Discovery of Lipophilic Bisphosphonates That Target Bacterial Cell Wall and Quinone Biosynthesis

Abstract

We report that alkyl-substituted bisphosphonates have activity against Bacillus anthracis Sterne (0.40 μg/mL), Mycobacterium smegmatis (1.4 μg/mL), Bacillus subtilis (1.0 μg/mL), and Staphylococcus aureus (13 μg/mL). In many cases, there is no effect of serum binding, as well as low activity against a human embryonic kidney cell line. Targeting of isoprenoid biosynthesis is involved with 74 having IC₅₀ values of ∼100 nM against heptaprenyl diphosphate synthase and 200 nM against farnesyl diphosphate synthase. B. subtilis growth inhibition was rescued by addition of farnesyl diphosphate, menaquinone-4 (MK-4), or undecaprenyl phosphate (UP), and the combination of MK-4 and UP resulted in a 25× increase in ED₅₀, indicating targeting of both quinone and cell wall biosynthesis. Clostridioides difficile was inhibited by 74, and since this organism does not synthesize quinones, cell wall biosynthesis is the likely target. We also solved three X-ray structures of inhibitors bound to octaprenyl diphosphate and/or undecaprenyl diphosphate synthases.

Figure 1.

Schematic illustration of some of the enzymes involved in cell wall and quinone biosynthesis in many bacteria, together with chemical structures of substrates and intermediates and sites of action of some inhibitors (red) and “rescue” agents (blue). (a) Enzymes, products, inhibitors and rescue agents. UP = undecaprenyl phosphate; MK-4 = menaquinone-4. (b) Chemical structures of selected enzyme substrates and products discussed in the Text. HepPPS (heptaprenyl diphosphate synthase) is a heterodimeric enzyme. Some bacteria such as E. coli use the homodimeric octaprenyl diphosphate synthase (OPPS), and also produce ubiquinones (not shown). DXP = the 1-deoxy-D-xylulose 5-phosphate pathway, found in most bacteria; MEV = the mevalonate pathway, found in e.g. S. aureus. In some bacteria, e.g. Listeria monocytogenes, both the DXP and MEV pathways are present.

Figure 2.

Structures of some compounds that inhibit isoprenoid biosynthesis enzymes such as FPPS, GGPPS, UPPS, and UPPP that are discussed in the Text.

Figure 3.

Structures of bisphosphonates synthesized. Compounds are rank-ordered by activity against B. subtilis from most active (74, top-left) to least active (97, bottom). Most of the active compounds contain meta-substituted pyridinium rings and a medium-size side-chain (cyan). Short or long chain substituents (red) are inactive.

Figure 4.

Typical dose-response curves for SaHepPPS inhibition. The most potent inhibitors also have potent activity (~10–20 nM) against EcOPPS (Figure S3), but were not active E. coli or other gram-negative bacteria. Results shown represent three pooled data sets taken on different days, fit to single dose-response curves.

Figure 5.

Cartoon illustration of the similarity between a putative FPP transition state/reactive intermediate (top) and a potent SaHepPPS inhibitor, 74 (bottom). Note that this mechanistic proposal would only apply to “long-chain” (~C₃₀, C₃₅, C₄₀) trans prenyltransferases (which use FPP as a substrate) and not to short-chain prenyl transferases, such as FPPS, since FPP is the product and presumably would have only weak binding to FPPS. However, FPP is also known (in human FPPS) to bind to the allosteric (i.e. non-catalytic) FPPS site and acts as an FPPS inhibitor, and it is possible that FPP-analogs may also bind in this way.

Figure 6.

Effects of MK-4, UP, HMBPP and FPP as well as pairwise combinations on B. subtilis growth inhibition by 74. (a) Effects of MK-4, UP and MK-4 plus UP on growth inhibition by 74. (b) Effects of HMBPP, FPP with or without MK-4 or UP as well as HMBPP+FPP on 74 inhibition of B. subtilis cell growth. All rescue agents were at 50 µM. (c) Schematic illustration of the x-fold rescues by compounds or pairs of compounds on B. subtilis growth inhibition by 74. The largest effect (25x) is found with UP (50 µM) + MK-4 (50 µM). Cell growth inhibition assays were carried out in duplicate. The largest effects (~>9x) are seen with MK-4 or UP combinations and are colored cyan. d) Summary of IC₅₀ values from data in a) and b).

Figure 7.

Structure of 69 bound to EcOPPS and a comparison with S. cerevisae GGPPS and P. vivax F/GGPPPS ligand-bound structures. (a) Structure superimposition EcOPPS•69 (PDB ID code 5ZLF) with ScGGPPS•69 (PDB ID code 2E93) blue = OPPS; yellow = GGPPS. (b) Superimposition of 69 ligands shown in a) pink = GGPPS; green = OPPS. (c) Superimposition of EcOPPS•69 with EcOPPS•FSPP (PDB ID code 3WJN). Pink = OPPS; cyan = FSPP. (d) Superimposition of 69 (yellow) and FSPP (cyan) ligands from (c). (e) Superimposition of EcOPPS•69 (orange) with P. vivax—F/GGPPS•105 (color; PDB ID code 3RBM). (f) Superimposition of 69 (brown) and 104 (blue) ligands from (e). 69 binds to just the allylic site (ab) in EcOPPS) and coordinates to 1 Mg²⁺.

Figure 8.

Structure of 70 and FSPP bound to EcOPPS. (a) Superimposition of EcOPPS•70 (blue; Chain B; PDB ID code 5ZE6) with EcOPPS•FSPP (yellow; PDB ID code 3WJN). (b) Illustration of 70 penetrating the dimer interface in EcOPPS•70. The ligand (cyan) is only present in Chain B and is close to the monomer surface, but is buried in the dimer interface.

Figure 9.

Structure of 70 bound to EcUPPS together with comparisons with pyrazole, clomiphene, 69 and 105 bound structures. (a) Structure of EcUPPS•70 (pink; PDB ID code 5ZHE) superimposed on a UPPS•pyrazole [N-(3-amino-3-oxopropyl)-5-(benzo[b]thiophen-5-yl)-1-benzyl-N-(4-isopropoxybenzyl)-1H-pyrazol e-4-carboxamide] (gold; PDB ID code 5KH5) structure (from S. pneumoniae). (b) Zoomed-in view of the ligand-binding region in (a) 70 in cyan. (c) Superimposition of EcUPPS•70 (blue) with EcUPPS•clomiphene (not all clomiphene atoms were resolved; green; PDB ID code 5CGJ). (d) Zoomed-in view of (c) 70 in yellow. (e) Superimposition of EcUPPS•70 (blue) with EcUPPS•69 (cyan; PDB ID code 2E98). (f) Zoomed-in view of (e) 70 in pink. (g) Superimposition of EcUPPS•70 (green) with EcUPPS•105 (pink; PDB ID code 3SH0). (h) Zoomed-in view of (g).