New N-phenylpyrrolamide inhibitors of DNA gyrase with improved antibacterial activity.

This study presents the discovery of a new series of N-phenylpyrrolamide inhibitors of bacterial DNA gyrase with improved antibacterial activity. The most potent inhibitors had low nanomolar IC50 values against Escherichia coli DNA gyrase (IC50; 2-20 nM) and E. coli topoisomerase IV (22i, IC50 = 143 nM). Importantly, none of the compounds showed activity against human DNA topoisomerase IIα, indicating selectivity for bacterial targets. Among the tested compounds, 22e emerged as the most effective against Gram-positive bacteria with minimum inhibitory concentration (MIC) values of 0.25 µg mL-1 against Staphylococcus aureus ATCC 29213 and MRSA, and 0.125 µg mL-1 against Enterococcus faecalis ATCC 29212. For Gram-negative bacteria, compounds 23b and 23c showed the greatest efficacy with MIC values ranging from 4 to 32 µg mL-1 against E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 17978 and A. baumannii ATCC 19606. Notably, compound 23b showed promising activity against the clinically relevant Gram-negative pathogen Klebsiella pneumoniae ATCC 10031, with an MIC of 0.0625 µg mL-1. Furthermore, compounds 23a and 23c exhibited significantly lower susceptibility to resistance development compared to novobiocin in S. aureus ATCC 29213 and K. pneumoniae ATCC 10031. Overall, the most promising compounds of this series showed excellent on-target potency, marking a significant improvement over previous N-phenylpyrrolamide inhibitors.

Development of narrow-spectrum topoisomerase-targeting antibacterials against mycobacteria.

New 2-pyrrolamidobenzothiazole-based inhibitors of mycobacterial DNA gyrase were discovered. Among these, compounds 49 and 51, show excellent antibacterial activity against Mycobacterium tuberculosis and Mycobacterium abscessus with a notable preference for mycobacteria. Both compounds can penetrate infected macrophages and reduce intracellular M. tuberculosis load. Compound 51 is a potent inhibitor of DNA gyrase (M. tuberculosis DNA gyrase IC50 = 4.1 nM, Escherichia coli DNA gyrase IC50 of <10 nM), selective for bacterial topoisomerases. It displays low MIC90 values (M. tuberculosis: 0.63 µM; M. abscessus: 2.5 µM), showing specificity for mycobacteria, and no apparent toxicity. Compound 49 not only displays potent antimycobacterial activity (MIC90 values of 2.5 µM for M. tuberculosis and 0.63 µM for M. abscessus) and selectivity for mycobacteria but also exhibits favorable solubility (kinetic solubility = 55 µM) and plasma protein binding (with a fraction unbound of 2.9 % for human and 4.7 % for mouse). These findings underscore the potential of fine-tuning molecular properties to develop DNA gyrase B inhibitors that specifically target the mycobacterial chemical space, mitigating the risk of resistance development in non-target pathogens and minimizing harm to the microbiome.

Identification of a Novel Structural Class of HV1 Inhibitors by Structure-Based Virtual Screening.

The human voltage-gated proton channel, hHV1, is highly expressed in various cell types including macrophages, B lymphocytes, microglia, sperm cells and also in various cancer cells. Overexpression of HV1 has been shown to promote tumor formation by highly metastatic cancer cells, and has been associated with neuroinflammatory diseases, immune response disorders and infertility, suggesting a potential use of hHV1 inhibitors in numerous therapeutic areas. To identify compounds targeting this channel, we performed a structure-based virtual screening on an open structure of the human HV1 channel. Twenty selected virtual screening hits were tested on Chinese hamster ovary (CHO) cells transiently expressing hHV1, with compound 13 showing strong block of the proton current with an IC50 value of 8.5 µM. Biological evaluation of twenty-three additional analogs of 13 led to the discovery of six other compounds that blocked the proton current by more than 50% at 50 µM concentration. This allowed for an investigation of structure-activity relationships. The antiproliferative activity of the selected promising hHV1 inhibitors was investigated in the cell lines MDA-MB-231 and THP-1, where compound 13 inhibited growth with an IC50 value of 9.0 and 8.1 µM, respectively. The identification of a new structural class of HV1 inhibitors contributes to our understanding of the structural requirements for inhibition of this ion channel and opens up the possibility of investigating the role of HV1 inhibitors in various pathological conditions and in cancer therapy.

A synthetic flavonoid derivate in the plasma membrane transforms the voltage-clamp fluorometry signal of CiHv1.

Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.