Topoisomerase Research Articles

New Potential Pharmacological Targets of Plant-Derived Hydroxyanthraquinones from Rubia spp.

The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening of DrugBank outlined pseudopurpurin, a hydroxyanthraquinone from Rubia cordifolia spp., as similar to gatifloxacin, a synthetic antibacterial agent. This suggested the bacterial DNA gyrase and DNA topoisomerase IV as potential pharmacological targets of pseudopurpurin. In this study, estimation of structural similarity to referent antibacterial agents and molecular docking in the DNA gyrase and DNA topoisomerase IV complexes were performed for a homologous series of four hydroxyanthraquinones. Estimation of shape- and chemical feature-based similarity with (S)-gatifloxacin, a DNA gyrase inhibitor, and (S)-levofloxacin, a DNA topoisomerase IV inhibitor, outlined pseudopurpurin and munjistin as the most similar structures. The docking simulations supported the hypothesis for a plausible antibacterial activity of hydroxyanthraquinones. The predicted docking poses were grouped into 13 binding modes based on spatial similarities in the active site. The simultaneous presence of 1-OH and 3-COOH substituents in the anthraquinone scaffold were emphasized as relevant features for the binding modes’ variability and ability of the compounds to strongly bind in the DNA-enzyme complexes. The results reveal new potential pharmacological targets of the studied polyphenols and help in their prioritization as drug candidates and dietary supplements.

Design, synthesis of DNA-interactive 4-thiazolidinone-based indolo-/pyrroloazepinone conjugates as potential cytotoxic and topoisomerase I inhibitors

With the rising cancer incidence and mortality globally, there is a prerequisite for effective design strategies towards the discovery of newer small molecular entities in chemotherapy. Hence, a series of new thiazolidinone-based indolo-/pyrroloazepinone conjugates was designed, synthesized via molecular hybridization, and evaluated for their in vitro cytotoxicity potential and DNA topoisomerase I and II inhibition. Among this series, conjugate 11g emerged as the most active compound with an IC50 value of 1.24 μM against A549 and 3.02–10.91 μM in the other tested cancer cell lines. Gratifyingly, 11g displayed 43-fold higher selectivity towards A549 cancer cells as compared to the non-cancer cells. Subsequently, conjugate 12g also demonstrated significant cytotoxicity against SK-MEL-28 cells. Basing the in vitro cytotoxicity results, SAR was established. Later, the conjugates 11g and 12g were further evaluated for their apoptosis-inducing ability, which was quantified by flow cytometric analysis, DNA-binding, Topo I inhibitory activity and IC50 value calculation. Molecular modeling studies provided profound insights about the binding nature of these compounds with DNA-Topo I complex. In silico ADME/T and prediction studies corroborated the drug-likeness of the two investigated compounds. TOPKAT toxicity profiling studies demonstrated the compounds’ safety in many animal models with a minimal toxicological profile. Encouraging results obtained from in vitro and in silico studies could put this series of conjugates at the forefront of cancer drug discovery.

Epigenetic Modification Impacts on Gene Regulation in Streptococcus pneumoniae

Methylation by the type I restriction modification system (RMS) SpnIII in Streptococcus pneumoniae is hypothesised to regulate gene expression via epigenetic changes (Manos). The phase variable SpnIII generates six host specificity determinants (hsdS) alleles through site-specific recombination (DeSteCroix), each allele correspond with different methylation pattern. In addition to known functions of the RMS in restricting phage infection (Furi) and transformation (Kwun), we have now tested impact on gene expression. RNAseq was used to analyse S. pneumoniae strains expressing a single spnIII allele (spnIIIA, spnIIIB or spnIIIE) to determine differences in gene expression profiles. The data have identified six genes which show differential expression and have a methylation site mapping to their predicted promoter region. Three synthetic promoters with the wild type and altered methylation target site were cloned in front of a luciferase gene in strains expressing a single spnIII alleles. For the first three sets of constructs analysed, data indicate that the methylated promoters show a three- to twenty-fold higher activity compared to non-methylated promoters. Results obtained were further confirmed by qPCR analysis. Preliminary data using drugs targeting DNA topoisomerases indicate that the mechanism by which methylation impact gene expression could be related to DNA topology. These data demonstrate for the first-time RMS dependent variations in gene expression and propose an alternative mechanism for this epigenetic regulation.

Design, Synthesis, Antibacterial Evaluations and In Silico Studies of Novel Thiosemicarbazides and 1,3,4-Thiadiazoles.

The emergence of drug-resistant bacterial strains continues to be one of the major challenges of medicine. For this reason, the importance of searching for novel structures of antibacterial drugs chemically different from the currently known antibiotics is still of great importance. In this study, we synthesized the thiosemicarbazide and 1,3,4-thiadiazole derivatives and tested them for antibacterial activity. In in vitro tests, we examined the activity of the synthesized substances against Gram-positive and Gram-negative bacteria strains. While all 1,3,4-thiadiazoles tested lacked significant activity, the antimicrobial response of the thiosemicarbazides was moderate and it was also dependent on the type and position of the substituent on the phenyl ring. The highest activity towards all Gram-positive bacteria strains was shown by all three linear compounds containing the trifluoromethylphenyl group in the structure. The MIC (minimum inhibitory concentration) values were in the range of 3.9–250 µg/mL. Additionally, we try to explain the mechanism of the antibacterial activity of the tested compounds using the molecular docking to DNA gyrase and topoisomerase IV, following previous reports on the molecular basis of the activity of thiosemicarbazides. Docking simulations allow the purposing dual mechanism of the antibacterial activity of the synthesized compounds through inhibition of topoisomerase IV DNA gyrase with the moderate prevalence of the topoisomerase pathway.

Recent Developments in Oxazole Derivatives as Anticancer Agents: Review on Synthetic Strategies, Mechanism of Action and SAR Studies.

Cancer is the world's third deadliest disease. Despite the availability of numerous treatments, researchers are focusing on the development of new drugs with no resistance and toxicity issues. Many newly synthesized drugs fail to reach clinical trials due to poor pharmacokinetic properties. Therefore, there is an imperative requisite to expand novel anticancer agents with in vivo efficacy. This review emphasizes synthetic methods, contemporary strategies used for the inclusion of oxazole moiety, mechanistic targets, along with comprehensive structure-activity relationship studies to provide perspective into the rational design of highly efficient oxazole-based anticancer drugs. Literature related to oxazole derivatives engaged in cancer research is reviewed. This article gives a detailed account of synthetic strategies, targets of oxazole in cancer, including STAT3, Microtubules, G-quadruplex, DNA topoisomerases, DNA damage, protein kinases, miscellaneous targets, in vitro studies, and some SAR studies. Oxazole derivatives possess potent anticancer activity by inhibiting novel targets such as STAT3 and Gquadruplex. Oxazoles also inhibit tubulin protein to induce apoptosis in cancer cells. Some other targets such as DNA topoisomerase enzyme, protein kinases, and miscellaneous targets including Cdc25, mitochondrial enzymes, HDAC, LSD1, HPV E2 TAD, NQO1, Aromatase, BCl-6, Estrogen receptor, GRP-78, and Keap-Nrf2 pathway are inhibited by oxazole derivatives. Many derivatives showed excellent potencies on various cancer cell lines with IC50 values in nanomolar concentrations. Oxazole is a five-membered heterocycle, with oxygen and nitrogen at 1 and 3 positions, respectively. It is often combined with other pharmacophores in the expansion of novel anticancer drugs. In summary, oxazole is a promising entity to develop new anticancer drugs.

Discovery, enantioselective synthesis of myrtucommulone E analogues as tyrosyl-DNA phosphodiesterase 2 inhibitors and their biological activities

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 2 (TDP2) can specifically repair topoisomerase 2 (TOP2)-mediated DNA damage, resulting in cancer cell resistance to TOP2 inhibitors. Its inhibitors can enhance the anticancer efficacy of TOP2 inhibitors. In this work, we report the discovery of natural product myrtucommulone E as selective TDP2 inhibitor and its first enantioselective total synthesis through a pivotal CPA-catalyzed Michael addition reaction. A series of myrtucommulone E analogues were asymmetrically synthesized and evaluated for TDP2 and TDP1 inhibitions, and cytotoxicity. Analogue (+)-29 shows good TDP2 inhibition potency (5.4 ± 0.25 μM), but no TDP1 inhibition at 100 μM concentration, and can significantly enhance the cytotoxicity of TOP2 inhibitor etoposide in both DU145 (CI = 0.26) and DT40 hTDP2 cells (CI = 0.48).

Septins promote caspase activity and coordinate mitochondrial apoptosis.

Apoptosis is a form of regulated cell death essential for tissue homeostasis and embryonic development. Apoptosis also plays a key role during bacterial infection, yet some intracellular bacterial pathogens (such as Shigella flexneri, whose lipopolysaccharide can block apoptosis) can manipulate cell death programs as an important survival strategy. Septins are a component of the cytoskeleton essential for mitochondrial dynamics and host defense, however, the role of septins in regulated cell death is mostly unknown. Here, we discover that septins promote mitochondrial (i.e., intrinsic) apoptosis in response to treatment with staurosporine (a pan-kinase inhibitor) or etoposide (a DNA topoisomerase inhibitor). Consistent with a role for septins in mitochondrial dynamics, septins promote the release of mitochondrial protein cytochrome c in apoptotic cells and are required for the proteolytic activation of caspase-3, caspase-7, and caspase-9 (core components of the apoptotic machinery). Apoptosis of HeLa cells induced in response to infection by S. flexneri ΔgalU (a lipopolysaccharide mutant unable to block apoptosis) is also septin-dependent. In vivo, zebrafish larvae are significantly more susceptible to infection with S. flexneri ΔgalU (as compared to infection with wildtype S. flexneri), yet septin deficient larvae are equally susceptible to infection with S. flexneri ΔgalU and wildtype S. flexneri. These data provide a new molecular framework to understand the complexity of mitochondrial apoptosis and its ability to combat bacterial infection.

Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.

During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association withtwo phospho-isoforms of the helicase component MCM2.During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin.In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation.In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

Targeted Delivery of DNA Topoisomerase Inhibitor SN38 to Intracranial Tumors of Glioblastoma Using Sub-5 Ultrafine Iron Oxide Nanoparticles.

Effectively delivering therapeutics for treating brain tumors is hindered by the physical and biological barriers in the brain. Even with the compromised blood-brain barrier and highly angiogenic blood-tumor barrier seen in glioblastoma (GBM), most drugs, including nanomaterial-based formulations, hardly reach intracranial tumors. This work investigates sub-5nm ultrafine iron oxide nanoparticles (uIONP) with 3.5nm core diameter as a carrier for delivering DNA topoisomerase inhibitor 7-ethyl-10-hydroxyl camptothecin (SN38) to treat GBM. Given a higher surface-to-volume ratio, uIONP shows one- or three-folds higher SN38 loading efficiency (48.3± 6.1%, mg/mg Fe) than those with core sizes of 10 or 20nm. SN38 encapsulated in the coating polymer exhibits pH sensitive release with <10%over 48 h at pH 7.4, but 86% at pH 5, thus being protected from converting to inactive glucuronide by UDP-glucuronosyltransferase 1A1. Conjugating αv β3 -integrin-targeted cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGD) as ligands, RGD-uIONP/SN38 demonstrates targeted cytotoxicity to αv β3 -integrin-overexpressed U87MG GBM cells with a half-maximal inhibitory concentration (IC50 ) of 30.9± 2.2nm. The efficacy study using an orthotopic mouse model of GBM reveals tumor-specific delivery of 11.5% injected RGD-uIONP/SN38 (10mg Fe kg-1 ), significantly prolonging the survival in mice by 41%, comparing to those treated with SN38 alone (p <0.001).

The Structural Features of Novel Bacterial Topoisomerase Inhibitors That Define Their Activity on Topoisomerase IV.

The continued emergence of bacterial resistance has created an urgent need for new and effective antibacterial agents. Bacterial type II topoisomerases, such as DNA gyrase and topoisomerase IV (topoIV), are well-validated targets for antibacterial chemotherapy. The novel bacterial topoisomerase inhibitors (NBTIs) represent one of the new promising classes of antibacterial agents. They can inhibit both of these bacterial targets; however, their potencies differ on the targets among species, making topoIV probably a primary target of NBTIs in Gram-negative bacteria. Therefore, it is important to gain an insight into the NBTIs key structural features that govern the topoIV inhibition. However, in Gram-positive bacteria, topoIV is also a significant target for achieving dual-targeting, which in turn contributes to avoiding bacterial resistance caused by single-target mutations. In this perspective, we address the structure–activity relationship guidelines for NBTIs that target the topoIV enzyme in Gram-positive and Gram-negative bacteria.

CRISPR/Cas9 with Homology‐Directed Repair Effectively Silences the Human Topoisomerase IIα Intron‐19 5′ Splice Site and Results in Etoposide Resistance in Human Leukemia K562 Cells

DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 a target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170. Our laboratory recently demonstrated that reduced levels of TOP2α/170 in acquired resistance to etoposide is associated with alternative RNA processing of the TOP2α gene and intron retention. Intron 19 retention in an etoposide‐resistant human leukemia K562 subline, K/VP.5, resulted not only in decreased TOP2α/170 but also translation and overexpression of a C‐terminal truncated 90‐kDa isoform of TOP2α, TOP2α/90. We found that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Splice site analysis revealed that the exon 19/intron 19 boundary of the TOP2α gene is suboptimal, providing a potential explanation for intron 19 retention. To further test the hypothesis that intron 19 retention is a determinant of acquired drug resistance, CRISPR/Cas9 with homology‐directed repair (HDR) was used to silence the intron 19 5′ splice site of the TOP2α gene in parental K562 cells (etoposide‐sensitive) by making two nucleotide changes to the splice site (GAG//GTAAAC→GAA//CTAAAC) thereby forcing intron 19 retention by abrogating spliceosome function. Gene‐edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. Clones with TOP2α edited alleles contained decreased TOP2α/170 mRNA/protein resulting in resistance to etoposide as assessed by reduced etoposide‐induced DNA damage (γH2AX, Comet assays) and growth inhibition. Forced expression of TOP2α/90 in the gene‐edited K562 cells further decreased etoposide‐induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the role of alternative TOP2α mRNA splicing as a determinant of resistance to TOP2α‐targeting agents.

Evaluation of the DNA topoisomerase inhibitor irinotecan (CPT-11) as an alternative to irradiation for pretreatment of NK-92s prior to adoptive cell immunotherapy.

Abstract Irradiation damages both proteins and DNA, which reduces the cytotoxicity of NK-92 cells as well as preventing their growth and engraftment when used for adoptive immunotherapies of cancer. We evaluated the DNA topoisomerase inhibitor irinotecan (CPT-11) as an alternative method to 10 Gy irradiation that might better preserve the perforin-granzyme cytotoxicity mediated by NK-92 cells towards tumor cells. Irinotecan is enzymatically converted into an active metabolite SN-38 which promotes its potential to become a delayed inhibitor in comparison to camptothecin which is a structurally similar topoisomerase inhibitor that is immediately active. We pretreated NK-92 cells for 4 hours with different concentrations of CPT-11, and measured NK-92 cell death and the effects on cell-mediated cytotoxicity towards Raji tumor cells (measured by 51Cr release one day after pretreatment). Previous experiments with 10 Gy irradiation were used for comparative purposes. Irinotecan had a dose response on cell death which progressed for 5 days. We found that one day after pretreatment with as little as 80 – 100 uM CPT-11, viable cell recoveries were similar to 10 Gy irradiated with over 50% compared to the counts of control cells. Activity of the viable cells after CPT-11 pretreatment, compared by lytic units, was over 30% conserved whereas irradiated cell activity was 2.9%, compared to controls. This data demonstrate that the negative effects of anti-proliferative treatment are less pronounced with irinotecan, than for irradiation. We conclude that optimization of dosage and exposure times to irinotecan may offer a means to prolong the life and function of NK-92 cells that are used for adoptive immunotherapies. Supported by the Nevada INBRE NIH GM103440, for student summer research scholarship and the UNR Foundation C.E. Hudig Fund.

Effects of hsa‐miR‐9‐3p and hsa‐miR‐9‐5p on Topoisomerase IIβ Expression in Human Leukemia K562 Cells with Acquired Resistance to Etoposide

DNA topoisomerase IIα (TOP2α/170; 170kDa) and Topoisomerase IIβ (TOP2β/180; 180kDa) are important targets for DNA damage‐stabilizing anticancer drugs, whose clinical efficacy is often attenuated by chemoresistance. Our laboratory has characterized an etoposide‐resistant human leukemia K562 clonal subline, designated K/VP.5. These cells show a decrease in both TOP2α/170 and TOP2β/180 expression. We recently demonstrated that a microRNA (miRNA)‐mediated post transcriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein levels in K/VP.5 cells. We hypothesize that a miRNA mechanism is similarly responsible for decreased TOP2β/180 levels in K/VP.5 cells. Both miR‐9‐3p and miR‐9‐5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by miRNA sequencing, and validated by quantitative polymerase chain reaction (qPCR). The 3’‐untranslated region (UTR) of TOP2β/180 contains a well‐defined miRNA recognition element (MRE) for miR‐9‐5p and putative MREs for miR‐9‐3p. Co‐transfection of K562 cells with a luciferase reporter plasmid harboring TOP2β/180‐3’UTR plus miR‐9‐3p and/or miR‐9‐5p mimics resulted in a statistically significant decrease in luciferase expression. miR‐9‐5p MRE mutation prevented this decrease validating direct interaction between this miRNA and TOP2β/180 mRNA. Transfection of K562 cells with miR‐9‐3p or miR‐9‐5p mimics led to decreased TOP2β protein levels without a change in TOP2β/180 mRNA. Inversely, K/VP.5 cells transfected with miR‐9‐3p or miR‐9‐5p inhibitors led to increased TOP2β/180 protein and no change in TOP2β/180 mRNA. Taken together, these results strongly suggest that TOP2β/180 mRNA is translationally repressed by miR‐9‐3p and miR‐9‐5p, and that these miRNAs play a role in acquired resistance to etoposide via regulation of TOP2β/180. Further experiments are underway to characterize miR‐9‐3p and miR‐9‐5p effects on TOP2β/180‐specific cytotoxicity.

POSSIBLE CARDIOPROTECTIVE MECHANISM OF ACTION OF DEXRAZOXANE, AND PROBABLE HUMAN TOPOISOMERASE IIβ INHIBITORS: AN IN SILICO ANALYSIS

Objective: The aim of this study was to determine which metabolite plays a role in the cardioprotective effect of dexrazoxane, and also to identify alternative compounds to dexrazoxane since clinical use of dexrazoxane is limited. For this purpose, the interactions of dexrazoxane and its three metabolites (B, C, and ADR-925), as well as the compounds, which reported to be inhibitors for topoisomerase VI (prototype of human DNA topoisomerase II beta), with human DNA topoisomerase II beta were investigated by molecular docking. Afterwards, the theoretical ADMET properties of all these compounds were determined Material and Method: The molecular structures were optimized by Gaussview 05 and Gaussian 03 package programs. AutoDock 4.2 software was used for molecular docking studies and the docking complexes were analyzed in 2D and 3D using the Discovery Studio Client 4.1 program. The pkCSM online program was used to calculate the theoretical ADMET parameters. Result and Discussion: As a result of molecular docking studies, it was determined that the B metabolite of dexrazoxane has a higher binding potential to human DNA topoisomerase II beta compared to both dexrazoxane and its other metabolites. The binding potentials of other compounds reported in the literature to human DNA topoisomerase II beta were radicicol>quinacrine>purpurin>9-Aminoacridine>hexylresorcinol, respectively. The results showed that the B metabolite of dexrazoxane plays an important role in the cardioprotective mechanism of action of dexrazoxane against anthracycline cardiotoxicity. In addition, it has been determined that other compounds, except purpurin, have the potential to cause toxicity.

Identification of the genetic elements involved in biofilm formation by Salmonella enterica serovar Tennessee using mini-Tn10 mutagenesis and DNA sequencing

Biofilms are known to contribute to bacterial tolerance to desiccation and survival in low moisture foods. However, the molecular mechanisms underlying biofilm formation have not been fully elucidated. This study identified some of the genes that are implicated in biofilm formation by Salmonella enterica serovar Tennessee, the “peanut butter outbreak” strain. Mini-Tn10 mutagenesis was used in the study to generate random transposon insertion libraries. The ability of selected mutants in forming biofilms was compared with their wildtype parent using the crystal violet binding assay. Mutants forming significantly less (P ≤ 0.05) biofilm compared to their wildtype parent were selected for whole-genome sequencing. Mini-Tn10 insertion sites on mutant genomes were identified by comparing the acquired sequencing data with those in the Genbank using the BLAST search. In total, 56 mutants were obtained, and five were selected for further analysis according to the result of the biofilm assay. Sequencing analysis revealed that the mini-Tn10 interrupted the S. enterica genes that encode bacterial cell membrane lipoprotein, DNA topoisomerase III, attachment and invasion locus protein, bacteriocin immunity protein, and cell division protein. The information generated from the research should be useful in the control fo S. enterica in low-moisture foods and their production environments.

Mitoxantrone modulates a heparan sulfate-spike complex to inhibit SARS-CoV-2 infection

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a heparan sulfate-spike complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar heparan sulfate-binding activities but with reduced affinity for DNA topoisomerases may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.

YM155 Induces DNA Damage and Cell Death in Anaplastic Thyroid Cancer Cells by Inhibiting DNA Topoisomerase IIα at the ATP-Binding Site.

Anaplastic thyroid cancer (ATC) is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing ATC cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, ATC patient samples show overexpression of topoisomerase Top2α compared with benign thyroid samples and to differentiated thyroid cancers. ATC cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which shows decreased expression of Top2α and is resensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP-binding site and identifies key amino acids for YM155-Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of ATC.

Activating transcription factor 2 promotes the progression of hepatocellular carcinoma by inducing the activation of the WHSC1-mediated TOP2A/PI3K/AKT axis.

Activating transcription factor 2 (ATF2) is a tumor driver gene implicated in several human malignancies. This study aimed to determine the roles of ATF2 and its related molecules in the tumorigenesis of hepatocellular carcinoma (HCC). According to the Pan-cancer bioinformatics system, ATF2 is highly expressed in HCC. An increase in the expression of ATF2 was identified in clinically collected tumor tissues and procured HCC cells. The silencing of ATF2 reduced the viability, colony formation, invasion, and death resistance of HepG2 and SNU-398 cells in vitro. ATF2 promoted the transcription of Wolf-Hirschhorn syndrome candidate 1 (WHSC1) by binding to its promoter. WHSC1 further increased the expression of DNA topoisomerase II alpha (TOP2A) in HCC by inducing the dimethylation of histone H3 lysine 36 (H3K36me2) in the TOP2A promoter region. TOP2A activated the oncogenic PI3K/AKT signaling pathway. Further overexpression of WHSC1 activated the TOP2A/PI3K/AKT axis and restored the malignant behaviors of HCC cells suppressed by ATF2 silencing in vitro. In summary, this study demonstrated that, depending on WHSC1, ATF2 can activate the TOP2A/PI3K/AKT signaling cascade to promote the tumorigenesis of HCC. ATF2, WHSC1, and TOP2A may serve as potential targets in managing HCC.

Characterization of Mutations in DNA Gyrase and Topoisomerase IV in Field Strains and In Vitro Selected Quinolone-Resistant Mycoplasma hyorhinis Mutants.

Mycoplasma hyorhinis is ubiquitous in swine, and it is a common pathogen of swine that causes polyserositis, arthritis, and maybe pneumonia. Fluoroquinolones are effective antimicrobials used for the treatment of mycoplasmal infection. However, a decrease in fluoroquinolones susceptibility in mycoplasma was observed. The molecular mechanisms have been studied in many mycoplasma species, while the mechanism in M. hyorhinis is still unknown. This study aimed to illustrate the in vitro development of fluoroquinolone resistance in M. hyorhinis and unveil the resistance mechanisms in both in vitro selected mutants and field strains. Seven ciprofloxacin-sensitive M. hyorhinis isolates were chosen to induce the fluoroquinolone resistance in vitro, and the point mutations in the quinolone resistance-determining regions (QRDRs) were characterized. The substitutions first occurred in ParC, resulting in a 2- to 8-fold increase in resistance, followed by additional mutations in GyrA and/or ParE to achieve a 32-fold increase. The mutations occurred in hot spots of QRDRs, and they were diverse and variable, including five in ParC (Ser80Phe, Ser80Tyr, Phe80Tyr, Glu84Gly, and Glu84Lys), four in GyrA (Ala83Val, Ser84Pro, Asp87Tyr, and Asp87Asn) and one in ParE (Glu470Lys). Target mutations in field strains were observed in the ParC (Ser80Phe, Ser81Pro, and Glu84Gln) of isolates with MICCIP = 2 μg/mL. This study characterized the point mutations in the QRDRs of M. hyorhinis and could be useful for the rapid detection of fluoroquinolone resistance in M. hyorhinis field isolates.

Synthesis and in vitro antibacterial activity of N-acylarylhydrazone-ciprofloxacin hybrids as novel fluoroquinolone derivatives

Twelve novel fluoroquinolone derivatives were rationally designed and synthesized by introducing N-acylarylhydrazone to the C-7 position of ciprofloxacin. Antibacterial evaluation revealed that compound 3k was over 4-folds more potent than ciprofloxacin against S. aureus, with a MIC value ≤0.125 µg/mL. Compound 3g showed activity against methicillin-resistant S. aureus (MRSA) with a MIC value of 32 µg/mL. Time-killing assays showed that compound 3g eradicated S. aureus in 4 h and E. coli in 2 h. Compound 3g had a high affinity towards DNA topoisomerase IV, with a least binding energy of −9.9 kcal/mol, which was better than ciprofloxacin (−8.0 kcal/mol). The binding modes of compound 3g to DNA topoisomerase IV differed from that of ciprofloxacin. Molecular dynamics simulation results support that the interaction between the receptor 3rae and the compound 3g is stable. Besides, cytotoxicity and hemolysis assays demonstrated that compound 3g and 3k have negligible risks of toxic effects.