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Principal Investigator: Demonta Coleman
Organization: TEXAS A&M UNIVERSITY
Fiscal Year: 2024
Award: $39,353
Funding agency: National Cancer Institute
Project Summary
The current cancer treatment bottleneck showcases the need for more innovative approaches in drug discovery
efforts. As of recent, immune-checkpoint inhibitors (ICHs) have shown promise in cancer therapeutics, and work
by manipulating immunoregulatory pathways involved in tumor detection and elimination. Many cancer types
overexpress the PD-L1 receptor to inhibit T cell activation and subsequently evade the immune response.
Though proven effective, the PD-1/PD-L1 pathway targeting ICHs are pharmacologically limited by occupancy-
based inhibition. A new drug paradigm has emerged through the development of proteolysis targeting chimeras
(PROTACs) that are not limited by such a mechanism. PROTACs are heterobifunctional molecules consisting of
an E3 ubiquitin (Ub) ligase ligand, a target protein warhead, and a linker connecting the two and work by bringing
an E3 Ub ligase and the target protein together through bivalent binding. The formation of a ternary structure
through this bivalent interaction drives the polyubiquitination and subsequent degradation of the target via the
ubiquitin proteasome system (UPS). Although this drug design has shown great medicinal promise through
research findings and clinical trials, there is seldom literature on the use of transmembrane E3 Ub ligase
recruiting PROTACs to degrade cancer-promoting membrane proteins such as PD-L1. The proposed project
is aimed at investigating the applicability of such PD-L1 PROTACs by recruitment of the transmembrane
E3 Ub ligase ZNRF3. In doing so, three specific aims will be investigated: Aim 1. Identifying cyclic peptide
binders of the ZNRF3 extracellular domain. Previous research in the Liu lab utilized a cyanobenzothiazole based
linker that allows for cyclization of peptides on a phage surface with applications in phage selections against
CD44 and SARS-CoV-2 spike protein epitopes. Recombinant bacterial expression of biotinylated ZNRF3 ECD
has been developed, and the binding of selected cyclic peptides will be validated through BLI and flow cytometry.
Aim 2. Synthesis and in-vitro characterization of PD-L1 PROTACs. Reported binders of PD-L1 will be
incorporated in the PROTAC design via polyethylene glycol (PEG) and polyether linkages of varying lengths.
The optimal positioning of linkers will be determined through structure-activity relationships (SAR) analysis,
binding assays, co-cocrystal structures, and molecular docking studies of ligand-protein interactions of the
ZNRF3 ECD and PD-L1. Aim 3. In-vivo characterization of PD-L1 PROTACs. Degradation potency will be
evaluated with western-blotting of residual PD-L1 and ZNRF3 dependency on the induced degradation of PD-L1
and mode of action (proteosome vs lysosome) will be assessed. In addition, whole-cell proteomics studies will
be conducted to investigate any consequences on normal cellular activities due to PROTAC activity. By
accomplishing these aims, new and important conclusions can be made about the efficacy and mode of action
of our designed PROTACs that we hope will have a lasting impact on the cancer therapeutics bottleneck and
general drug discovery efforts.
Terms: <2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><APF-1><ATP-Dependent Proteolysis Factor 1><Affinity><Anti-Cancer Agents><Antigenic Determinants><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Assay><B7-H1><B7H1><Bacteriophages><Binding><Binding Determinants><Bioassay><Biological Assay><Biotinylation><CD274><CD44><CD44 gene><COVID-19 S protein><COVID-19 spike><COVID-19 spike glycoprotein><COVID-19 spike protein><Cancer Drug><Cancer Treatment><Cancer cell line><Cancers><Cell Body><Cell Line><CellLine><Cells><Cellular Regulation><Checkpoint inhibitor><Clinical Trials><Colorectal Carcinomas><Consensus><Consensus Sequence><Cyclic Peptides><Cyclization><Dependence><Detection><Development><Dimensions><Docking><Drug Design><E coli><E. coli><E3 Ligase><E3 Ubiquitin Ligase><Enzyme Gene><Enzymes><Epitopes><Escherichia coli><Event><External Domain><Extracellular Domain><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><HMG-20><HPLC><High Mobility Protein 20><High Performance Liquid Chromatography><High Pressure Liquid Chromatography><High Speed Liquid Chromatography><Immobilization><Immune Evasion><Immune Targeting><Immune checkpoint inhibitor><Immune response><Immunoblotting><Immunological response><Immunomodulation><In Vitro><Incubated><Investigators><Kinetics><Large Bowel Carcinoma><Large Intestine Carcinoma><Length><Libraries><Ligand Binding><Ligands><Literature><Lysosomes><MDA MB 231><MDA-231><MDA-MB231><MDU3><Macrogols><Macropain><Macroxyproteinase><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Medicine><Membrane><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Metabolic Protein Degradation><Methods><Modeling><Molecular><Molecular Interaction><Multicatalytic Proteinase><NSCLC><NSCLC - Non-Small Cell Lung Cancer><Neoplastic Disease Chemotherapeutic Agents><Non-Small Cell Lung Cancer><Non-Small-Cell Lung Carcinoma><Oncology><Oncology Cancer><Organic Synthesis><PD 1><PD-1><PD-1/PD-L1><PD-1/PDL1><PD-L1><PD-L1 pathway><PD1><PD1-PD-L1><PD1/PD-L1><PD1/PDL1><PDL-1><PDL1><PDL1 pathway><Pathway interactions><Peptide Synthesis><Peptides><Pgp1><Phage Display><Phages><Phase><Polyethylene Glycols><Polyethylene Oxide><Polyethyleneoxide><Polyoxyethylenes><Polyubiquitination><Position><Positioning Attribute><Programmed Cell Death 1 Ligand 1><Programmed Death Ligand 1><Proliferating><Prosome><Protac><Proteasome><Proteasome Endopeptidase Complex><Protein Turnover><Proteins><Proteolysis targeting chimeric><Proteomics><Proteosome><Receptor Protein><Recombinants><Regulatory Protein Degradation><Reporting><Research><Research Personnel><Researchers><Residual><Residual state><Resolution><Route><SARS-CoV-2 S><SARS-CoV-2 S protein><SARS-CoV-2 spike><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Solid><Strains Cell Lines><Strepavidin><Streptavidin><Structure><Structure-Activity Relationship><Surface><Surface Proteins><System><T-Cell Activation><T-Cells><T-Lymphocyte><TNBC><Testing><Therapeutic><Time><Tumor Cell><Tumor-Specific Treatment Agents><Ubiquitin><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><Western Blotting><Western Immunoblotting><Work><activate T cells><anti-cancer drug><anti-cancer therapy><bacterial virus><cancer therapy><cancer type><cancer-directed therapy><cell growth regulation><chemical structure function><coronavirus disease 2019 S protein><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><cultured cell line><design><designing><developmental><drug development><drug discovery><extracellular><flow cytophotometry><frontier><host response><immune check point inhibitor><immune evasive><immune modulation><immune regulation><immune system response><immunologic reactivity control><immunomodulatory><immunoregulation><immunoregulatory><immunoresponse><in vivo><innovate><innovation><innovative><insight><malignancy><member><membrane structure><multicatalytic endopeptidase complex><neoplasm/cancer><neoplastic cell><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><orthopedic freezing><overexpress><overexpression><pathway><peptide aminoacid sequence><peptide sequence><pharmacologic><programmed cell death 1><programmed cell death ligand 1><programmed cell death ligand 1 pathway><programmed cell death protein 1><programmed cell death protein ligand 1><programmed cell death protein ligand 1 pathway><programmed death 1><protein aminoacid sequence><protein blotting><protein death-ligand 1><protein degradation><proteolysis targeting chimera><receptor><recruit><resolutions><sle2><small molecule><spike proteins on SARS-CoV-2><structure function relationship><systemic lupus erythematosus susceptibility 2><thymus derived lymphocyte><triple-negative breast cancer><triple-negative invasive breast carcinoma><tumor><ubiquitin-protein ligase>