RESUMO
Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds.
Assuntos
Instabilidade de Microssatélites , Proteólise , Helicase da Síndrome de Werner , Humanos , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Instabilidade de Microssatélites/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.
Assuntos
Replicação do DNA , Instabilidade Genômica , Helicase da Síndrome de Werner , Síndrome de Werner , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Humanos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Animais , Telômero/metabolismo , Telômero/genéticaRESUMO
Whether small nucleolar RNAs (snoRNAs) are involved in the regulation of liver cancer stem cells (CSCs) self-renewal and serve as therapeutic targets remains largely unclear. Here we show that a functional snoRNA (SNORD88B) is robustly expressed in Hepatocellular carcinoma (HCC) tumors and liver CSCs. SNORD88B deficiency abolishes the self-renewal of liver CSCs and hepatocarcinogenesis. Mechanistically, SNORD88B anchors WRN in the nucleolus, promoting XRCC5 interacts with STK4 promoter to suppress its transcription, leading to inactivation of Hippo signaling. Moreover, low expression of STK4 and high expression of XRCC5 are positively correlated with HCC poor prognosis. Additionally, snord88b knockout suppresses mouse liver tumorigenesis. Notably, co-administration of SNORD88B antisense oligonucleotides (ASOs) with MST1 agonist adapalene (ADA) exert synergistic antitumor effects and increase overall murine survival. Our findings delineate that SNORD88B drives self-renewal of liver CSCs and accelerates HCC tumorigenesis via non-canonical mechanism, providing potential targets for liver cancer therapy by eliminating liver CSCs.
Assuntos
Carcinogênese , Carcinoma Hepatocelular , Neoplasias Hepáticas , Células-Tronco Neoplásicas , RNA Nucleolar Pequeno , Animais , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Camundongos , RNA Nucleolar Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , Carcinogênese/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Nucléolo Celular/metabolismo , Linhagem Celular Tumoral , Autorrenovação Celular , Regulação Neoplásica da Expressão Gênica , Masculino , Via de Sinalização Hippo , Oligonucleotídeos Antissenso/farmacologia , Transdução de SinaisRESUMO
Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.
Assuntos
Cromatina , Proteínas Inibidoras de STAT Ativados , Proteína com Valosina , Helicase da Síndrome de Werner , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Humanos , Animais , Cromatina/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Camundongos , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Instabilidade de Microssatélites , Proteólise/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , FemininoRESUMO
The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.
Assuntos
Cricetulus , Replicação do DNA , Autoantígeno Ku , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Animais , Células CHO , Humanos , Cricetinae , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Reparo do DNA por Junção de Extremidades , Ligação Proteica , Camptotecina/farmacologia , Hidroxiureia/farmacologiaRESUMO
Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.
Assuntos
Envelhecimento , Modelos Animais de Doenças , Fígado , Proteômica , Caracteres Sexuais , Helicase da Síndrome de Werner , Síndrome de Werner , Animais , Masculino , Feminino , Síndrome de Werner/genética , Fígado/metabolismo , Camundongos , Envelhecimento/imunologia , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Assuntos
Antineoplásicos , Descoberta de Drogas , Inibidores Enzimáticos , Instabilidade de Microssatélites , Neoplasias , Mutações Sintéticas Letais , Helicase da Síndrome de Werner , Animais , Feminino , Humanos , Camundongos , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Dano ao DNA/efeitos dos fármacos , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Domínios Proteicos , Reprodutibilidade dos Testes , Supressão Genética , Mutações Sintéticas Letais/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Assuntos
Regulação Alostérica , Descoberta de Drogas , Inibidores Enzimáticos , Proteômica , Helicase da Síndrome de Werner , Animais , Feminino , Humanos , Masculino , Camundongos , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Cisteína/efeitos dos fármacos , Cisteína/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Instabilidade de Microssatélites , Modelos Moleculares , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Morte Celular/efeitos dos fármacos , Trifosfato de Adenosina/metabolismoRESUMO
Purified translesion synthesis (TLS) DNA polymerases (Pols) replicate through DNA lesions with a low fidelity; however, TLS operates in a predominantly error-free manner in normal human cells. To explain this incongruity, here we determine whether Y family Pols, which play an eminent role in replication through a diversity of DNA lesions, are incorporated into a multiprotein ensemble and whether the intrinsically high error rate of the TLS Pol is ameliorated by the components in the ensemble. To this end, we provide evidence for an indispensable role of Werner syndrome protein (WRN) and WRN-interacting protein 1 (WRNIP1) in Rev1-dependent TLS by Y family Polη, Polι, or Polκ and show that WRN, WRNIP1, and Rev1 assemble together with Y family Pols in response to DNA damage. Importantly, we identify a crucial role of WRN's 3' â 5' exonuclease activity in imparting high fidelity on TLS by Y family Pols in human cells, as the Y family Pols that accomplish TLS in an error-free manner manifest high mutagenicity in the absence of WRN's exonuclease function. Thus, by enforcing high fidelity on TLS Pols, TLS mechanisms have been adapted to safeguard against genome instability and tumorigenesis.
Assuntos
DNA Polimerase Dirigida por DNA , Síntese de DNA Translesão , Helicase da Síndrome de Werner , Humanos , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo , Síntese de DNA Translesão/genética , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
Microsatellite instability (MSI) is a relatively common feature associated with multiple cancers, and Werner syndrome (WRN) ATP-dependent helicase has been recognized as a novel target for treating MSI cancers, such as colorectal cancer. A small-molecule inhibitor targeting WRN would be a promising strategy for treating colorectal cancer with high MSI expression. In this study, we employed a computer-assisted drug discovery strategy to screen over 30,000 natural product molecules. By using a combination of docking, ligand efficiency, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), and thermodynamic integration (TI) calculations, we identified MOL008980, MOL010740, MOL011832, T4743, TN1166, and TNP-002173 as potential WRN inhibitors. Subsequent molecular dynamics simulation revealed that these screened natural products possessed better binding dynamic characteristics than ATP substrate and were capable of inhibiting the dynamic process of WRN, making them potential strong ATP competitive inhibitors. In conclusion, our computational approach revealed potential WRN inhibitors from a natural product database, providing a theoretical basis for future research.
Assuntos
Produtos Biológicos , Neoplasias Colorretais , Síndrome de Werner , Humanos , Helicase da Síndrome de Werner/metabolismo , Trifosfato de Adenosina , Produtos Biológicos/farmacologiaRESUMO
The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.
Assuntos
DNA Helicases , Tiadiazóis , Ureia , DNA Helicases/metabolismo , Helicase da Síndrome de Werner/metabolismoRESUMO
Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.
Assuntos
Instabilidade de Microssatélites , Neoplasias , Tiofenos , Humanos , Cicloexanonas , Neoplasias/tratamento farmacológico , Neoplasias/genética , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/metabolismo , Tiofenos/química , Tiofenos/farmacologiaRESUMO
Leukemia is a type of malignant hematological disease that is generally resistant to chemotherapy and has poor therapeutic outcomes. Werner (WRN) DNA helicase, an important member of the RecQ family of helicases, plays an important role in DNA repair and telomere stability maintenance. WRN gene dysfunction leads to premature aging and predisposes humans to various types of cancers. However, the biological function of WRN in cancer remains unknown. In this study, the expression of this RecQ family helicase was investigated in different types of leukemia cells, and the leukemia cell line K562 with high WRN expression was selected to construct a WRN knockdown cell line. The results showed that WRN knockdown inhibited leukemia occurrence and development by regulating the proliferation, cell cycle, differentiation, and aging of cells and other biological processes. The results of transcriptome sequencing revealed that WRN promoted the sensitivity of leukemia cells to the DNA damage inducer Etoposide by regulating cell cycle-related proteins, such as CDC2, cyclin B1, p16, and p21, as well as key proteins in DNA damage repair pathways, such as p53, RAD50, RAD51, and MER11. Our findings show that WRN helicase is a promising potential target for leukemia treatment, providing new ideas for the development of targeted drugs against leukemia.
Assuntos
Exodesoxirribonucleases , Leucemia , Humanos , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Ciclo Celular/genética , Reparo do DNA , Dano ao DNA , Leucemia/genéticaRESUMO
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.
Assuntos
Replicação do DNA , Neoplasias , Humanos , Replicação do DNA/genética , DNA Helicases/metabolismo , Repetições de Microssatélites , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 â¼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.
Assuntos
Neoplasias , Síndrome de Werner , Humanos , Exodesoxirribonucleases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Ensaios de Triagem em Larga Escala , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/metabolismoRESUMO
Helicases are crucial enzymes in DNA and RNA metabolism and function by unwinding particular nucleic acid structures. However, most convenient and high-throughput helicase assays are limited to the typical duplex DNA. Herein, we developed an immunosorbent assay to monitor the Werner syndrome (WRN) helicase unwinding a wide range of DNA structures, such as a replication fork, a bubble, Holliday junction, G-quadruplex and hairpin. This assay could sensitively detect the unwinding of DNA structures with detection limits around 0.1 nM, and accurately monitor the substrate-specificity of WRN with a comparatively less time-consuming and high throughput process. Remarkably, we have established that this new assay was compatible in evaluating helicase inhibitors and revealed that the inhibitory effect was substrate-dependent, suggesting that diverse substrate structures other than duplex structures should be considered in discovering new inhibitors. Our study provided a foundational example for using this new assay as a powerful tool to study helicase functions and discover potent inhibitors.
Assuntos
RecQ Helicases , Síndrome de Werner , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismo , Imunoadsorventes , Replicação do DNA , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Exodesoxirribonucleases/metabolismo , DNA/química , Síndrome de Werner/genéticaRESUMO
Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168-333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168-333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168-333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.
Assuntos
Exodesoxirribonucleases , Proteína 2 de Ligação a Repetições Teloméricas , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Exodesoxirribonucleases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Interstitial lung disease (ILD) is a condition affecting the lung parenchyma by inflammation and fibrosis and can be caused by various exposures, connective tissue diseases (CTD), and genetic disorders. In this report, a family with five patients having progressive respiratory failure that begins with coughing in adolescence, followed by dyspnea and recurrent spontaneous pneumothorax, and death in early adulthood is presented. The patients were diagnosed to have ILD through clinical and radiological evaluations. Molecular genetic analyses of the family provided two homozygous rare variants in the WRN and SFXN5 genes, co-segregating with the phenotype. The network analyses pointed out that the variant in the WRN, rather than that in the SFXN5 gene, could be the main factor in the existence of the ILD phenotype, putatively through the altered DNA repair and telomere maintenance pathways. In silico analyses suggested that the variant could affect the exonuclease activity or the stability of the WRN protein. Moreover, the adolescent-onset pulmonary phenotype described in the case has not been reported in Werner Syndrome, the only disease known to be associated with biallelic WRN pathogenic variants. Thus, the present phenotype could be either a very atypical presentation of Werner syndrome or a new clinical entity associated with the WRN gene.
Assuntos
Doenças Pulmonares Intersticiais , Pneumotórax , Síndrome de Werner , Humanos , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/genética , Pneumotórax/diagnóstico , Pneumotórax/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologia , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
The determination of the oligomeric state of functional enzymes is essential for the mechanistic understanding of their catalytic activities. RecQ helicases have diverse biochemical activities, but it is still unclear how their activities are related to their oligomeric states. We use single-molecule multi-color fluorescence imaging to determine the oligomeric states of Werner syndrome protein (WRN) during its unwinding and replication fork regression activities. We reveal that WRN binds to a forked DNA as a dimer, and unwinds it without any change of its oligomeric state. In contrast, WRN binds to a replication fork as a tetramer, and is dimerized during activation of replication fork regression. By selectively inhibiting the helicase activity of WRN on specific strands, we reveal how the active dimers of WRN distinctly use the energy of ATP hydrolysis for repetitive unwinding and replication fork regression.
Assuntos
Helicase da Síndrome de Werner , Humanos , Replicação do DNA , Exodesoxirribonucleases/metabolismo , RecQ Helicases/metabolismo , Helicase da Síndrome de Werner/metabolismoRESUMO
The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.