Type IA Topoisomerases as Targets for Infectious Disease Treatments

Infectious diseases are one of the main causes of death all over the world, with antimicrobial resistance presenting a great challenge. New antibiotics need to be developed to provide therapeutic treatment options, requiring novel drug targets to be identified and pursued. DNA topoisomerases control the topology of DNA via DNA cleavage-rejoining coupled to DNA strand passage. The change in DNA topological features must be controlled in vital processes including DNA replication, transcription, and DNA repair. Type IIA topoisomerases are well established targets for antibiotics. In this review, type IA topoisomerases in bacteria are discussed as potential targets for new antibiotics. In certain bacterial pathogens, topoisomerase I is the only type IA topoisomerase present, which makes it a valuable antibiotic target. This review will summarize recent attempts that have been made to identify inhibitors of bacterial topoisomerase I as potential leads for antibiotics and use of these inhibitors as molecular probes in cellular studies. Crystal structures of inhibitor-enzyme complexes and more in-depth knowledge of their mechanisms of actions will help to establish the structure-activity relationship of potential drug leads and develop potent and selective therapeutics that can aid in combating the drug resistant bacterial infections that threaten public health

Type IA topoisomerases as targets for infectious disease treatments

Infectious diseases are one of the main causes of death all over the world, with antimicrobial resistance presenting a great challenge. New antibiotics need to be developed to provide therapeutic treatment options, requiring novel drug targets to be identified and pursued. DNA topoisomerases control the topology of DNA via DNA cleavage–rejoining coupled to DNA strand passage. The change in DNA topological features must be controlled in vital processes including DNA replication, transcription, and DNA repair. Type IIA topoisomerases are well established targets for antibiotics. In this review, type IA topoisomerases in bacteria are discussed as potential targets for new antibiotics. In certain bacterial pathogens, topoisomerase I is the only type IA topoisomerase present, which makes it a valuable antibiotic target. This review will summarize recent attempts that have been made to identify inhibitors of bacterial topoisomerase I as potential leads for antibiotics and use of these inhibitors as molecular probes in cellular studies. Crystal structures of inhibitor–enzyme complexes and more in-depth knowledge of their mechanisms of actions will help to establish the structure–activity relationship of potential drug leads and develop potent and selective therapeutics that can aid in combating the drug resistant bacterial infections that threaten public health

Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of <it>Escherichia coli </it>and <it>M. tuberculosis </it>DNA topoisomerase I proteins.</p> <p>Results</p> <p>A new protocol for expression and purification of recombinant <it>M. tuberculosis </it>DNA topoisomerase I (MtTOP) has been developed to produce enzyme of much higher specific activity than previously characterized recombinant enzyme. MtTOP was found to be less efficient than <it>E. coli </it>DNA topoisomerase I (EcTOP) in removal of remaining negative supercoils from partially relaxed DNA. DNA cleavage by MtTOP was characterized for the first time. Comparison of DNA cleavage site selectivity with EcTOP showed differences in cleavage site preferences, but the preferred sites of both enzymes have a C nucleotide in the -4 position.</p> <p>Conclusion</p> <p>Recombinant <it>M. tuberculosis </it>DNA topoisomerase I can be expressed as a soluble protein and purified in high yield from <it>E. coli </it>host with a new protocol. Analysis of DNA cleavage with <it>M. tuberculosis </it>DNA substrate showed that the preferred DNA cleavage sites have a C nucleotide in the -4 position.</p

Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme\u27s multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target

Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains

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

Covalent Complex of DNA and Bacterial Topoisomerase: Implications in Antibacterial Drug Development

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

Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial topoisomerase IA inhibitors

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-NH and/or 6-substituted amine functionalities generally exhibited good to excellent inhibitory activities against topoisomerase I with ICs 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 (IC = 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 (q = 0.688 and r = 0.806). The predictive power of the developed model was obtained using a test set of 7 compounds, giving a predictive correlation coefficient r 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

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4

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. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb’s toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM)

Identification of Anziaic Acid, a Lichen Depside from Hypotrachyna sp., as a New Topoisomerase Poison Inhibitor

Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds

A diverse global fungal library for drug discovery

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 was able to block transmission to , and the fungal extract from was able to kill myelogenous leukemia cell line K562. We also identified a set of candidate fungal extracts against bacterial pathogens