EXO1

本文为转化医学网原创，转载请注明出处 作者：Rainbow 导读： DNA损伤在疾病的肿瘤发生和进展中起着重要作用。DNA修复异常会影响癌症的治疗和预后。 5月27日，同济大学医学院袁健、李昀辉共同通讯在期刊《ADVANCED SCIENCE》上在线发表题为“USP25 Elevates SHLD2-Mediated DNA Double-Strand Break Repair and Regulates Chemoresponse in Cancer”的研究论文， 研究提出了一个机制模型，即USP25在K64位点用K63连接的多泛素链去泛素化和修饰SHLD2，从而促进NHEJ修复，从而降低癌细胞的化学敏感性。然后，研究人员开发了基于破坏USP25-SHLD2结合的策略的肽，并证明肽与化疗药物的组合提高了结肠癌细胞和PDX模型的杀伤效率，这意味着一种潜在的治疗策略，以提高化疗敏感性并实现更好的结果。 https://onlinelibrary-wiley-com.libproxy1.nus.edu.sg/doi/10.1002/advs.202403485 研究背景 01 DNA损伤反应（DDR）系统对于维持基因组完整性和防止DNA损伤至关重要。DDR通路功能障碍导致基因组不稳定，这是肿瘤发生开始和进展的关键驱动力。 DNA 双链断裂（DSB）是最致命的病变，可引发一系列细胞DNA损伤反应。DSBs通过两种主要途径进行修复，即同源重组（HR）和非同源末端连接（NHEJ）。NHEJ通路修复增殖细胞中75%的DSB，并在整个细胞周期中发生。屏蔽DNA末端是启动DSB NHEJ修复的核心过程。最近的报道已将shieldin复合物确定为53BP1相关活性的新效应子，可促进NHEJ修复并限制DNA末端切除。shieldin复合物由C20orf196（SHLD1）、FAM35A（SHLD2）、CTC-534A2.2（SHLD3）和REV7组成，SHLD2是该复合体中的关键调节因子。SHLD2的N端与REV7结合，促进SHLD2募集到DSB。SHLD2的C末端与ssDNA末端结合，通过与EXO1和DNA2竞争来防止DNA末端的切除。SHLD2对于促进NHEJ和抗体类别开关重组（CSR）至关重要。SHLD2的高表达与乳腺癌患者的不良预后相关。然而，对控制SHLD2活性的调控机制知之甚少。 翻译后修饰（PTM）在调节蛋白质功能中起着至关重要的作用。NHEJ通路中关键调节因子的许多PTM已被证明与肿瘤发生和癌症治疗有关。具体来说，ATM对53BP1 N末端的S/T-Q位点进行磷酸化是DNA末端保护和免疫球蛋白类转换重组（CSR）所必需的，促进其与RIF1的相互作用，激活NHEJ修复和癌细胞存活。此外，RNF168对K1268的53BP1泛素化对NHEJ修复、维持基因组稳定性和放射敏感性至关重要。然而，调节SHLD2的PTM尚未得到广泛的表征。 研究发现 02 在这项研究中，证明去泛素化酶USP25促进非同源末端连接（NHEJ），从而促进癌症的化疗耐药性。结果表明，USP25在K64位点使SHLD2去泛素化，从而增强其与REV7的结合并促进NHEJ。此外，USP25缺陷会损害NHEJ介导的DNA修复并降低 USP25 缺陷小鼠的类别转换重组（CSR）。USP25在结肠癌的一个亚群中过表达。USP25的耗竭使结肠癌细胞对IR、5-Fu和顺铂敏感。TRIM25也被鉴定为一种E3连接酶，负责降解USP25 。TRIM25的下调导致USP25水平升高，进而诱导结肠癌细胞的化疗耐药性。最后，在PDX模型中，成功鉴定了一种破坏USP25-SHLD2相互作用的肽，损害了NHEJ并增加了对化疗的敏感性。 总体而言，这些发现揭示了USP25是SHLD2调节NHEJ修复途径的关键效应因子，并表明其作为癌症治疗靶点的潜力。 研究结论 03 综上所述， 研究提出了一个机制模型，即USP25在K64位点用K63连接的多泛素链去泛素化和修饰SHLD2，从而促进NHEJ修复，从而降低癌细胞的化学敏感性。然后，研究人员开发了基于破坏USP25-SHLD2结合的策略的肽，并证明肽与化疗药物的组合提高了结肠癌细胞和PDX模型的杀伤效率，这意味着一种潜在的治疗策略，以提高化疗敏感性并实现更好的结果。 参考资料： https://onlinelibrary-wiley-com.libproxy1.nus.edu.sg/doi/10.1002/advs.202403485 注：本文旨在介绍医学研究进展，不能作为治疗方案参考。如需获得健康指导，请至正规医院就诊。 热门·直播/活动 🕓 上海｜06月14日-16日 ▶ 第六届上海国际癌症大会将于6月14-16日在上海举办，学科交叉，共同推进肿瘤研究与诊治多模态！ 🕓 北京｜09月20日-21日 ▶ 第五届单细胞技术应用研讨会暨空间组学前沿研讨会欢迎您的参与！ 点击对应文字 查看详情

SAN DIEGO, April 10, 2024 /PRNewswire/ -- Blacksmith Medicines, Inc. (Blacksmith), a leading biopharma dedicated to discovering and developing medicines targeting metalloenzymes, today announced preclinical data on its oncology program targeting flap endonuclease 1 (FEN1), a structure-specific metallonuclease that cleaves 5' DNA flaps during replication and repair, at the American Association for Cancer Research (AACR) Annual Meeting 2024, taking place April 5-10 at the San Diego Convention Center, San Diego CA. "FEN1 has been identified as an important therapeutic metalloenzyme involved in DNA replication and repair but previous drug discovery efforts have been hampered by chemistry limitations resulting in inhibitors lacking potency and selectivity," said Zachary Zimmerman, Ph.D. CEO of Blacksmith. "Using our metalloenzyme fragment-based drug discovery approach, we have identified a highly potent and selective inhibitor of FEN1 having synergies with multiple DDR drug classes that include inhibitors of PARP, PARG, USP1, and ATR." The Blacksmith fragment-based drug discovery platform identified a novel metal-binding pharmacophore that binds to the two magnesium ions in the FEN1 active site. Further elaboration using fragment growth strategies resulted in highly potent and selective inhibitors. The current lead (BSM-1516) is ~65-fold more potent against FEN1 than its related enzyme Exonuclease 1 (Exo1) in biochemical assays (IC50 of 7 nM and 460 nM, respectively), an improvement of more than an order of magnitude in selectivity compared to earlier efforts. FEN1 target engagement in live cells was validated by cellular thermal shift assay (CETSA EC50 of 24 nM). Inhibition of FEN1 led to its increased association with chromatin in S-phase cells and recruitment of PARP1 enzyme. In clonogenic assay, BRCA2-deficient DLD1 cells were ~15-fold more sensitive to FEN1 inhibition than their isogenic BRCA2-wild-type counterparts (EC50 of 350 nM and 5 µM, respectively), confirming the increased susceptibility of HR deficient cancer cells to FEN1 inhibition. Treatment of BRCA2-deficient but not wild-type DLD1 cells with BSM-1516 resulted in cell cycle arrest accompanied by DNA damage signaling and accumulation of chromatin-bound RPA32, a marker of ssDNA. In FEN1-inhibitor-anchored CRISPR screen, it was observed that perturbations in EXO1, USP1, PARP1 and HR pathway genes sensitized cells to FEN1 inhibition. Synergistic relationships of BSM-1516 and its combination potential were further explored in viability studies with a panel of DDR inhibitors (n=25) in BRCA2-proficient and deficient cell lines. Strong synergy was identified with multiple drug classes that included inhibitors of USP1 (KSQ-4279), PARP (Olaparib, Niraparib, Talazoparib, AZD5305), PARG (PDD 00017273) and ATR (AZD6738, VE-822, Elimusertib). In vitro ADME assays and in vivo PK studies showed that BSM-1516 has properties suitable for in vivo testing, either as a single agent or in combination with synergistic DDR inhibitors, an investigation that is currently underway. Poster presentation details Abstract Number: 7148 Title: "Small molecule inhibitor of FEN1 nuclease utilizing a novel metal binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR" Session Category: Experimental and Molecular Therapeutics Session Title: Novel Antitumor Agents 6 Session Date and Time: Wednesday April 10, 2024 9:00 AM - 12:30 PM Location: Poster Section 23 Poster Board Number: 10 About FEN1 Flap endonuclease 1 (FEN1) is a structure-specific di-magnesium metallonuclease that cleaves 5' DNA flaps during replication and repair. FEN1 is an attractive target for development of anticancer therapeutics because it is overexpressed in many tumor types and has a large number of synthetic lethality partners including genes in Homologous Recombination (HR) pathway. About metalloenzymes and the Blacksmith platform Metalloenzymes utilize a metal ion cofactor in the enzyme active site to perform essential biological functions. This diverse class of targets has historically been difficult to drug due to small molecule chemistry limitations that have plagued the industry. The Blacksmith metalloenzyme platform has solved this problem by leveraging the following: A large proprietary fragment library of metal-binding pharmacophores (MBPs); A comprehensive database containing a full characterization of the metalloenzyme genome including functions, metal cofactors, and associations to disease; A first-of-its-kind metallo-CRISPR library of custom single guide RNAs; An industry-leading metalloenzyme computational toolkit for docking, modeling and structure-based drug design; and A robust and blocking intellectual property estate covering bioinorganic, medicinal, and computational chemistry approaches for metalloenzyme-targeted medicines. About Blacksmith Medicines At Blacksmith Medicines, we are developing medicines targeting metal-dependent enzymes. Over 30% of known enzymes are metalloenzymes, covering all major enzyme classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Metal ions, including magnesium, zinc, iron, manganese and copper, are the essential ingredient in these metalloenzymes. We recognized a large unmet need for new chemical matter and innovative approaches to drug this important class of enzymes. Our purpose-built platform for metalloenzyme-targeted medicines combines, for the first time in industry, a focused library of metal-binding pharmacophores with proprietary computational modeling approaches to rapidly and rationally design small molecule inhibitors that interact with key metal ions in the enzyme's active site. Our comprehensive knowledge of the metal environment and key active site interactions enables Blacksmith to rapidly build potent and selective inhibitors in a stepwise and predictable manner. Blacksmith has executed strategic drug discovery collaborations with Basilea Pharmaceutica International Ltd., Cyteir Therapeutics Inc., Eli Lilly and Company (Lilly), Hoffmann-La Roche Ltd., and Zoetis LLC., and has been awarded non-dilutive Federal funding agreements with CARB-X and NIH/NIAID. Blacksmith investors include Lilly, Evotec A.G., MP Healthcare Partners, MagnaSci Ventures, and Alexandria Venture Investments. For further information, please visit the company's website and LinkedIn Media Contact: Amy Conrad Juniper Point [email protected] 858-366-3243 SOURCE Blacksmith Medicines

Cells zealously protect the integrity of their genomes, because damage can lead to cancer or cell death. The genome - a cell's complete set of DNA - is most vulnerable while it is being duplicated before a cell divides. Cancer cells constantly are dividing, so their genomes are constantly in jeopardy. Researchers at Washington University School of Medicine in St. Louis have identified a previously unknown signaling pathway cells use to protect their DNA while it is being copied. The findings, published Jan. 24 in the journal Molecular Cell, suggest that targeting this pathway potentially could boost the potency of cancer therapeutics. "A cell that can't protect its genome is going to die," said senior author Zhongsheng You, PhD, a professor of cell biology & physiology. "This entire pathway we found exists to protect the genome so the cell can survive in the face of replication stress. By combining inhibitors of this pathway with chemotherapy drugs that target the DNA replication process, we potentially could make such drugs more effective." Replication stress occurs when the cells DNA duplication machinery runs into problems copying the genome. Certain stretches of DNA are inherently difficult to copy, because they contain many repeated sequences. Factors that damage the DNA, such as radiation and toxic molecules, also cause replication stress, as does the activation of cancer-causing genes. Dozens of cancer drugs, including widely used medications such as cisplatin and doxorubicin, work by damaging the DNA and increasing replication stress. You studies how cells protect their genomes while they are being duplicated. Early in his career, he worked on the ATR-Chk1 genome-protection pathway - a pathway that controls the cell-division cycle and prevents stalled replication machinery from failing entirely and causing breaks in the DNA. For the past eight years, he and his team painstakingly have been piecing together another previously unknown genome-protection pathway. With this new study, the final piece of the puzzle has clicked into place. The process they discovered goes like this: When the DNA-duplicating machinery stalls, a protein called Exo1 that normally follows behind the machinery gets a little out of hand. Exo1s job is to perform quality control by cutting out incorrectly copied pieces of DNA, but when the machinery stops moving forward, Exo1 starts snipping away haphazardly, cleaving off bits of DNA that then make their way out of the nucleus and into the main part of the cell. DNA is not found outside the nucleus under normal conditions, so its presence in the main part of the cell sets off an alarm. Upon encountering a fragment of DNA, a sensor molecule triggers a cascade of molecular events, including the release of the calcium ion from a cellular organelle known as the endoplasmic reticulum, which in turn shuts down Exo1, preventing it from dicing up the genome any further until the problem with the machinery can be fixed. This newest study describes the discovery of DNA fragments as the warning signal that sets off the whole genome-protection response. The study was led by first author Shan Li, PhD, as a postdoctoral researcher and then a staff scientist in Yous lab. Li is now an assistant professor at Zhejiang University School of Medicine in Hangzhou, China. Co-author Lingzhen Kong, a graduate student, also made important contributions to the study. Over the years, You and colleagues have identified eight protein factors involved in this genome-protection pathway. Most of them already have inhibitors under development that could be repurposed for cancer studies. "Now that we have the pathway, we want to know whether it can be targeted for cancer treatment," You said. "Lung, ovarian and breast cancer are intrinsically under replication stress. Other cancers are put under replication stress by chemotherapy drugs. This pathway protects cells from replication stress, so if we could block the pathway, it might improve patients response to cancer therapies." Several of the proteins in this pathway also play a role in other critical biological processes, including immunity, metabolism and autophagy, the process by which cells break down their own unwanted materials. "One of the most exciting things about this pathway is how it intersects with so many other pathways," You said. "I've been focusing on cancer, but much of this could also apply to autoimmune diseases. Two of the proteins we identified have been linked to chronic activation of the immune response and autoimmune disease. We want to understand the relationship between this replication-stress response pathway and the innate immune response pathway. The work we do is very basic, and it is so exciting to connect the dots between these fundamental processes and see how they relate to human health and disease." Shan Li, Lingzhen Kong, Ying Meng, Chen Cheng, Delphine Sangotokun Lemacon, Zheng Yang, Ke Tan, Abigael Cheruiyot, Zhimin Lu, Zhongsheng You. Cytosolic DNA sensing by cGAS/STING promotes TRPV2-mediated Ca2+ release to protect stressed replication forks. Molecular Cell, 2023. doi: 10.1016/j.molcel.2022.12.034