RESUMO
Bacterial RNA polymerase (RNAP), the core enzyme responsible for bacterial transcription, requires the NusG factor for efficient transcription elongation and termination. As the primary binding site for NusG, the RNAP clamp-helix (CH) domain represents a potential protein-protein interaction (PPI) target for novel antimicrobial agent design and discovery. In this study, we designed a pharmacophore model based on the essential amino acids of the CH for binding to NusG, such as R270, R278, and R281 (Escherichia coli numbering), and identified a hit compound with mild antimicrobial activity. Subsequent rational design and synthesis of this hit compound led to improved antimicrobial activity against Streptococcus pneumoniae, with the minimum inhibitory concentration (MIC) reduced from 128 to 1 µg/mL. Additional characterization of the antimicrobial activity, inhibitory activity against RNAP-NusG interaction, and cell-based transcription and fluorescent assays of the optimized compounds demonstrated their potential for further lead optimization.
Assuntos
Antibacterianos , RNA Polimerases Dirigidas por DNA , Testes de Sensibilidade Microbiana , Streptococcus pneumoniae , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Descoberta de Drogas , Ligação Proteica , Relação Estrutura-Atividade , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Escherichia coli/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/antagonistas & inibidoresRESUMO
Mechanistic investigations are of paramount importance in elucidating the modes of action of antibiotics and facilitating the discovery of novel drugs. We reported a luciferase-based reporter system using bacterial cells to unveil mechanisms of antimicrobials targeting transcription and translation. The reporter gene Nluc encoding NanoLuciferase (NanoLuc) was integrated into the genome of the Gram-positive model organism, Bacillus subtilis, to generate a reporter strain BS2019. Cellular transcription and translation levels were assessed by quantifying the amount of Nluc mRNA as well as the luminescence catalyzed by the enzyme NanoLuc. We validated this system using three known inhibitors of transcription (rifampicin), translation (chloramphenicol), and cell wall synthesis (ampicillin). The B. subtilis reporter strain BS2019 successfully revealed a decline in Nluc expression by rifampicin and NanoLuc enzyme activity by chloramphenicol, while ampicillin produced no observable effect. The assay was employed to characterize a previously discovered bacterial transcription inhibitor, CUHK242, with known antimicrobial activity against drug-resistant Staphylococcus aureus. Production of Nluc mRNA in our reporter BS2019 was suppressed in the presence of CUHK242, demonstrating the usefulness of the construct, which provides a simple way to study the mechanism of potential antibiotic candidates at early stages of drug discovery. The reporter system can also be modified by adopting different promoters and reporter genes to extend its scope of contribution to other fields of work. IMPORTANCE: Discovering new classes of antibiotics is desperately needed to combat the emergence of multidrug-resistant pathogens. To facilitate the drug discovery process, a simple cell-based assay for mechanistic studies is essential to characterize antimicrobial candidates. In this work, we developed a luciferase-based reporter system to quantify the transcriptional and translational effects of potential compounds and validated our system using two currently marketed drugs. Reporter strains generated in this study provide readily available means for identifying bacterial transcription inhibitors as prospective novel antibacterials. We also provided a series of plasmids for characterizing promoters under various conditions such as stress.
Assuntos
Antibacterianos , Bacillus subtilis , Genes Reporter , Luciferases , Bacillus subtilis/genética , Bacillus subtilis/efeitos dos fármacos , Antibacterianos/farmacologia , Luciferases/genética , Testes de Sensibilidade MicrobianaRESUMO
The increasing prevalence of multidrug-resistant pathogens necessitates the urgent development of new antimicrobial agents with innovative modes of action for the next generation of antimicrobial therapy. Bacterial transcription has been identified and widely studied as a viable target for antimicrobial development. The main focus of these studies has been the discovery of inhibitors that bind directly to the core enzyme of RNA polymerase (RNAP). Over the past two decades, substantial advancements have been made in understanding the properties of protein-protein interactions (PPIs) and gaining structural insights into bacterial RNAP and its associated factors. This has led to the crucial role of computational methods in aiding the identification of new PPI inhibitors to affect the RNAP function. In this context, bacterial transcriptional PPIs present promising, albeit challenging, targets for the creation of new antimicrobials. This review will succinctly outline the structural foundation of bacterial transcription networks and provide a summary of the known small molecules that target transcription PPIs.
RESUMO
RNA polymerase is an essential enzyme involved in bacterial transcription, playing a crucial role in RNA synthesis. However, it requires the association with sigma factors to initiate this process. In our previous work, we utilized a structure-based drug discovery approach to create benzoyl and benzyl benzoic acid compounds. These compounds were designed based on the amino acid residues within the key binding site of sigma factors, which are crucial for their interaction with RNA polymerase. By inhibiting bacterial transcription, these compounds exhibited notable antimicrobial activity, and we coined them as sigmacidins to highlight their resemblance to sigma factors and the benzoic acid structure. In this study, we further modified the compound scaffolds and developed a series of sulfonamidyl benzoic acid derivatives. These derivatives displayed potent antimicrobial activity, with minimum inhibitory concentrations (MICs) as low as 1 µg/mL, demonstrating their efficacy against bacteria. Furthermore, these compounds demonstrated low cytotoxicity, indicating their potential as safe antimicrobial agents. To ascertain their mechanism of action in interfering with bacterial transcription, we conducted biochemical and cellular assays. Overall, this study showcases the effectiveness of sulfonamidyl benzoic acid derivatives as antimicrobial agents by targeting protein-protein interactions involving RNA polymerase and sigma factors. Their strong antimicrobial activity and low cytotoxicity implicate their potential in combating antibiotic-resistant bacteria.