Substrate S-adenosylmethionine Research Articles

Abstract 4598: Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers

Abstract Homozygous deletions of the p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus, which is responsible for regulating the cell cycle, are commonly found in cancer and often involve the deletion of adjacent genes. One such adjacent gene is methylthioadenosine phosphorylase (MTAP), involved in metabolism, located on chromosome 9p21 in close proximity to the p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP is observed in approximately 80-90% of tumors with homozygous deletion of CDKN2A, representing 10-15% of all human tumors. These tumor types, including non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and mesothelioma, have a poor prognosis, highlighting the significant unmet medical need in this area. Deletion of MTAP leads to a significant accumulation of methylthioadenosine (MTA) in cells. MTA, at high concentrations, selectively inhibits the PRMT5 methyltransferase enzyme, competing with the substrate S-adenosylmethionine (SAM) required for methylation reactions. As a result, the overall level of symmetric arginine dimethylation throughout the proteome is reduced. This heightened sensitivity to modulation of methylosome activity makes cells with MTAP deletion more susceptible to therapeutic targeting of PRMT5. Hence, selective targeting of PRMT5 in cancers with homozygous MTAP deletion represents a promising strategy for specifically eliminating cancer cells with this genetic alteration. Ryvu has developed MTA-cooperative PRMT5 inhibitors characterized by good drug-like physicochemical properties and inhibition of methyltransferase activity with IC50 values in the low nanomolar range. A structure-based lead optimization delivered compounds coming from two independent series with high selective potency in MTAP-deleted cell lines and DMPK profiles allowing an oral administration. The antitumor activities were compared in vitro and in vivo to MRTX1719 and AMG193 in MTAP null tumors such as HCT116 MTAP KO, DoHH-2 and Lu99. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies. Taken together, these studies confirm that MTA cooperative PRMT5 inhibitors exert strong synthetic lethal phenotype in MTAP deleted cancers and offer an exciting therapeutic opportunity for a large patient population. Citation Format: Adam Radzimierski, Aneta Bobowska, Agata Stachowicz, Kamil Kuś, Kamila Kozłowska-Tomczyk, Agnieszka Ludwig-Słomczyńska, Paulina Podkalicka-Gołda, Aniela Gołas, Monika Żukowska, Pavlo Lebed, Przemysław Wyrębek, Daria Szukiel, Matylda Stefaniak, Oleksandr Popika, Julia Krzywik, Sujit Sasmal, Paulina Niedziejko-Ćwiertnia, Klara Korta-Piątek, Karolina Fijołkowska, Aleksandra Więckowska, Marta Olszak-Płachta, Swapnil Nipunge, Mateusz Świrski, Marek Wronowski, Henryk Pawlik, Quỳnh Vũ, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Igor Tomczyk, Ewelina Gabor-Worwa, Nilesh Gaud, Anna Kowal-Chwast, Dawid Gogola, Magdalena Miodek, Róża Starczak, Agata Dudek, Jacek Faber, Bożena Winnik, Sanja Novak-Ratajczak, Agnieszka Świrska, Karolina Gluza, Paweł Guzik, Katarzyna Banaszak, Nicolas Boutard, Grzegorz Ćwiertnia, Krzysztof Brzózka, Mateusz Nowak, Anna Bartosik, Didier Pez. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4598.

Abstract C135: Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers

Abstract Homozygous deletions of the p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus, which is responsible for regulating the cell cycle, are commonly found in cancer and often involve the deletion of adjacent genes. One such adjacent gene is methylthioadenosine phosphorylase (MTAP), involved in metabolism, located on chromosome 9p21 in close proximity to the p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP is observed in approximately 80-90% of tumors with homozygous deletion of CDKN2A, representing 10-15% of all human tumors. These tumor types, including non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and mesothelioma, have a poor prognosis, highlighting the significant unmet medical need in this area. Deletion of MTAP leads to a significant accumulation of methylthioadenosine (MTA) in cells. MTA, at high concentrations, selectively inhibits the PRMT5 methyltransferase enzyme, competing with the substrate S-adenosylmethionine (SAM) required for methylation reactions. As a result, the overall level of symmetric arginine dimethylation throughout the proteome is reduced. This heightened sensitivity to modulation of methylosome activity makes cells with MTAP deletion more susceptible to therapeutic targeting of PRMT5. Hence, selective targeting of PRMT5 in cancers with homozygous MTAP deletion represents a promising strategy for specifically eliminating cancer cells with this genetic alteration. Ryvu is developing two independent series of PRMT5 inhibitors characterized by good drug-like physicochemical properties and inhibition of methyltransferase activity with IC50 values in the low nanomolar range. Structure-based lead optimization enabled rapid expansion and delivery of a compound library with novel intellectual property (IP), high target engagement in cells and selective potency in MTAP-deleted cell lines. In long-term three-dimensional (3D) cell culture, Ryvu compounds selectively inhibit the growth of MTAP-deficient cancer cells, which correlate strongly with the inhibition of PRMT5-dependent symmetric protein dimethylation (SDMA) in these cells. The synthesized library allowed the identification of compounds with more than 350-fold selectivity between the effects observed in MTAP-deficient and WT cells for both SDMA and growth inhibition. The DMPK profile of these compounds allows for oral administration, which enables testing of antitumor activity in MTAP null tumor xenograft bearing mice. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies. Taken together, these studies provide a rationale for the further development of our chemical series of MTA cooperative PRMT5 inhibitors toward a clinical candidate. Citation Format: Adam Radzimierski, Aneta Bobowska, Agata Stachowicz, Kamil Kus, Kamila Kozłowska-Tomczyk, Agnieszka Ludwig-Słomczyńska, Aniela Gołas, Paulina Podkalicka, Andrzej Gondela, Nicolas Boutard, Grzegorz Ćwiertnia, Krzysztof Brzózka, Anna Bartosik, Mateusz Nowak, Didier Pez. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C135.

Coronavirus Inhibitors Targeting nsp16.

During the past three decades, humans have been confronted with different new coronavirus outbreaks. Since the end of the year 2019, COVID-19 threatens the world as a rapidly spreading infectious disease. For this work, we targeted the non-structural protein 16 (nsp16) as a key protein of SARS-CoV-2, SARS-CoV-1 and MERS-CoV to develop broad-spectrum inhibitors of nsp16. Computational methods were used to filter candidates from a natural product-based library of 224,205 compounds obtained from the ZINC database. The binding of the candidates to nsp16 was assessed using virtual screening with VINA LC, and molecular docking with AutoDock 4.2.6. The top 9 compounds were bound to the nsp16 protein of SARS-CoV-2, SARS-CoV-1, and MERS-CoV with the lowest binding energies (LBEs) in the range of -9.0 to -13.0 kcal with VINA LC. The AutoDock-based LBEs for nsp16 of SARS-CoV-2 ranged from -11.42 to -16.11 kcal/mol with predicted inhibition constants (pKi) from 0.002 to 4.51 nM, the natural substrate S-adenosyl methionine (SAM) was used as control. In silico results were verified by microscale thermophoresis as in vitro assay. The candidates were investigated further for their cytotoxicity in normal MRC-5 lung fibroblasts to determine their therapeutic indices. Here, the IC50 values of all three compounds were >10 µM. In summary, we identified three novel SARS-CoV-2 inhibitors, two of which showed broad-spectrum activity to nsp16 in SARS-CoV-2, SARS-CoV-1, and MERS-CoV. All three compounds are coumarin derivatives that contain chromen-2-one in their scaffolds.

Abstract 1025: Genomic and metabolomic analysis of MAT2A inhibition reveals increased RNA splicing, lipid metabolism and cell cycle arrest in MTAP deleted tumor models

Abstract Background: Approximately 15% of solid tumors harbor deletions in Methylthioadenosine phosphorylase (MTAP) (1). MTAP deletion confers enhanced sensitivity to Methionine Adenosyltransferase 2A (MAT2A) inhibition, which synthesizes the universal methyl donor and PRMT5 substrate S-Adenosyl Methionine (SAM) (2). MTAP loss results in the accumulation of its substrate methylthioadenosine (MTA) which partially inhibits PRMT5. PRMT5 symmetrically di-methylates arginine (SDMA) residues on proteins that are key to genomic integrity and proteostasis. Therefore, MTA accumulation and MAT2A inhibitor-driven abrogation of SAM significantly inhibit PRMT5 activity and key oncogenic signaling in MTAP deleted cancers. In this study, we sought to investigate the mechanistic and phenotypic implications of this synthetic lethal relationship. Methods: Using the HCT116 MTAP deleted isogenic pair and a panel of MTAP deleted cell lines, we evaluated the genomic and metabolomic effects of a proprietary MAT2A inhibitor using RNA sequencing (RNA-seq) and untargeted metabolomics respectively. Further, we investigated the effects of MAT2A inhibition across a large panel of MTAP WT and deleted cell lines in a multiplexed assay that assessed proliferation, apoptosis and cell cycle status. Results: MAT2A inhibition decreased SAM, and selectively decreased the proliferation and expression of SDMA in MTAP deleted cell lines. From RNA-seq data, MAT2A inhibition notably affected genes involved in p53 and plasma membrane signaling in the HCT116 MTAP deleted cell line compared to its parental counterpart. RNA-seq data also identified elevated frequency of alternative spliced transcripts that are involved in cell cycle, RNA processing and lipid metabolism pathways. In alignment with these observations, untargeted metabolomic analysis of MAT2A inhibition identified robust and selective changes in polyamine synthesis, oxidative stress and lipid metabolism in the HCT116 MTAP deleted cell line. With the changes in cell cycle related genes and previous reports indicating a role of lipid metabolism in cell cycle arrest , MAT2A inhibition increased expression of the G2/M cell cycle marker phospho-histone H3 and decreased proliferation in a larger percentage of MTAP deleted cell lines compared to MTAP WT cell lines. We further confirmed RNA-Seq data and observed increased p53 expression and a concordant decrease in the p53-regulated G2/M transition marker Cyclin B1 upon MAT2A inhibition in the HCT116 MTAP-deleted cell line. Conclusion: We have shown that MAT2A blockade results in distinct genomic and metabolomic effects that drives G2/M cell cycle arrest in MTAP deleted cell lines. Further analysis of these changes will help identify potential biomarkers that can further inform on the activity of MAT2A inhibition. We are currently targeting to enter clinical trials in 2021. Citation Format: Neil Bhola, Atieh Givmanesh, John Faulhaber, Melissa Fleury, Mark Lackner, Zineb Mounir, Divya Pankajakshan. Genomic and metabolomic analysis of MAT2A inhibition reveals increased RNA splicing, lipid metabolism and cell cycle arrest in MTAP deleted tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1025.

Abstract 1278: MAT2A inhibitor, IDE397, displays broad anti-tumor activity across a panel of MTAP-deleted patient-derived xenografts

Abstract Background: Approximately 15% of all solid tumors harbor deletions in methylthioadenosine phosphorylase (MTAP) (1). MTAP deficiencies have been reported to sensitize tumor cells to methionine adenosyltransferase 2A (MAT2A) modulation (2). MAT2A synthesizes the universal methyl donor and PRMT5 substrate S-adenosylmethionine (SAM) from methionine. PRMT5 symmetrically di-methylates arginine (SDMA) residues on proteins that are key to genomic integrity and proteostasis. We evaluated the anti-tumor activity of the small molecule MAT2A inhibitor IDE397 in multiple CDX models and in an ongoing study of 45 MTAP-deleted PDX models to inform clinical strategy for a First-in-Human clinical trial evaluating IDE397. Methods: CDX models utilized included the HCT116 isogenic pair and endogenous MTAP-deleted models. A series of dose response and time-course studies were performed to inform the efficacy-PD relationship. SAM was quantified by LC-MS and SDMA was visualized by western blot (WB) and further quantified by immunohistochemistry (IHC). PDX models with MTAP homozygous deletions were selected from bladder, esophageal, gastric, head and neck, NSCLC and pancreatic cancers. MTAP deletion was determined by WES and/or SNP 6.0 microarray and verified by absence of significant mRNA. Tumors were subcutaneously implanted into immunocompromised mice by serial transplantation of tumor fragments. Each PDX model included a vehicle control and an IDE397-treated group (n=5/group). IDE397 was administered orally once per day (QD). Tumor growth inhibition (TGI) was calculated using the formula TGI = (TV controlfinal - TV treatedfinal)/(TV controlfinal - TV controlinitial) x 100. Results: Administration of IDE397 resulted in TGI and/or tumor regression in MTAP-deleted CDX and PDX models. The HCT116 MTAP-deleted CDX model was more sensitive to IDE397 compared to the HCT116 MTAP-WT model. IDE397 produced a dose- and time-dependent modulation of the proximal and distal PD biomarkers SAM and SDMA. SAM was decreased in plasma and tumors irrespective of MTAP status. In contrast, SDMA was selectively modulated in MTAP-null models only. In a NSCLC CDX model, a dose dependent TGI was observed, with the higher doses leading to tumor regression. Anti-tumor activity is observed in MTAP-deleted PDX models, where IDE397 administration has resulted in TGI and tumor regressions. Conclusion: Xenograft studies indicate that IDE397 exhibits anti-tumor activity as a single agent in MTAP-deleted CDX models and in MTAP-deleted PDX models of NSCLC, pancreatic, bladder, head and neck, esophageal and gastric cancer. This data supports the clinical evaluation of IDE397 across a wide range of solid tumor types with MTAP-deletion.

Biosynthesis of a Novel Bioactive Metabolite of Spermidine from Bacillus amyloliquefaciens: Gene Mining, Sequence Analysis, and Combined Expression

Spermidine is a biologically active polyamine with extensive application potential in functional foods. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders its industrial application. To improve the spermidine titer, key genes affecting the spermidine production were mined to modify Bacillus amyloliquefaciens. Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Gene and amino acid sequence analysis further confirmed the function of speD and speE. Then, these two genes were co-expressed to generate a recombinant strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 105.2 mg/L, improving by 11.0-fold compared with the control (HSAM2). Through optimization of the fermentation medium, the spermidine titer was increased to 227.4 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding its synthesis pattern. In conclusion, two critical genes for spermidine synthesis were obtained, and an engineering B. amyloliquefaciens strain was constructed for enhanced spermidine production.

Abstract 631: Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells

Abstract Deletions in Methylthioadenosine phosphorylase (MTAP) are observed in 15% of all cancers including pancreatic, lung, bladder and GBM cancers (1). MTAP loss results in an accumulation of MTA which partially inhibits Protein Arginine Methyltransferase 5 (PRMT5). MTAP deletions confer enhanced sensitivity to inhibition of Methionine Adenosyltransferase 2A (Mat2A), which is involved in the synthesis of the universal methyl donor and PRMT5 substrate S-Adenosyl Methionine (SAM). In combination with MTA accumulation, Mat2A inhibition depletes SAM levels resulting in further inhibition of PRMT5 activity and a synthetic lethal (SL) relationship in MTAP null cancers. Inhibition of PRMT5 activity as a result increases RNA splicing defects and chromatin dysregulation which impacts tumor cell viability in different models. We developed MAT2A inhibitors for treatment of MTAP-deleted tumors, and in this study sought to determine the mechanism of action of these inhibitors in MTAP null models. These inhibitors were assessed for activity against both MTAP-wt and MTAP-null tumor models using cellular proliferation assays. MTAP-null (KP4, BxPC3, RT112/84, MiaPaCa-2, H647) models were more sensitive to Mat2A inhibition than MTAP-wt (HCT116, NCI-H460) models. Inhibition of Mat2A increased the expression of markers associated with cellular senescence and cell cycle arrest. Cellular SAM levels were diminished in both MTAP-wt and MTAP-null cells and xenografts while selective reduction of Symmetric Dimethyl Arginine (SDMA) levels were observed in MTAP-null models. Cellular response to Mat2A inhibitors is unaltered by RNAi-mediated knockdown of Mat1A. Signaling changes that were observed due to decreased PRMT5 activity included increased stability of p53, which may be due to the inefficient splicing of MDM4 mRNA, and was seen across multiple MTAP-null models but not in MTAP WT. Furthermore, increased protein and mRNA expression of the p53 target gene p21 was also observed in MTAP-null models. In conclusion, Mat2A inhibition effectively decreases SAM and SDMA levels, leading to increased stability of p53 and its targets and resulting in decreased cellular proliferation. Ongoing exploratory studies will further delineate the molecular mechanisms mediated by Mat2A inhibition in MTAP-null cell lines. The genes and metabolites identified from these studies will be validated in Mat2A-dependent and non-dependent MTAP-null cell lines identified from DepMap studies to inform further preclinical and clinical studies. Citation Format: Neil Bhola, Atieh Givmanesh, Marie-Claire Wagle, Candy Garcia, Kedar Vaidya, Leah Cleary, Zhonghua Pei, Mark Lackner, Zineb Mounir. Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 631.

Abstract 652: Exploiting PRMT5, a metabolic and epigenetic regulator, as a novel therapeutic target in mantle cell lymphoma

Abstract Distinct genetic and epigenetic alterations as well as metabolic reprogramming are among the hallmarks of mantle cell lymphoma (MCL), an incurable cancer. Deletion of MTAP, encoding 5-Methylthioadenosine phosphorylase (MTAP), results in the accumulation of metabolite methylthioadenosine (MTA), a selective inhibitor of the catalytic activity of protein arginine methyltransferase 5 (PRMT5). MTAP loss therefore presents a passenger vulnerability to PRMT5 inhibition. PRMT5 is a type II protein arginine methyltransferase that is involved in cellular processes via the regulation of the active status of transcription by symmetric dimethylation of arginine in histones, and the post-translational modification by symmetric dimethylation of arginine in nuclear and cytoplasmic oncoproteins and tumor suppressors. We recently reported that CDKN2A and MTAP were commonly co-deleted in ibrutinib-resistant MCL tumors. Consistently, PRMT5 was found to be selectively over expressed in ibrutinib-resistant MCL cells. Metabolomics analysis revealed that both the substrate (S-Adenosylmethionine) and the product (S-adenosylhomocysteine) of the PRMT5 enzyme are elevated in ibrutinib-resistant MCL, suggesting an increase in PRMT5 enzymatic activity. Importantly, our clinical data analysis indicates that the expression levels of PRMT5 in MCL patients are inversely correlated with patient responses to ibrutinib treatment. Moreover, pharmacological blockade with the PRMT5 specific inhibitor, GSK3226595, significantly inhibited tumor growth in ibrutinib-resistant MCL PDX models, demonstrating that PRMT5 is an actionable target in MCL. Mechanistically, PRMTs play an important role in mRNA splicing, among which MDM4 mRNA was identified as one of the key mRNAs that senses defects in the spliceosomal machinery during PRMT5 deficiency. Alternative splicing of MDM4 mRNA reduces the expression of Mdm2 and Mdm4 E3 ligases, leading to the upregulation of p53. Selective overexpression of PRMT5 in MCL may drive repressive epigenetic marks on chromatin to silence regulatory and tumor suppressor genes. Inhibition of PRMT5 reverses PRMT5-catalyzed epigenetic marks, restores regulatory pathways and induces cell death in ibrutinib-resistant primary MCL. Taken together, sustained PRMT5 expression and activity presents a vulnerability in ibrutinib-resistant MCL where MTAP is frequently deleted. As metabolic reprogramming towards OXPHOS is one of the hallmarks of ibrutinib-resistant MCL, combinatory inhibition of OXPHOS and PRMT5 may represent a rational therapeutic strategy that ultimately eradicates MCL. Citation Format: Yixin Yao, Lingzhi Li, Liang Zhang, Shaojun Zhang, Hui Guo, Hui Zhang, Maria Badillo, Zhihong Chen, Kimberly Hartig, Preetesh Jain, Krystle Nomie, Linghua Wang, Michael Wang. Exploiting PRMT5, a metabolic and epigenetic regulator, as a novel therapeutic target in mantle cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 652.

High-Density CRISPR Scan Identifies Functional Regions of DOT1L That Mediate Therapeutic Response in MLL-r Leukemia

Leukemias containing Mixed Lineage Leukemiagene rearrangement (MLL-r) account for 5-10% of human acute leukemia cases and are associated with a poor prognosis. The unmet clinical need and the lack of an effective targeted therapy emphasizes the need for novel approaches for these malignancies.Recent studies have discovered an essential role for the histone H3 lysine 79 (H3K79) methyltransferase DOT1L in the maintenance of MLL-r leukemias. Phase-I clinical trials (NCT01684150 and NCT02141828) have demonstrated the safety and clinical activity of the DOT1L-specific small molecule inhibitor EPZ5676 (Pinometostat, Epizyme Inc.). However, the variable response of the MLL-r patients in these clinical trials indicates the need to further understand the mechanism of action and resistance to DOT1L inhibitors.To address this issue, we designed a high-density CRISPR screen approach (Fig 1) to dissect the function of Dot1l coding regions in mouse MLL-AF9 leukemic cells, a well-established mouse model that mimics human MLL-r acute myeloid leukemia (AML). We constructed a library of 602 sgRNA that targets most of the “NGG” protospacer adjacent motifs (PAM) within Dot1l exons. The average targeting density is 7.7 bp per sgRNA (i.e. 2.5 a.a. per sgRNA). We delivered this sgRNA library into Cas9-expressing MLL-AF9 AML cells using lentiviral transduction, and then compared the frequencies of each integrated sgRNA sequence before vs. after 12 days of culture using high-throughput sequencing (NextSeq, Illumina Inc.). The results revealed a significant depletion of clusters of sgRNA targeting the known functional regions including the lysine methyltransferase (KMT) core and the AF9-binding domains of DOT1L. We mapped the CRISPR scan score to the previously solved three-dimensional structures of DOT1L and found the high-density CRISPR scan clearly distinguishes the substrate binding pocket from the less important regions within the KMT domain. Furthermore, we identified CRISPR hotspots that coincide with amino acid residues directly contacting the enzymatic substrate S-adenosylmethionine (SAM). Similarly, CRISPR scan identifies amino acid hot spots in DOT1L that establish a direct interaction with the cofactor AF9. These results implicate the utility of the high-density CRISPR scan to pinpoint the functional elements within a protein to a sub-domain resolution.To identify regions in DOT1L that mediate response of MLL-r leukemia to DOT1L-inhibitors, we conducted parallel CRISPR scans by culturing the Dot1l sgRNA library transduced MLL-AF9-Cas9+cells in either DMSO (vehicle) or the DOT1L inhibitor (1 uM EPZ5676) for 12 days. These paired screens revealed distinct patterns of the CRISPR scan scores between the two culture conditions. Surprisingly, a cluster of 31 sgRNA targeting the amino acid residues T560 - S661 of DOT1L was significantly enriched only in the presence of the DOT1L inhibitor.Expression of individual sgRNA targeting this region confirmed the EPZ5676-resistant phenotype observed in the CRISPR scans. Computational modeling of the structure of DOT1L suggests this newly identified region overlaps with two alpha-helixes motifs in a predicted coiled-coil domain. Finally, we examined eight clinically observed DOT1L missense mutations in this region (cBioPortal database; 54,510 patients) and foundthree DOT1L-mutant constructs (A591V, G594S and L626P) when expressed in the MLL-AF9 leukemia cells can confer resistance to EPZ5676. These findings suggest the utility of the high-density CRISPR scan for de novoidentification of drug-resistant mutant alleles in the human population, which maydirect future clinical decisions and benefit patients with specific genomic backgrounds. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Crystallographic and Computational Characterization of Methyl Tetrel Bonding in S-Adenosylmethionine-Dependent Methyltransferases.

Tetrel bonds represent a category of non-bonding interaction wherein an electronegative atom donates a lone pair of electrons into the sigma antibonding orbital of an atom in the carbon group of the periodic table. Prior computational studies have implicated tetrel bonding in the stabilization of a preliminary state that precedes the transition state in SN2 reactions, including methyl transfer. Notably, the angles between the tetrel bond donor and acceptor atoms coincide with the prerequisite geometry for the SN2 reaction. Prompted by these findings, we surveyed crystal structures of methyltransferases in the Protein Data Bank and discovered multiple instances of carbon tetrel bonding between the methyl group of the substrate S-adenosylmethionine (AdoMet) and electronegative atoms of small molecule inhibitors, ions, and solvent molecules. The majority of these interactions involve oxygen atoms as the Lewis base, with the exception of one structure in which a chlorine atom of an inhibitor functions as the electron donor. Quantum mechanical analyses of a representative subset of the methyltransferase structures from the survey revealed that the calculated interaction energies and spectral properties are consistent with the values for bona fide carbon tetrel bonds. The discovery of methyl tetrel bonding offers new insights into the mechanism underlying the SN2 reaction catalyzed by AdoMet-dependent methyltransferases. These findings highlight the potential of exploiting these interactions in developing new methyltransferase inhibitors.

An in silico Approach Towards Crop Improvement by ACC Synthase Inhibition Declining Ethylene Production

Introduction: The increased level of ethylene inhibits root elongation and causes physiological damage, thereby reduces ethylene level imparts a positive support against various biotic and abiotic stresses viz. phytopathogens, extreme temperatures, hyper salinity, flooding, drought, metal/organic contaminants and insect predation. The metabolic pathways showed the involvement of ACC synthase inhibition for ethylene suppression in plants. Objective: The primary objective of this study focused towards the use of In-silico approach to assess the inhibitory effect of S-adenosyl methionine (SAM) analogue on ACC synthase activity. Methods: The 3D structure of ACC synthase of Pisum sativum was constructed using modeler 9v11 software. The reliability of developed model was evaluated by PROCHECK, ERRAT and ProSA web servers. Furthermore the molecular interactions between substrate SAM and inhibitors were performed. Result: The docking study demonstrated that the binding energy of the substrate SAM is -5.37 Kcal/mol. The SAM analogue (Inhibitors) considered in this study were 3-dzSAHC, SAHC, sinefungin, SIBA, 7-dz-SIBA, 1-dz-SIBA,3-dz-SIBA and S-n-Butyladenosine. Among these analogues, 7-dz-SIBA was found to be most effective on ACC synthase as inhibitor due to lowest binding energy (- 5.51Kcal/mol) and strong Ki value (91.74M). The LYS276 amino acid residue of ACC synthase was observed in the interaction with both substrates SAM and 7-dz-SIBA (S-isobutyl-7-deazaadenosine) demonstrates as most crucial catalytic residue for molecular interaction. Conclusion: This study successfully screened most potent inhibitor for ACC synthase which have indicated the compounds 7-dz- SIBA as effective inhibitor with lowest binding energy, better hydrogen bond interaction and strong inhibition constant compared to others compounds studied. Thus 7-dz-SIBA can be projected to use as a growth enhancer for overall crop improvement. It may help in plant growth promotion, prevents the plants from various environmental stress and phytopathogenic infections etc.

Steric Clash in the SET Domain of Histone Methyltransferase NSD1 as a Cause of Sotos Syndrome and Its Genetic Heterogeneity in a Brazilian Cohort.

Most histone methyltransferases (HMTase) harbor a predicted Su(var)3–9, Enhancer-of-zeste, Trithorax (SET) domain, which transfers a methyl group to a lysine residue in their substrates. Mutations of the SET domains were reported to cause intellectual disability syndromes such as Sotos, Weaver, or Kabuki syndromes. Sotos syndrome is an overgrowth syndrome with intellectual disability caused by haploinsufficiency of the nuclear receptor binding SET domain protein 1 (NSD1) gene, an HMTase at 5q35.2–35.3. Here, we analyzed NSD1 in 34 Brazilian Sotos patients and identified three novel and eight known mutations. Using protein modeling and bioinformatic approaches, we evaluated the effects of one novel (I2007F) and 21 previously reported missense mutations in the SET domain. For the I2007F mutation, we observed conformational change and loss of structural stability in Molecular Dynamics (MD) simulations which may lead to loss-of-function of the SET domain. For six mutations near the ligand-binding site we observed in simulations steric clashes with neighboring side chains near the substrate S-Adenosyl methionine (SAM) binding site, which may disrupt the enzymatic activity of NSD1. These results point to a structural mechanism underlying the pathology of the NSD1 missense mutations in the SET domain in Sotos syndrome. NSD1 mutations were identified in only 32% of the Brazilian Sotos patients in our study cohort suggesting other genes (including unknown disease genes) underlie the molecular etiology for the majority of these patients. Our studies also found NSD1 expression to be profound in human fetal brain and cerebellum, accounting for prenatal onset and hypoplasia of cerebellar vermis seen in Sotos syndrome.

MTAP Deletions in Cancer Create Vulnerability to Targeting of the MAT2A/PRMT5/RIOK1 Axis.

Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

Biological evaluation of tanshindiols as EZH2 histone methyltransferase inhibitors

EZH2 is the core subunit of Polycomb repressive complex 2 catalyzing the methylation of histone H3 lysine-27 and closely involved in tumorigenesis. To discover small molecule inhibitors for EZH2 methyltransferase activity, we performed an inhibitor screen with catalytically active EZH2 protein complex and identified tanshindiols as EZH2 inhibitors. Tanshindiol B and C potently inhibited the methyltransferase activity in in vitro enzymatic assay with IC50 values of 0.52μM and 0.55μM, respectively. Tanshindiol C exhibited growth inhibition of several cancer cells including Pfeiffer cell line, a diffuse large B cell lymphoma harboring EZH2 A677G activating mutation. Tanshindiol treatment in Pfeiffer cells significantly decreased the tri-methylated form of histone H3 lysine-27, a substrate of EZH2, as revealed by Western blot analysis and histone methylation ELISA. Based on enzyme kinetics and docking studies, we propose that tanshindiol-mediated inhibition of EZH2 activity is competitive for the substrate S-adenosylmethionine. Taken together, our findings strongly suggest that tanshindiols possess a unique anti-cancer activity whose mechanism involves the inhibition of EZH2 activity and would provide chemically valuable information for designing a new class of potent EZH2 inhibitors.

High Specificity in CheR Methyltransferase Function: CheR2 OF PSEUDOMONAS PUTIDA IS ESSENTIAL FOR CHEMOTAXIS, WHEREAS CheR1 IS INVOLVED IN BIOFILM FORMATION

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14-2.2 μM) than the substrate S-adenosylmethionine (KD of 22-43 μM), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in <i>Xenopus laevis</i> embryogenesis

To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.

Chemogenetic Analysis of Human Protein Methyltransferases

A survey of the human genome was performed to understand the constituency of protein methyltransferases (both protein arginine and lysine methyltransferases) and the relatedness of their catalytic domains. We identified 51 protein lysine methyltransferase proteins based on similarity to the canonical Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain. Disruptor of telomeric silencing-1-like, a known protein lysine methyltransferase, did not fit within the protein lysine methyltransferase family, but did group with the protein arginine methyltransferases, along with 44 other proteins, including the METTL and NOP2/Sun domain family proteins. We show that a representative METTL, METTL11A, demonstrates catalytic activity as a histone methyltransferase. We also solved the co-crystal structures of disruptor of telomeric silencing-1-like with S-adenosylmethionine and S-adenosylhomocysteine bound in its active site. The conformation of both ligands is virtually identical to that found in known protein arginine methyltransferases, METTL and NOP2/Sun domain family proteins and is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain protein lysine methyltransferases. We have developed biochemical assays for 11 members of the protein methyltransferase target class and have profiled the affinity of three ligands for these enzymes: the common methyl-donating substrate S-adenosylmethionine; the common reaction product S-adenosylhomocysteine; and the natural product sinefungin. The affinity of each of these ligands is mapped onto the family trees of the protein lysine methyltransferases and protein arginine methyltransferases to reveal patterns of ligand recognition by these enzymes.

544 DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy in breast cancer

Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

Native liver glycine N-methyltransferase (GNMT) is N-acetylated while the recombinant enzyme is not. We show here that acetylation of the N-terminal valine affects several kinetic parameters of the enzyme. Glycine N-methyltransferase is a regulatory enzyme mediating the availability of methyl groups by virtue of being inhibited by folate. N-acetylation does not affect the overall structure of the protein and does not affect basal enzyme activity of GNMT. Binding of both the mono- and pentaglutamate forms of 5-methyltetrahydrofolate is the same for the acetylated and non-acetylated forms of the enzyme, however the pentaglutamate form is bound more tightly than the monoglutamate form in both cases. Although binding of the folates is similar for the acetylated and non-acetylated forms of the enzyme, inhibition of enzyme activity differs significantly. The native, N-acetylated form of the enzyme shows 50% inhibition at 1.3 µM concentration of the pentaglutamate while the recombinant non-acetylated form shows 50% inhibition at 590 µM. In addition, the binding of folate results in cooperativity of the substrate S-adenosylmethionine (AdoMet), with a Hill coefficient of 1.5 for 5-methyltetrahydrofolate pentaglutamate.

Crystal Structures of 1-Aminocyclopropane-1-carboxylate (ACC) Synthase in Complex with Aminoethoxyvinylglycine and Pyridoxal-5′-Phosphate Provide New Insight into Catalytic Mechanisms

The structures of tomato 1-aminocyclopropane-1-carboxylate synthase (ACS) in complex with either cofactor pyridoxal-5'-phosphate (PLP) or both PLP and inhibitor aminoethoxyvinylglycine have been determined by x-ray crystallography. The structures showed good conservation of the catalytic residues, suggesting a similar catalytic mechanism for ACS and other PLP-dependent enzymes. However, the proximity of Tyr152 to the C-gamma-S bond of model substrate S-adenosylmethionine implies its critical role in the catalysis. The concerted accomplishment of catalysis by cofactor PLP and a protein residue, as proposed on the basis of the ACS structures in this paper, may represent a general scheme for the diversity of PLP-dependent catalyses. PLP-dependent enzymes have been categorized into four types of folds. A structural comparison revealed that a core fragment of ACS in fold type I is superimposable over tryptophan synthase beta subunit in fold type II and mouse ornithine decarboxylase in fold type III, thus suggesting a divergent evolution of PLP-dependent enzymes.