DNA Supercoiling Assay Research Articles

RNA interacts with topoisomerase I to adjust DNA topology

Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and invitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA invitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.

Topoisomerase-modulated genome-wide DNA supercoiling domains colocalize with nuclear compartments and regulate human gene expression.

DNA supercoiling is a biophysical feature of the double helix with a pivotal role in biological processes. However, understanding of DNA supercoiling in the chromatin remains limited. Here, we developed azide-trimethylpsoralen sequencing (ATMP-seq), a DNA supercoiling assay offering quantitative accuracy while minimizing genomic bias and background noise. Using ATMP-seq, we directly visualized transcription-dependent negative and positive twin-supercoiled domains around genes and mapped kilobase-resolution DNA supercoiling throughout the human genome. Remarkably, we discovered megabase-scale supercoiling domains (SDs) across all chromosomes that are modulated mainly by topoisomerases I and IIβ. Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to SD formation, indicating the long-range propagation of transcription-generated supercoiling. Genome-wide SDs colocalize with A/B compartments in both human and Drosophila cells but are distinct from topologically associating domains (TADs), with negative supercoiling accumulation at TAD boundaries. Furthermore, genome-wide DNA supercoiling varies between cell states and types and regulates human gene expression, underscoring the importance of supercoiling dynamics in chromatin regulation and function.

Synthesis and Biological Evaluation of Diphenyl Ether‐Linked Quinolone Derivatives as Antibacterial Agents

AbstractThe increase in the prevalence of multi‐drug‐resistant bacteria poses a significant healthcare challenge. The urgent need to combat the resistant microbes necessitates discovering new antibacterial agents capable of circumventing the existing resistance mechanisms. Targeting DNA gyrase by suitably modifying the fluoroquinolones can lead to antibiotics with better activity and lower incidence of resistance. The substituted diphenyl ethers like triclosan are known to have potent antibacterial activity. In the current study, the hybridisation of diphenyl ether moiety with fluoroquinolones led to the design and synthesis of new compounds with potent inhibitory activity against staphylococcus aureus with MIC 0.5–64 μg/mL and moderately active against mycobacteria with MIC 2–64 μg/mL. The compounds are non‐toxic to Vero cells with a selectivity index >10 to 200. The compounds also inhibited the resistant strains of S. aureus with a MIC ranging from 0.5–64 μg/mL. The synthesised compounds also exhibited potent anti‐biofilm activity against S. aureus. Furthermore, the DNA supercoiling assay revealed the compounds 7 i, 7 o, 7 p and 7 q showed concentration‐dependant DNA‐Gyrase inhibition at 1 μg/mL.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Synthesis, antimycobacterial screening, molecular docking, ADMET prediction and pharmacological evaluation on novel pyran-4-one bearing hydrazone, triazole and isoxazole moieties: Potential inhibitors of SARS CoV-2

The respiratory infection tuberculosis is caused by the bacteria Mycobacterium tuberculosis and its unrelenting spread caused millions of deaths around the world. Hence, it is needed to explore potential and less toxic anti-tubercular drugs. In the present work, we report the synthesis and antitubercular activity of four different (hydrazones 7–12, O-ethynyl oximes 19–24, triazoles 25–30, and isoxazoles 31–36) hybrids. Among these hybrids 9, 10, 33, and 34, displayed high antitubercular activity at 3.12 g/mL with >90% of inhibitions. The hybrids also showed good docking energies between -6.8 and -7.8 kcal/mol. Further, most active molecules were assayed for their DNA gyrase reduction ability towards M. tuberculosis and E.coli DNA gyrase by the DNA supercoiling and ATPase gyrase assay methods. All four hybrids showed good IC50 values comparable to that of the reference drug. In addition, the targets were also predicted as a potential binder for papain-like protease (SARS CoV-2 PLpro) by molecular docking and a good interaction result was observed. Besides, all targets were predicted for their absorption, distribution, metabolism, and excretion - toxicity (ADMET) profile and found a significant amount of ADMET and bioavailability

CID12261165, a flavonoid compound as antibacterial agents against quinolone-resistant Staphylococcus aureus

Flavonoids are plant-produced secondary metabolites that are found ubiquitously. We have previously reported that apigenin, a class of flavonoid, has unique antimicrobial activity against Staphylococcus aureus (S. aureus), one of the major human pathogens. Apigenin inhibited fluoroquinolone-resistant S. aureus with DNA gyrase harboring the quinolone-resistant S84L mutation but did not inhibit wild-type DNA gyrase. In this study, we describe five flavonoids, quercetin, luteolin, kaempferol, baicalein, and commercially available CID12261165, that show similar antimicrobial activity against fluoroquinolone-resistant S. aureus. Among them, CID12261165 was the most effective with MIC values of ≤ 4 mg/L against quinolone-resistant S. aureus strains. In vitro DNA cleavage and supercoiling assays demonstrated inhibitory activity of CID12261165 against mutated DNA gyrase, whereas activity against wild-type DNA gyrase was not observed. CID12261165 also inhibited quinolone-resistant Enterococci with an MIC value of 8 mg/L. While fluoroquinolone-resistant amino acid replacements can improve the fitness of bacterial cells, it is unknown why quinolone-susceptible S. aureus strains were predominant before the introduction of fluoroquinolone. The present study discusses the current discrepancies in the interpretation of antimicrobial activities of flavonoids, as well as the possible reasons for the preservation of wild-type DNA gyrase wherein the environmental flavonoids cannot be ignored.

Novel fluoroquinolones containing 2‐arylamino‐2‐oxoethyl fragment: Design, synthesis, evaluation of antibacterial and antituberculosis activities and molecular modeling studies

AbstractNovel substituted fluoroquinolone derivatives, compounds 6–20 were designed, synthesized, and evaluated for antituberculosis and antibacterial activity. Antibacterial activities of the compounds were determined and compound 14 was found to be the most potent antimicrobial agent owing to minimal inhibitory concentration (MIC) value of <1.16 μg/μl for all tested bacteria. Further, compounds were tested in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Most of the compounds showed antimycobacterial effects with 1.56–25.00 μg/ml MIC values. Compounds 14 and 18 were found to be the most active derivatives due to their MIC at 1.56 μg/ml. Selected compounds 11, 14, 17, and 18 were tested for M. tuberculosis DNA supercoiling assay and they had IC50 values within a range of 6.35–15 μM. Mechanism of binding to DNA gyrase enzymes was evaluated using in silico molecular modeling studies and it was shown that compounds 6–20 adopt a similar binding mode as already known for fluoroquinolone drugs.

Effectiveness of Fluoroquinolones with Difluoropyridine Derivatives as R1 Groups on the Salmonella DNA Gyrase in the Presence and Absence of Plasmid-Encoded Quinolone Resistance Protein QnrB19.

Aims: WQ-3810 has strong inhibitory activity against Salmonella and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to 6-amino-3,5-difluoropyridine-2-yl at R1 group. The aim of this study was to examine WQ-3810 and its derivatives WQ-3334 and WQ-4065 as the new drug candidate for wild-type Salmonella and that carrying QnrB19. Materials and Methods: The half maximal inhibitory concentrations (IC50s) of WQ-3810, WQ-3334 (Br atom in place of methyl group at R8), and WQ-4065 (6-ethylamino-3,5-difluoropyridine-2-yl in place of 6-amino-3,5-difluoropyridine-2-yl group at R1) in the presence or absence of QnrB19 were assessed by in vitro DNA supercoiling assay utilizing recombinant DNA gyrase and QnrB19. Results: IC50s of WQ-3810, WQ-3334, and WQ-4065 against Salmonella DNA gyrase were 0.031 ± 0.003, 0.068 ± 0.016, and 0.72 ± 0.39 μg/mL, respectively, while QnrB19 increased IC50s of WQ-3810, WQ-3334, and WQ-4065 to 0.44 ± 0.05, 0.92 ± 0.34, and 9.16 ± 2.21 μg/mL, respectively. Conclusion: WQ-3810 and WQ-3334 showed stronger inhibitory activity against Salmonella Typhimurium DNA gyrases than WQ-4065 even in the presence of QnrB19. The results suggest that 6-amino-3,5-difluoropyridine-2-yl group at R1 is playing an important role and WQ-3810 and WQ-3334 to be good candidates for Salmonella carrying QnrB19.

Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

BackgroundElaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies.ResultsWe describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications.ConclusionsThe method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.

Structure-based drug repurposing to inhibit the DNA gyrase of Mycobacterium tuberculosis.

Drug repurposing is an alternative avenue for identifying new drugs to treat tuberculosis (TB). Despite the broad-range of anti-tubercular drugs, the emergence of multi-drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb) H37Rv, as well as the significant death toll globally, necessitates the development of new and effective drugs to treat TB. In this study, we have employed a drug repurposing approach to address this drug resistance problem by screening the drugbank database to identify novel inhibitors of the Mtb target enzyme, DNA gyrase. The compounds were screened against the ATPase domain of the gyrase B subunit (MtbGyrB47), and the docking results showed that echinacoside, doxorubicin, epirubicin, and idarubicin possess high binding affinities against MtbGyrB47. Comprehensive assessment using fluorescence spectroscopy, surface plasmon resonance spectroscopy (SPR), and circular dichroism (CD) titration studies revealed echinacoside as a potent binder of MtbGyrB47. Furthermore, ATPase, and DNA supercoiling assays exhibited an IC50 values of 2.1-4.7 µM for echinacoside, doxorubicin, epirubicin, and idarubicin. Among these compounds, the least MIC90 of 6.3 and 12 μM were observed for epirubicin and echinacoside, respectively, against Mtb. Our findings indicate that echinacoside and epirubicin targets mycobacterial DNA gyrase, inhibit its catalytic cycle, and retard mycobacterium growth. Further, these compounds exhibit potential scaffolds for optimizing novel anti-mycobacterial agents that can act on drug-resistant strains.

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

ObjectivesTo evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches.MethodsRedx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863.ResultsBoth compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive.ConclusionsRedx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion.

Optical tweezers are flexible and powerful single-molecule tools that have been extensively utilized in biophysical studies. With their ability to stretch and twist DNA, and measure its force and torque simultaneously, they provide excellent opportunities to gain novel insights into the function of protein motors and protein-DNA interactions. Recently, a novel DNA supercoiling assay using an angular optical tweezers (AOT) has been developed to investigate torque generation during transcription. Here, we provide a detailed and practical guide to performing this technique. Using bacterial RNA polymerase (RNAP) as an example, we present protocols for constructing and calibrating an AOT instrument, preparing DNA templates, and acquiring and analyzing real-time data for transcription under DNA supercoiling. While these protocols were initially developed with E. coli RNAP, they can be readily adapted to study other DNA-based motor proteins.

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5'-TA-3' is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5' end to a 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae.

BackgroundFluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined.Methodology/Principal FindingsTo gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8- and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone–mediated cleavage assay showed that potencies of those drugs were at most 13.5- and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae.Conclusions/SignificanceThe results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans.

Benzothiazinone-piperazine derivatives as efficient Mycobacterium tuberculosis DNA gyrase inhibitors

Background and objectivesBacterial DNA topoisomerases are unique in maintaining the DNA topology for cell viability. Mycobacterium tuberculosis (MTB) DNA gyrase, a sole type II topoisomerase has a larger scope as a target for developing novel therapeutics. In this study, an effort was made towards the design and synthesis of benzothiazinone-piperazine hybrid analogues to obtain the possibility of it to lead development through the molecular hybridization technique. MethodsA five-step scheme was followed to obtain a series of 36 benzothiazinone-piperazine derivatives and to evaluate them for MTB DNA gyrase inhibition, antimycobacterial and cytotoxicity studies. ResultsCompound N-(4-chlorophenyl)-4-(6-nitro-4-oxo-4H-benzo[e][1,3]thiazin-2-yl)piperazine-1-carbothioamide (18) showed greater inhibitory potential with an IC50 of 0.51±0.16μM in the DNA supercoiling assay of MTB with a moderate anti-tubercular activity of 4.41μM. The compound even passed the safety profile of eukaryotic cell cytotoxicity with a 1.81% inhibition in the RAW 264.7 cell line at 100μM concentration. ConclusionsThis study describes the discovery of benzothiazinone as gyrase inhibitors with potent MTB MIC and inhibitory profiles of the gyrase enzyme with less cytotoxic effect. Furthermore, it is believed that this class of compounds has the potential to be further developed as an anti-TB drug candidate.

Novel lead generation of an anti-tuberculosis agent active against non-replicating mycobacteria: exploring hybridization of pyrazinamide with multiple fragments

The key to shortening tuberculosis (TB) drug regimen lies in eliminating the reservoir of non-replicating persistent (NRP) Mycobacterium tuberculosis (Mtb). Pyrazinamide (PZA) is the only known drug used as part of a combination therapy that is believed to kill NRP Mtb and achieve sterilization. PZA is active only under low pH screening conditions. Screening and identification of NRP-active anti-TB compounds are severely limited because compounds are usually inactive under regular assay conditions. In an effort to design novel NRP-active anti-TB compounds, we used pyrazinamide as a core and hybridized it with the fragments derived from marketed drugs. One of these designs, compound 8, was a hybrid with fluoroquinolone. This compound exhibited >10 fold improvement in NRP activity under low pH condition as compared to pyrazinamide and a modest activity (0.8 log10 kill) under nutritionally starved NRP condition. Furthermore, compound 8 was active against fluoroquinolone-resistant strains and did not show any activity in a DNA supercoiling assay (gyrase inhibition), suggesting that its mechanism of action is not that of the parent fluoroquinolone. These results provide a novel avenue in the exploration of new chemotypes that are active against non-replicating Mtb.

Action mechanism of 6, 6′-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities

BackgroundMultidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. MethodsAn antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. ResultsWe isolated and identified 6,6′-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1–4μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10–15μM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6′-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. ConclusionsDTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. General significanceDTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.

Design, synthesis, and evaluation of novel fluoroquinolone–flavonoid hybrids as potent antibiotics against drug-resistant microorganisms

Based on a rationally conceived pharmacophore model to build a multi-target bacterial topoisomerase inhibitor, twenty-one fluoroquinolone–flavonoid hybrids were synthesized. Some obtained hybrids show excellent antibacterial activity against drug-resistant microorganisms with narigenin–ciprofloxacin being the most active, showing 8, 43, 23 and 88 times better activity than ciprofloxacin against Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 90873, respectively. Drug accumulation and DNA supercoiling assays of two active analogues revealed potent inhibition of both the DNA gyrase and efflux pump, confirming the desired dual mode of action. Molecular docking study disclosed that the introduced flavonoid moiety not only provides several additional interactions but also does not disturb the binding mode of the floxacin moiety. Our data also demonstrated that development of antifungals is possible from fluoroquinolones modified at C-7 position.

<i>In-Vitro</i> Inhibitory Effect of Methanol Extracts of Chinese Herbal Drugs on Supercoiling Activity of Bacterial DNA Gyrase

A large number of Chinese herbal drugs (CHDs) exhibit antibacterial activities both in vivo and in vitro, but until now little is known regarding their inhibitory mechanisms. Bacterial DNA gyrase is a proven target for antibacterial agents. Aim of this study was to investigate the in-vitro inhibitory effect of methanol extracts of CHDs against supercoiling activity of bacterial DNA gyrase. Fifteen CHDs were selected and extracted with methanol, respectively. Inhibitory effect of the extracts on DNA gyrase was tested using gel-based DNA supercoiling assay. Among fifteen CHDs tested, methanol extracts of Lonicerae Japonicae Flos (S2), Taraxaci Herba (S7), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (S8) demonstrated an obvious inhibitory effect against supercoiling activity of DNA gyrase, and the others were either less active or could not be determined with the present method. Moreover, it was likely that S7 and S8 inhibit gyrase in a concentration-dependent manner. In conclusion, DNA supercoiling assay is a promising method to study the inhibitory activity of CHDs on bacterial DNA gyrase. Some CHDs do have gyrase-inhibitory activity as proposed. Further investigations are needed to elucidate the inhibition mechanism of these CHDs on supercoiling activity of gyrase.