Discovery of N-quinazolinone-4-hydroxy-2-quinolone-3-carboxamides as DNA gyrase B-targeted antibacterial agents

Abstract

Emerging drug resistance is generating an urgent need for novel and effective antibiotics. A promising target that has not yet been addressed by approved antibiotics is the bacterial DNA gyrase subunit B (GyrB), and GyrB inhibitors could be effective against drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA). Here, we used the 4-hydroxy-2-quinolone fragment to search the Specs database of purchasable compounds for potential inhibitors of GyrB and identified AG-690/11765367, or f1, as a novel and potent inhibitor of the target protein (IC₅₀: 1.21 µM). Structural modification was used to further identify two more potent GyrB inhibitors: f4 (IC₅₀: 0.31 µM) and f14 (IC₅₀: 0.28 µM). Additional experiments indicated that compound f1 is more potent than the others in terms of antibacterial activity against MRSA (MICs: 4-8 µg/mL), non-toxic to HUVEC and HepG2 (CC₅₀: approximately 50 µM), and metabolically stable (t_1/2: > 372.8 min for plasma; 24.5 min for liver microsomes). In summary, this study showed that the discovered N-quinazolinone-4-hydroxy-2-quinolone-3-carboxamides are novel GyrB-targeted antibacterial agents; compound f1 is promising for further development.

Keywords: Antibiotic resistance; DNA Gyrase inhibitors; MRSA; antibacterial agent; computer-aided drug design.

Figure 1.

Overview of representative bacterial DNA Gyrase B inhibitors.

Figure 2.

Computer-guided search for novel GyrB inhibitors. (a) Essential fragment for the binding of g37 to the ATPase domain of S. aureus GyrB. The image was generated with LigPlot + in which hydrogen bonds and names of the interacting residues are coloured in green. (b) Structures selected from the Specs compound library and their GyrB inhibition rates (%) at 10 μM. Novobiocin was set as the positive control (inhibition rate at 10 μM: 99%). (c) Concentration-dependent ATPase inhibition of S. aureus Gyrase B (GyrB). The calculated IC₅₀ value of AG-690/11765367 was 1.21 μM. Novobiocin was set as the positive control (IC₅₀: 0.02 μM). (d) Likely binding mode of the hit compound AG-690/11765367 to the ATP binding site of S. aureus GyrB as derived by molecular docking. The interacting residues and the hit compound are shown in stick representations; the binding site is shown as a surface representation.

Scheme 1.

Reagents and conditions: (i) DIPEA, 45 °C, DMF, CH₃CH₂I, 10 h; (ii) NaH, DMF, diethyl malonate, 70 °C, 8 h; (iii) 12 N HCl, MeOH, 65 °C, 10 h; (iv) R²COCl, TEA, DCM, r.t.; (v) hydrazine hydrate, EtOH, 78 °C, 10 h; and (vi) HATU, DIPEA, DMF, r.t, 48 h.

Figure 3.

Predicted binding modes of f4 and f14 to the ATP binding site of S. aureus GyrB as derived from molecular docking experiments. (a) Compounds f4 and f14 superimposed with f1. (b) Interactions formed between f4 and S. aureus GyrB. (c) Interactions formed between f14 and S. aureus GyrB. The interacting residues and the ligands are shown in stick representation. Colour code: pink, compound f4; blue, compound f14.