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Principal Investigator: Ari Benjamin Fitzsimmons
Organization: BOSTON UNIVERSITY MEDICAL CAMPUS
Fiscal Year: 2024
Award: $48,974
Funding agency: National Heart Lung and Blood Institute
SIGNIFICANCE: G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in the human
genome and are implicated in a wide array of cardiovascular and respiratory diseases. Notably, GPCRs are the
targets of one third of FDA approved therapeutics, including drugs prescribed for coronary heart disease,
hypertension, and asthma. However, of the ~800 known GPCRs, ~120 are orphan receptors (oGPCRs), which
are considered druggable targets but remain understudied in basic research efforts as they lack a known ligand.
Over the past two decades, extensive efforts to deorphanize oGPCRs have resulted in little turnout. It is evident
there is a need to increase the rate of discovery of ligands for oGPCRs. This proposal outlines a yeast directed
evolution platform for systematic, high-throughput discovery of antibody-based ligands for oGPCRs.
Deorphanizing a single oGPCR alone would open the door to understanding new biology and development of a
potential therapeutic agent.
BACKGROUND: oGPCR deorphanization efforts have been historically slow and low-throughput. Conventional
screens for oGPCR ligands can handle many targets, but due to the sheer amount of possibilities, including
protons, photons, lipids, hormones, peptides, and larger proteins, success has been limited. Nanobodies, or
small antibody fragments, offer distinct advantages for ligand screening, namely their suitability to directed
evolution. Directed evolution allows for billions of nanobodies to be screened for binding and modulation of
oGPCRs with a simple growth selection. Using a yeast-based hypermutation directed evolution platform, and a
precise, scalable, high-throughput continuous culture device, directed evolution campaigns can be scaled up to
ensure a deep search of nanobody sequence space.
SYNOPSIS OF AIMS: In this proposal are two related, but not interdependent aims. AIM1 establishes a
systematic workflow for the functional reconstitution of human GPCRs in yeast, which will (1) create a library of
yeast strains with orphan and non-orphan GPCRs, and (2) elucidate general guidelines to increase success
rates of GPCR expression in yeast. AIM2 takes oGPCRs found to be functional in yeast and uses them to run
directed evolution campaigns to discover nanobody ligands and validate these ligands in human cells. Both aims
can be developed in parallel, with results from AIM1 being progressively integrated into AIM2. Together, these
innovative platforms will lead to the deorphanization of many oGPCRs, thereby enabling the exploration of new
GPCR biology and pharmacology.
Terms: <Agonist><Anti-Hypertensive Agents><Anti-Hypertensive Drugs><Anti-Hypertensives><Antibodies><Antibody Fragments><Area><Asthma><Autocrine Communication><Autocrine Signaling><Autocrine Systems><Bacteria><Baker's Yeast><Basic Research><Basic Science><Benchmarking><Best Practice Analysis><Binding><Biological Agent><Biological Products><Biology><Bioreactors><Biosensor><Brewer's Yeast><Bronchial Asthma><CMKRL2><Cardiovascular><Cardiovascular Body System><Cardiovascular Diseases><Cardiovascular Organ System><Cardiovascular Physiology><Cardiovascular system><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell surface><Cells><Chemicals><Chemokine Receptor-Like 2><Clinic><Cloning><Collection><Coronary Disease><Coronary heart disease><Data><Development><Devices><Directed Molecular Evolution><Drug Prescribing><Drug Prescriptions><Drugs><Endocrine Gland Secretion><Ensure><Evolution><FDA approved><Family><G Protein-Complex Receptor><G Protein-Coupled Estrogen Receptor><G Protein-Coupled Receptor 30><G Protein-Coupled Receptor Genes><G Protein-Coupled Receptor Signaling><G-Protein-Coupled Receptors><G-Proteins><GPCR><GPCR Signaling><GPER><GPER gene><GPR30><GTP-Binding Proteins><GTP-Regulatory Proteins><Generalized Growth><Goals><Growth><Guanine Nucleotide Coupling Protein><Guanine Nucleotide Regulatory Proteins><Guidelines><H+ element><Heart Vascular><Heart failure><Hormones><Human><Human Genome><Hydrogen Ions><Hypertension><Hypotensive Agent><Hypotensive Drugs><Immunoglobulin Fragments><Intracellular Communication and Signaling><Libraries><Ligands><Link><Lipids><Mammalian Cell><Medication><Membrane><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Modern Man><Molecular Evolution><Molecular Interaction><Optics><Orphan><Peptides><Pharmaceutical Preparations><Pharmacology><Photons><Proteins><Protons><Receptor Protein><Reporter><Research><Respiratory Disease><Respiratory System Disease><Respiratory System Disorder><Role><Running><S cerevisiae><S. cerevisiae><Saccharomyces cerevisiae><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Specificity><Surface Proteins><System><Testing><Therapeutic><Therapeutic Agents><Therapeutic Hormone><Thrombosis><Tissue Growth><Variant><Variation><Vascular Hypertensive Disease><Vascular Hypertensive Disorder><Yeasts><antagonist G><anti-hypertension><atherosclerotic heart disease><autocrine><benchmark><biological sensor><biological signal transduction><biologics><biopharmaceutical><biotherapeutic agent><cardiac failure><cardiovascular disorder><cardiovascular function><circulatory system><coronary disorder><determine efficacy><developmental><directed evolution><drug/agent><druggable target><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><evaluate efficacy><examine efficacy><high blood pressure><human whole genome><hyperpiesia><hyperpiesis><hypertensive disease><hypertensive disorder><in silico><innovate><innovation><innovative><medication prescription><membrane structure><microbioreactor><nanobodies><nanobody><new drug treatments><new drugs><new pharmacological therapeutic><new technology><new therapeutics><new therapy><next generation therapeutics><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel technologies><novel therapeutics><novel therapy><ontogeny><optical><pharmacologic><prescribed medication><promoter><promotor><prototype><receptor><receptor binding><receptor bound><receptor expression><reconstitute><reconstitution><scale up><screening><screenings><sdAb><single domain antibodies><small molecule><social role><success><synthetic protein><therapeutic candidate><thrombotic disease><thrombotic disorder><tool>