Antibacterial drug leads targeting isoprenoid biosynthesis

Abstract

With the rise in resistance to antibiotics such as methicillin, there is a need for new drugs. We report here the discovery and X-ray crystallographic structures of 10 chemically diverse compounds (benzoic, diketo, and phosphonic acids, as well as a bisamidine and a bisamine) that inhibit bacterial undecaprenyl diphosphate synthase, an essential enzyme involved in cell wall biosynthesis. The inhibitors bind to one or more of the four undecaprenyl diphosphate synthase inhibitor binding sites identified previously, with the most active leads binding to site 4, outside the catalytic center. The most potent leads are active against Staphylococcus aureus [minimal inhibitory concentration (MIC)(90) ∼0.25 µg/mL], and one potently synergizes with methicillin (fractional inhibitory concentration index = 0.25) and is protective in a mouse infection model. These results provide numerous leads for antibacterial development and open up the possibility of restoring sensitivity to drugs such as methicillin, using combination therapies.

Fig. 1.

Schematic outline of cell wall biosynthesis (in most bacteria) showing involvement of isoprenoid biosynthesis in the early stages of peptidoglycan formation.

Fig. 2.

Chemical structures of UPPS inhibitors and drug leads of interest.

Fig. 3.

X-ray structures of E. coli UPPS showing substrate and inhibitor binding sites. (A) FSPP (yellow) binds to site 1 (PDB ID code 1X06) and FPP (green) binds to sites 1 and 4 (PDB ID code 1V7U). (B) A bisphosphonate (5) binds to sites 1–4 (PDB ID code 2E98). (C) Benzoic acid inhibitor 8 binds to site 3 (cyan, PDB ID code 3SGT), superimposed on FPP-bound structure (green, PDB ID code 1V7U). (D) Benzoic acid inhibitor 9 binds to sites 1–3 (cyan, PDB ID code 3SGV), superimposed on FPP-bound structure (green, PDB ID code 1V7U). The large red numbers indicate sites 1–4.

Fig. 4.

Crystal structures of the more potent benzoic acids and a phosphonate inhibitor. (A) 10 (PDB ID code 3SGX). (B) 11 (PDB ID code 3SH0). (C) 12 (PDB ID code 4H2O). (D) 13 (PDB ID code 4H38). In each case, site 4 is occupied, together with either site 1, 2, or 3, indicating the likely importance of site 4 binding for good activity. The values shown are the IC₅₀s for E. coli UPPS inhibition (Ec) or S. aureus UPPS inhibition (Sa).

Fig. 5.

Crystal structures of diketo acids and two dicationic inhibitors bound to E. coli UPPS. (A) 14 (PDB ID code 4H3C). (B) 15 (PDB ID code 4H3A). (C) 16 (PDB ID code 4H2J). (D) 18 (PDB ID code 4H2M). The common feature in each case is binding to site 4. The values shown are the IC₅₀s for E. coli UPPS inhibition (Ec) or S. aureus UPPS inhibition (Sa).

Fig. 6.

UPPS as a missing link. Models and cartoons. (A) Pharmacophore model for UPPS inhibition by benzoic acids. (B) Pharmacophore model for S. aureus growth inhibition by benzoic acids. Common features are benzoic acid carboxylates (cyan) with electron-withdrawing meta substituents (red); an x–y spacer (dark pink); two aromatic features (orange); and more-distal hydrophobic features (green). (C) Cationic-hydrophobic-cationic inhibitor binding to DNA. (D) Cationic-hydrophobic-cationic inhibitor binding to anionic lipids in a membrane. (E) Cationic-hydrophobic-cationic inhibitor binding to a protein.

Fig. 7.

In vitro synergy and in vivo results with 17. (A) Isobologram for 17 + methicillin inhibition of S. aureus (USA300) cell growth. FICI = 0.25. (B) Activity of 17 in a mouse model of S. aureus (USA200) infection. Shown is one representative experiment repeated twice (n = 10 mice per group per experiment). No mice in the group treated once daily with 10 mg/kg of 17 (three doses total) died during either experiment.

Fig. 8.

Computational analysis of UPPS structural results. (A) FTMap computational solvent mapping of UPPS structures (PDB ID codes 2E98 and 3QAS) suggest that site 4 is druggable in either inhibitor-bound complexes or unbound. UPPS is represented as a cartoon; small probes are colored spheres; black wireframe outlines site 4. (B) Principal component analysis of E. coli UPPS structures. Substrate-bound structures (yellow) are closed (33); bisphosphonates (blue) are open (33); the apo and nonbisphosphonate structures (red) are all ajar (slightly open). (C) ROC/AUC analysis of most-predictive UPPS structures in terms of initial enrichment for actives under 100 μM (Fig. S6).