Optimization of Acetazolamide-Based Scaffold as Potent Inhibitors of Vancomycin-Resistant Enterococcus

Abstract

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL (22) and 1 μg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Figure 1.

FDA-approved carbonic anhydrase inhibitors with reported efficacy against VRE.

Figure 2.

Analogs 33 – 35 have MICs > 64 μg/mL against E. faecium HM-965.

Figure 3.

Cell viability for Caco-2 cells dosed at 64 μg/mL (blue bars) and 128 μg/mL (red bars) for representative analogs.

Figure 4.

Time-kill assay for analogs 22 and 26 along with AZM and linezolid compared to DMSO-treated controls over 24 hours at 37 °C. Limit of detection is 100 CFU/mL. The data were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons. Asterisks (*) denote statistically significant different (p < 0.05) between treatments with either 22, 26, AZM or linezolid in comparison to DMSO-treated cells.

Figure 5.

(A) Homology model of Efα-CA (gray ribbons) built with H. pylori α-CA crystal structure (PDB: 4YGF) as the template. Catalytic Zn²⁺ shown with dark grey sphere. (B) Zoom in of active site for Efα-CA model (light gray) overlaid with H. pylori α-CA crystal structure (magenta). Conserved residues and/or residues important to AZM binding shown in sticks. Amino acid numbers for H. pylori shown in labels. E. faecium amino acids that are different from H. pylori in parentheses. All images generated using PyMol.

Figure 6.

Representations of Efγ-CA homology model. (A) Side view ribbon representation, Ni²⁺ (gray spheres); (B) Top view representation shows trimeric catalytic unit. (C) Side view surface representation, black arrow orients viewer to Ni²⁺. (D) Overlay of C. difficile γ-CA crystal structure (purple, PDB: 4MFG) and Efγ-CA homology model (gold). Overlaid active site histidine residues in sticks. Residue numbers correspond to of C. difficile γ-CA. H79 from one monomeric unit while H64’ and H84’ from adjacent monomer. Images generated using PyMol.

Figure 7.

Ligand poses for AZM, 22 and 26 generated by MD simulation in the active site of the Efα-CA. Ligands, residues and waters important for ligand interactions shown as sticks. Polar hydrogens shown for clarity of proposed hydrogen bond interactions (yellow dashed lines). (A) Generated pose for AZM (green sticks) in the Efα-CA site. (B) Generated pose for 22 (cyan sticks). (C) Predicted binding pose for 26 (yellow sticks). (D) Overlaid ligands show the alternative positions adopted by analogs 22 and 26 compared to AZM. (E) Surface representation of Efα-CA with 22 (cyan sticks) and 26 (yellow sticks). The hydrophobic patch lined with Leu178, Pro181, and Pro182 shown as red surface. Polar patch lined by Glu67, Lys69, and Asn70 shown as blue surface. Images generated using PyMol.

Figure 8.

Proposed binding poses of analogs from MD simulation with Efγ-CA. (A) Binding pose for AZM (green sticks). Residues associated with binding interactions are in sticks and labeled. Water molecules associated with binding interactions shown as red and white sticks. Predicted interactions shown as yellow dashed lines. Ni²⁺ ion shown as gray sphere. (B) Predicted binding pose for 22 (cyan sticks). (C) Predicted binding pose for 26 (yellow sticks). (D) Surface electrostatic map for Efγ-CA with ligands overlaid to show different in predicted binding modes. Blue surface indicates net positive charge and red surface indicates net negative charge. Images generated using PyMol.

Figure 9.

In vivo pharmacokinetic analysis of plasma concentrations for analog 22 in mice dosed orally. Seven time points shown, final time point at 1440 min was 0 ng/mL and not included on plot. Error bars indicate standard deviation at each time point (n = 3 per time point). Green dotted line represents the MIC concentration of 22 (0.007 μg/mL).

Figure 10.

Summary of SAR for sulfonamide anti-VRE scaffold.

Scheme 1.

Synthetic route for analogs 3 – 28. Reagents and conditions: a) concentrated HCl (13 eq.), 95 °C, 24 hr, 74%; b) 1 (1.0 eq.), 2-chloroacetyl chloride (1.1 eq to yield 2a) or 3-chloropropanoyl chloride (1.1 eq to yield 2b), TEA (1.2 eq.), MeCN, 0 °C – rt 19 hr, 46 and 39%, respectively; c) R-COOH (1.1 eq.), oxalyl chloride (1.2 eq.), DMF (1 drop), DCM, 0 °C – rt, 2 hr, crude product carried into the next step; d). 1 (1.0 eq.), R²-COCl (from previous step or commercial source, 1.1 eq.), TEA (1.2 eq.), MeCN, 0 °C – rt, 14 hr, 4.5 – 71%; e) 2a or 2b (1.0 eq.), cyclic amine (2.0 eq.), TEA (2.0 eq.), anhydrous THF, 0 °C – rt, 20 hr, 82%.

Scheme 2.

Synthesis of analogs 29 - 31. Reagents and conditions: a) i. 1 (1.0 eq.), R-CHO (1.5 eq.), CH₂Cl₂, rt, 1 hr; ii. sodium cyanoborohydride (1.5 eq.), 0 °C – rt, 18 hr, 8 – 21%.

Scheme 3.

Synthesis of analog 33. Reagents and conditions: a) 32 (1.0 eq.), acetic anhydride (1.5 eq.), acetic acid, 60 °C, 1 hr, 42%.