New Insights into the Bacterial RNA Polymerase Inhibitor CBR703 as a Starting Point for Optimization as an Anti-Infective Agent

Abstract

c CBR703 was reported to inhibit bacterial RNA polymerase (RNAP) and biofilm formation, considering it to be a good candidate for further optimization. While synthesized derivatives of CBR703 did not result in more-active RNAP inhibitors, we observed promising antibacterial activities. These again correlated with a significant cytotoxicity toward mammalian cells. Furthermore, we suspect the promising effects on biofilm formation to be artifacts. Consequently, this class of compounds can be considered unattractive as antibacterial agents. Bacterial RNA polymerase (RNAP) is essential for bacterial growth and survival and is thus an attractive target for drug development (1, 2). Along with the recently FDA-approved fidaxomycin (3), the rifamycins, applied as first-line antituberculosis drugs, are the only RNAP inhibitors that are in clinical use (2). However, similarly to other anti-infectives, the use of rifamycins resulted in the occurrence of resistant bacterial strains (1, 4-7), which represents a remarkable threat to public health (8, 9). Consequently, there is a need to focus on novel promising inhibitors. Recently, interesting peptidic and peptidomimetic (10-12) as well as nonpeptidic (13-18) small-molecule RNAP inhibitors have been described. Another example is CBR703 (Fig. 1), whose mechanism of action is reported to be different from that of the rifamycins (19,20). This compound has been identified in a highthroughput screening searching for small-molecule inhibitors of RNAP (19). Two more-potent analogs in that report reveal the potential of optimizing CBR703 by structural enlargement. Furthermore, pursuing the hypothesis that RNAP is of particular importance for bacterial survival in biofilms, Villain-Guillot et al. showed CBR703 to significantly reduce Staphylococcus epidermidis biofilm mass (21). We therefore considered CBR703 to be a promising starting point for drug development. Consequently, we focused on CBR703 to perform systematic modifications on its core structure, aiming to obtain a more appropriate starting point for further structural optimization.Detailed information concerning the materials and methods used in synthesis and biology can be found in the supplemental material.In total, 30 final compounds and 24 intermediates were obtained and tested for Escherichia coli RNAP inhibition and their ability to inhibit the growth of E. coli TolC (see Table S1 to S3 in the supplemental material). According to their structures, the synthesized derivatives can be divided into three groups with modifications in part A, B, or C (Fig. 1). Compounds 1 to 25 (see Scheme S1 in the supplemental material) with introduction of substituents into the aromatic moieties (part A or B) were prepared by condensation of an intermediate amide with hydroxylamine (22, 23). In order to ensure an appropriate coverage of lipophilic and electronic properties, the substituents were chosen rationally from all quadrants of a Craig plot (e.g., Hansch-Fujita versus constant) (24). The results (see Table S1) showed that compound...

The transcription-repair coupling factor Mfd associates with RNA polymerase in the absence of exogenous damage

“…For experiments involving drug treatment, growth medium was supplemented with rifampicin (50 µg per mL, Sigma-Aldrich, USA) or CBR703 (75 µg per mL, Thermo Fisher Scientific, UK). The minimal inhibitory concentrations of rifampicin for E. coli K-12 and CBR703 for E. coli tolC − were determined to be 7 µg per mL and 14 µg per mL respectively 52 .…”

Structural Basis of Transcription Inhibition by CBR Hydroxamidines and CBR Pyrazoles

“…However, the potential development of CBR hydroxamidines and pyrazoles as Gram-negative antibacterial drugs has been thwarted by the low potencies of known CBR hydroxamidines and pyrazoles against wild-type, efflux-proficient Gram-negative bacterial strains (MICs > 50 μg/ml; legend to Figure 1; Zhu et al, 2014), and by the absence of structural information on RNAP-CBR interactions that could be used to inform and direct medicinal chemistry efforts to improve potency (Li et al, 2001a,b; Zhu et al, 2014). …”

Spray-drying of inhalable, multifunctional formulations for the treatment of biofilms formed in cystic fibrosis

“…Determination of growth inhibitory effects and minimum inhibitory concentrations (MICs) for the multifunctional DPI formulations and the antibiotics were performed as described in literature [46] [47]. Briefly, MIC values against P. aeruginosa PA14 were determined in 96-well plates (Sarstedt, Nümbrecht, Germany).…”

“…To investigate whether the interaction of NAC with the antibiotics would modulate their antibacterial activity, we tested the antibiotic/NAC formulations, the neat antibiotics, and NAC against P. aeruginosa PA14 using the bacterial growth-inhibition assay [46,47]. Pure NAC did not show any inhibition of P. aeruginosa PA14 growth in the concentration range of 0.049 to 0.52 µg/mL corresponding to those in the tested formulations.…”