ABSTRACT
Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Nucleotides, Cyclic , Ribonucleases , Ribonucleases/metabolism , Allosteric Regulation , RNA/metabolismABSTRACT
In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.
Subject(s)
Bacterial Proteins , CRISPR-Associated Proteins , CRISPR-Cas Systems , Adenine Nucleotides/metabolism , Adenine Nucleotides/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/genetics , Models, Molecular , Oligoribonucleotides , Protein Binding , Protein Domains , Protein Multimerization , Ribonucleases/metabolism , Ribonucleases/chemistry , Ribonucleases/geneticsABSTRACT
The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.
Subject(s)
CRISPR-Associated Proteins , Clustered Regularly Interspaced Short Palindromic Repeats , Second Messenger Systems , Signal Transduction , Endonucleases/metabolism , Ions/metabolism , CRISPR-Cas Systems , CRISPR-Associated Proteins/metabolismABSTRACT
The quantitative detection of drug-resistance mutations in Mycobacterium tuberculosis (MTB) is critical for determining the drug resistance status of a sample. We developed a drop-off droplet digital PCR (ddPCR) assay targeting all major isoniazid (INH)-resistant mutations. The ddPCR assay consisted of three reactions: reaction A detects mutations at katG S315; reaction B detects inhA promoter mutations; and reaction C detects ahpC promoter mutations. All reactions could quantify 1%-50% of mutants in the presence of the wild-type, ranging from 100 to 50,000 copies/reaction. Clinical evaluation with 338 clinical isolates yielded clinical sensitivity of 94.5% (95% confidence interval [CI] = 89.1%-97.3%) and clinical specificity of 97.6% (95% CI = 94.6%-99.0%) compared with the traditional drug susceptibility testing (DST). Further clinical evaluation using 194 nucleic acid-positive MTB sputum samples revealed clinical sensitivity of 87.8% (95% CI = 75.8%-94.3%) and clinical specificity of 96.5% (95% CI = 92.2%-98.5%) in comparison with DST. All the mutant and heteroresistant samples detected by the ddPCR assay but susceptible by DST were confirmed by combined molecular assays, including Sanger sequencing, mutant-enriched Sanger sequencing and a commercial melting curve analysis-based assay. Finally, the ddPCR assay was used to monitor longitudinally the INH-resistance status and the bacterial load in nine patients undergoing treatment. Overall, the developed ddPCR assay could be an indispensable tool for quantification of INH-resistant mutations in MTB and bacterial loads in patients.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Humans , Isoniazid/pharmacology , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , Microbial Sensitivity Tests , Sensitivity and Specificity , Polymerase Chain Reaction , Mutation , Tuberculosis, Multidrug-Resistant/microbiology , Bacterial Proteins/geneticsABSTRACT
The MUS81-EME1/2 structure-specific endonucleases play a crucial role in the processing of stalled replication forks and recombination intermediates, and have been recognized as an attractive drug target to potentiate the anti-cancer efficacy of DNA-damaging agents. Currently, no bioactive small-molecule inhibitors of MUS81 are available. Here, we performed a high-throughput small-molecule inhibitors screening, using the FRET-based DNA cleavage assay. From 7920 compounds, we identified dyngo-4a as a potent inhibitor of MUS81 complexes. Dyngo-4a effectively inhibits the endonuclease activities of both MUS81-EME1 and MUS81-EME2 complexes, with IC50 values of 0.57 µM and 2.90 µM, respectively. Surface plasmon resonance (SPR) and electrophoretic mobility shift assay (EMSA) assays reveal that dyngo-4a directly binds to MUS81 complexes (KD â¼ 0.61 µM) and prevents them from binding to DNA substrates. In HeLa cells, dyngo-4a significantly suppresses bleomycin-triggered H2AX serine 139 phosphorylation (γH2AX). Together, our results demonstrate that dyngo-4a is a potent MUS81 inhibitor, which could be further developed as a potentially valuable chemical tool to explore more physiological roles of MUS81 in the cells.
Subject(s)
DNA-Binding Proteins , Endodeoxyribonucleases , Humans , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , DNA-Binding Proteins/chemistry , HeLa Cells , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Endonucleases/metabolism , DNA Replication , DNA/metabolismABSTRACT
The Holliday junction (HJ) branch migrator RuvAB complex plays a fundamental role during homologous recombination and DNA damage repair, and therefore, is an attractive target for the treatment of bacterial pathogens. Pseudomonas aeruginosa (P. aeruginosa, Pa) is one of the most common clinical opportunistic bacterial pathogens, which can cause a series of life-threatening acute or chronic infections. Here, we performed a high throughput small-molecule screening targeting PaRuvAB using the FRET-based HJ branch migration assay. We identified that corilagin, bardoxolone methyl (BM) and 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1) could efficiently inhibit the branch migration activity of PaRuvAB, with IC50 values of 0.40 ± 0.04 µM, 0.38 ± 0.05 µM and 4.64 ± 0.27 µM, respectively. Further biochemical and molecular docking analyses demonstrated that corilagin directly bound to PaRuvB at the ATPase domain, and thus prevented ATP hydrolysis. In contrast, BM and SKQ1 acted through blocking the interactions between PaRuvA and HJ DNA. Finally, these compounds were shown to increase the susceptibility of P. aeruginosa to UV-C irradiation. Our work, for the first time, reports the small-molecule inhibitors of RuvA and RuvB from any species, providing valuable chemical tools to dissect the functional role of each individual protein in vivo.
Subject(s)
Escherichia coli Proteins , Adenosine Triphosphate/metabolism , Bacterial Proteins/metabolism , DNA Helicases , DNA Repair , DNA, Bacterial , DNA, Cruciform/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Glucosides , Hydrolyzable Tannins , Molecular Docking Simulation , Oleanolic Acid/analogs & derivatives , Pseudomonas aeruginosa/metabolism , Recombination, GeneticABSTRACT
Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin, and by phosphorylation of both the enzyme and substrates. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here we report crystal structures of the separase protease domain from the thermophilic fungus Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study, mutating two securin residues in a conserved motif that partly matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin.
Subject(s)
Cell Cycle Proteins/metabolism , Chaetomium/enzymology , Chromosomal Proteins, Non-Histone/metabolism , Separase/chemistry , Separase/metabolism , Amino Acid Sequence , Binding, Competitive/drug effects , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Chromosome Segregation , Crystallography, X-Ray , Models, Molecular , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Proteolysis , Proto-Oncogene Proteins/metabolism , Securin/chemistry , Securin/genetics , Securin/metabolism , Securin/pharmacology , Separase/antagonists & inhibitors , Structure-Activity Relationship , Substrate Specificity/genetics , Cohesins , Polo-Like Kinase 1ABSTRACT
Invasive fungal infections including Candidiasis and Aspergillosis are associated with considerable morbidity and mortality in immunocompromised individuals, such as cancer patients. Aurora B is a key mitotic kinase required for the cell division of eukaryotes from fungus to man. Here, we identified a novel Aurora B inhibitor GSK650394 that can inhibit the recombinant Aurora B from human and Aspergillus fumigatus, with IC50 values of 5.68 and 1.29 µM, respectively. In HeLa and HepG2 cells, GSK650394 diminishes the endogenous Aurora B activity and causes cell cycle arrest in G2/M phase. Further cell-based assays demonstrate that GSK650394 efficiently suppresses the proliferation of both cancer cells and Aspergillus fumigatus. Finally, the molecular docking calculation and site-directed mutagenesis analyses reveal the molecular mechanism of Aurora B inhibition by GSK650394. Our work is expected to provide new insight into the combinational therapy of cancer and Aspergillus fumigatus infection.
Subject(s)
Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Aspergillus fumigatus/drug effects , Aurora Kinase B/antagonists & inhibitors , Benzoates/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Drug Discovery , Antifungal Agents/chemistry , Antineoplastic Agents/chemistry , Aurora Kinase B/metabolism , Benzoates/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
The Holliday junction (HJ) is a key intermediate during homologous recombination and DNA double-strand break repair. Timely HJ resolution by resolvases is critical for maintaining genome stability. The mechanisms underlying sequence-specific substrate recognition and cleavage by resolvases remain elusive. The monokaryotic chloroplast 1 protein (MOC1) specifically cleaves four-way DNA junctions in a sequence-specific manner. Here, we report the crystal structures of MOC1 from Zea mays, alone or bound to HJ DNA. MOC1 uses a unique ß-hairpin to embrace the DNA junction. A base-recognition motif specifically interacts with the junction center, inducing base flipping and pseudobase-pair formation at the strand-exchanging points. Structures of MOC1 bound to HJ and different metal ions support a two-metal ion catalysis mechanism. Further molecular dynamics simulations and biochemical analyses reveal a communication between specific substrate recognition and metal ion-dependent catalysis. Our study thus provides a mechanism for how a resolvase turns substrate specificity into catalytic efficiency.
Subject(s)
Chloroplasts/metabolism , Holliday Junction Resolvases/metabolism , Plant Proteins/metabolism , Holliday Junction Resolvases/chemistry , Molecular Dynamics Simulation , Protein Conformation , Substrate SpecificityABSTRACT
The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5'-TTC-3'. PaRuvC uses Mg2+ as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0-9.0. Elevated temperatures (37-60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system.
Subject(s)
Crystallography, X-Ray/methods , DNA, Cruciform/metabolism , Holliday Junction Resolvases/chemistry , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Biocatalysis , Holliday Junction Resolvases/genetics , Holliday Junction Resolvases/metabolism , Homologous Recombination , Mutagenesis , Protein Multimerization , Protein StabilityABSTRACT
Fibrinolysis is a process responsible for the dissolution of formed thrombi to re-establish blood flow after thrombus formation. Plasminogen activator inhibitor-1 (PAI-1) inhibits urokinase-type and tissue-type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI-1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI-1 and identified a potent PAI-1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active-site mutation (S195A) and four additional mutations (G37bR-R217L-C122A-N145Q). PAItrap inhibits human recombinant PAI-1 with high potency (Kd = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI-1. In addition, PAItrap inhibits both human and murine PAI-1, allowing the evaluation in murine models. In vivo, using a laser-induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI-1 inhibitors using inactivated urokinase.
Subject(s)
Peptide Fragments/pharmacology , Plasminogen Activator Inhibitor 1/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Catalytic Domain , Crystallography, X-Ray , Disease Models, Animal , Fibrinolysis , Humans , Inhibitory Concentration 50 , Kinetics , Mice, Inbred C57BL , Models, Molecular , Mutant Proteins/chemistry , Peptide Fragments/chemistry , Protein Binding , Thrombosis/pathology , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/pharmacologyABSTRACT
The natural product embelin was found to have PAI-1 inhibitory activity with the IC50 value of 4.94µM. Based on the structure of embelin, a series of analogues were designed, synthesized, and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory potency with the IC50 value of 0.18µM.
Subject(s)
Benzoquinones/chemistry , Benzoquinones/pharmacology , Plasminogen Activator Inhibitor 1/chemistry , Plasminogen Activator Inhibitor 1/metabolism , Benzoquinones/chemical synthesis , Binding Sites , Drug Design , Models, Molecular , Structure-Activity RelationshipABSTRACT
Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase.
Subject(s)
DNA, Cruciform , DNA, Cruciform/metabolism , DNA, Cruciform/chemistry , DNA, Cruciform/genetics , Metals/metabolism , Metals/chemistry , Holliday Junction Resolvases/metabolism , Holliday Junction Resolvases/chemistry , Catalytic Domain , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Crystallography, X-Ray , Ions/metabolism , DNA Breaks, Double-Stranded , Models, Molecular , Allosteric RegulationABSTRACT
Holliday junction (HJ) is a four-way structured DNA intermediate in processes of homologous recombination and DNA double-stranded break (DSB) repair. In bacteria, HJs are processed via either the RuvABC or RecG-dependent pathways. In addition, RecG also plays a critical role in the reactivation of stalled replication forks, making it an attractive target for antibacterial drug development. Here, we conducted a high-throughput screening targeting the RecG helicase from a common opportunistic pathogen Pseudomonas aeruginosa (Pa). From a library containing 7920 compounds, we identified Ebselen and TPI-1 (2',5'-Dichloro-[1,1'-biphenyl]-2,5-dione) as two potent PaRecG inhibitors, with IC50 values of 0.31 ± 0.02 µM and 1.16 ± 0.06 µM, respectively. Further biochemical analyses suggested that both Ebselen and TPI-1 inhibited the ATPase activity of PaRecG, and hindered its binding to HJ DNA with high selectivity. These compounds, when combined with our previously reported RuvAB inhibitors, resulted in more severe DNA repair defects than the individual treatment, and potently enhanced the susceptibility of P. aeruginosa to the DNA damage agents. This work reports novel small molecule inhibitors of RecG, offering valuable chemical tools for advancing our understanding of RecG's function and mechanism. Additionally, these inhibitors might be further developed as promising antibacterial agents in the fight against P. aeruginosa infections.
Subject(s)
Escherichia coli Proteins , Isoindoles , Organoselenium Compounds , Pseudomonas aeruginosa , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Bacterial Proteins , DNA Helicases/metabolism , DNA Repair , DNA Damage , DNA, Cruciform , DNA ReplicationABSTRACT
Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC3.4.22.49), a cysteine protease, plays a critical role in mitosis by cleaving the kleisin subunit of cohesin, thereby presenting a promising target for cancer therapy. However, challenges in isolating active human separase suitable for high-throughput screening have limited the identification of effective inhibitors. Here, we conducted a high-throughput screening of small-molecule inhibitors using the protease domain of Chaetomium thermophilum separase (ctSPD), which not only shares significant sequence similarity with human separase but is also readily available. After conducting a primary screening of a library containing 9,172 compounds and subsequent validation using human separase, we identified walrycin B and its analogs, toxoflavin, 3-methyltoxoflavin, and 3-phenyltoxoflavin, as potent inhibitors of human separase. Subsequent microscale thermophoresis assays and molecular dynamics simulations revealed that walrycin B binds to the active site of separase and competes with substrates for binding. Additionally, cell-based studies showed that walrycin B and its analogs effectively induce cell cycle arrest at the M phase, activate apoptosis, and ultimately lead to cell death in mitosis. Finally, in a mouse xenograft model, walrycin B exhibited significant antitumor efficacy with minimal side effects. Together, these findings highlight the therapeutic potential of walrycin B for cancer treatment and its utility as a chemical tool in future studies involving separase.
ABSTRACT
Sleep is crucial for wellness, and emerging research reveals a profound connection to gut microbiota. This review explores the bidirectional relationship between gut microbiota and sleep, exploring the mechanisms involved and the therapeutic opportunities it presents. The gut-brain axis serves as a conduit for the crosstalk between gut microbiota and the central nervous system, with dysbiosis in the microbiota impairing sleep quality and vice versa. Diet, circadian rhythms, and immune modulation all play a part. Specific gut bacteria, like Lactobacillus and Bifidobacterium, enhance sleep through serotonin and gamma-aminobutyric acid production, exemplifying direct microbiome influence. Conversely, sleep deprivation reduces beneficial bacteria, exacerbating dysbiosis. Probiotics, prebiotics, postbiotics, and fecal transplants show therapeutic potential, backed by animal and human research, yet require further study on safety and long-term effects. Unraveling this intricate link paves the way for tailored sleep therapies, utilizing microbiome manipulation to improve sleep and health. Accelerated research is essential to fully tap into this promising field for sleep disorder management.
ABSTRACT
The emergence of chemoresistance poses a significant challenge to the efficacy of DNA-damaging agents in cancer treatment, in part due to the inherent DNA repair capabilities of cancer cells. The Ku70/80 protein complex (Ku) plays a central role in double-strand breaks (DSBs) repair through the classical non-homologous end joining (c-NHEJ) pathway, and has proven to be one of the most promising drug target for cancer treatment when combined with radiotherapy or chemotherapy. In this study, we conducted a high-throughput screening of small-molecule inhibitors targeting the Ku complex by using a fluorescence polarization-based DNA binding assay. From a library of 11,745 small molecules, UMI-77 was identified as a potent Ku inhibitor, with an IC50 value of 2.3 µM. Surface plasmon resonance and molecular docking analyses revealed that UMI-77 directly bound the inner side of Ku ring, thereby disrupting Ku binding with DNA. In addition, UMI-77 also displayed potent inhibition against MUS81-EME1, a key player in homologous recombination (HR), demonstrating its potential for blocking both NHEJ- and HR-mediated DSB repair pathways. Further cell-based studies showed that UMI-77 could impair bleomycin-induced DNA damage repair, and significantly sensitized multiple cancer cell lines to the DNA-damaging agents. Finally, in a mouse xenograft tumor model, UMI-77 significantly enhanced the chemotherapeutic efficacy of etoposide with little adverse physiological effects. Our work offers a new avenue to combat chemoresistance in cancer treatment, and suggests that UMI-77 could be further developed as a promising candidate in cancer treatment.
Subject(s)
Antineoplastic Agents , Ku Autoantigen , Humans , Ku Autoantigen/metabolism , Animals , Cell Line, Tumor , Mice , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , DNA Damage/drug effects , Molecular Docking Simulation , Xenograft Model Antitumor Assays , DNA End-Joining Repair/drug effects , Etoposide/pharmacology , Drug Discovery , DNA Breaks, Double-Stranded/drug effects , Drug Resistance, Neoplasm/drug effectsABSTRACT
AIM: des-O-methyllasiodiplodin (DML) from Cerbera manghas has shown antagonistic activity against mineralocorticoid receptor (MR). Considering the involvement of MR in the insulin tolerance, we attempted to investigate the potential of DML in the treatment of type 2 diabetes mellitus (T2DM). METHODS: Surface plasmon resonance (SPR) technology and reporter gene-based assays were used to study protein-small molecule interactions. HepG2 and 3T3-L1 cells were treated with H2O2 (0.2 mmol/L) or aldosterone (10 nmol/L) for 24 h. The expression of MR in the cells was downregulated with siRNA. The anti-inflammatory effect of the compound was evaluated, respectively. db/db mice were administered DML (30 mg·kg(-1)·d(-1)) for 4 weeks. Serum biochemical parameters and insulin sensitivity were examined. The expression levels of pro-inflammatory cytokines (MCP-1, TNF-α and IL-6) and ROS-related genes (NADPH p47 subunit and transcriptional factor PU.1) in adipose tissues and livers were analyzed using real-time RT-PCR. RESULTS: In HepG2 and 3T3-L1 cells, both H2O2 and aldosterone markedly stimulates the expression of MCP-1, TNFα, IL-6, p47 and PU.1 genes. Co-treatment with DML (10 µmol/L) significantly reduced the H2O2- or aldosterone-induced expression of these genes. SPR-based assay confirmed the antagonistic activity of DML against the interaction between SRC-1 and MR-LBD. Furthermore, DML decreased aldosterone-induced MR transcriptional activity in a dose-dependent manner. Downregulation of MR with siRNA in the cells prevented or significantly attenuated aldosterone-stimulated expression of these genes, whereas DML did no longer affect the expression of these genes except that of IL-6. Oral administration of DML effectively reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) in db/db mice. The treatment also rectified the expression of pro-inflammatory factor and ROS-related genes in db/db mice. CONCLUSION: DML effectively lowers the blood glucose level in db/db mice possibly via ameliorating the expression of obesity-related pro-inflammatory cytokines, highlighting the potential of the marine natural product as a drug lead for the treatment of metabolic disorders.
Subject(s)
Apocynaceae/chemistry , Blood Glucose/drug effects , Inflammation/drug therapy , Macrolides/pharmacology , 3T3-L1 Cells , Aldosterone/pharmacology , Animals , Cytokines/metabolism , Diabetes Mellitus, Type 2/drug therapy , Dose-Response Relationship, Drug , Genes, Reporter , Glycated Hemoglobin/metabolism , Hep G2 Cells , Humans , Hydrogen Peroxide/pharmacology , Inflammation/pathology , Macrolides/administration & dosage , Male , Mice , Obesity/drug therapy , Obesity/physiopathology , Reverse Transcriptase Polymerase Chain Reaction , Surface Plasmon ResonanceABSTRACT
The DNA repair gene X-ray cross-complementary group 4 (XRCC4), an important caretaker of the overall genome stability, is thought to play a major role in human tumorigenesis. We investigated the association between an important polymorphic variant of this gene at codon 247 (rs373409) and diffusely infiltrating astrocytoma (DIA) risk and prognosis. This hospital-based case-control study investigated this association in the Guangxi population. In total, 242 cases with DIA and 358 age-, sex-, and race-matched healthy controls were genotyped using TaqMan-PCR technique. We found a significant difference in the frequency of XRCC4 genotypes between cases and controls. Compared with the homozygote of XRCC4 codon 247 Ala alleles (XRCC4-AA), the genotypes of XRCC4 codon 247 Ser alleles (namely XRCC4-AS or -SS) increased DIA risk (odds ratios [OR], 1.82 and 2.89, respectively). Furthermore, XRCC4 polymorphism was correlated with tumor dedifferentiation of DIA (r = 0.261, p < 0.01). Additionally, this polymorphism modified the overall survival of DIA patients (the median survival times were 26, 14, and 8 months for patients with XRCC4-AA, -AS, and -SS, respectively). Like tumor grade, XRCC4 codon 247 polymorphism was an independent prognostic factor influencing the survival of DIA. These results suggest that XRCC4 codon 247 polymorphism may be associated with DIA risk and prognosis among the Guangxi population.
Subject(s)
Astrocytoma/genetics , Brain Neoplasms/genetics , DNA-Binding Proteins/genetics , Polymorphism, Single Nucleotide , Adult , Age Factors , Aged , Alleles , Astrocytoma/diagnosis , Astrocytoma/mortality , Brain Neoplasms/diagnosis , Brain Neoplasms/mortality , Case-Control Studies , Codon , DNA-Binding Proteins/metabolism , Female , Gene Frequency , Genotype , Homozygote , Humans , Male , Middle Aged , Neoplasm Staging , Prognosis , Risk FactorsABSTRACT
Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa (P. aeruginosa, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa. The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal ß-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA.