC.00170-11. Epub 2011 Apr 4.

T Schneider, M Elliott, W R P Scott, J Pan, C Anklin, H Yang, D Dugourd, A Müller, K Gries, S K Straus, H G Sahl, R E W Hancock

Affiliations

Mechanism of action and limited cross-resistance of new lipopeptide MX-2401

E Rubinchik et al. Antimicrob Agents Chemother. 2011 Jun.

Abstract

MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca²⁺-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C₅₅-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C₅₅-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

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Figures

Fig. 1.

Fig. 1.

MX-2401 structure.

Fig. 2.

Fig. 2.

Intracellular accumulation of UDP-_N_-acetylmuramyl pentapeptide in untreated and vancomycin-treated (A) and amphomycin-treated and MX-2401-treated (B) S. simulans 22 cells. The experiment was performed with 10× MIC of the respective antibiotic compounds for 45 min. Subsequently, cells were extracted with boiling water, and the intracellular nucleotide pool was analyzed by reverse-phase HPLC. The identity of UDP-MurNAc-pentapeptide was confirmed by MALDI-TOF mass spectrometry, yielding a molecular mass of 1,148.72 Da for the indicated peaks (arrows).

Fig. 3.

Fig. 3.

Impacts of amphomycin and MX-2401 on the overall in vitro lipid II synthesis by cytoplasmic membrane preparations of M. luteus. (A) The synthesis of lipid II in the presence of increasing concentrations of amphomycin and MX2401 was qualitatively analyzed using TLC and PMA staining. (B) Quantitative analysis was carried out using [14C]UDP-GlcNAc as described in Materials and Methods. Peptides were added at molar ratios of 0.25 to 2 with respect to the amount of the substrate C55-P. The reaction product of the untreated control was taken as 100%. The error bars indicate standard deviations.

Fig. 4.

Fig. 4.

Impacts of amphomycin and MX-2401 on bactoprenol-phosphate-consuming reactions catalyzed by MraY (A) and TagO (B). The incorporation of [14C]UDP-MurNAc-pentapeptide into lipid I (A) and [14C]UDP-GlcNAc into lipid III (B) was analyzed in the presence of increasing peptide concentrations. Peptides were added in molar ratios of 0.25 to 2 with respect to the substrate C55-P. Daptomycin was added in 2-fold molar excess with respect to C55-P. Quantification was carried out using phosphorimaging. The error bars indicate standard deviations.

Fig. 5.

Fig. 5.

Dose-dependent membrane depolarization by MX-2401, daptomycin, and amphomycin. Cells were incubated with antibiotics for 60 min before depolarization was measured.

Fig. 6.

Fig. 6.

Membrane depolarization and cell death in S. epidermidis following incubation with sub- and supra-MIC concentrations of antibiotics in the presence or absence of surfactant. (A) Cells incubated with MX-2401 for 90 min. (B) Cells incubated with amphomycin for 90 min. (C) Cells incubated with daptomycin for 60 min.

Fig. 7.

Fig. 7.

Effects of MX-2401 and daptomycin on bacterial membrane permeability.

Fig. 8.

Fig. 8.

Ability of MX-2401 to interact with model membranes. (A) Dose-dependent lipid flip-flop of C6-NBD-PC asymmetrically labeled POPC/POPG liposomes treated with MX-2401, daptomycin, and GMS in the presence of 5 mM CaCl2. (B) Dose-dependent calcein release from POPC/POPG liposomes treated with MX-2401, daptomycin, and GMS in the presence of 5 mM CaCl2. The error bars indicate standard deviations.

Fig. 9.

Fig. 9.

Effect of MX-2401 and daptomycin on membrane fusion as determined by light scattering. Daptomycin (black bars) induces fusion in the presence of POPC, POPG, and Ca2+ as observed by the increase in vesicle size, whereas MX-2401 (white bars) does not, similarly to the control (gray bars; lipid only). The error bars represent the error associated with three repeats.

Fig. 10.

Fig. 10.

Representative structures of MX-2401 as obtained from the refinement simulations. The backbone trace on the right represents residues Asp1 → Pro11 (carbon, gray; nitrogen, blue; and oxygen, red), while the traces on the left (yellow) represent the acyl chain and linker connected to the N-terminal residue Asp1. The cyclic part is well defined (relative to daptomycin) and appears to adopt two major conformations. The image was created using UCSF CHIMERA (26).

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