Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase

Abstract

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED₅₀ ∼0.15 μg mL^-1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition.

Keywords: Staphylococcus aureus; benzoic acids; cell-wall biosynthesis; drug discovery; membrane proteins.

Figure 1

Schematic outline of cell wall biosynthesis (in most bacteria) delineating the role of isoprenoid biosynthesis in the early stages of peptidoglycan formation, together with the reactions targeted by several compounds discussed in the Text.

Figure 2

Representative dose-response curves for three inhibitors (7, 13 and 18) against B. subtilis and S. aureus. Data points are reported as mean±SD for duplicate experiments.

Figure 3

Representative isobolograms for 7 with antibiotics having known mechanisms of action. a) 7+fosmidomycin in B. subtilis showing synergy (FICI=0.17) of 7 with a cell wall biosynthesis inhibitor (that targets DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, in the non-mevalonate pathway); b) 7+sulfamethoxazole in B. subtilis showing an indifferent effect (FICI=1.72) of 7 with a nucleic acid biosynthesis inhibitor (that targets dihydropteroate synthase); c) 7+bacitracin in S. aureus showing synergy (FICI=0.20) of 7 with a cell wall biosynthesis inhibitor (that targets UPPP); d) 7+ kanamycin in S. aureus showing an indifferent effect (FICI=1.72) of 7 with a protein biosynthesis inhibitor (that targets ribosome function).

Figure 4

Dose-response curves of various benzoic acid and phenyl phosphonic acid derivatives against SaUPPS and EcUPPP. The benzoic acids are up to ~40× more potent UPPP inhibitors than bacitracin, a known UPPP inhibitor used as a topical antibiotic. Data points are reported as mean±SD, for duplicate experiments.

Figure 5

Correlations between cell growth and enzyme inhibition results. a) Correlation between S. aureus and B. subtilis cell growth inhibition based on pED₅₀ (=−log₁₀ED₅₀ [μM]) results; b) Correlation between S. aureus cell growth inhibition and SaUPPS inhibition; c) Pearson r-value correlation matrix/heat map for S. aureus cell growth inhibition, B. subtilis cell growth inhibition, SaUPPS and EcUPPP enzyme inhibition (all based on pED₅₀ or pIC₅₀ values), and logD. The Pearson r-values are indicated and red/orange=high correlation, green=low correlation.

Scheme 1

General synthesis methods. a) for benzoic acids; b) for phenylphosphonates.