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. 2018 Jul 23;26(12):3453-3460.
doi: 10.1016/j.bmc.2018.05.017. Epub 2018 May 24.

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus

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Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus

Hai Liang et al. Bioorg Med Chem. .

Abstract

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline's antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA-the enzyme that catalyzes the first step of peptidoglycan biosynthesis-over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC50 of 480 μM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.

Keywords: Albocycline; MRSA; MurA; Peptidoglycan biosynthesis inhibition; VRSA.

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Figures

Figure 1
Figure 1
First Biosynthetic step of peptidoglycan biosynthesis and proposed inhibitor: (A) Structure of (−)-albocycline (1), that is proposed to inhibit MurA. (B) MurA catalyzes the addition of phosphoenolpyruvate to UDP-N-Acetyl-Glucosamine to form enolypruvl-UDP-N-Acetyl-Glucosamine. UDP is urdine diphopshate shown in blue.
Figure 2
Figure 2
Projection of the simulation trajectory data onto the first two tICA components shows three main conformational basins sampled by pseudo-apo MurA (E. coli): a closed-form region (top left), half-open-form region (bottom right) and open-form region (top right). Black circles denote the 100 microstate centers obtained by k-means conformational clustering.
Figure 3
Figure 3
Albocycline conformers in solution are a mixture of roughly equal populations of conformers A and B (calculated by BICePs and confirmed by 1H NMR).
Figure 4
Figure 4
(A) DOCK 6.7 correctly predicts the pose of UDP-GlcNAc substrate upon re-docking into the MurA (E. coli) crystal structure (PDB: 1UAE), to within 1.4 Å rmsd of heavy-atom positions. The crystal poses of fosfomycin and UDP-GlcNAc are shown in blue, and the lowest-energy re-docked pose of UDP-GlcNAc is shown in gray. (B) The lowest-energy docked pose of albocycline (conformer A, magenta) to the Mur A (E. coli) crystal structure overlaps with molecular volume available in the UDP-GlcNAc (blue) binding site. (C) Distributions of dock scores for conformational clusters of bound poses, shown here as simplified Gaussians with the same mean and standard deviation to aid the viewer. (D) Average locations of clustered poses (shown as red, yellow, green and blue is surfaces) display notably deeper bound poses for MurA (S. aureus, red arrow).
Figure 5
Figure 5
Sequence variation of residues near the substrate binding site for MurA (E. coli, blue), MurA (S. aureus, tan) and MurZ (S. aureus, lavender). Side chains are shown for all non-conserved residues less than 5 Å from the native bound ligands fosfomycin (gray, left) and UDP-GlcNAc (gray, right) in the MurA (E. coli) crystal structure (PDB: 1UAE).
Figure 6
Figure 6
HPLC-based IC50 study of albocycline against S. aureus MurA. (a) HPLC trace of the MurA reaction in the presence 250 μM albocycline showing the separation of UDP-GlcNAc and EP-UDP-GlcNAc. (b) IC50 data for albocycline and S. aureus MurA. Data were fitted using a non-linear regression curve fit in Graph Pad Prism 6.

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