Sulfonamidyl derivatives of sigmacidin: Protein-protein interaction inhibitors targeting bacterial RNA polymerase and sigma factor interaction exhibiting antimicrobial activity against antibiotic-resistant bacteria.

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.

Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.

The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

Synthesis and biological evaluation of nusbiarylin derivatives as bacterial rRNA synthesis inhibitor with potent antimicrobial activity against MRSA and VRSA.

Bacterial transcription is a valid but underutilized target for antimicrobial agent discovery because of its function of bacterial RNA synthesis. Bacterial transcription factors NusB and NusE form a transcription complex with RNA polymerase for bacterial ribosomal RNA synthesis. We previously identified a series of diarylimine and -amine inhibitors capable of inhibiting the interaction between NusB and NusE and exhibiting good antimicrobial activity. To further explore the structural viability of these inhibitors, coined "nusbiarylins", 36 new derivatives containing diverse substituents at the left benzene ring of inhibitors were synthesized based upon isosteric replacement and the structure-activity relationship concluded from earlier studies. Some of the derivatives displayed good to excellent antibacterial efficacy towards a panel of clinically significant pathogens including methicillin-resistance Staphylococcus aureus (MRSA) and vancomycin-resistance S. aureus (VRSA). In particular, compound 22r exhibited the best antimicrobial activity with a minimum inhibitory concentration (MIC) of 0.5 µg/mL. Diverse mechanistic studies validated the capability of 22r inhibiting the function of NusB protein and bacterial rRNA synthesis. In silico study of drug-like properties also provided promising results. Overall, this series of derivatives showed potential antimicrobial activity and drug-likeness and provided guidance for further optimization.

Chimeric crRNA improves CRISPR-Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2.

Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.

Nusbiarylins, a new class of antimicrobial agents: Rational design of bacterial transcription inhibitors targeting the interaction between the NusB and NusE proteins.

Discovery of antibiotics of a novel mode of action is highly required in the fierce battlefield with multi-drug resistant bacterial infections. Previously we have validated the protein-protein interaction between bacterial NusB and NusE proteins as an unprecedented antimicrobial target and reported the identification of a first-in-class inhibitor of bacterial ribosomal RNA synthesis with antimicrobial activities. In this paper, derivatives of the hit compound were rationally designed based on the pharmacophore model for chemical synthesis, followed by biological evaluations. Some of the derivatives demonstrated the improved antimicrobial activity with the minimum inhibitory concentration (MIC) at 1-2 µg/mL against clinically significant bacterial pathogens. Time-kill kinetics, confocal microscope, ATP production, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells of a representative compound were also measured. This series of compounds were named "nusbiarylins" based on their target protein NusB and the biaryl structure and were expected to be further developed towards novel antimicrobial drug candidates in the near future.

Design, synthesis and biological evaluation of antimicrobial diarylimine and -amine compounds targeting the interaction between the bacterial NusB and NusE proteins.

Discovery of antimicrobial agents with a novel model of action is in urgent need for the clinical management of multidrug-resistant bacterial infections. Recently, we reported the identification of a first-in-class bacterial ribosomal RNA synthesis inhibitor, which interrupted the interaction between the bacterial transcription factor NusB and NusE. In this study, a series of diaryl derivatives were rationally designed and synthesized based on the previously established pharmacophore model. Inhibitory activity against the NusB-NusE binding, circular dichroism of compound treated NusB, antimicrobial activity, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells were measured. Structure-activity relationship and quantitative structure-activity relationship were also concluded and discussed. Some of the derivatives demonstrated improved antimicrobial activity than the hit compound against a panel of clinically important pathogens, lowering the minimum inhibition concentration to 1-2 µg/mL against Staphylococcus aureus, including clinical strains of methicillin-resistant Staphylococcus aureus at a level comparable to some of the marketed antibiotics. Given the improved antimicrobial activity, specific inhibition of target protein-protein interaction and promising pharmacokinetic properties without significant cytotoxicity, this series of diaryl compounds have high potentials and deserve for further studies towards a new class of antimicrobial agents in the future.

Simple Method for Studying in Vitro Protein-Protein Interactions Based on Protein Complementation and Its Application in Drug Screening Targeting Bacterial Transcription.

Protein-protein interactions (PPIs) underpin essential cellular processes of all organisms and are increasingly considered as drug targets. A number of techniques have been established to study PPIs; however, development of a simple and cost-effective method for in vitro high throughput screening of PPI inhibitors is still in demand or desirable. We report herein a simple method based on protein complementation for the in vitro study of PPIs, as well as screening of inhibitors against the PPI of interest. We have validated this system utilizing bacterial transcription factors NusB and NusE. Three derivatives of an inhibitor targeting the NusB-NusE interaction were synthesized and characterized with the system, which showed specific inhibition and antimicrobial activities. We have further confirmed the system with the RNA polymerase-σ interaction and an inhibitor. This system is expected to be suitable for more extensive high throughput screening of large chemical libraries. Additionally, our vector system can be easily adapted to study other PPI pairs, followed by inhibitor screening for hit identification in the application of early stage drug discovery.