Minkui Luo
Sloan-kettering Institute For Cancer Res
Project start date: 2011-02-01
Project end date: 2015-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Minkui Luo
DEVELOPING HIGH THROUGHPUT ASSAY TO IDENTIFY PROTEIN METHYLTRANSFERASE INHIBITORS
Minkui Luo
Sloan-kettering Institute For Cancer Res, 1275 York Ave, New York, Ny 10065
Grant 1R21NS071520-01A1 from Office Of The Director, National Institutes Of Health
Abstract: Epigenetic regulations participate in numerous biological processes and their errors have been implicated in many diseases including cancer. Among the key modulators in epigenetic are protein methyltransferases (PMTs). Strong evidence showed that PMTs function through methylating histone and nonhistone targets. theless, fully understanding the biological roles of PMTs is restricted by the inability to profile the PMT targets and to characterize the downstream functions. PMT-specific inhibitors can readily hijack PMT- involved epigenetics and thus serve as valuable chemical genetic tools to investigate these processes. However, the lack of high throughput screening (HTS) methods represents a significant barrier in identifying such small-molecule entities. The situation, if not addressed, greatly limits the application of chemical genetic tools to define, perturb and manipulate the epigenetic functions. The objective of this proposal is to develop generalizable HTS assays of PMTs for identifying target-specific PMT inhibitors. The critical analysis of PMTs structures formulated the central hypothesis that the distinct substrate/cofactor-binding pockets of PMTs can be exploited for developing target-specific inhibitors. The goal of this proposal will be achieved by pursing two specific aims. A 384-well mix-and-measure scintillation proximity assay (SPA) has been developed and is expected to be transformed into a 1536-well AlphaScreen format (Aim 1). These HTS assays will be then validated with a pilot compound library. With the aid of counter-screening and secondary assays (Aim 1), target-specific PMT inhibitors are expected to be unambiguously identified and characterized in vitro and in vivo (Aim 2). These inhibitors can be used to generate PMT-specific hypomethylation proteome for target profiling and to disrupt specific methylations for function characterization. In conjunction with two NIH Roadmap missions of epigenomics and molecular libraries & imaging, developing the HTS assay and identifying PMT inhibitors will vertically advance epigenetic research and facilitate the use of PMT inhibitors for pharmacological intervention. Deregulated protein methyltransferases (PMTs) have been implicated in many diseases including cancer. The impact of this proposed research is to develop an efficient approach for identifying PMT inhibitors. These molecules can be used as pharmacological probes to investigate the mechanism of PMT-involved diseases. Additionally, novel therapeutic reagents can be developed from PMT inhibitors
Keywords: Address; Antioncogene Protein p53; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological; Biological Assay; Biological Function; Biological Process; Biomedical Research; Cancers; Cellular Tumor Antigen P53; Chemicals; Collection; Data; Disease; Disorder; Enzymes; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Estrogen Receptors; Future; Goals; High Throughput Assay; Histone H3; Histones; Image; In Vitro; Individual; Intervention; Intervention Strategies; L-Lysine; Libraries; Lysine; Malignant Neoplasms; Malignant Tumor; Measures; Methods; Methylation; Mission; Molecular Bank; Molecular Interaction; NIH; National Institutes of Health; National Institutes of Health (U.S.); Oncoprotein p53; Outcome; Phosphoprotein P53; Phosphoprotein pp53; Physiologic; Physiological; Process; Protein Methylases; Protein Methylation; Protein Methyltransferases; Protein TP53; Protein p53; Proteome; Radioactive; Reagent; Regulation; Reporting; Research; Role; Screening procedure; Structure; TP53; Time; Tumor Protein p53; United States National Institutes of Health; VEGF Receptors; VEGFR; VPF Receptor; Validation; Vascular Endothelial Cell Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor; Vascular Permeability Factor Receptor; ing; base; chemical genetics; cofactor; cost; disease/disorder; epigenomics; experience; high throughput screening; imaging; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; interventional strategy; malignancy; methyl-B12-CoM methyltransferase; methylcobalamin-CoM methyltransferase; methylcobalamin-coenzyme M methyltransferase; methyltransferase 2; neoplasm/cancer; new therapeutics; next generation therapeutics; notch; notch protein; notch receptors; novel; novel therapeutics; p53 Antigen; p53 Tumor Suppressor; screening; screenings; small molecule; social role; tool
Relevance: Deregulated protein methyltransferases (PMTs) have been implicated in many diseases including cancer. The impact of this proposed research is to develop an efficient approach for identifying PMT inhibitors. These molecules can be used as pharmacological probes to investigate the mechanism of PMT-involved diseases. Additionally, novel therapeutic reagents can be developed from PMT inhibitors
Project start date: 2010-09-27
Project end date: 2011-08-31
Budget start date: 27-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PAR-08-024
1R21NS071520-01A1 (2010): $190900
ENZYME-ENGINEERING APPROACHES TO DISSECT PROTEIN METHYLATION PROFILES
Minkui Luo
Sloan-kettering Institute For Cancer Res, 1275 York Ave, New York, Ny 10065
Grant 1DP2OD007335-01 from Office Of The Director, National Institutes Of Health
Abstract: Epigenetic regulations are involved in numerous biological processes and errors in these processes have been implicated in many diseases such as cancer. Among the key enzymes that orchestrate epigenetics are over 60 human protein methyltransferases. Accumulated evidence showed that epigenetic diversity requires protein methyltransferases to act on histone and nonhistone targets. theless, elucidating the physiological and pathological roles of these processes has been significantly hindered by our inability to unambiguously profile the targets of the over 60 human protein methyltransferases in vivo. To address this formidable challenge and thus vertically advance the epigenetic research, we propose to develop a highly innovative technology, which we termed Bioorthogonal Profiling of Protein Methylation (BPPM) and, as a paradigm, apply the approach for target profiling of cancer-relevant protein methyltransferases. In conjunction with our supportive preliminary results, we envisioned that protein methyltransferases can be rationally engineered to exploit S-adenosyl-L-methionine analogues as alternate cofactors and thus label their targets with distinct chemical groups. The distinct modifications can be selectively enriched and unambiguously characterized with respective reporters for BPPM. We further plan to leverage the approach for in vivo BPPM by incorporating a creative approach for in situ production of SAM analogues. With regard to expected outcomes, the proposed work is aimed at providing transformative technological advancement and generally applicable reagents for target profiling of protein methyltransferases. Compared with conventional approaches, our BPPM is featured as the high integrity by using intact methyltransferases in context of cellular proteome, the high specificity by linking cell-type-specific methylation profiles to designated methyltransferases, and the high sensitivity by selectively enriching modified targets from proteome. The results are expected to reveal how the methylation profiles differ between diverse protein methyltransferases, in normal versus pathological stages or in non-aggressive versus aggressive diseases. Deregulated protein methylation can cause developmental abnormalities, neurological disorders and cancer. The successful completion of this proposal is expected to provide the valuable tools to understand the molecular mechanisms involved in these disease processes. The resultant knowledge is expected to enhance our understanding of aberrant methylation pathways in these diseases and likely lead to the identification of novel biomarkers for disease diagnosis or targets for pharmacological intervention. Additionally, this research will contribute to a broader understanding of stem cell differentiation, for which protein-methylationinvolved epigenetic regulations have not been well understood
Keywords: Address; Biological Function; Biological Process; Cancers; Disease; Disorder; EC 2.1.1; Engineering; Engineerings; Enzymes; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Histones; Human; Human, General; In Situ; L-Methionine; Label; Link; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Methionine; Methionine, L-Isomer; Methylation; Methyltransferase; Modification; Outcome; Pathological Staging; Physiologic; Physiological; Process; Production; Protein Methylases; Protein Methylation; Protein Methyltransferases; Proteome; Reagent; Regulation; Reporter; Research; Role; Specificity; Work; ing; analog; cell type; chemical group; cofactor; disease/disorder; in vivo; innovative technologies; malignancy; methylase; neoplasm/cancer; social role; transmethylase
Project start date: 2010-09-30
Project end date: 2015-08-31
Budget start date: 30-SEP-2010
Budget end date: 31-AUG-2015
PFA/PA: RFA-RM-09-011
1DP2OD007335-01 (2010): $2863500