The overall goal of this proposal is to develop a non-invasive, real-time, quantifiable cell-based assay to detect and report on furin-like protease activity to identify small molecule inhibitors of furin-like proteases by high throughput screening (HTS). Furin-like proteolytic enzymes are members of the Proprotein Convertase (PC) family that serve to process immature latent proteins, including growth factors and hormones, receptors, plasma proteins, and matrix metalloproteases containing a specific recognition cleavage motif (RX(K/R)R?), to their mature or functional forms. Processing by furin-like protease family members, such as furin, PACE4, PC5/6, and PC7/8, contributes to development of several degenerative diseases, such as Alzheimer's disease, arteriolosclerosis, and arthritis. Furin-like protease expression and activity is necessary for processing substrates that enhance the cancer phenotype, contributing to cell transformation, tumor progression, metastasis, and angiogenesis. Further, furin-like proteolytic processing of viral coat glycoproteins is required for propagation of infectious viruses such as H5N1 avian influenza, HIV-1, human papillomavirus, ebola, yellow fever, and SARS-CoV. Furin-like proteases activate bacterial toxins found in anthrax, shigella, botulinum, pseudomonas, and diphtheria. Inhibition of furin-like proteolytic activity has been shown to halt toxicity of bacterial toxins, infectivity of viruses, and motility of cancer cells. We hypothesize that inhibiting furin-like proteolytic activity may lead to development of a therapeutic drug that inhibits a broad-spectrum of furin-like protease mediated disease. To aid in experimentation of this hypothesis, in specific aim 1A, we will develop a furin-like protease reporter, which non-invasively and quantitatively senses furin-like protease activity in real time and characterize its specificity and sensitivity to furin-like protease activity. In specific aim 1B, we will miniaturize this assay to adapt it to HTS. In specific aim 1C, we will perform HTS of several specialized small molecule libraries containing 71K compounds to identify furin-like protease inhibitory molecules. In specific aim 2A, a secondary screen will be employed to eliminate false positives, cytotoxic, and non-specific inhibitory molecules. Potency will be assessed by exposing the furin-reporter cells to various concentrations of the candidate compound to determine pIC50 values. In specific aim 2B, we subject the five most efficacious compounds to further validation by determining inhibition (IC50 value) of furin processing of physiological substrates using western blot analysis. Additionally, cytotoxicity will be gauged using cell proliferation assays. In specific aim 2C, the compound's ability to inhibit furin will be confirmed using purified furin in vitro. We will also investigate the molecule's specificity by performing in vitro inhibition assays with other serine proteases. At the conclusion of phase I, we expect to have identified at least one compound or derivative with IC50 < 1uM that will be the subject of further analysis and targeted for drug development to treat furin-mediated diseases such as anthrax and cancer in subsequent years.
Public Health Relevance: Millions of people worldwide are exposed to and/or contract furin-like protease mediated diseases such as HIV-1, ebola, avian influenza, human papillomavirus, yellow fever, SARS-CoV, anthrax, botulinum, measles, pseudomonas, shigella, diphtheria, arthritis, arteriosclerosis, Alzheimer's disease, and malignant cancer. Instead of searching for a therapeutic to address each pathogen and disease individually, targeting a single cellular protease may allow defeat of a broad spectrum of furin-like protease mediated disease. The studies described here will result in identification of a molecule that inhibits furin-like proteases and thus may be used to treat the diseases listed above.
Public Health Relevance Statement: Project Narrative Millions of people worldwide are exposed to and/or contract furin-like protease mediated diseases such as HIV-1, ebola, avian influenza, human papillomavirus, yellow fever, SARS-CoV, anthrax, botulinum, measles, pseudomonas, shigella, diphtheria, arthritis, arteriosclerosis, Alzheimer's disease, and malignant cancer. Instead of searching for a therapeutic to address each pathogen and disease individually, targeting a single cellular protease may allow defeat of a broad spectrum of furin-like protease mediated disease. The studies described here will result in identification of a molecule that inhibits furin-like proteases and thus may be used to treat the diseases listed above.
Project Terms: AIDS Virus; ATGN; Address; Alkaline Phosphatase; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers disease; Anthrax; Anthrax disease; Antigens; Antiproteases; Arterioloscleroses; Arteriosclerosis; Arthritis; Assay; Avian Influenza; Bacillus anthracis, protective antigen; Bacterial Toxins; Bioassay; Biologic Assays; Biological Assay; Bird Flu; Blotting, Western; Cancers; Cell Growth in Number; Cell Line; Cell Lines, Strains; Cell Locomotion; Cell Migration; Cell Movement; Cell Multiplication; Cell Proliferation; CellLine; Cells; Cellular Assay; Cellular Migration; Cellular Proliferation; Chemicals; Chrysemonas; Cleaved cell; Collection; Contracting Opportunities; Contracts; Degenerative Disorder; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Development; Diphtheria; Disease; Disorder; Dose; Drug usage; Drugs; Endopeptidase Inhibitors; Ensure; Esteroproteases; Family; Family member; Flavimonas; Fowl Pest; Fowl Plague; GFAC; Glycoproteins; Goals; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; H5N1; H5N1 Subtype Influenza A Virus; H5N1 virus; HIV-1; HIV-I; HIV1; HPV; High Throughput Assay; Hormone Receptor; Human Papillomavirus; Human immunodeficiency virus 1; IC50; Immunodeficiency Virus Type 1, Human; In Vitro; Infectious Human Wart Virus; Influenza A Virus, H5N1 Subtype; Influenza, Avian; Inhibitory Concentration 50; Lead; Life; Lytotoxicity; MMP-X1; MMP14 gene; MMPs; MT1-MMP; MTMMP1; Malignant; Malignant - descriptor; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Matrix Metalloproteinases; Measles; Mediating; Medication; Metallopeptidases; Metalloproteases; Metalloproteinases; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Monitor; Motility; Motility, Cellular; Neoplasm Metastasis; Oranges; Organism-Level Process; Organismal Process; Orthophosphoric-monoester phosphohydrolase (alkaline optimum); PA antigen, Bacillus anthracis; Papilloma Virus, Human; Papillomavirus, Human; Pathway interactions; Pb element; Peptidase Inhibitors; Peptidases; Peptide Hydrolase Inhibitors; Peptide Hydrolases; Peptide Peptidohydrolase Inhibitors; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Phenotype; Physiologic; Physiologic Processes; Physiological; Physiological Processes; Plasma Proteins; Poisons; Primary Senile Degenerative Dementia; Process; Property; Property, LOINC Axis 2; Proprotein Convertases; Protease Antagonists; Protease Inhibitor; Proteases; Proteinase Inhibitors; Proteinases; Proteins; Proteolytic Clipping; Proteolytic Enzymes; Proteolytic Processing; Pseudomonas; Reporter; Reporting; Rubeola; SARS Virus; SARS coronavirus; SARS-Associated Coronavirus; SARS-CoV; SARS-Related Coronavirus; Secondary Neoplasm; Secondary Tumor; Sensitivity and Specificity; Serine Endopeptidases; Serine Protease; Serine Protein Hydrolases; Serine Proteinases; Severe Acute Respiratory Syndrome Virus; Shigella; Solubility; Specificity; System; System, LOINC Axis 4; Testing; Therapeutic; Time; Toxic Chemical; Toxic Substance; Toxic effect; Toxicities; Tumor Cell Migration; Urbani SARS-Associated Coronavirus; Validation; Viral; Viral Diseases; Virus; Virus Diseases; Viruses, General; Western Blotting; Western Blottings; Western Immunoblotting; Yellow Fever; alkaline phosphomonoesterase; angiogenesis; anthrax protective antigen; anthrax protective factor; anthrax toxin PA; anthrax toxins translocating protein, Bacillus anthracis; arthritic; avian flu; base; botulinum; cancer cell; cancer metastasis; cancer progression; cell motility; cell transformation; cleaved; cultured cell line; cytotoxic; cytotoxicity; degenerative condition; degenerative disease; dementia of the Alzheimer type; disease/disorder; drug development; drug use; drug/agent; experiment; experimental research; experimental study; gene product; glycerophosphatase; heavy metal Pb; heavy metal lead; high throughput screening; human T cell leukemia virus III; human T lymphotropic virus III; immunogen; improved; influenza in birds; inhibitor; inhibitor/antagonist; malignancy; member; metalloproteinase (general); miniaturize; morbilli; neoplasm progression; neoplasm/cancer; neoplastic progression; pag protein, Bacillus anthracis; pagA protein, Bacillus anthracis; pathogen; pathway; poison; primary degenerative dementia; protein blotting; public health relevance; research study; scaffold; scaffolding; senile dementia of the Alzheimer type; small molecule; small molecule libraries; therapeutic development; toxic compound; transformed cells; tumor progression; viral infection; virus infection; wart virus
