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Each catalytic cycle of type IA topoisomerases has been proposed to comprise multistep reactions. The capture of the transport-segment DNA (T-segment) into the central cavity of the N-terminal toroidal structure is an important action, which is preceded by transient gate-segment (G-segment) cleavage and succeeded by G-segment religation for the relaxation of negatively supercoiled DNA and decatenation of DNA. The T-segment passage in and out of the central cavity requires significant domain–domain rearrangements, including the movement of D3 relative to D1 and D4 for the opening and closing of the gate towards the central cavity. Here we report a direct observation of the interaction of a duplex DNA in the central cavity of a type IA topoisomerase and its associated domain–domain conformational changes in a crystal structure of a Mycobacterium tuberculosis topoisomerase I complex that also has a bound G-segment. The duplex DNA within the central cavity illustrates the non-sequence-specific interplay between the T-segment DNA and the enzyme. The rich structural information revealed from the novel topoisomerase–DNA complex, in combination with targeted mutagenesis studies, provides new insights into the mechanism of the topoisomerase IA catalytic cycle.

Mycobacterial infectious diseases, including tuberculosis (TB), severely threaten global public health. Nonreplicating

Bacterial DNA gyrase, an essential enzyme, is a validated target for discovering and developing new antibiotics. Here we screened a pool of polyphenols and discovered that digallic acid is a potent DNA gyrase inhibitor. We also found that several food additives based on gallate, such as dodecyl gallate, potently inhibit bacterial DNA gyrase. Interestingly, the IC

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, β clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 A2 with

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER using a fluorescence-based assay suitable for HTS. A gel-based secondary assay confirmed the reduction of BER capacity by compounds identified in the primary screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase β (pol β) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 μM concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies.

Inhibitors targeting human topoisomerase I and topoisomerase II alpha have provided a useful chemotherapy option for the treatment of many patients suffering from a variety of cancers. While the treatment can be effective in many patient cases, use of these human topoisomerase inhibitors is limited by side-effects that can be severe. A strategy of employing the topoisomerase inhibitors in combination with other treatments can potentially sensitize the cancer to increase the therapeutic efficacy and reduce resistance or adverse side effects. The combination strategies reviewed here include inhibitors of DNA repair, epigenetic modifications, signaling modulators and immunotherapy. The ongoing investigations on cellular response to topoisomerase inhibitors and newly initiated clinical trials may lead to adoption of novel cancer therapy regimens that can effectively stop the proliferation of cancer cells while limiting the development of resistance.

Exonuclease VII excises trapped DNA gyrase and provides a new strategy to overcome resistance to quinolones., The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5′-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

A topoisomerase-DNA transient covalent complex can be a druggable target for novel topoisomerase poison inhibitors that represent a new class of antibacterial or anticancer drugs. Herein, we have investigated molecular features of the functionally important Escherichia coli topoisomerase I (EctopoI)-DNA covalent complex (EctopoIcc) for molecular simulations, which is very useful in the development of new antibacterial drugs. To demonstrate the usefulness of our approach, we used a model small molecule (SM), NSC76027, obtained from virtual screening. We examined the direct binding of NSC76027 to EctopoI as well as inhibition of EctopoI relaxation activity of this SM via experimental techniques. We then performed molecular dynamics (MD) simulations to investigate the dynamics and stability of EctopoIcc and EctopoI-NSC76027-DNA ternary complex. Our simulation results show that NSC76027 forms a stable ternary complex with EctopoIcc. EctopoI investigated here also serves as a model system for investigating a complex of topoisomerase and DNA in which DNA is covalently attached to the protein.

SUMMARYGenetic inactivation of TOP3B is linked with schizophrenia, autism, intellectual disability and cancer. The present study demonstrates that in vivo TOP3B forms both RNA and DNA cleavage complexes (TOP3Bccs) and reveals a pathway for repairing TOP3Bccs. For detecting cellular TOP3Bccs, we engineered a “self-trapping” mutant of TOP3B (R338W TOP3B) and to determine how human cells repair TOP3Bccs, we depleted tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells produced elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowered cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 cannot excise the native form of TOP3Bccs. Hypothesizing that TDP2 cannot access phosphotyrosyl linkage unless TOP3B is either proteolyzed or denatured, we found that cellular TOP3Bccs are ubiquitinated by the E3 Ubiquitin Ligase TRIM41 before undergoing proteasomal degradation and excision by TDP2.HIGHLIGHTSMethod for in vivo detection of TOP3B cleavage complexes (TOP3Bccs) formed both in DNA and RNA, using a religation defective “self-trapping” R338W TOP3B mutant.First evidence that TDP2 excises TOPccs produced by a type IA topoisomerase.TDP2 processes both RNA and DNA TOP3Bccs following their ubiquitylation and proteasomal degradation inside cell.TRIM41 is the first reported E3 ubiquitin ligase for TOP3Bcc ubiquitylation and proteasomal degradation.

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA. Single-molecule magnetic tweezers analyses and supporting MD simulations provide evidence for protein-gate opening of type IA topoisomerases.

Bacterial infections are serious health threats. Emerging drug resistance in bacteria further poses serious challenges to the treatment options involving traditional antibiotics. Antimicrobial polymers disrupt the physical cell membrane integrity of bacteria to address the drug resistance problems. Here, we introduce a conceptually new class of antimicrobial polymers containing positively charged guanylurea backbones for enhanced antimicrobial effects. The initial structure-activity relationship studies demonstrate that poly(guanylurea piperazine)s (PGU-Ps) exhibit excellent antimicrobial activity against different types of bacteria with high selectivity. The new design concept of using a positively charged guanylurea backbone will contribute to the development of future biocompatible, specific, and selective antimicrobial polymers.

In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (−) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (−) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (−) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (−) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (−) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (−) sc DNA.

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC50 values 32 μg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2−23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. ...

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation–deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase.

THE EMERGING FIELD OF PRECISION MEDICINE EMPHASIZES AN INDIVIDUALIZED APPROACH TO PREVENTION, DIAGNOSIS, AND TREATMENT OF DISEASE. USING INFORMATION FROM AN INDIVIDUAL'S GENETICS, ENVIRONMENT AND LIFESTYLE, EPIGENETICS, AND OTHER FIELDS IN PRECISION MEDICINE, PRACTITIONERS CAN MAXIMIZE RESOU

Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma. Synthetic RNA topological structures are significant for understanding the physical and biological properties pertaining to RNA topology, and these properties in turn could facilitate identifying naturally occurring topologically nontrivial RNA molecules. Here we show that topological structures containing single-stranded RNA (ssRNA) free of strong base pairing interactions can be created either by configuring RNA–DNA hybrid four-way junctions or by template-directed synthesis with a single-stranded DNA (ssDNA) topological structure. By using a constructed ssRNA knot as a highly sensitive topological probe, we find that Escherichia coli DNA topoisomerase I has low RNA topoisomerase activity and that the R173A point mutation abolishes the unknotting activity for ssRNA, but not for ssDNA. Furthermore, we discover the topological inhibition of reverse transcription (RT) and obtain different RT–PCR patterns for an ssRNA knot and circle of the same sequence., In vivo, complex topologies have been identified in proteins and DNA, while their existence in RNA is still unclear. Here, the authors design synthetic topological structures containing single stranded RNA, offering tools for investigating biologically relevant questions about RNA topology.

New antibacterial agents with novel target and mechanism of action are urgently needed to combat problematic bacterial infections and mounting antibiotic resistances. Topoisomerase IA represents an attractive and underexplored antibacterial target, as such, there is a growing interest in developing selective and potent topoisomerase I inhibitors for antibacterial therapy. Based on our initial biological screening, fluoroquinophenoxazine 1 was discovered as a low micromolar inhibitor against E. coli topoisomerase IA. In the literature, fluoroquinophenoxazine analogs have been investigated as antibacterial and anticancer agents, however, their topoisomerase I inhibition was relatively underexplored and there is little structure-activity relationship (SAR) available. The good topoisomerase I inhibitory activity of 1 and the lack of SAR prompted us to design and synthesize a series of fluoroquinophenoxazine analogs to systematically evaluate the SAR and to probe the structural elements of the fluoroquinophenoxazine core toward topoisomerase I enzyme target recognition. In this study, a series of fluoroquinophenoxazine analogs was designed, synthesized, and evaluated as topoisomerase I inhibitors and antibacterial agents. Target-based assays revealed that the fluoroquinophenoxazine derivatives with 9-NH2 and/or 6-substituted amine functionalities generally exhibited good to excellent inhibitory activities against topoisomerase I with IC50s ranging from 0.24 to 3.9 μM. Notably, 11a bearing the 6-methylpiperazinyl and 9-amino motifs was identified as one of the most potent topoisomerase I inhibitors (IC50 = 0.48 μM), and showed broad spectrum antibacterial activity (MICs = 0.78-7.6 μM) against all the bacteria strains tested. Compound 11g with the 6-bipiperidinyl lipophilic side chain exhibited the most potent antituberculosis activity (MIC = 2.5 μM, SI = 9.8). In addition, CoMFA analysis was performed to investigate the 3D-QSAR of this class of fluoroquinophenoxazine derivatives. The constructed CoMFA model produced reasonable statistics (q2 = 0.688 and r2 = 0.806). The predictive power of the developed model was obtained using a test set of 7 compounds, giving a predictive correlation coefficient r2pred of 0.767. Collectively, these promising data demonstrated that fluoroquinophenoxazine derivatives have the potential to be developed as a new chemotype of potent topoisomerase IA inhibitors with antibacterial therapeutic potential.

Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle. This article is protected by copyright. All rights reserved.

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)(2)(O(2)CO)]Cl·6H(2)O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)(2)(O(2)CO)]Cl·6H(2)O 2 was used to produce anhydrous [Co(phen)(2)(H(2)O)(2)](NO(3))(3) 3. Subsequently, anhydrous [Co(phen)(2)(H(2)O)(2)](NO(3))(3) 3 was reacted with MeATSC 1 to produce [Co(phen)(2)(MeATSC)](NO(3))(3)·1.5H(2)O·C(2)H(5)OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV–visible, and multinuclear NMR ((1)H, (13)C, and (59)Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV–visible spectrophotometric studies. UV–visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (K(b) = 8.1 × 10(5) and 1.6 × 10(4) M(−1), respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC(50) = 34.4 ± 5.2 μM when compared to IC(50) = 13.75 ± 1.08 μM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨ(m)). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨ(m), studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

A novel complex, [Cu(acetylethTSC)Cl]Cl•0.25C2H5OH 1 (where acetylethTSC = (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide), was shown to have anti-proliferative activity against various colon and aggressive breast cancer cell lines. In vitro studies showed that complex 1 acted as a poison inhibitor of human topoisomerase IIα, which may account for the observed anti-cancer effects.

Background Secondary fungal metabolites are important sources for new drugs against infectious diseases and cancers. Methods To obtain a library with enough diversity, we collected about 2,395 soil samples and 2,324 plant samples from 36 regions in Africa, Asia, and North America. The collection areas covered various climate zones in the world. We examined the usability of the global fungal extract library (GFEL) against parasitic malaria transmission, Gram-positive and negative bacterial pathogens, and leukemia cells. Results Nearly ten thousand fungal strains were isolated. Sequences of nuclear ribosomal internal transcribed spacer (ITS) from 40 randomly selected strains showed that over 80% were unique. Screening GFEL, we found that the fungal extract from Penicillium thomii was able to block Plasmodium falciparum transmission to Anopheles gambiae, and the fungal extract from Tolypocladium album was able to kill myelogenous leukemia cell line K562. We also identified a set of candidate fungal extracts against bacterial pathogens.

SUMMARY The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a “self-trapping” mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2., Graphical Abstract, In Brief Saha et al. introduce an approach to generate and detect the catalytic intermediates of TOP3B in DNA and RNA by engineering a self-poisoning enzyme, R338W-TOP3B. They reveal the cellular consequences of abortive TOP3Bcc formation and a repair pathway involving TRIM41, the proteasome, and TDP2 for processing of TOP3Bcc.

We have previously reported the inhibition of bacterial topoisomerase I activity by a fluoroquinophenoxazine compound (FP-11g) with a 6-bipiperidinyl lipophilic side chain that exhibited promising antituberculosis activity (MIC = 2.5 μM againstMycobacterium tuberculosis, SI = 9.8). Here, we found that the compound is bactericidal towardsMycobacterium smegmatis, resulting in greater than 5 Log10reduction in colony-forming units [cfu]/mL following a 10 h incubation at 1.25 μM (4X MIC) concentration. Growth inhibition (MIC = 50 μM) and reduction in cfu could also be observed against a clinical isolate ofMycobacterium abscessus.Stepwise isolation of resistant mutants ofM. smegmatiswas conducted to explore the mechanism of resistance. Mutations in the resistant isolates were identified by direct comparison of whole-genome sequencing data from mutant and wild-type isolates. These include mutations in genes likely to affect the entry and retention of the compound. FP-11g inhibitsMtbtopoisomerase I andMtbgyrase with IC50of 0.24 and 31.5 μM, respectively. Biophysical analysis showed that FP-11g binds DNA as an intercalator but the IC50for inhibition ofMtbtopoisomerase I activity is >10 fold lower than the compound concentrations required for producing negatively supercoiled DNA during ligation of nicked circular DNA. Thus, the DNA-binding property of FP-11g may contribute to its antimycobacterial mechanism, but that alone cannot account for the observed inhibition of Mtb topoisomerase I.

Topoisomerases are important complex enzymes that modulate DNA topology to maintain chromosome superstructure and integrity. These enzymes are involved in many cellular processes that resolve specific DNA superstructures and intermediates. The low abundance combined with the biological heterogeneity of relevant intermediates of topoisomerases makes their structural information not readily accessible using traditional structural biology tools (e.g., NMR and X-ray crystallography). In the present work, a second-generation trapped ion mobility spectrometry–mass spectrometry (TIMS–MS) was used to study Escherichia coli topoisomerase IA (EcTopIA) and variola virus topoisomerase IB (vTopIB) as well as their complexes with a single-stranded DNA and a stem-loop DNA under native conditions. The higher trapping efficiency and extended mass range of the new, convex TIMS geometry allowed for the separation and identification of multiple conformational states for the two topoisomerases and their DNA complexes. Inspection of the conformational space of EcTopIA and vTopIB in complex with DNA showed that upon DNA binding, the number of conformational states is significantly reduced, suggesting that the DNA binding selects for a narrow range of conformers restricted by the interaction with the DNA substrate. The large microheterogeneity observed for the two DNA binding proteins suggests that they can have multiple biological functions. This work highlights the potential of TIMS–MS for the structural investigations of intrinsically disordered proteins (e.g., DNA binding proteins) as a way to gain a better understanding of the mechanisms involved in DNA substrate recognition, binding, and assembly of the catalytically active enzyme–DNA complex.