Exploring the interaction of N-(benzothiazol-2-yl)pyrrolamide DNA gyrase inhibitors with the GyrB ATP-binding site lipophilic floor: A medicinal chemistry and QTAIM study.

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.

Rational design of balanced dual-targeting antibiotics with limited resistance.

Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins. ULD1 and ULD2 are excellently potent against a broad range of gram-positive bacteria. Notably, the efficacy of these compounds was tested against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. Antibiotics with clinical relevance against staphylococcal infections fail to inhibit a significant fraction of these isolates, whereas both ULD1 and ULD2 inhibit all of them (minimum inhibitory concentration [MIC] ≤1 µg/mL). Resistance mutations against these compounds are rare, have limited impact on compound susceptibility, and substantially reduce bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant bacterial infections.

A New Cell-Based AI-2-Mediated Quorum Sensing Interference Assay in Screening of LsrK-Targeted Inhibitors.

Quorum sensing (QS), a bacterial communication strategy, has been recognized as one of the control mechanisms of virulence in bacteria. Thus, targeting QS offers an interesting opportunity to impair bacterial pathogenicity and develop antivirulence agents. Aiming to enhance the discovery of QS inhibitors, we developed a bioreporter Escherichia coli JW5505 pET-Plsrlux and set up a cell-based assay for identifying inhibitors of autoinducer-2 (AI-2)-mediated QS. A comparative study on the performance of target- versus cell-based assays was performed, and 91 compounds selected with the potential to target the ATP binding pocket of LsrK, a key enzyme in AI-2 processing, were tested in an LsrK inhibition assay, providing 36 hits. The same set of compounds was tested by the AI-2-mediated QS interference assay, resulting in 24 active compounds. Among those, six were also found to be active against LsrK, whereas 18 might target other components of the pathway. Thus, this AI-2-mediated QS interference cell-based assay is an effective tool for complementing target-based assays, yet also stands as an independent assay for primary screening.

Anti-influenza virus activity of benzo[d]thiazoles that target heat shock protein 90.

Seasonal or pandemic influenza virus infections are a worldwide health problem requiring antiviral therapy. Since virus resistance to the established neuraminidase inhibitors and novel polymerase inhibitors is growing, new drug targets are needed. Heat shock protein 90 (Hsp90) is associated with several aspects of the influenza virus life cycle, and is considered a relevant host cell target. We report here on a series of benzo[d]thiazole and 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives with robust and selective activities against influenza A (H1N1, H3N2) and influenza B viruses. Two compounds, 1 and 4, have low micromolar EC50 values and show high binding affinities for Hsp90, which suggests that inhibition of Hsp90 is the mechanism underlying their antiviral effects. These compounds represent suitable scaffolds for designing novel Hsp90 inhibitors with favourable activities against influenza virus.

Discovery of new ATP-competitive inhibitors of human DNA topoisomerase IIα through screening of bacterial topoisomerase inhibitors.

Human DNA topoisomerase II is one of the major targets in anticancer therapy, however ATP-competitive inhibitors of this target have not yet reached their full potential. ATPase domain of human DNA topoisomerase II belongs to the GHKL ATPase superfamily and shares a very high 3D structural similarity with other superfamily members, including bacterial topoisomerases. In this work we report the discovery of a new chemotype of ATP-competitive inhibitors of human DNA topoisomerase IIα that were discovered through screening of in-house library of ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV. Systematic screening of this library provided us with 20 hit compounds. 1,2,4-Substituted N-phenylpyrrolamides were selected for a further exploration which resulted in 13 new analogues, including 52 with potent activity in relaxation assay (IC50 = 3.2 µM) and ATPase assay (IC50 = 0.43 µM). Cytotoxic activity of all hits was determined in MCF-7 cancer cell line and the most potent compounds, 16 and 20, showed an IC50 value of 8.7 and 8.2 µM, respectively.

Design, synthesis and biological evaluation of novel DNA gyrase inhibitors and their siderophore mimic conjugates.

Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor - siderophore mimic conjugates.

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria.

Bacterial infections are an increasingly serious issue worldwide. The inability of existing therapies to treat multidrug-resistant pathogens has been recognized as an important challenge of the 21st century. Efflux pumps are important in both intrinsic and acquired bacterial resistance and identification of small molecule efflux pump inhibitors (EPIs), capable of restoring the effectiveness of available antibiotics, is an active research field. In the last two decades, much effort has been made to identify novel EPIs. However, none of them has so far been approved for therapeutic use. In this article, we explore different structural families of currently known EPIs for multidrug resistance efflux systems in the most extensively studied pathogens (NorA in Staphylococcus aureus, AcrAB-TolC in Escherichia coli, and MexAB-OprM in Pseudomonas aeruginosa). Both synthetic and natural compounds are described, with structure-activity relationship studies and optimization processes presented systematically for each family individually. In vitro activities against selected test strains are presented in a unifying manner for all the EPIs described, together with the most important toxicity, pharmacokinetic and in vivo efficacy data. A critical evaluation of lead-likeness characteristics and the potential for clinical development of the most promising inhibitors of the three efflux systems is described. This overview of EPIs is a good starting point for the identification of novel effective antibacterial drugs.

Searching new structural scaffolds for BRAF inhibitors. An integrative study using theoretical and experimental techniques.

The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes. Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.

Synthesis and Evaluation of Spumigin Analogues Library with Thrombin Inhibitory Activity.

Spumigins are marine natural products derived from cyanobacteria Nodularia spumigena, which mimics the structure of the d-Phe-Pro-Arg sequence and is crucial for binding to the active site of serine proteases thrombin and factor Xa. Biological evaluation of spumigins showed that spumigins with a (2S,4S)-4-methylproline central core represent potential lead compounds for the development of a new structural type of direct thrombin inhibitors. Herein, we represent synthesis and thrombin inhibitory activity of a focused library of spumigins analogues with indoline ring or l-proline as a central core. Novel compounds show additional insight into the structure and biological effects of spumigins. The most active analogue was found to be a derivative containing l-proline central core with low micromolar thrombin inhibitory activity.

Design, synthesis, and biological evaluation of 1-ethyl-3-(thiazol-2-yl)urea derivatives as Escherichia coli DNA gyrase inhibitors.

Discovery of novel DNA gyrase B inhibitors remains an attractive field in the search for new antibacterial drugs to overcome the known bacterial resistance mechanisms. In the present study, we designed and synthesized novel ethylurea derivatives of 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine, 2-(2-aminothiazol-4-yl)acetic acid, and benzo[1,2-d]thiazole-2,6-diamine and evaluated their Escherichia coli DNA gyrase inhibition. The most potent DNA gyrase inhibitors in the prepared library of compounds were benzo[1,2-d]thiazoles 32-34, 36, and 37 with IC50 values in the low micromolar range. The most promising inhibitors identified were evaluated against selected Gram-positive and Gram-negative bacterial strains. Compound 33 showed a MIC of 50 µM against an E. coli efflux pump-defective strain, which suggests that efflux decreases the on-target concentrations of these compounds.

Design, synthesis and biological evaluation of 4,5-dibromo-N-(thiazol-2-yl)-1H-pyrrole-2-carboxamide derivatives as novel DNA gyrase inhibitors.

Development of novel DNA gyrase B inhibitors is an important field of antibacterial drug discovery whose aim is to introduce a more effective representative of this mechanistic class into the clinic. In the present study, two new series of Escherichia coli DNA gyrase inhibitors bearing the 4,5-dibromopyrrolamide moiety have been designed and synthesized. 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives inhibited E. coli DNA gyrase in the submicromolar to low micromolar range (IC50 values between 0.891 and 10.4µM). Their "ring-opened" analogues, based on the 2-(2-aminothiazol-4-yl)acetic acid scaffold, displayed weaker DNA gyrase inhibition with IC50 values between 15.9 and 169µM. Molecular docking experiments were conducted to study the binding modes of inhibitors.

Design, Synthesis, and Evaluation of Novel Tyrosine-Based DNA Gyrase B Inhibitors.

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC90 ) of 14 and 28 µg/mL, respectively.

Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin.

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC50 = 20 µM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.

Antimicrobial activity of the marine alkaloids, clathrodin and oroidin, and their synthetic analogues.

Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC90 (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound's potential as an antimicrobial lead, was found to be 2.9 for compound 6h.

Action of clathrodin and analogues on voltage-gated sodium channels.

Clathrodin is a marine alkaloid and believed to be a modulator of voltage-gated sodium (Na(V)) channels. Since there is an urgent need for small molecule Na(V) channel ligands as novel therapeutics, clathrodin could represent an interesting lead compound. Therefore, clathrodin was reinvestigated for its potency and Na(V) channel subtype selectivity. Clathrodin and its synthetic analogues were subjected to screening on a broad range of Na(V) channel isoforms, both in voltage clamp and patch clamp conditions. Even though clathrodin was not found to exert any activity, some analogues were capable of modulating the Na(V) channels, hereby validating the pyrrole-2-aminoimidazole alkaloid structure as a core structure for future small molecule-based Na(V) channel modulators.

Evaluation of Michael-type acceptor reactivity of 5-benzylidenebarbiturates, 5-benzylidenerhodanines, and related heterocycles using NMR.

Despite existing experimental and computational tools to assess the risk, the non-specific chemical modification of protein thiol groups remains a significant source of false-positive hits, particularly in academic drug discovery. Herein, we describe the application of a simple NMR method in a systematic study on the reactivity of 5-benzylidenebarbiturates, 5-benzylidenerhodanines, and their related oxo-heterocycles, which have been associated with numerous biological activities and have recently gained a reputation as unselective promiscuous binders. Using this method, we confirmed the reactivity of 5-benzylidenebarbiturates, which are known to easily form Michael adducts with nucleophiles. In contrast, 5-benzylidene five-membered oxo-heterocycles revealed almost insignificant reactivity. We can conclude that the distinct binding profile of the most controversial compounds, 5-benzylidenerhodanines, is not necessarily related to their unspecific Michael acceptor reactivity.

New ATP-competitive inhibitors of E. coli GyrB obtained from the mapping of the hydrophobic floor at the binding site: synthesis and biological evaluation.

We mapped the hydrophobic floor, an interesting subsite at the active site of DNA gyrase B (GyrB) from E. coli. We synthesized three new compounds with pendant groups targeting the hydrophobic floor and evaluated their inhibitory activities on DNA gyrase. A new benzothiazole derivative with a benzyl substituent at position 3 of the benzothiazole ring exhibited strong inhibitory activity against E. coli DNA gyrase (IC50 = 19 ± 3 nM). An exhaustive conformational study using potential energy surfaces (PESs) allowed us to map the new subsite evaluating all critical points on the surface and conformational interconversion pathways. We analyzed the molecular interactions using QTAIM calculations. Our data provide insights into the mechanism of action of these new ligands at the molecular level. Theoretical and experimental data suggest that new ligand optimization strategies should focus on strengthening interactions at the hydrophobic floor while preserving the binding mode of the main scaffold.

Estimation of passive gastrointestinal absorption of new dual DNA gyrase and topoisomerase IV inhibitors using PAMPA and biopartitioning micellar chromatography and quantitative structure-retention relationship analysis.

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.

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.

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.