Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth

Abstract

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron-withdrawing aryl-alkyl side chains which inhibited the growth of Gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ∼1-4 μg mL^-1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially "rescued" by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (∼2-6 μg mL^-1 ) against Gram-positive but not Gram-negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ∼1-2 μg mL^-1 .

Keywords: Gram-negative pathogens; Staphylococcus aureus; cell wall biosynthesis; drug discovery; membrane proteins.

Figure 1

Illustration of selected molecules involved in cell wall biosynthesis in most bacteria. Also shown are sites of action of some antibiotics and potential targets, discussed in the Text.

Figure 2

Structures of substrates and inhibitors discussed in the Text.

Figure 3

ClustalW alignments of T. brucei, human, S. mansoni and six bacterial FPPSs. The YS pair is present in all of the bacterial FPPSs and the Tyr is proposed here to interact with electron-deficient aryl groups in bisphosphonate inhibitors. FARM = first aspartate-rich motif; SARM = second aspartate-rich motif. The squiggly lines represent residues omitted from the alignment, for clarity.

Figure 4

Stereo representation of superimposition of TbFPPS•6 (PDB code 2P1C) with EcFPPS (PDB ID code 1RQJ). Side chains of HY (red) and YS (yellow) amino acids are shown in stick format. 6 is shown in purple. Aspartate-rich motif (FARM) is shown in magenta. Mg²⁺ are orange spheres.

Figure 5

Isobolograms for bacterial cell growth inhibition by 11 and fosmidomycin (7) against a) E. coli, b) A. baumannii, c) K. pneumoniae and d) P. aeruginosa. The FICI (fractional inhibitory concentration indices) are shown in Table 2. The mean FICI value is 0.42 ± 0.17 indicating synergistic activity (in most cases).

Figure 6

Partial rescue of cell growth inhibition by farnesol or PaFPPS overexpression. Addition of 200 μm farnesol (FOH) to the growth medium increases the IC₅₀ of 11 and 12 for cell growth inhibition by a factor of ~8. a) E. coli K-12 with FOH; b) A. baumannii with FOH; c) K. pneumoniae with FOH; and d) P. aeruginosa with FOH; e) E. coli BL21(DE3) with PaFPPS overexpression.

Figure 7

Representative isobolograms for 32 with antibiotics having known mechanisms of action. a) 32+fosmidomycin in B. subtilis showing synergy (FICI=0.27) of 32 with a cell wall biosynthesis inhibitor (that targets DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, in the non-mevalonate pathway); b) 32+sulfamethoxazole in B. subtilis showing an indifferent effect (FICI=1.78) of 32 with a nucleic acid biosynthesis inhibitor (that targets dihydropteroate synthase); c) 32+bacitracin in S. aureus showing synergy (FICI=0.24) of 32 and a cell wall biosynthesis inhibitor (that targets UPPP); d) 32+tetracycline in S. aureus showing an indifferent effect (FICI=1.61) of 32 with a protein synthesis inhibitor (that targets ribosome function).

Figure 8

Enzyme and cell growth inhibition by the dihydroxyacid 20 and hydroxyphosphonates 32 and 41. a) SaUPPS inhibition; b) EcUPPP inhibition together with result for bacitracin; c) partial rescue of E. coli cell growth inhibition by EcUPPS overexpression.

Scheme 1

Synthesis route to FPPS inhibitors 11-13.

Scheme 2

General methods for synthesis of a) 2,3-dihydroxy acids; b) 1-hydroxyphosphonic acids; c) 2-hydroxyphosphonic acids.