EXO1

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

SOUTH SAN FRANCISCO, Calif.--( BUSINESS WIRE )-- Cellworks Group Inc. , a leader in Precision Drug Development and Personalized Therapy Biosimulation, today announced results from a Cellworks biosimulation pilot study, which examined the impact of mismatch repair deficiency (MRD) on survival of temozolomide (TMZ)-treated patients with MGMT methylated (m-MGMT) glioblastoma (GBM). The biosimulation study validated that TMZ does not trigger apoptosis in mismatch repair deficiency cancers and uncovered promising new biomarkers for TMZ resistance. The study also found inferior overall survival (OS) for MRD compared to the historical experience of unmethylated-MGMT patients, suggesting that TMZ-induced hypermutation may compromise survival. Results from the study were featured in a poster presentation at the 27th Annual Meeting and Education Day of the Society for Neuro-Oncology on November 18, 2022 and available online at Neuro-Oncology Journal Article EPCO-06 . “It has been a surprise to most oncologists that DNA mismatch repair (MMR) deficiency determines temozolomide success against GBM and that MMR is much more complicated than merely mutation or deletion of the four MMR genes,” said Dr. Michael Castro M.D ., Neuro-oncologist, Medical Oncologist at Beverly Hills Cancer Center; Chief Medical Officer at Cellworks, Group Inc. and Principal Investigator of the study. “In fact, Cellworks’ biosimulation identifies MMR deficiency due to epigenetic, translational and proteomic silencing that leads to early progression on temozolomide despite methylated MGMT. To a significant extent, these other mechanisms of MMR deficiency explain the long observed anomaly that many patients expected to respond to temozolomide based on methylated MGMT have no chance to benefit. Crucially, they could be treated instead with lomustine which does not require MMR for efficacy. Hopefully, biosimulation of MMR deficiency should emerge as an important new biomarker in neurooncology that allows physicians to make more effective treatment decisions in the front-line management of GBM.” Cellworks Therapy Biosimulation The Cellworks Platform biosimulates how a patient's personalized genomic disease model will respond to therapies prior to treatment and can also identify novel drug combinations for treatment-refractory patients. The platform is powered by the groundbreaking Cellworks Computational Biology Model (CBM), a highly curated mechanistic network of 6,000+ human genes, 30,000 molecular species and 600,000+ molecular interactions. As part of the biosimulation process, personalized disease models are created for each patient using their cytogenetic and molecular data as input to the Cellworks CBM. The Cellworks platform analyzes the impact of specific therapies on the patient’s personalized disease model and predicts the efficacy of specific chemotherapies. Biosimulation Study The purpose of the study was to examine the impact of mismatch repair deficiency (MRD) on survival of temozolomide (TMZ)-treated patients with MGMT methylated (m-MGMT) glioblastoma (GBM) using Cellworks computational biosimulation. Background TMZ-induced G:T mismatches trigger mismatch repair (MMR) to perform futile repair of O 6 -meG necessary to achieve apoptosis. Without intact MMR, the G:T mutation does not cause cell death, but instead is genomically incorporated to produce DNA mutation signature #11. Hypermutation ensues and may compromise survival without the redemptive benefit of immunotherapy. MMR genes are infrequently mutated or deleted in GBM. More commonly, MRD results from epigenetic, translational and proteomic silencing. Methods Comprehensive genomic profiling and Cellworks biosimulation was utilized to diagnose MRD and correlated with survival in 38 TCGA patients with newly diagnosed IDH wildtype, m-MGMT GBM treated with adjuvant TMZ. The integrity of MMR (MutSa and MutLa) was biosimulated for each patient using a computational model of signaling pathway behavior. Kaplan-Meier curves were constructed for progression-free survival (PFS) and overall survival (OS) using the treatment start date for calculation. Results Biosimulation results indicate that TMZ does not trigger apoptosis in mismatch repair deficiency cancers. Overall Survival (OS) in MRD patients was worse than historical u-MGMT patients who benefit little from TMZ. MMR had higher MMR biosimulation scores compared to patients with one or more abnormalities resulting in MRD (p = 0.00082). Half of MRD patients had classical COSMIC MMR mutation signatures representing a portion of the mutations in the tumor. Five-year survivors were only found in the MMR proficient group. Partial MRD from epigenetic silencing can result in MMR mutation signatures, but did not cause hypermutation and MSI. Nineteen of the 38 patients with MMR deficiency had 1 to 9 pathways compromised, impacting MMR function. Six patients had deletions of MLH1, HSH2 or MSH6. Other abnormalities included loss of EP300, CREBBP, KMT2A-D, ARID1A, HUS or EXO1. One of the 38 patients had MSI from germline MRD. Hypermutation was not identified in 37 of the 38 patients. Conclusions This study shows that biosimulation of MRD can identify m-MGMT patients who develop early progression on TMZ, echoing the long-established knowledge that TMZ does not trigger apoptosis in MRD cancers. Lomustine does not rely on intact MMR for efficacy and therefore it may blunt the impact of MMR on OS or could produce better disease control if used as an initial therapy. The observed deleterious impact of MRD on survival for TMZ treated m-MGMT patients should be examined and confirmed in a larger patient cohort. About Cellworks Group Cellworks Group, Inc. is a leader in Precision Drug Development and Personalized Therapy Biosimulation. The Cellworks Platform predicts therapy response for individual patients and patient cohorts using a breakthrough Computational Biology Model (CBM) and biosimulation technology. The Cellworks CBM is a mechanistic network model representation of biological knowledge curated from heterogeneous datasets that uses patient-specific genomic data to produce therapy response predictions. Backed by UnitedHealth Group, Sequoia Capital, Agilent and Artiman, Cellworks has the world’s strongest trans-disciplinary team of molecular biologists, cellular pathway modelers and software technologists working toward a common goal – attacking serious diseases to improve the lives of patients. The company is based in South San Francisco, California with a research and development facility in Bangalore, India. For more information, visit www.cellworks.life . All trademarks and registered trademarks in this document are the properties of their respective owners.