Targeting class A and C serine β-lactamases with a broad-spectrum boronic acid derivative

Abstract

Production of β-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight β-lactam antibiotics. The substrate spectrum of BLs has become increasingly broad, posing a serious health problem. Thus, there is an urgent need for novel BL inhibitors. Boronic acid transition-state analogues are able to reverse the resistance conferred by class A and C BLs. We describe a boronic acid analogue possessing interesting and potent broad-spectrum activity vs class A and C serine-based BLs. Starting from benzo(b)thiophene-2-boronic acid (BZBTH2B), a nanomolar non-β-lactam inhibitor of AmpC that can potentiate the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria, we designed a novel broad-spectrum nanomolar inhibitor of class A and C BLs. Structure-based drug design (SBDD), synthesis, enzymology data, and X-ray crystallography results are discussed. We clarified the inhibitor binding geometry responsible for broad-spectrum activity vs serine-active BLs using double mutant thermodynamic cycle studies.

Figure 1

β-Lactamases inhibitors and β-lactam antibiotics. (A) Starting lead compound 1, BZBTH2B, benzo(b)thiophene-2-boronic acid. (B) The BL inhibitor clavulanic acid. (C) Cefotaxime, III generation cephalosporin. (D) The last generation BL inhibitor avibactam. (E) Broad spectrum inhibitor compound 5, 3-(2-carboxyvinyl)benzo(b)thiophene-2-boronic acid. (F) Inhibitor SMS2 ((1R)-1-(2-thienylacetylamino)-1-(3-carboxyphenyl)methylboronic acid.

Figure 2

(a–c). 3-D comparative analysis of class A and C β-lactamases guided inhibitor design. (a) Binary complex of BZB2THB in AmpC BL. Key residues and interactions are highlighted. Carbon atoms are in green, nitrogen atoms are colored blue, oxygen atoms red, and sulfur atoms yellow. Red spheres represent water molecules. Picture was prepared using CCP4mg. (b) Glycylboronic acid inhibitor SMS2 complexes with CTM-M-9 (carbon atoms in blue) and TEM-1 (carbon atoms in orange) are superimposed. The interactions with C3(4′)carboxylate binding pocket of β-lactams are highlighted. Nitrogen atoms are colored blue, oxygen atoms red, and sulfur atoms yellow. Red spheres represent water molecules. Picture was prepared using CCP4mg. (c) Molecular surface of the superimposed active site region of CTX-M-9, TEM-1, and AmpC. Glycylboronic acid inhibitor SMS2 complexes with CTM-M-9 (carbon atoms in blue) and TEM-1 (carbon atoms in orange) were superimposed with BZB2THB in complex with AmpC (carbon atoms in green). The C3(4′)carboxylate binding pocket of β-lactams and the C3 position in BZB2THB selected for chemical derivatization are highlighted. The surface contributed by nitrogen atoms is colored blue, oxygen atoms are colored red, and carbon atoms are colored gray. Picture was prepared using CCP4mg.

Scheme 1

Figure 3

(a) Electron density of compound 5 in complex with CTM-M-9. The 2F_o – F_c electron density map is represented by the green cage and is contoured at 1.0σ. The inhibitor is covalently attached to Ser70. Carbon atoms of CTM-M-9 and compound 5 are colored green, Nitrogen atoms are colored blue, oxygen atoms red, and sulfur atoms yellow. Picture was prepared using Pymol. (b) Key interactions observed in the complex of 5 with CTM-M-9, site B. The inhibitor is covalently attached to Ser70. Carbon atoms of CTX are colored gray, carbon atoms for 5 are colored green. Nitrogen atoms are colored blue, oxygen atoms red, and sulfur atoms yellow. Red spheres represent water molecules. Residues Ser130 and Ser237 are in double conformation. Picture was prepared using CCP4mg.

Scheme 2. Double Perturbation Analysis: (a) Ligands, (b) Double Perturbation Analysis Cycle

Thermodynamic cycle to quantify the hydrogen bond energy between Arg244 and 5. E = wild-type enzyme, E′ = mutant enzyme. L = wild-type ligand, L′ = “mutant” ligand. ΔΔΔG = (ΔΔG₁ – ΔΔG₄) or (ΔΔG₂ – ΔΔG₃). E = Tem-1, E′ = Tem-30 R244S. L = 5, L′ = 4. ΔΔΔG_1–4 = 2.73 kcal/mol. ΔΔΔG_2–3 = 2.75 kcal/mol.

Figure 4

Interpreting DPA results. Compound 5 (carbon atoms in green) in binary complex with CTX-M-9 (carbon atoms in gray) is superimposed TEM-1 (carbon atoms in orange). Key residue Arg276, which in CTM-M-9 interacts with compound 5 carboxylate group, is replaced by Asn276 in TEM-1. However, in TEM-1, as suggested from DPA, key interaction could be picked up from Arg244, oriented at H-bond distance from compound 5. Picture was prepared using CCP4mg.