Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents

Sandhaus, Shayna; Annamalai, Thirunavukkarasu; Welmaker, Greg; Houghten, Richard A.; Paz, Carlos; Garcia, Pamela K.; Andres, Angelo; Narula, Gagandeep; Felix, Carolina Rodrigues; Geden, Sandra; Netherton, Mandy; Gupta, Rashmi; Rohde, Kyle H.; Giulianotti, Marc A.; Tse‐Dinh, Yuk‐Ching

doi:10.1128/aac.00288-16

Cited by 33 publications

(37 citation statements)

References 45 publications

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“…Because of their importance in genome maintenance and replication, topoisomerases are a potential target for both anti-bacterial and anti-cancer drugs [53][54][55] .…”

Section: Discussionmentioning

confidence: 99%

Direct Observation of Topoisomerase IA Gate Dynamics

Mills

Tse‐Dinh

Neuman

2018

Preprint

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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 it is assumed type IA topoisomerases 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 E. coli type IA topoisomerases I and III. We found that following cleavage of singlestranded 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 singlemolecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.religate the DNA backbone, and open again to release the trapped DNA ( Fig. 1b).Although structural, 31,32 biochemical, 6,33 and single-molecule experiments 7 support this model, direct evidence for the gate mechanism has proved elusive. No structures exist of a type IA topoisomerase in the open state and no measurements have definitivelyshown that an open state exists, leaving open the possibility that the strand passage is coupled to some other conformational change 34 .Here we describe the direct observation of type IA topoisomerase gate opening dynamics in single-molecule magnetic tweezers based measurements of the two E. coli type IA topoisomerases, topo I and topo III, interacting with ssDNA ( Fig. 2a and 3a). By analyzing the force-and magnesium-dependent kinetics of the gate opening and closing, we delineate the catalytic cycle of type IA topoisomerases. These results show key differences in the gate dynamics of topo I and topo III that may underlie the differences in their biochemical activities. Molecular dynamics simulations reveal the potential structure of the open conformation and confirm that the experimentally observed extension changes are consistent with opening of the gate between domains I and III. Our results provide a detailed description of the kinetics of type IA topoisomerase-ssDNA interactions and establish an experimental paradigm to study the conformational dynamics of type IA topoisomerases. ResultsCleavage by type IA topoisomerases increases ssDNA extension by greater than 6 nm.

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“…Because of their importance in genome maintenance and replication, topoisomerases are a potential target for both anti-bacterial and anti-cancer drugs [53][54][55] .…”

Section: Discussionmentioning

confidence: 99%

Direct Observation of Topoisomerase IA Gate Dynamics

Mills

Tse‐Dinh

Neuman

2018

Preprint

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“…After 30 min at 37°C, reactions were stopped with 4 µL of 6% SDS, 0.3% bromophenol blue, 30% glycerol, and analyzed by agarose gel electrophoresis. 27 DNA in the gel was stained with EtBr and photographed over UV light. AlphaView (ProteinSimple) was used to analyze the fraction of supercoiled DNA substrate converted into relaxed DNA.…”

Section: Methodsmentioning

confidence: 99%

Tyrosyl-DNA phosphodiesterase 1 and topoisomerase I activities as predictive indicators for Glioblastoma susceptibility to genotoxic agents

Wang

Rodriguez-Silva

Rocha

et al. 2019

Preprint

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34Background. Glioblastoma (GBM) patients have an estimated survival of ~15 months with 35 treatment, and the standard of care only modestly enhances patient survival. Identifying 36 biomarkers representing vulnerabilities may allow for selection of efficacious chemotherapy 37 options to address personalized variations in GBM tumors. Irinotecan, currently in clinical trials 38 for GBM, targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex 39 (TOP1cc) inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair 40 enzyme that may reduce the effectiveness of irinotecan. 41 42 3 Methods. We treated GBM cell lines with increasing concentrations of irinotecan and compared 43 the IC50 values. TOP1 and TDP1 protein levels from each cell type as well as GBM patient tumors 44 were determined by Western blot analysis, while activity levels were ascertained by specific 45 enzymatic assays. Cellular TDP1 was elevated by ectopic expression of wild-type or mutant TDP1. 46 47 Results. After comparing cellular susceptibility to TDP1 and TOP1 concentrations and activities, 48we found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation 49 coefficient R = 0.92) with IC50 values following irinotecan treatment. Increasing the TDP1/TOP1 50 activity ratio by ectopic expression of wild-type TDP1 increased in irinotecan IC50, while 51 expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan. 52 53 Conclusions. TDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability 54 to irinotecan, allowing for selection of individual patients for irinotecan treatment based on risk-55 benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to 56 improve GBM patient responsiveness to genotoxic chemotherapies. 57 58 Keywords: glioblastoma, DNA repair, TOP1, TDP1, irinotecan 59 60 Key Points 61 1. TDP1/TOP1 activity ratio correlates with irinotecan sensitivity in GBM cell lines. 62

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“…We were interested in how the compounds' antibacterial activity would be affected by the overexpression of the hypothesized target. To determine whether overexpression of the target would have any effect, antibacterial studies were conducted using the M+ and Mnol strains; the M+ strain expresses MtbTopI at least 6-fold higher than the Mnol strain, even in the absence of the tetracycline inducer (Sandhaus et al, 2016a The four compounds are also selective in their inhibition of bacterial topoisomerase I-concentrations more than 10-fold higher were required to inhibit the activity of DNA gyrase, human topoisomerase I, and human topoisomerase IIα ( Figure 1.3, Table 1.4). These results indicate that the mixture-based screening was also successful at discovering selective inhibitors; the polyamine compounds are not promiscuous inhibitors for just any DNA-binding enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…The samples were loaded into a 1% agarose gel containing 0.5 µg/mL ethidium bromide, and were run in 1x TAE buffer containing 0.5 µg/mL ethidium bromide as well. The gels were then photographed under UV light (Sandhaus et al, 2016a). (Sandhaus et al, 2016a).…”

Section: Human Topoisomerase Iiα Decatenation Inhibition Assaymentioning

confidence: 99%

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Drug Candidate Discovery: Targeting Bacterial Topoisomerase I Enzymes for Novel Antibiotic Leads

Sandhaus¹

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This work could not have been done without the help of many people, scientists and non-scientists alike. First, I would like to thank my committee members for sticking with me through the years. Their input was invaluable, and I learned so much from all of them. Secondly, I want to thank my lab mates. Everyone working in Dr. Tse-Dinh's lab was helpful to me in some way, be it with help setting up an experiment, starting a cell culture over the weekend, or just having a friendly face to talk to. To our lab manager Arasu: his guidance keeps this whole lab running. Without him, I am sure we would all have been lost. Next, I would like to thank the FIU Research Initiative for Scientific Enhancement (RISE) fellowship for offering me funding in my last year of graduate work.

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Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents

Cited by 33 publications

References 45 publications

Direct Observation of Topoisomerase IA Gate Dynamics

Direct Observation of Topoisomerase IA Gate Dynamics

Tyrosyl-DNA phosphodiesterase 1 and topoisomerase I activities as predictive indicators for Glioblastoma susceptibility to genotoxic agents

Drug Candidate Discovery: Targeting Bacterial Topoisomerase I Enzymes for Novel Antibiotic Leads

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