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. 2015 Oct 27;54(42):6514-6524.
doi: 10.1021/acs.biochem.5b00966. Epub 2015 Oct 15.

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents

Joe S Matarlo #  1   2 , Christopher E Evans #  3 , Indrajeet Sharma  4 , Lubens J Lavaud  5 , Stephen C Ngo  5 , Roger Shek  2 , Kanagalaghatta R Rajashankar  6 , Jarrod B French  1   5   2 , Derek S Tan  3   4 , Peter J Tonge  1   5
Affiliations

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents

Joe S Matarlo et al. Biochemistry. .

Abstract

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

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Figures

Figure 1
Figure 1. OSB-AMS (1) analogs
Open keto-acid and closed lactol isomers of OSB-AMS (1) are in equilibrium at physiological pH. We synthesized analogues (3–11) of both conformations to test the active pharmacophore. Compounds 3–8 are keto-acid analogues which favor the open-ring conformation and compounds 9–11 are lactol analogues which favor the close-ring structure. Compound 2 is a meta-carboxylate analogue to test the importance of the ortho position of the carboxylate. Synthesis of these analogues can be found in the supplemental section.
Figure 2
Figure 2. Sequence alignment of MenE homologs from pathogenic bacteria
E. coli (K-12), S. aureus (MRSA, Rosenbach), M. tuberculosis (H37Rv) were aligned using INRA hierarchical clustering[53] and Alignment Annotator[54]. Blue arrow indicates the conserved arginine identified in the active site of saMenE (R222) by docking OSB-AMS into the crystal structure of apo saMenE[44]. The homologous residue in ecMenE is R195 and R90 in mtMenE. Red arrows indicate relevant residues interacting with OSB-AMS seen in the ecMenE crystal structure and conserved in saMenE and mtMenE.
Figure 3
Figure 3. Overlaid structures of OSB-AMS: R195K ecMenE and apo saMenE
Structure overlay of the OSB-AMS:ecMenE complex with apo saMenE (3IPL.pdb). These structures differ in the relative orientation of large domain 1 (silver) and small domain 2 (beige for E. coli and cyan for S. aureus), but represent the adenylate-bound conformation in which G358/G402 (yellow) in the A8 core motif is removed from the active site whereas the K437/K483 (red) is located in the active site. G358 and K437 are residues from E. coli MenE. G402 and K483 are residues from S. aureus. K483 is disordered in the S. aureus structure.
Figure 4
Figure 4. X-ray structure of OSB-AMS: R195K ecMenE showing interactions with OSB-AMS (A)
The overall structure of ecMenE:OSB-AMS is shown with the larger N-terminal (domain I) and the smaller C-terminal domain (domain II) highlighted by transparent surface representations in blue and green, respectively. The ligand is provided in ball-and-stick representation. (B) The structure of the bound ligand, OSB-AMS, is shown in the active site. The ligand with green carbon atoms in ball-and-stick representation and side chains that hydrogen bond with the ligand are shown with grey carbon atoms. (C) A schematic of OSB-AMS in the ecMenE active site. The ecMenE sidechains interacting with OSB-AMS are shown in black, the ligand is shown in red, and hydrogen bonding interactions are illustrated with dashed lines.
Figure 5
Figure 5. Effect of OSB-AMS on menaquinone levels in MRSA
Menaquinone levels were quantified by LC-MS/MS using standard curves generated with MK4 and MK9 (see experimental methods section for more details). A distribution of MKs are present in untreated MRSA with MK8 the most abundant, consistent with other reports[72]. Treatment with 15.6 μM OSB-AMS (half-MIC) results in a ~60% decrease in MK levels consistent with MenE inhibition. Error bar shown as experimental triplicates.
Scheme 1
Scheme 1. Classical de novo menaquinone biosynthesis pathway
This pathway consists of at least nine enzymes that catalyze the formation of menaquinone from chorismate[12, 13, 16]. The fifth enzyme, MenE, is an acyl-CoA ligase which ligates CoA to OSB via an OSB-AMP intermediate. (OSB = o-succinylbenzoate; AMP = adenosine monophosphate; DHNA-CoA = 1,4-dihydroxy-2-napthoyl-CoA)
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