Discovery of antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one

Abstract

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.

Figure 1

Macromolecular synthesis assays. Antibiotic 2 at 1/2 the MIC. Positive controls for DNA, RNA, protein, and peptidoglycan synthesis are ciprofloxacin (0.5 μg/mL), rifampicin (8 ng/mL), tetracycline (31 ng/mL), and fosfomycin (16 μg/mL), respectively. The maximum inhibition observed for antibiotic 2 is reported at 120 min of incubation in all cases, except for DNA synthesis, which was at 80 min.

Figure 2

Inhibition of Bocillin FL binding to S. aureus PBP2a and PBP1. (A) Fluorescence labeling of recombinant purified PBP2a (1 μM) by Bocillin FL (20 μM) in the presence of increasing amounts of antibiotic 2 (μg/mL). (B) Fluorescence labeling of S. aureus membrane preparation (150 μg) by Bocillin FL (30 μM) in the presence of increasing amounts of antibiotic 2 (μg/mL). For data fitting, consult Figure S7.

Figure 3

Crystal structure of S. aureus PBP2a in complex with quinazolinone 2. (A) Ribbon representation of PBP2a showing antibiotic 2 (in cyan for carbons) bound to the allosteric site. The allosteric domain spans residues 27–326, where the N-terminal domain (residues 27–138) is shown in green and the remaining allosteric domain is colored yellow. The transpeptidase domain (residues 327–668) is shown in purple. (B) Ribbon superimposition at the active site of apo PBP2a (PDB ID: 1VQQ) colored in orange and complex colored in purple. Structural changes occur at loops α9-β3, β3-β4, and β5-α10. (C) Key interactions of antibiotic 2 at the allosteric site. Salt-bridge interactions with K273 and K316 are shown as black dashed lines, the distances are 2.9 Å. π-Stacking interactions are observed with Y105 and Y297. Stereoview showing the refined electron density (2F_o − F_c feature-enhanced map) for the ligand (blue mesh) contoured at 1.0 σ.

Figure 4

Binding of quinazolinone 2 to PBP2a allosteric site, determined using a previously described procedure. (A) The change in the maximum fluorescence intensity gave a K_d of 6.9 ± 2 μg/mL (average of three experiments; nonlinear regression for data fitting with an R² of 0.9997). (B) Emission scans of PBP2a intrinsic fluorescence with excitation at 280 nm. Antibiotic 2 was titrated in to give the final concentrations shown.

Scheme 1

Synthesis of Quinazolinone 2