Cleavage Complex Research Articles

Silymarin protects against doxorubicin induced cardiotoxicity by down-regulating topoisomerase IIβ expression in mice

ABSTRACT We investigated the potential protective effects of silymarin (SLY) on doxorubicin (DOX) induced chronic cardiotoxicity in mice. We used 30 male BALB/c mice assigned randomly to four experimental groups: control group administered normal saline orally daily and intraperitoneally (i.p.) twice/week during weeks 1, 2, 5 and 6; SLY group (was administered 100 mg/kg SLY daily by oral gavage for 6 weeks; DOX group was administered 3 mg/kg DOX i.p. twice/week during weeks 1, 2, 5 and 6; DOX + SLY group administered DOX and SLY corresponding to the DOX and SLY groups. At the end of the experiment, heart tissues were collected for analysis. Cardiomyopathy was observed in the DOX group; this damage was reduced by SLY treatment. SLY administration in DOX treated mice decreased topoisomerase IIβ (TopIIβ) expression as indicated by qPCR and immunostaining. Immunohistochemistry and western blot analysis revealed decreased phosphorylated histone-2AX (γH2Ax) expression in the SLY + DOX group. SLY administration combined with DOX increased cardiac troponin T and I (cTnT and cTnI) expression based on immunohistochemical and western blot analyses. SLY administration with DOX was cardioprotective by reducing double-strand DNA breakage by blocking the DOX-TopIIβ-DNA cleavage complex in response to down-regulation of TopIIβ expression. SLY also preserved the contractility of the heart by decreasing DOX related loss of cTnT and cTnI expression.

Phase 1 study of indenoisoquinoline LMP744 in adults with relapsed solid tumors and lymphomas.

3022 Background: Indenoisoquinolines (IIQ) are non-camptothecin inhibitors of topoisomerase 1 (TOP1) that have improved characteristics over camptothecin TOP1 inhibitors. IIQs demonstrate better chemical stability, produce more persistent DNA breaks induced by trapping DNA-TOP1 cleavage complexes, induce DNA breaks at different sites than camptothecins, and are not substrates for cellular export proteins. We present data from a trial of LMP744 (NSC 706744), which was the most efficacious IIQ in a canine lymphoma trial, designed to establish the safety, efficacy, and maximum tolerated dose (MTD) of LMP744. Methods: We conducted a multicenter phase 1 trial in adult patients (pts) with dose escalations following design 3 of the Simon accelerated titration designs. LMP744 was administered intravenously daily on d1-5 in 28-day cycles (C), starting at dose level (DL) 1 (6 mg/m2/d). Responses and adverse events (AEs) were defined by RECIST 1.1 and CTCAE v4.0, respectively. Plasma concentrations were determined by LC-MS/MS with a validated assay over a range of 1-3,000 ng/mL. Paired biopsies (baseline and C1D2) were analyzed for DNA damage response markers (γH2AX, pNBS1, and Rad51) by IFA. Results: A total of 36 pts were enrolled, median age was 61 years (range 35-81), median prior lines of systemic treatment was 5 (range 2-10) and 26 pts (72%) had received prior TOP1 inhibitor therapy. One pt on DL6 (190 mg/m2/d) experienced a dose-limiting toxicity (DLT) of grade 3 hypokalemia during C1, reverting the study to a 3+3 design. MTD was established at DL6. Of 24 patients evaluable for response, 1 pt with small cell lung cancer on DL6 achieved a partial response (PR), remaining on treatment for 7C. 17 pts had stable disease (including 1 pt with an unconfirmed PR), and 6 had progressive disease. Median duration of response among pts with SD was 4.5C (range 1-28). A total of 6 pts on DL6 with colorectal cancer (n=4, all had received prior irinotecan), mesothelioma (n=1), and low-grade appendiceal mucinous neoplasm (n=1) remained on study for >6C. Treatment-related grade 3/4 AEs included leukopenia (n=9), anemia (n=4), dehydration (n=2), neutropenia (n=2) and nausea (n=2). Pharmacokinetic (PK) data for the first 17 patients suggests dose-linear PK, best fit to a 2-compartment model. Approximate half-life, clearance, central and peripheral volume values were 30 h, 90 L/h, 200 L, and 2500 L, respectively. Induction of γH2AX and Rad51 was measured in the pt with PR. Conclusions: The MTD of LMP744 was established at 190 mg/m2/d on d1-5 in a 28d C. The single agent activity of LMP744 in this heavily pretreated population was limited; however, 4 colorectal cancer pts had prolonged stabilization of disease with prior progression on irinotecan. Analyses of secondary and exploratory correlatives are ongoing. Clinical trial information: NCT03030417 .

Integrative transcriptomics and cell systems analyses reveal protective pathways controlled by Igfbp-3 in anthracycline-induced cardiotoxicity.

Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents; however, their use is hampered by subsequent cardiotoxicity risk. Our understanding of cardiomyocyte protective pathways activated following anthracycline-induced cardiotoxicity (AIC) remains incomplete. Insulin-like growth factor binding protein (IGFBP) 3 (Igfbp-3), the most abundant IGFBP family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cells. Whereas Igfbp-3 is induced by Dox in the heart, its role in AIC is ill-defined. We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human-induced pluripotent stem cell-derived cardiomyocytes. Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 reduces DNA damage, impedes topoisomerase IIβ expression (Top2β) which forms Top2β-Dox-DNA cleavage complex leading to DNA double-strand breaks (DSB), alleviates detyrosinated microtubule accumulation-a hallmark of increased cardiomyocyte stiffness and heart failure-and favorably affects contractility following Dox treatment. These results indicate that Igfbp-3 is induced by cardiomyocytes in an effort to mitigate AIC.

Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes

Topoisomerase IIIα (TOP3A) belongs to the conserved Type IA family of DNA topoisomerases. Here we report that human TOP3A is associated with DNA replication forks and that a “self-trapping” TOP3A mutant (TOP3A-R364W) generates cellular TOP3A DNA cleavage complexes (TOP3Accs). We show that trapped TOP3Accs that interfere with replication, induce DNA damage and genome instability. To elucidate how TOP3Accs are repaired, we explored the role of Spartan (SPRTN), the metalloprotease associated with DNA replication, which digests proteins forming DNA-protein crosslinks (DPCs). We find that SPRTN-deficient cells show elevated TOP3Accs, whereas overexpression of SPRTN lowers cellular TOP3Accs. SPRTN is deubiquitinated and epistatic with TDP2 in response to TOP3Accs. In addition, we found that MRE11 can excise TOP3Accs, and that cell cycle determines the preference for the SPRTN-TDP2 vs. the ATM-MRE11 pathways, in S vs. G2, respectively. Our study highlights the prevalence of TOP3Accs repair mechanisms to ensure normal DNA replication.

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases.

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIβ, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.

Regulation of the hypertonic stress response by the 3' mRNA cleavage and polyadenylation complex.

Maintenance of osmotic homeostasis is one of the most aggressively defended homeostatic set points in physiology. One major mechanism of osmotic homeostasis involves the upregulation of proteins that catalyze the accumulation of solutes called organic osmolytes. To better understand how osmolyte accumulation proteins are regulated, we conducted a forward genetic screen in Caenorhabditis elegans for mutants with no induction of osmolyte biosynthesis gene expression (Nio mutants). The nio-3 mutant encoded a missense mutation in cpf-2/CstF64, while the nio-7 mutant encoded a missense mutation in symk-1/Symplekin. Both cpf-2 and symk-1 are nuclear components of the highly conserved 3' mRNA cleavage and polyadenylation complex. cpf-2 and symk-1 block the hypertonic induction of gpdh-1 and other osmotically induced mRNAs, suggesting they act at the transcriptional level. We generated a functional auxin-inducible degron (AID) allele for symk-1 and found that acute, post-developmental degradation in the intestine and hypodermis was sufficient to cause the Nio phenotype. symk-1 and cpf-2 exhibit genetic interactions that strongly suggest they function through alterations in 3' mRNA cleavage and/or alternative polyadenylation. Consistent with this hypothesis, we find that inhibition of several other components of the mRNA cleavage complex also cause a Nio phenotype. cpf-2 and symk-1 specifically affect the osmotic stress response since heat shock-induced upregulation of a hsp-16.2::GFP reporter is normal in these mutants. Our data suggest a model in which alternative polyadenylation of 1 or more mRNAs is essential to regulate the hypertonic stress response.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Developments in Non-Intercalating Bacterial Topoisomerase Inhibitors: Allosteric and ATPase Inhibitors of DNA Gyrase and Topoisomerase IV.

Increases in antibiotic usage and antimicrobial resistance occurrence have caused a dramatic reduction in the effectiveness of many frontline antimicrobial treatments. Topoisomerase inhibitors including fluoroquinolones are broad-spectrum antibiotics used to treat a range of infections, which stabilise a topoisomerase-DNA cleavage complex via intercalation of the bound DNA. However, these are subject to bacterial resistance, predominantly in the form of single-nucleotide polymorphisms in the active site. Significant research has been undertaken searching for novel bioactive molecules capable of inhibiting bacterial topoisomerases at sites distal to the fluoroquinolone binding site. Notably, researchers have undertaken searches for anti-infective agents that can inhibit topoisomerases through alternate mechanisms. This review summarises work looking at the inhibition of topoisomerases predominantly through non-intercalating agents, including those acting at a novel allosteric site, ATPase domain inhibitors, and those offering unique binding modes and mechanisms of action.

Unraveling the molecular mechanism of type II topoisomerase poisons with magnetic tweezers.

Topoisomerases are an essential class of enzymes that resolve topological problems on DNA (e.g., supercoils, knots, and links) that arise during transcription, replication, and recombination. As part of their reaction cycle, type II topoisomerases transiently cleave double-stranded DNA and form a covalent protein-DNA cleavage complex. Typically, these cleavage complexes are tightly regulated in vivo because they are highly vulnerable and can generate cytotoxic DNA breaks, yet some of the most efficacious and broad-spectrum antibacterials are topoisomerase poisons that exploit this vulnerability by disrupting cleavage complex regulation. Due to their biological and clinical importance, the effects of these poisons have been studied extensively with a variety of in vitro ensemble assays. These assays have shown that antibacterials increase overall levels of cleavage complexes and inhibit enzyme activity. However, the kinetics of individual poisoned cleavage complexes under physiologically relevant conditions is unknown. Therefore, we carried out single-molecule magnetic tweezer measurements of E. coli topo IV activity on supercoiled DNA. In the presence of topoisomerase poisons, we observed long pauses in enzyme activity that correspond to poison-stabilized cleavage complexes. We studied the effects of DNA tension, as well as supercoil handedness and extent, determining poison on-rates, cleavage-complex lifetimes, and topoisomerase activity with several common antibacterials under physiologically relevant conditions. Together, these results provide new mechanistic insight into how topoisomerase poisons disrupt cleavage complex regulation that is likely to be relevant to their action in vivo.

ATM inhibitor KU60019 synergistically sensitizes lung cancer cells to topoisomerase II poisons by multiple mechanisms

Type II topoisomerases (TOP2) poisons represent one class of the most successful and widely prescribed chemotherapeutics, which is frontline therapy for a myriad of systemic cancers and solid tumors, including lymphomas, leukemias, and lung cancer. Despite this, treatment with this class of drugs induces unwanted side effects (including cardiovascular morbidity and secondary malignancies). Additionally, the emergence of drug resistance also greatly compromises the clinical use of these drugs. To enhance therapeutic efficiency while lowering unwanted side effects, new insights into effective combination therapy are required. In this study we found that KU60019, a novel, and highly specific ATM kinase inhibitor interferes with the association of ATM with TOP2β and stabilizes TOP2β-DNA cleavage complex, thereby impairing the repair of TOP2 poison-induced DSBs and contributes to genome stability, leading to accelerated cell death. In H1299 as well as in A549 lung cancer cell lines, biologically, KU60019 combined with VP-16 (one of the TOP2 poisons) synergistically suppressed the growth of cells and survival and triggered a much higher apoptosis rate. In summary, we provide a proof-of-concept strategy that ATM inhibitors combined with TOP2 poison would synergistically suppresses lung cancer cell survival as well as reduce DNA damage responses, thus may lowering the possibility of cardiotoxicity and secondary malignancy linked to therapy.

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

Nanoplatform for the Delivery of Topotecan in the Cancer Milieu: An Appraisal of its Therapeutic Efficacy

Chemotherapy has been the predominant treatment modality for cancer patients, but its overall performance is still modest. Difficulty in penetration of tumor tissues, a toxic profile in high doses, multidrug resistance in an array of tumor types, and the differential architecture of tumor cells as they grow are some of the bottlenecks associated with the clinical usage of chemotherapeutics. Recent advances in tumor biology understanding and the emergence of novel targeted drug delivery tools leveraging various nanosystems offer hope for developing effective cancer treatments. Topotecan is a topoisomerase I inhibitor that stabilizes the transient TOPO I-DNA cleavable complex, leading to single-stranded breaks in DNA. Due to its novel mechanism of action, TOPO is reported to be active against various carcinomas, namely small cell lung cancer, cervical cancer, breast cancer, and ovarian cancer. Issues of cross-resistance with numerous drugs, rapid conversion to its inactive form in biological systems, appended adverse effects, and higher water solubility limit its therapeutic efficacy in clinical settings. Topotecan nanoformulations offer several benefits for enhancing the therapeutic action of this significant class of chemotherapeutics. The likelihood that the target cancer cells will be exposed to the chemotherapeutic drug while in the drug-sensitive s-phase is increased due to the slow and sustained release of the chemotherapeutic, which could provide for a sustained duration of exposure of the target cancer cells to the bioavailable drug and result in the desired therapeutic outcome. This article explores nanoenabled active and passive targeting strategies and combinatorial therapy employing topotecan to ameliorate various cancers, along with a glimpse of the clinical studies utilizing the said molecule.

Bifunctional G-Quadruplex Aptamer Targeting Nucleolin and Topoisomerase 1: Antiproliferative Activity and Synergistic Effect of Conjugated Drugs.

As a counterpart to antibody-drug conjugates (ADCs), aptamer-drug conjugates (ApDCs) have been considered a promising strategy for targeted therapy due to the various benefits of aptamers. However, an aptamer merely serves as a targeting ligand in ApDCs, whereas the antibody enables the unexpected therapeutic efficacy of ADCs through antibody-dependent cellular cytotoxicity (ADCC). In this study, we developed a tumor-specific aptamer with an effector function and used it to confirm the feasibility of more potent ApDCs. First, we designed a nucleolin (NCL)-binding G-quadruplex (GQ) library based on the ability of NCL to bind to telomeric sequences. We then identified a bifunctional GQ aptamer (BGA) inhibiting the catalytic activity of topoisomerase 1 (TOP1) by forming an irreversible cleavage complex. Our BGA specifically targeted NCL-positive MCF-7 cells, exhibiting antiproliferative activity, and this suggested that tumor-specific therapeutic aptamers can be developed by using a biased library to screen aptamer candidates for functional targets. Finally, we utilized DM1, which has a synergistic interaction with TOP1 inhibitors, as a conjugated drug. BGA-DM1 exerted an anticancer effect 20-fold stronger than free DM1 and even 10-fold stronger than AS1411 (NCL aptamer)-DM1, highlighting our approach to develop synergistic ApDCs. Therefore, we anticipate that our library might be utilized for the identification of aptamers with effector functions. Furthermore, by employing such aptamers and appropriate drugs, synergistic ApDCs can be developed for targeted cancer therapy in a manner distinct from how ADCs exhibit additional therapeutic efficacy.

Birth of a poly(A) tail: mechanisms and control of mRNA polyadenylation.

During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the transcribing RNA polymerase II and a polyadenosine (poly(A)) tail is added to the newly formed 3'-end. These biochemical reactions might appear simple at first sight (endonucleolytic RNA cleavage and synthesis of a homopolymeric tail), but their catalysis requires a multi-faceted enzymatic machinery, the cleavage and polyadenylation complex (CPAC), which is composed of more than 20 individual protein subunits. The activity of CPAC is further orchestrated by Poly(A) Binding Proteins (PABPs), which decorate the poly(A) tail during its synthesis and guide the mRNA through subsequent gene expression steps. Here, we review the structure, molecular mechanism, and regulation of eukaryotic mRNA 3'-end processing machineries with a focus on the polyadenylation step. We concentrate on the CPAC and PABPs from mammals and the budding yeast, Saccharomyces cerevisiae, because these systems are the best-characterized at present. Comparison of their functions provides valuable insights into the principles of mRNA 3'-end processing.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

Mitochondrial topoisomerase 1 inhibition induces topological DNA damage and T cell dysfunction in patients with chronic viral infection.

T cells are crucial for controlling viral infections; however, the mechanisms that dampen their responses during viral infections remain incompletely understood. Here, we studied the role and mechanisms of mitochondrial topoisomerase 1 (Top1mt) inhibition in mitochondrial dysfunction and T cell dysregulation using CD4 T cells from patients infected with HCV or HIV and compared it with CD4 T cells from healthy individuals following treatment with Top1 inhibitor - camptothecin (CPT). We found that Top1mt protein levels and enzymatic activity are significantly decreased, along with Top1 cleavage complex (Top1cc) formation, in mitochondria of CD4 T cells from HCV- and HIV-infected patients. Notably, treatment of healthy CD4 T cells with CPT caused similar changes, including inhibition of Top1mt, accumulation of Top1cc in mitochondria, increase in PARP1 cleavage, and decrease in mtDNA copy numbers. These molecular changes resulted in mitochondrial dysfunction, T cell dysregulation, and programmed cell death through multiple signaling pathways, recapitulating the phenotype we detected in CD4 T cells from HCV- and HIV-infected patients. Moreover, treatment of CD4 T cells from HCV or HIV patients with CPT further increased cellular and mitochondrial reactive oxygen species (ROS) production and cell apoptosis, demonstrating a critical role for Top1 in preventing mtDNA damage and cell death. These results provide new insights into the molecular mechanisms underlying immune dysregulation during viral infection and indicate that Top1 inhibition during chronic HCV or HIV infection can induce mtDNA damage and T cell dysfunction. Thus, reconstituting Top1mt protein may restore the mtDNA topology and T cell functions in humans with chronic viral infection.

Novel and Structurally Diversified Bacterial DNA Gyrase Inhibitors Discovered through a Fluorescence-Based High-Throughput Screening Assay.

Bacterial DNA gyrase, a type IIA DNA topoisomerase that plays an essential role in bacterial DNA replication and transcription, is a clinically validated target for discovering and developing new antibiotics. In this article, based on a supercoiling-dependent fluorescence quenching (SDFQ) method, we developed a high-throughput screening (HTS) assay to identify inhibitors targeting bacterial DNA gyrase and screened the National Institutes of Health's Molecular Libraries Small Molecule Repository library containing 370,620 compounds in which 2891 potential gyrase inhibitors have been identified. According to these screening results, we acquired 235 compounds to analyze their inhibition activities against bacterial DNA gyrase using gel- and SDFQ-based DNA gyrase inhibition assays and discovered 155 new bacterial DNA gyrase inhibitors with a wide structural diversity. Several of them have potent antibacterial activities. These newly discovered gyrase inhibitors include several DNA gyrase poisons that stabilize the gyrase-DNA cleavage complexes and provide new chemical scaffolds for the design and synthesis of bacterial DNA gyrase inhibitors that may be used to combat multidrug-resistant bacterial pathogens. Additionally, this HTS assay can be applied to screen inhibitors against other DNA topoisomerases.

The androgen receptor couples promoter recruitment of RNA processing factors to regulation of alternative polyadenylation at the 3' end of transcripts.

Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.

The Potential of Topoisomerase Inhibitor-Based Antibody-Drug Conjugates.

DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody–drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs.