S -Adenosylhomocysteine Analogs Selectively Suppress Pan-Coronavirus Replication by Inhibition of nsp14 Methyltransferase

Discovering drug-like molecules for hard-to-target proteins remains a significant challenge. Methyltransferases (DNA, RNA, and protein) are among such targets. At Zafrens, we have developed an ultra-high-throughput discovery platform that leverages large arrays of isolated nano-wells and single-bead-single-compound DNA-encoded library (DELs) to uncover target-specific inhibitory impacts on cells. Using a combination of next-generation sequencing and spatially indexed optical imaging of biomarkers and cell paint probes, we successfully linked transcriptomic signatures and phenotypic responses to a combinatorial chemical library of over 15, 000 compounds, achieving approximately fivefold hit coverage. Incorporation of known inhibitors of a methyltransferase into the chemical library revealed that both transcriptome-driven clustering and machine learning-driven linkage of compound SMILES with cell phenotype could successfully identify reproducible signatures that were aligned well with public databases, including expected methyltransferase-dependent genes such as MTF1. The robustness and consistency of our platform have allowed us to identify several novel compounds that mirrored the transcriptomic and phenotypic profiles of the control compounds. Subsequent biochemical assays confirmed that many of these were bona fide inhibitors of the target methyltransferase. Among these, ZF165 emerged as a promising candidate due to its high hit frequency across multiple screens and across two cell lines. ZF165 was then resynthesized and further validated by both immunofluorescence- and western blot analyses for its impact on biomarker responses. Bulk transcriptomic mRNA sequencing analysis confirmed common profiles between ZF165 and a control methyltransferase inhibitor. Furthermore, ZF165 demonstrated initial anti-tumor cell activity with a potency comparable to known pre-clinical compounds, further supporting its potential as a lead molecule. With ZF165 as the new series lead, we designed a secondary DEL library featuring novel warheads and improved physicochemical properties. The secondary library, ZEL29, was synthesized and screened, yielding new hits as additional (or) providing SAR, with enhanced potency and better drug-like properties relative to ZF165. These results support the utility of our platform in both the initial hit discovery and subsequent hit optimization toward lead compounds. Validation-design cycles for lead optimization are currently ongoing. By coupling this powerful screening platform with robust data analytics, we aim to revolutionize the discovery of novel therapeutics to address other challenging drug targets. Citation Format: Nathan G. Hedrick, Maina Ndungu, Ivy Nai-Jung Hung, Devin K. Porter, Elliot Imler, Gopi Chandran RaviChandran, Aurelien Lauerre, Chris M. Glinkerman, Martin Smrcina, Mark R. Hansen, Allan Nojadera, Alexandra Cole, Cindy Huynh, Michael Miller, Dmitri rozanov, Logan Van Meter, Nicholas Sm-Soon, Steven Brown, Yi Zhang, Vikas K. Goel. An ultrahigh throughput phenotypic screening platform to discover novel inhibitors of methyl transferases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 456.

CN02-02: Epigenetic targets in endometrial cancer

Advanced cutaneous melanoma is considered to be the most aggressive type of skin cancer and has variable rates of treatment response. Currently, there are some classes of immunotherapy and target therapies for its treatment. Immunotherapy can inhibit tumor growth and its recurrence by triggering the host's immune system, whereas targeted therapy inhibits specific molecules or signaling pathways. However, melanoma responses to these treatments are highly heterogeneous, and patients can develop resistance. Epigenomics (DNA/histone modifications) contribute to cancer initiation and progression. Epigenetic alterations are divided into four levels of gene expression regulation: DNA methylation, histone modification, chromatin remodeling, and non-coding RNA regulation. Deregulation of lysine methyltransferase enzymes is associated with tumor initiation, invasion, development of metastases, changes in the immune microenvironment, and drug resistance. The study of lysine histone methyltransferase (KMT) and nicotinamide N-methyltransferase (NNMT) inhibitors is important for understanding cancer epigenetic mechanisms and biological processes. In addition to immunotherapy and target therapy, the research and development of KMT and NNMT inhibitors is ongoing. Many studies are exploring the therapeutic implications and possible side effects of these compounds, in addition to their adjuvant potential to the approved current therapies. Importantly, as with any drug development, safety, efficacy, and specificity are crucial considerations when developing methyltransferase inhibitors for clinical applications. Thus, this review article presents the recently available therapies and those in development for advanced cutaneous melanoma therapy.

Krüppel-Like Factor 10 Expression as a Prognostic Indicator for Pancreatic Adenocarcinoma

We have been interested in the biochemical mechanisms by which elevated plasma total homocysteine concentrations are associated with premature cardiovascular disease and neurological impairment in humans (1). In this paper, we would like to consider the possibility that a part of the pathology may result from the inhibition of one or more members of a class of S-adenosylmethionine (AdoMet)-dependent methyltransferases. These intracellular enzymes are often subject to potent inhibition by their S-adenosylhomocysteine (AdoHcy) product (2). This product is normally metabolized by AdoHcy hydrolase, which gives rise to adenosine and homocysteine. However, the equilibrium of this reaction strongly favors AdoHcy formation (3), thus linking increases in intracellular homocysteine to increases in AdoHcy and resulting in the inhibition of methyltransferases.

Activation and inhibition of DNA methyltransferases by S-adenosyl- l-homocysteine analogues

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Three new compounds have been synthesized and tested as in vitro inhibitors of normal and tumor tRNA methyltransferases. These compounds are 5'-methylethyl(5'adenosyl) sulfonium chloride (MEAS), 5'-methylpropyl-(5'adenosyl)sulfonium chloride (MPAS), and 5'-ethylpropyl(5'-adenosyl)sulfonium chloride (EPAS) They were prepared by reacting an alkyl iodide with the appropriate alkyladenosyl thioether. Inhibition assays revealed all three compounds to be inhibitors of normal and tumor tRNA methyltransferases. The propyl compounds were slightly better inhibitors of the tumor tRNA methyl transferases. MPAS, EPAS, and MEAS had KI's of 58.5, 61.7, and 24.5, respectively, for the normal tRNA methyltransferases and 15.3, 13.8, and 44.3, respectively, for the tumor tRNA methyltransferases.

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.

DNA methylation by DNA methyltransferases in CpG-rich promoter regions of genes is a well-described component of epigenetic silencing in human cells. Dysregulation of this process in cancer cells may lead to hypermethylation of promoter CpG islands, thus disabling transcription initiation of certain genes, such as tumor suppressor genes. Reversing epigenetic silencing and up-regulating genes involved in preventing or reversing the malignant phenotype has become a new, important targeted approach for cancer prevention and treatment. Therefore, methyltransferase inhibitors (MTI) have emerged recently as promising chemotherapeutic or preventive agents. The potent MTI 5-aza-2-deoxycytidine (5-Azadc) causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. The present study shows that low micromolar concentrations of 5-Azadc induce the expression of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer cells. The expression of 15-LOX-1 correlates with 5-Azadc-induced increases in 13-S-hydroxyoctadecadienoic acid levels, growth inhibition, and apoptosis in these cells. Furthermore, specific inhibition of 15-LOX-1 by pharmacologic means or small interfering RNA significantly reduced the 5-Azadc-induced effects. These novel findings are the first demonstration of a mechanistic link between the induction of 15-LOX-1 by a MTI and apoptosis in cancer cells. This result has important implications for the study of 5-Azadc and other MTIs in the prevention and therapy of colorectal cancer and supports future investigations of the mechanisms by which MTIs up-regulate 15-LOX-1.

Combination Methyltransferase and Histone Deacetylase Inhibition in Elderly Patients with Secondary Acute Myelogenous Leukemia.

BACKGROUND: Solid tumors employ multiple mechanisms to evade an immune response. However, the potential to enhance the immune response to cancer has been proven in several malignancie and is under investigation in many others, including primary CNS tumors. Brain tumors in particular lack robust T-cell infiltration. Recent studies have found that certain tumors can be induced to express T-cell attracting chemokines, CXCL9 and CXCL10, by interferon gamma (IFNg). This response is further amplified using methyltransferase inhibitors (Peng, et al. Nature 2015. Vol 527: 249-253.), resulting in increased Tcell trafficking to tumors both in vitro and in vivo. We hypothesized that T-cell trafficking to brain tumors could likewise be enhanced with DNA and histone methyltransferase inhibitors to induce CXCL9 and CXCL10 transcription. METHODS: Assays were performed on 7 human glioma brain tumor cell lines. CXCL9 and CXCL10 expression were measured by real-time PCR. Two commercially available methyltransferase inhibitors, 5-AZA-dC and GSK126, were utilized to demethylate DNA and histone H3 (K9 and K27), respectively. Histone methylation status was examined using Western blot. T-cell migration was measured using transwell migration assays. RESULTS: IFNg increased CXCL9 and CXCL10 transcription in brain tumor lines. GSK126 and 5-AZA-dC enhanced expression of CXCL9 and CXCL10 compared to IFNg alone. Migration assays confirmed T-cell trafficking towards chemokines produced by tumor cells in response to methyltransferase inhibitors. CONCLUSIONS: These studies demonstrate that brain tumors express T-cell attracting chemokines CXCL9 and CXCL10 in response to IFNg. Further, GSK126 and the combination of GSK126 and 5-AZA-dC enhanced expression of CXCL9 and CXCL10 transcription by real-time PCR and T-cell trafficking by migration assay. Together, these data provide a potential means to increase T-cell trafficking into tumors and potentially enhances the efficacy of immune therapies for brain tumors. Citation Format: Heather M. Sonnemann, Amber J. Giles, Caitlin M. Reid, Marsha-Kay N. Hutchinson, Deric M. Park, Mark R. Gilbert. Alerting the immune system by removing epigenetic silencing of Th1 chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3998. doi:10.1158/1538-7445.AM2017-3998

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.

Because of the putative rule of phospholipid methyltransferase reactions in many important membrane and receptor translocation processes, we studied the effect of methyltransferase inhibitors on acetylcholine receptor (AcChoR) turnover in cultured rat skeletal muscle. Inhibition of methyltransferase significantly reduced the normal rate of degradation of AcChoRs, a process that involves endocytosis. Further, under conditions that greatly accelerate the rate of degradation of AcChoRs--i.e., by addition of anti-AcChoR antibody--methyltransferase inhibitors again significantly reduced receptor turnover. AcChoR synthesis was unaffected. Thus, the net effect of this treatment was slowing of the antibody-induced loss of surface AcChoRs. That this drug effect was mediated specifically by inhibition of methylation reactions was suggested by certain additional pharmacologic features: partial reversibility of the effect by methionine, enhancement by homocysteine, and correspondence with marked inhibition of phospholipid methylation. The substrate specificity of the methyltransferase inhibitors capable of reducing AcChoR degradation suggests that phospholipid methylation reactions may be most relevant. Methyltransferase inhibitor drugs may provide a therapeutic strategy in receptor disorders such as myasthenia gravis, in which accelerated receptor endocytosis plays a major role.

Methylation is a fundamental mechanism used in Nature to modify the structure and function of biomolecules, including proteins, DNA, RNA, and metabolites. Methyl groups are predominantly installed into biomolecules by a large and diverse class of S-adenosyl methionine (SAM)-dependent methyltransferases (MTs), of which there are ∼200 known or putative members in the human proteome. Deregulated MT activity contributes to numerous diseases, including cancer, and several MT inhibitors are in clinical development. Nonetheless, a large fraction of the human MT family remains poorly characterized, underscoring the need for new technologies to characterize MTs and their inhibitors in native biological systems. Here, we describe a suite of S-adenosyl homocysteine (SAH) photoreactive probes and their application in chemical proteomic experiments to profile and enrich a large number of MTs (>50) from human cancer cell lysates with remarkable specificity over other classes of proteins. We further demonstrate that the SAH probes can enrich MT-associated proteins and be used to screen for and assess the selectivity of MT inhibitors, leading to the discovery of a covalent inhibitor of nicotinamide N-methyltransferase (NNMT), an enzyme implicated in cancer and metabolic disorders. The chemical proteomics probes and methods for their utilization reported herein should prove of value for the functional characterization of MTs, MT complexes, and MT inhibitors in mammalian biology and disease.