Gyrase and topoisomerase IV are the cellular targets for fluoroquinolones, a critically important class of antibacterial agents used to treat a broad spectrum of human infections. Unfortunately, the clinical efficacy of the fluoroquinolones has been curtailed by the emergence of target-mediated resistance. This is especially true for Neisseria gonorrhoeae, the causative pathogen of the sexually transmitted infection gonorrhea. Spiropyrimidinetriones (SPTs), a new class of antibacterials, were developed to combat the growing antibacterial resistance crisis. Zoliflodacin is the most clinically advanced SPT and displays efficacy against uncomplicated urogenital gonorrhea in human trials. Like fluoroquinolones, the primary target of zoliflodacin in N. gonorrhoeae is gyrase, and topoisomerase IV is a secondary target. Because unbalanced gyrase/topoisomerase IV targeting has facilitated the evolution of fluoroquinolone-resistant bacteria, it is important to understand the underlying basis for the differential targeting of zoliflodacin in N. gonorrhoeae. Therefore, we assessed the effects of this SPT on the catalytic and DNA cleavage activities of N. gonorrhoeae gyrase and topoisomerase IV. In all reactions examined, zoliflodacin displayed higher potency against gyrase than topoisomerase IV. Moreover, zoliflodacin generated more DNA cleavage and formed more stable enzyme-cleaved DNA-SPT complexes with gyrase. The SPT also maintained higher activity against fluoroquinolone-resistant gyrase than topoisomerase IV. Finally, when compared to zoliflodacin, the novel SPT H3D-005722 induced more balanced double-stranded DNA cleavage with gyrase and topoisomerase IV from N. gonorrhoeae, Escherichia coli, and Bacillus anthracis. This finding suggests that further development of the SPT class could yield compounds with a more balanced targeting against clinically important bacterial infections.

Bacterial DNA gyrase and topoisomerase IV inhibition has emerged as a promising strategy for the cure of infections caused by antibiotic-resistant bacteria. The Novel Bacterial Topoisomerase Inhibitors (NBTIs) bind to a different site from that of the quinolones with novel mechanism of action. This evades the existing target-mediated bacterial resistance associated with quinolones. This article presents our efforts to identify in vitro potent and broad-spectrum antibacterial agent 4l.

We have conducted an experimental and computational evaluation of new doxorubicin (4a-c) and β-lapachone (5a-c) analogs. These novel anticancer analogs were previously synthesized, but had not been tested or characterized until now. We have evaluated their antiproliferative and DNA cleavage inhibition properties using breast (MCF-7 and MDA-MB-231) and prostate (PC3) cancer cell lines. Additionally, cell cycle analysis was performed using flow cytometry. Computational studies, including molecular docking, pharmacokinetic properties, and an analysis of DFT and QTAIM chemical descriptors, were performed to gain insights into the electronic structure and elucidate the molecular binding of the new β-lapachone and doxorubicin analogs with a DNA sequence and Topoisomerase II (Topo II)α. Our results show that 4a analog displays the highest antiproliferative activity in cancer cell lines by inducing cell death. We observed that stacking interactions and hydrogen bonding are essential to stabilize the molecule-DNA-Topo IIα complex. Moreover, 4a and 5a analogs inhibited Topo\'s DNA cleavage activity. Pharmacodynamic results indicated that studied molecules have favorable adsorption and permeability properties. The calculated chemical descriptors indicate that electron accumulation in quinone rings is relevant to the reactivity and biological activity. Based on our results, 4a is a strong candidate for becoming an anticancer drug.

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.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 μM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 μg/mL, respectively, superior to novobiocin (MIC > 100.0 μg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.

DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.

N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.

UNASSIGNED: Neisseria gonorrhoeae is a common sexually transmitted disease connected with extensive drug resistance to many antibiotics. Presently, only expanded spectrum cephalosporins (ceftriaxone and cefixime) and azithromycin remain useful for its management.
UNASSIGNED: New chemotypes for the classical antibiotic drug target gyrase/topoisomerase IV afforded inhibitors with potent binding to these enzymes, with an inhibition mechanism distinct from that of fluoroquinolones, and thus less prone to mutations. The α-carbonic anhydrase from the genome of this bacterium (NgCAα) was also validated as an antibacterial target.
UNASSIGNED: By exploiting different subunits from the gyrase/topoisomerase IV as well as new chemotypes, two new antibiotics reached Phase II/III clinical trials, zoliflodacin and gepotidacin. They possess a novel inhibition mechanism, binding in distinct parts of the enzyme compared to the fluoroquinolones. Other chemotypes with inhibitory activity in these enzymes were also reported. NgCAα inhibitors belonging to a variety of classes were obtained, with several sulfonamides showing MIC values in the range of 0.25-4 µg/mL and significant activity in animal models of this infection. Acetazolamide and similar CA inhibitors might thus be repurposed as antiinfectives. The scientific/patent literature has been searched for on PubMed, ScienceDirect, Espacenet, and PatentGuru, from 2016 to 2024.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests - parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.