The Quinazolinone Allosteric Inhibitor of PBP 2a Synergizes with Piperacillin and Tazobactam against Methicillin-Resistant Staphylococcus aureus

Checkerboard assay to assess the interactions of compound 2 with β-lactams and non-β-lactam antibiotics against three Staphylococcus aureus strains. Synergy was seen only in MRSA strains (NRS70 and NRS123; both strains produce PBP 2a) and with β-lactams, such as oxacillin, piperacillin, imipenem, meropenem, and cefepime. NRS128 is an MSSA strain. OXA, oxacillin; PIP, piperacillin; IPM, imipenem; MEM, meropenem; FEP, cefepime; VAN, vancomycin; DOX, doxycycline; GEN, gentamicin; LZD, linezolid; AZI, azithromycin.

Time-kill assays with compound 2 in combination with PIP and TZP. (A) Combination of compound 2 at 0.5× MIC with PIP at 0.5× MIC in MRSA NRS70 showed no significant bactericidal synergy, while the combination with PIP at 0.25× and 0.125× MIC was clearly antagonistic. The orange line represents PIP at 0.5× MIC, and the purple lines represent combination of compound 2 at 0.5× MIC with PIP at 0.5× (solid line), 0.25× (dashed line), and 0.125× (dotted line). (B) Compound 2 at 0.5× MIC (solid line) and 0.25× MIC (dashed line) in combination with TZP at 0.5× MIC in MRSA NRS70. (C) Compound 2 at 0.5× MIC in combination with TZP at 0.5× MIC in MRSA NRS123. (D) Compound 2 at 0.5× MIC (solid line) and 1× MIC (dashed line) MIC in combination with TZP at 0.5× MIC in MRSA USA300. (E) Compound 2 at 0.5× MIC in combination with 0.5× MIC of PIP in NRS100 (COL), a β-lactamase-negative MRSA strain.

Compound 2 in combination with TZP damages MRSA NRS70, as documented by SEM. (A) Control cells without antibiotic treatment. (B) Cells treated with compound 2 at 4× MIC overnight. (C) Cells treated with TZP at 0.25× MIC. (D) Cells treated with the triple combination of compound 2 with TZP. Magnification is ×35,000. Scale bar is 2 μm.

Compound 2 in combination with TZP shows efficacy in the neutropenic thigh infection model. Mice (n = 8 per group) were infected intramuscularly in the right thigh with MRSA strain NRS70 (10⁵ CFU per thigh). Three doses of compound 2 (40 mg/kg), TZP (32 mg/kg of TZB and 4 mg/kg of PIP), the triple combination, or the vehicle were administered subcutaneously starting at 1 h after infection and every 8 h, for a total of three doses. The triple combination of compound 2 with TZP was significantly better than either compound 2 (P < 0.001) or TZP (P < 0.01) by itself. Values are means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney U test with two tails).

Three-dimensional structure of the ternary complex PBP 2a-compound 2-PIP (complex 1). Shown is the molecular surface of complex 1, with compound 2 and PIP in spheres (yellow and orange for carbon atoms, respectively). Right, detailed view of residues interacting with ligands at allosteric and active sites. Polar contacts represented as dotted lines.