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Principal Investigator: James Inglese
Organization: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
Fiscal Year: 2023
Award: $693,256
Funding agency: National Center for Advancing Translational Sciences
This project includes the development of biochemical and target-focused assays based on specific protein or nucleic acid targets implicated in disease. The assay designs are considered in the context of analysis and progression strategies for evaluation of a wide range of compound classes using high throughput screening technologies. There is a strong emphasis on methods development research to advance assay and lead discovery efficiency. Complementing these activities, we also explore and devise approaches for the interrogation of complex chemical libraries (e.g., natural product extracts, mRNA display). The work from this program is used to support a range of grant applications, program objectives and prototype projects. The following are on-going:
Targeting protein palmitoylation with small molecules. In collaboration with A. Banerjee (NICHD, NIH) we have developed 1536-well compatible protein palmitoyl acyl transferase assays to evaluate chemical libraries for potential inhibitors of the enzyme as possible therapeutic leads for the large number of diseases to which this class of enzyme have been linked. These compounds are also anticipated to have value as structural, functional, and pharmacological probes.
Assay development to enable discovery of novel small molecule antagonists of the receptor guanylate cyclase Npr1. In collaboration with M. Hoon (NIDCR, NIH) we have developed assays of the b-type natriuretic peptide (BNP) receptor, Npr1. Recently the agonist, BNP was shown to be required for the transmission of itch sensation between peripheral and spinal cord nerves. The Npr1 assays were employed in large-scale chemical library screening to identify novel natriuretic peptide receptor antagonists to investigate the potential of pharmacological treatments of chronic itch, a condition that results in long-term unremitting urge to scratch that significantly degrades the quality of life for sufferers (Solinski HJ et al., in press). Most recently, in collaboration with the NCATS ASPIRE program and Mytide Therapeutics we are assessing synthetic peptide analogs of the receptors natural cyclic peptide agonist as novel modulators of Npr1 signaling.
Chorismate mutase inhibitors. This project seeks to identify and develop inhibitors of chorismate mutase (CM). CM is an important enzyme found in plants and microorganisms required for the biosynthesis of the aromatic amino acids, phenylalanine and tyrosine. Mammals cannot carry out the de novo biosynthesis of aromatic amino acids and must rely on dietary sources. Thus, a potent and selective drug-like inhibitor of CM would be a valuable antimicrobial agent, particularly for antimicrobial resistant infections. Assay methods for CM have not progressed for decades and have been hampering the discovery of novel CM inhibitors. Thus, this project will serve as a model for the application of mass spectrophotometry-based screening with the RapidFire technology in collaboration with the Dingyin Tao in Analytical Chemistry. In collaboration with the Suga lab, mRNA-encoded macrocyclic peptide library-based affinity selection has resulted in the first novel active site and allosteric site macrocyclic peptide inhibitors.
Targeting G proteins with small molecules. Fibrous dysplasia of bone (McCune-Albright syndrome) is a hyperfunctioning endocrinopathy resulting from mis-sense mutations in the small -subunit of the G-protein, Gs leading to increased levels of cellular cAMP. The aim of this project is to develop biochemical and cell-based assays suitable for evaluating the activity and coupling of G proteins to their GPCRs and effector adenylyl cyclase (Getz RA et al., 2019). For example, by enabling a quantitative high throughput screening assay using the R201C mutant form of Gs to identify small molecules capable of antagonizing the R201C Gs adenylyl cyclase-activating conformation.
SARS CoV-2 Nsp1 Inhibitors. The nonstructural viral protein, nsp1 is a virulence factor of SARS-CoV2 that inhibits gene expression in infected cells. The resultant inhibition of host gene expression restricts antiviral signaling and the interferon response. In the closely related virus SARS-CoV1, mutations in Nsp1 create highly attenuated viruses, which have been used in vaccine trials. Here in collaboration with the Structural Virology Laboratory of Prof. Jeffrey Kieft at the University of Colorado School of Medicine, Prof. Anna-Lena Steckelberg, Ph.D. at Columbia University, and Prof. Hiro Suga, Ph.D. of The University of Tokyo we are developing a compound screening and validation assay series to identify inhibitors of SARS-CoV2 Nsp1 function.
Metallo beta-lactamases. With 2.8 million acquired antibiotic-resistant infections yearly in the US, leading to >35,000 deaths, antibiotic resistance remains a significant and increasing public health challenge. -lactamases which hydrolyze and thereby inactivate a broad range of clinically used -lactam drugs are a major global cause of antibiotic resistance. New Delhi metallo--lactamases (NDM) can hydrolyze the last-resort carbapenems. In collaboration with Prof. Michael W. Crowder, Ph.D. and colleagues we are developing machine leaning-enabled screening paradigms to discovery novel metallo--lactamases inhibitors chemotypes.
Inhibition of Hedgehog autoprocessing and cholesteroylation. The Hedgehog (Hh) signaling pathway is essential to embryonic cell differentiation as Hh protein concentration gradients are critical to proper embryonic morphologic development, where pathway malfunction can result in cancer. In a collaboration with Dr. Callahan, an expert in the biochemistry of Hh cholesteroylation, which occurs via the self-catalyzed endo-proteolysis of the hedgehog precursor protein by activated cholesterol bound to the precursor C-terminus, we are developing, optimizing, and validating cell-based qHTS assays for the identification of Hh autoprocessing inhibitors. Inhibitors of this process may have potential as anticancer agents .
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><3'5'-cyclic ester of AMP><3,5 cyclic AMP synthetase><ANF receptor B><ANPB><Active Sites><Adenosine Cyclic 3',5'-Monophosphate><Adenosine Cyclic Monophosphate><Adenosine, cyclic 3',5'-(hydrogen phosphate)><Adenyl Cyclase><Adenylate Cyclase><Adenylyl Cyclase><Affinity><Agonist><Albright Syndrome><Albright's syndrome><Allosteric Site><Anabolism><Analytic Chemistry><Analytical Chemistry><Anti-Cancer Agents><Antibiotic Resistance><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Applications Grants><Aromatic Amino Acids><Assay><Atrial Natriuretic Peptide B-Type Receptor><Atrial Natriuretic Peptide Receptor B><Atrionatriuretic Peptide Receptor B><Attenuated><B-type natriuretic peptide receptor><BNP Gene Product><BNP-32><Binding><Bioassay><Biochemical><Biochemistry><Biologic Assays><Biological Assay><Biological Chemistry><Brain Natriuretic Peptide-32><Brain natriuretic peptide><COVID-19 virus><COVID19 virus><Cancer Drug><Cancers><Carbapenems><Cell Body><Cell Communication and Signaling><Cell Differentiation><Cell Differentiation process><Cell Signaling><Cells><Cessation of life><Chemicals><Cholesterol><Chorismate Mutase><Chorismate Pyruvatemutase><Clinical><CoV-2><CoV2><Collaborations><Colorado><Complement><Complement Proteins><Complex><Coupling><Cyclic AMP><Cyclic Peptides><Death><Deoxyguanylate Cyclase><Development><Development and Research><Disease><Disorder><Doctor of Philosophy><Drugs><EXTMR><Embryo><Embryonic><Enzyme Antagonist><Enzyme Gene><Enzyme Inhibitor><Enzyme Inhibitor Agent><Enzyme Inhibitor Drugs><Enzymes><Erinaceidae><Evaluation><Extramural><Extramural Activities><Fibrous Dysplasia of Bone><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><G-Proteins><G24 protein><GC-B><GPCR><GTP-Binding Proteins><GTP-Regulatory Proteins><Gene Expression><Genetic Alteration><Genetic Change><Genetic defect><Grant Proposals><Guanine Nucleotide Coupling Protein><Guanine Nucleotide Regulatory Proteins><Guanyl Cyclase><Guanylate Cyclase><Guanylate Cyclase B><Hedgehogs><High Throughput Assay><IFN><Inosinate Cyclase><Interferons><Intracellular Communication and Signaling><Itching><Laboratories><Lactamase><Lactams><Lead><Leanness><Link><Malignant Neoplasms><Malignant Tumor><Mammalia><Mammals><McCune Albright's Syndrome><McCune-Albright Syndrome><Medication><Medulla Spinalis><Messenger RNA><Methods><Missense Mutation><Modeling><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Morphology><Mutation><NCATS><NICHD><NIDCR><NIDR><NIH><NS Proteins><National Center for Advancing Translational Sciences><National Institute of Child Health and Human Development><National Institute of Children's Health and Human Development><National Institute of Dental Research><National Institute of Dental and Craniofacial Research><National Institutes of Health><Natriuretic Factor-32><Natriuretic Peptide Hormones><Natriuretic Peptide Receptor B><Natriuretic Peptides><Natural Products><Neoplastic Disease Chemotherapeutic Agents><Nerve><Nesiritide><Nucleic Acids><Osteitis Fibrosa Disseminata><Pathogenicity Factors><Pathway interactions><Pb element><Peptide Library><Peptide Receptor><Peptides><Peripheral><Ph.D.><PhD><Pharmaceutic Preparations><Pharmaceutical Preparations><Pharmacological Treatment><Phenylalanine><Plants><Process><Protein Precursors><Proteins><Pruritic Disorder><Pruritis><Pruritus><Public Health><QOL><Quality of life><R & D><R&D><Receptor Protein><Resistance to antibiotics><Resistant to antibiotics><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS-Associated Coronavirus><SARS-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV-1><SARS-CoV-2><SARS-CoV-2 inhibitor><SARS-CoV2><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Scientist><Series><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 inhibitor><Severe acute respiratory syndrome related corona virus 2><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Source><Spectrophotometry><Spinal Cord><Technology><Therapeutic><Thinness><Tokyo><Transferase><Transferase Gene><Translations><Transmission><Tumor-Specific Treatment Agents><Type-B Natriuretic Peptide><Tyrosine><United States National Institutes of Health><Universities><Validation><Viral><Viral Nonstructural Proteins><Virulence Factors><Virus><Work><Wuhan coronavirus><adenosine 3'5' monophosphate><adenylcyclase><antagonism><antagonist><anti-cancer drug><anti-microbial agent><anti-microbial drug><anti-microbial resistant infection><antibiotic drug resistance><antibiotic resistant><antibiotic resistant infections><anticancer agent><anticancer drug><antimicrobial agent><antimicrobial drug><antimicrobial resistant infection><assay development><atrial natriuretic factor receptor B><attenuate><attenuates><beta lactam hydrolase><beta-Lactamase><beta-Lactamhydrolase><biological signal transduction><biosynthesis><block SARS-CoV-2><block severe acute respiratory syndrome coronavirus 2><brain Natriuretic factor><cAMP><chemical library><chronic itch><chronic pruritus><conformation><conformational><conformational state><conformationally><conformations><coronavirus disease 2019 virus><coronavirus disease-19 virus><design><designing><developmental><dietary><drug discovery><drug/agent><embryo cell><genome mutation><guanylyl cyclase><guanylyl cyclase-B receptor><hCoV19><heavy metal Pb><heavy metal lead><high throughput screening><inhibit SARS-CoV-2><inhibit severe acute respiratory syndrome coronavirus 2><inhibitor><itch sensation><mRNA><malignancy><medical college><medical schools><method development><microorganism><mutant><nCoV2><naturally occurring product><neoplasm/cancer><novel><palmitoylation><pathway><peptide analog><pharmacologic><programs><prototype><receptor><research and development><response><school of medicine><screening><screenings><severe acute respiratory syndrome-CoV><small molecule><small molecule libraries><spectrophotometer><synthetic peptide><translation><transmission process><vaccination study><vaccination trial><vaccine study><vaccine trial><validations><virology><β-Lactamase>