Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus

Abstract

The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.

Keywords: Antibiotic resistance; DNA repair; Drug synergy; IMP-1700; Target validation.

Graphical abstract

Scheme 1

Synthetic routes to analogues and structures. General procedures: (A) NaHCO₃, DMF, RT, overnight; (B) DMSO, microwave (115 °C, 3 h); (C) pyridine, reflux, overnight; (D) 4 M HCl, dioxane, RT, overnight.

Figure 1

Compounds do not inhibit DNA gyrase to exhibit synergy with ciprofloxacin. a) Effect of compounds on E. coli DNA gyrase supercoiling of pBR322. Gyrase is inhibited by Ciprofloxacin (CFX), but not 1, 5, 10 or 15 (OC = open-circular DNA; Rel = relaxed DNA; SC = supercoiled DNA). b) Densitometry analysis of DNA supercoiling. Data represent mean ± SEM (n = 3 technical replicates). c) Synergy quantification of 1, 5 and 10 with CFX using CompuSyn software simulation, displayed as combination index (CI) against fraction affected (Fa). d) Dose-reduction index (DRI) of compound or CFX against Fa. e) Summary of CI, compound and CFX DRI. Only compound 10 (IMP-1700) quantitatively synergizes with CFX. Data represent mean ± SEM (n = 3 biological replicates).

Figure 2

Mechanism of action of IMP-1700 (10). a) Growth inhibition of JE2 transformed to express GFP under control of the recA promoter, treated with 10 µM Ciprofloxacin (CFX) to induce DNA damage, plus titrations of 10 or CFX. b) GFP expression in response to titration of 10 or CFX. c) Normalized GFP fluorescence/cell, showing SOS activation increased with increased CFX but decreased with 10. Data represent mean ± SEM (n = 3 biological replicates). d) Synthesis of Sepharose resin functionalized with 10. Conditions (A) PyBOP, DIPEA, DMF, RT, overnight; (B) 4 M HCl in dioxane, RT, overnight; (C) NHS-Sepharose, 1:1 (v/v) DMSO:0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3, 4 °C, overnight. e) Pulldown from E. coli lysates co-expressing StrepII-AddA and His₆-AddB, demonstrating Sepharose functionalized with 10 allows co-isolation of the complex (n = 2 biological replicates).