A novel high-throughput screening method for identifying compounds that inhibit plasmid conjugation.

Plasmid conjugation is an important contributing factor to the spread of antibiotic resistance among bacteria, posing a significant global health threat. Our method introduces an innovative high-throughput screening approach to identify compounds that inhibit or reduce conjugation, addressing the need for new strategies against the spread of antimicrobial resistance. Using Escherichia coli strains as donor and recipient, we screened 3500 compounds from a library provided by ABAC Therapeutics. Each 96 -well plate was loaded with 88 different compounds and bacterial cultures. Every plate also included negative and positive controls of conjugation. After an hour, cultures from wells were spotted on agar plates and assessed visually. Compounds that showed a visible effect on conjugation were retested. Six compounds targeting conjugation were found, showing promise for further analysis.

Click chemistry-aided drug discovery: A retrospective and prospective outlook.

Click chemistry has emerged as a valuable tool for rapid compound synthesis, presenting notable advantages and convenience in the exploration of potential drug candidates. In particular, in situ click chemistry capitalizes on enzymes as reaction templates, leveraging their favorable conformation to selectively link individual building blocks and generate novel hits. This review comprehensively outlines and introduces the extensive use of click chemistry in compound library construction, and hit and lead discovery, supported by specific research examples. Additionally, it discusses the limitations and precautions associated with the application of click chemistry in drug discovery. Our intention for this review is to contribute to the development of a modular synthetic approach for the rapid identification of drug candidates.

Rapid Construction of Recombinant PDCoV Expressing an Enhanced Green Fluorescent Protein for the Antiviral Screening Assay Based on Transformation-Associated Recombination Cloning in Yeast.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that mainly causes diarrhea and death in suckling piglets and also has the potential for cross-species transmission, threatening public health. However, there is still no effective vaccine or drug to prevent PDCoV infection. In order to accelerate the development of antiviral drugs, we established a high-throughput screening platform using a novel genome editing technology called transformation-associated recombination cloning in yeast. The recombinant PDCoV and PDCoV reporter virus expressing enhanced green fluorescent protein were both rapidly rescued with stable genealogical characteristics during passage. Further study demonstrated that the reporter virus can be used for high-throughput screening of antiviral drugs with a Z-factor of 0.821-0.826. Then, a medicine food homology compound library was applied, and we found that three compounds were potential antiviral reagents. In summary, we have established a fast and efficient reverse genetic system of PDCoV, providing a powerful platform for the research of antiviral drugs.

In silico and structural investigation of sulfonamides targeting VraSR two component system in methicillin-resistant Staphylococcus aureus.

Drug-resistant Staphylococcus aureus strains are global health concerns. Several studies have shown that these strains can develop defences against cell wall antibiotics such as ß-lactams, glycopeptides and daptomycin which target cell wall biosynthesis. The coordination of these responses have been associated with two component system (TCS) regulated by histidine kinase protein (VraS) and its cognate regulator VraR which influences the target DNA upon signal recognition. Computer-based screening methods, predictions and simulations have emerged as more efficient and quick ways to identify promising new compound leads from large databases against emerging drug targets thus allowing prediction of small select set of molecules for further validations. These combined approaches conserve valuable time and resources. Due to methicillin resistance, sulfonamide-derivative medications have been found to be effective treatment strategy to treat S. aureus infections. The current study used ligand-based virtual screening (LBVS) to identify powerful sulfonamide derivative inhibitors from an antibacterial compound library against VraSR signaling components, VraS and VraR. We identified promising sulfonamide derivative [compound 5: (4-[(1-{[(3,5-Dimethoxyphenyl)Carbamoyl]Methyl}-2,4-Dioxo-1,2,3,4-Tetrahydroquinazolin-3-Yl)Methyl]-N-[(Furan-2-Yl)Methyl]Benzamide)] with reasonable binding parameters of -31.38 kJ/mol and ΔGbind score of -294.32 kJ/mol against ATP binding domain of sensor kinase VraS. We further identified four compounds N1 (PCID83276726), N3 (PCID83276757), N9 (PCID3672584), and N10 (PCID20900589) against VraR DNA binding domain (VraRC) with ΔGbind energies of -190.27, -237.54, -165.21, and -190.88 kJ/mol, respectively. Structural and simulation analyses further suggest their stable interactions with DNA interacting residues and potential to disrupt DNA binding domain dimerization; therefore, it is prudent to further investigate and characterize them as VraR dimer disruptors and inhibit other promoter binding site. Interestingly, the discovery of drugs that target VraS and VraR may open new therapeutic avenues for drug-resistant S. aureus. These predictions based on screening, simulations and binding affinities against VraSR components hold promise for opening novel therapeutic avenues against drug-resistant S. aureus and present opportunities for repositioning efforts. These efforts aim to create analogs with enhanced potency and selectivity against two-component signaling systems that significantly contribute to virulence in MRSA or VRSA. These analyses contribute valuable insights into potential avenues for combating antibiotic-resistant S. aureus through computationally driven drug discovery strategies.Communicated by Ramaswamy H. Sarma.

Use of in vitro methods combined with in silico analysis to identify potential skin sensitizers in the Tox21 10K compound library.

Introduction: Skin sensitization, which leads to allergic contact dermatitis, is a key toxicological endpoint with high occupational and consumer prevalence. This study optimized several in vitro assays listed in OECD skin sensitization test guidelines for use on a quantitative high-throughput screening (qHTS) platform and performed in silico model predictions to assess the skin sensitization potential of prioritized compounds from the Tox21 10K compound library. Methods: First, we screened the entire Tox21 10K compound library using a qHTS KeratinoSensTM (KS) assay and built a quantitative structure-activity relationship (QSAR) model based on the KS results. From the qHTS KS screening results, we prioritized 288 compounds to cover a wide range of structural chemotypes and tested them in the solid phase extraction-tandem mass spectrometry (SPE-MS/MS) direct peptide reactivity assay (DPRA), IL-8 homogeneous time-resolved fluorescence (HTRF) assay, CD86 and CD54 surface expression in THP1 cells, and predicted in silico sensitization potential using the OECD QSAR Toolbox (v4.5). Results: Interpreting tiered qHTS datasets using a defined approach showed the effectiveness and efficiency of in vitro methods. We selected structural chemotypes to present this diverse chemical collection and to explore previously unidentified structural contributions to sensitization potential. Discussion: Here, we provide a skin sensitization dataset of unprecedented size, along with associated tools, and analysis designed to support chemical assessments.