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学者姓名：林忠辉
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Abstract ：
The DdmDE antiplasmid system, consisting of the helicase-nuclease DdmD and the prokaryotic Argonaute (pAgo) protein DdmE, plays a crucial role in defending Vibrio cholerae against plasmids. Guided by DNA, DdmE specifically targets plasmids, disassembles the DdmD dimer, and forms a DdmD-DdmE handover complex to facilitate plasmid degradation. However, the precise ATP-dependent DNA translocation mechanism of DdmD has remained unclear. Here, we present cryo-EM structures of DdmD bound to single-stranded DNA (ssDNA) in nucleotide-free, ATP gamma S-bound, and ADP-bound states. These structures, combined with biochemical analysis, reveal a unique "gate-clamp" mechanism for ssDNA translocation by DdmD. Upon ATP binding, arginine finger residues R855 and R858 reorient to interact with the gamma-phosphate, triggering HD2 domain movement. This shift repositions the gate residue Q781, causing a flip of the 3 ' flank base, which is then clamped by residue F639. After ATP hydrolysis, the arginine finger releases the nucleotide, inducing HD2 to return to its open state. This conformational change enables DdmD to translocate along ssDNA by one nucleotide in the 5 ' to 3 ' direction. This study provides new insights into the ATP-dependent translocation of DdmD and contributes to understanding the mechanistic diversity within SF2 helicases.
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| GB/T 7714 | Li, Ruoyu , Liu, Yusong , Gao, Haishan et al. A gate-clamp mechanism for ssDNA translocation by DdmD in Vibrio cholerae plasmid defense [J]. | NUCLEIC ACIDS RESEARCH , 2025 , 53 (3) . |
| MLA | Li, Ruoyu et al. "A gate-clamp mechanism for ssDNA translocation by DdmD in Vibrio cholerae plasmid defense" . | NUCLEIC ACIDS RESEARCH 53 . 3 (2025) . |
| APA | Li, Ruoyu , Liu, Yusong , Gao, Haishan , Lin, Zhonghui . A gate-clamp mechanism for ssDNA translocation by DdmD in Vibrio cholerae plasmid defense . | NUCLEIC ACIDS RESEARCH , 2025 , 53 (3) . |
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Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC 3.4.22.49), 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. © 2024 Elsevier Inc.
Keyword ：
Anticancer Anticancer Inhibitor Inhibitor Separase Separase Toxoflavin Toxoflavin Walrycin B Walrycin B
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| GB/T 7714 | Zhu, Q. , Du, L. , Wu, J. et al. Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model [J]. | Biochemical Pharmacology , 2024 , 229 . |
| MLA | Zhu, Q. et al. "Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model" . | Biochemical Pharmacology 229 (2024) . |
| APA | Zhu, Q. , Du, L. , Wu, J. , Li, J. , Lin, Z. . Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model . | Biochemical Pharmacology , 2024 , 229 . |
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Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors. © 2024 Elsevier B.V.
Keyword ：
Anticancer Anticancer DNA repair DNA repair Inhibitor Inhibitor KPT-6566 KPT-6566 STAG1 STAG1 STAG2 STAG2
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| GB/T 7714 | Zhu, Q. , Chen, X. , Lin, Z. . Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro [J]. | DNA Repair , 2024 , 144 . |
| MLA | Zhu, Q. et al. "Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro" . | DNA Repair 144 (2024) . |
| APA | Zhu, Q. , Chen, X. , Lin, Z. . Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro . | DNA Repair , 2024 , 144 . |
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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 mu M. 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.
Keyword ：
Anticancer Anticancer Chemosensitivity Chemosensitivity DNA repair DNA repair Ku70 Ku70 Ku80 Ku80 NHEJ NHEJ Small-molecule inhibitor Small-molecule inhibitor UMI-77 UMI-77
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| GB/T 7714 | Chen, Xuening , Chen, Changkun , Luo, Chengmiao et al. Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo [J]. | EUROPEAN JOURNAL OF PHARMACOLOGY , 2024 , 975 . |
| MLA | Chen, Xuening et al. "Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo" . | EUROPEAN JOURNAL OF PHARMACOLOGY 975 (2024) . |
| APA | Chen, Xuening , Chen, Changkun , Luo, Chengmiao , Liu, Jianyong , Lin, Zhonghui . Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo . | EUROPEAN JOURNAL OF PHARMACOLOGY , 2024 , 975 . |
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Abstract ：
Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.
Keyword ：
Anticancer Anticancer DNA repair DNA repair Inhibitor Inhibitor KPT-6566 KPT-6566 STAG1 STAG1 STAG2 STAG2
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| GB/T 7714 | Zhu, Qinwei , Chen, Xuening , Lin, Zhonghui . Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro [J]. | DNA REPAIR , 2024 , 144 . |
| MLA | Zhu, Qinwei et al. "Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro" . | DNA REPAIR 144 (2024) . |
| APA | Zhu, Qinwei , Chen, Xuening , Lin, Zhonghui . Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro . | DNA REPAIR , 2024 , 144 . |
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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. Graphical Abstract
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| GB/T 7714 | Zhang, Danping , Du, Liyang , Gao, Haishan et al. Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system [J]. | NUCLEIC ACIDS RESEARCH , 2024 , 52 (14) : 8419-8430 . |
| MLA | Zhang, Danping et al. "Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system" . | NUCLEIC ACIDS RESEARCH 52 . 14 (2024) : 8419-8430 . |
| APA | Zhang, Danping , Du, Liyang , Gao, Haishan , Yuan, Cai , Lin, Zhonghui . Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system . | NUCLEIC ACIDS RESEARCH , 2024 , 52 (14) , 8419-8430 . |
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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. The precise mechanism of metal-ion catalysis in Holliday junction resolution remains elusive. Here, the authors describe a metal ion-mediated nick and counter-nick mechanism of Holliday junction cleavage by MOC1.
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| GB/T 7714 | Zhang, Danping , Xu, Shenjie , Luo, Zhipu et al. MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions [J]. | NATURE COMMUNICATIONS , 2024 , 15 (1) . |
| MLA | Zhang, Danping et al. "MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions" . | NATURE COMMUNICATIONS 15 . 1 (2024) . |
| APA | Zhang, Danping , Xu, Shenjie , Luo, Zhipu , Lin, Zhonghui . MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions . | NATURE COMMUNICATIONS , 2024 , 15 (1) . |
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Abstract ：
Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC 3.4.22.49), 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.
Keyword ：
Anticancer Anticancer Inhibitor Inhibitor Separase Separase Toxoflavin Toxoflavin Walrycin B Walrycin B
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| GB/T 7714 | Zhu, Qinwei , Du, Liyang , Wu, Juhong et al. Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model [J]. | BIOCHEMICAL PHARMACOLOGY , 2024 , 229 . |
| MLA | Zhu, Qinwei et al. "Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model" . | BIOCHEMICAL PHARMACOLOGY 229 (2024) . |
| APA | Zhu, Qinwei , Du, Liyang , Wu, Juhong , Li, Jinyu , Lin, Zhonghui . Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model . | BIOCHEMICAL PHARMACOLOGY , 2024 , 229 . |
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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. Type III CRISPR systems produce second messengers that activate ancillary nucleases for degradation of viral DNA. Here, structures of one such nuclease, TtCsm6, in various catalytic states reveal how cA4 binding allosterically activates its ribonuclease activity as well as cA4 cleavage for subsequent inactivation.Crystal structures reveal the mechanism of cA4 recognition and cleavage by TtCsm6 cA4 binding in the CARF domain allosterically activates the ribonuclease activity of the HEPN domain. cA4 cleavage by the CARF domain leads to the inactivation of the HEPN ribonuclease. TtCsm6 variants with sustained cA4-activated RNase activity may be applicable in genome editing and diagnostics. Crystal structures show the basis for ancillary nuclease activation and auto-inactivation via second messengers generated in Type III CRISPR systems.
Keyword ：
Allosteric Activation Allosteric Activation Ancillary Nuclease Ancillary Nuclease Csm6 Csm6 Cyclic Oligoadenylate Cyclic Oligoadenylate Type III CRISPR Type III CRISPR
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| GB/T 7714 | Du, Liyang , Zhu, Qinwei , Lin, Zhonghui . Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate [J]. | EMBO JOURNAL , 2024 , 43 (2) : 304-315 . |
| MLA | Du, Liyang et al. "Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate" . | EMBO JOURNAL 43 . 2 (2024) : 304-315 . |
| APA | Du, Liyang , Zhu, Qinwei , Lin, Zhonghui . Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate . | EMBO JOURNAL , 2024 , 43 (2) , 304-315 . |
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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 RecGdependent 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]-2,5dione) as two potent PaRecG inhibitors, with IC50 values of 0.31 +/- 0.02 mu M and 1.16 +/- 0.06 mu M, 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.
Keyword ：
Combined effect Combined effect DNA damage repair DNA damage repair Holliday junction Holliday junction Pseudomonas aeruginosa Pseudomonas aeruginosa RecG RecG Small-molecule inhibitors Small-molecule inhibitors
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| GB/T 7714 | Li, Longheng , Guo, Binbin , Dai, Lin et al. Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents [J]. | BIOCHEMICAL PHARMACOLOGY , 2024 , 222 . |
| MLA | Li, Longheng et al. "Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents" . | BIOCHEMICAL PHARMACOLOGY 222 (2024) . |
| APA | Li, Longheng , Guo, Binbin , Dai, Lin , Liu, Chun , Lin, Zhonghui . Ebselen and TPI-1, as RecG helicase inhibitors, potently enhance the susceptibility of Pseudomonas aeruginosa to DNA damage agents . | BIOCHEMICAL PHARMACOLOGY , 2024 , 222 . |
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