Benzyl and benzoyl benzoic acid inhibitors of bacterial RNA polymerase-sigma factor interaction

Abstract

Transcription is an essential biological process in bacteria requiring a core enzyme, RNA polymerase (RNAP). Bacterial RNAP is catalytically active but requires sigma (σ) factors for transcription of natural DNA templates. σ factor binds to RNAP to form a holoenzyme which specifically recognizes a promoter, melts the DNA duplex, and commences RNA synthesis. Inhibiting the binding of σ to RNAP is expected to inhibit bacterial transcription and growth. We previously identified a triaryl hit compound that mimics σ at its major binding site of RNAP, thereby inhibiting the RNAP holoenzyme formation. In this study, we modified this scaffold to provide a series of benzyl and benzoyl benzoic acid derivatives possessing improved antimicrobial activity. A representative compound demonstrated excellent activity against Staphylococcus epidermidis with minimum inhibitory concentrations reduced to 0.5 μg/mL, matching that of vancomycin. The molecular mechanism of inhibition was confirmed using biochemical and cellular assays. Low cytotoxicity and metabolic stability of compounds demonstrated the potential for further studies.

Keywords: Antimicrobial; Bacterial transcription; Inhibitor; RNA polymerase; Sigma factor.

Fig. 1.

Structure of A) the E. coli RNAP holoenzyme crystal complex (PDB: 4LJZ) and B) interactions at the interface of σ_2.2 and β’CH, with key amino acid side chains shown [18].

Fig. 2.

A) Structures of C3 and C3–005 and MICs against S. pneumoniae; B) Docking model of C3–005 (yellow) onto β’CH (semi-transparent surface showing hydrophobicity in blue).

Fig. 3.

Antimicrobial activity (MIC μg/mL) of compound 8e against clinically important Gram-positive pathogens. EFAE: Enterococcus faecalis ATCC 19433, SEPI: S. epidermidis ATCC 12228, SSAP: S. saprophyticus ATCC 15305, SPYO: S. pyogenes ATCC 19615, SAGA: S. agalactiae ATCC 12386, CDIFF: Clostridium difficile ATCC 9689; RT 002: C. difficile ribotype 002; RT 027: C. difficile ribotype 027; VAN: vancomycin.

Fig. 4.

Effects of test compound 8e and control drugs vancomycin and fidaxomicin at sub-inhibitory concentrations (1/2 ×, 1/4 ×, 1/8 ×, and 1/16 × MIC) on the levels of Toxin A (dark grey bars), Toxin B (light grey bars) and CFU count (black circles) in (A) C. difficile ATCC 9689, (B) ribotype 002, and (C) ribotype 027 after 48 hours of anaerobic incubation. All toxin levels were normalized against drug-free control (dotted horizontal line from left y-axis = 1). CFU counts are relative to drug-free control values denoted by each respective dashed horizontal line from right y-axes: (A) y = 7.23, (B) y = 6.79 and (C) y = 6.48.

Fig. 5.

Inhibition of the β’CH-σ interaction by representative compounds 5b, 5e, 8e and 5f using the NanoLuc PCA system.

Fig. 6.

Confocal microscopy of B. subtilis with RNAP fluorescence where 8e was added to the culture at 0.5, 1, and 2 MIC.

Fig. 7.

The effects of 8e on the levels of (A) DNA, (B) RNA and (C) protein of S. aureus ATCC 29213 when challenged at ¼ × (checkered bars) and ⅛ × (light grey bars) MICs compared to the control drug rifampicin (Rif) and the no-drug control (solid black bars).

Scheme 1.

Synthetic route for compounds 5a – j^{a a} Reagents and conditions: a) Arylboronic acid, Pd(OAc)₂, PPh₃, K₃PO₄, Tol, 80 °C; b) 3,4-Dichlorobenzenethiol, NaOAc, EtOH, reflux; (c) NaOH, H₂O, dioxane, 50 °C, overnight.

Scheme 2.

Synthetic route for compounds 8a – i^{a a} Reagents and conditions: (a) NBS, H₂O, CHCl₃, 60 ~ 85 °C; (b) 3,4-Dichlorobenzenethiol, NaOAc, EtOH, reflux; (c) NaOH, H₂O, dioxane, 50 °C, overnight.