Optimization of a novel potent and selective bacterial DNA helicase inhibitor scaffold from a high throughput screening hit

Abstract

Benzobisthiazole derivatives were identified as novel helicase inhibitors through high throughput screening against purified Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) replicative helicases. Chemical optimization has produced compound 59 with nanomolar potency against the DNA duplex strand unwinding activities of both B. anthracis and S. aureus helicases. Selectivity index (SI=CC50/IC50) values for 59 were greater than 500. Kinetic studies demonstrated that the benzobisthiazole-based bacterial helicase inhibitors act competitively with the DNA substrate. Therefore, benzobisthiazole helicase inhibitors represent a promising new scaffold for evaluation as antibacterial agents.

Figure 1

The structure of HTS hit 1.

Figure 2

Mode of inhibition of benzobisthiazole 49. A dilution series of compound 49 was examined in the FRET-based S. aureus helicase assay with varying concentrations of ATP (A) or DNA (B) to determine IC₅₀ values. Data were plotted in GraphPad Prism 5.0 using 4-parameter curve fitting. (C) The results from varying DNA are also shown in a Dixon plot (1/V vs [I]). (D) Compounds 49 and 59 failed to exhibit significant inhibition of the single-stranded DNA stimulated ATPase activity of S. aureus helicase as detected by malachite green.

Scheme 1

Reagents and conditions: a, benzoyl chlorides, CH₂Cl₂, yields 31–95%; b, aq. KMnO₄, glacial HOAc, dioxane, 73% for 47, and 44% for 48; c, R²R³NH, DMF, 80–100 °C, yields 20–83%.

Scheme 2

Reagents and conditions: a, 3,4,5-trimethoxybenzaldehyde, Na(OAc)₃BH, glacial HOAc, DMSO, r.t., 48%; b, 3,4,5-trimethoxybenzaldehyde, toluene, reflux, 67%.