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Principal Investigator: Jeffery Micheal Tharp
Organization: INDIANA UNIVERSITY INDIANAPOLIS
Fiscal Year: 2023
Award: $249,000
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
Bicyclic peptides are conformationally constrained peptides comprised of two macrocyclic rings. Owing to their
increased rigidity, bicyclic peptides can bind to protein targets with greater affinity and selectivity than their linear
and monocyclic counterparts. As a result, these molecules are highly desirable scaffolds for the development of
peptide-based therapeutics. Phage display is a laboratory evolution technique that enables the discovery of high-
affinity peptide ligands from large, combinatorial peptide libraries. Although initially limited to linear peptides, this
technique has been adapted for the discovery of bicyclic peptide ligands. Most often, phage-displayed bicyclic
peptides are generated by chemically modifying linear peptides using cysteine-reactive small molecules; how-
ever, this method is technically challenging. As a result, progress in this field has been limited. Recently, several
studies have used genetic code expansion to co-translationally install cysteine-reactive noncanonical amino
acids (ncAAs) into phage-displayed peptides to produce libraries of cyclic peptides. This strategy has significant
advantages over the chemical cyclization approach, but is currently limited to monocyclic peptides. The over-
arching objective of this proposal is to develop technology that enables phage display of bicyclic pep-
tides using genetic code expansion. Our central hypothesis is that bifunctional ncAAs, i.e. ncAAs containing
two cysteine-reactive functional groups, can be used to generate ribosomally synthesized bicyclic peptides by
intramolecular reaction with cysteine residues. To realize our objective, we will pursue three Specific Aims. In
Aim 1 (K99 Phase) we will engineer an aminoacyl-tRNA synthetase that recognizes bifunctional ncAAs contain-
ing two cysteine-reactive moieties. This will be accomplished using traditional and state-of-the-art methods of
directed evolution. In Aim 2 (K99/R00 Phase) we will develop a phage display system that is compatible with co-
translational installation of bifunctional ncAAs and we will optimize this system for bicyclic peptide formation. We
will then validate this system by selecting and characterizing ligands for model targets. In Aim 3 (R00 Phase) we
will use phage display to identify bicyclic peptides that bind to the spike protein of human coronaviruses and
inhibit virus-host membrane fusion. By targeting proteins from various coronaviruses, we will strive to identify
inhibitors with broad-spectrum antiviral activity. The proposed work will provide a facile route for generating bi-
cyclic peptide libraries thereby greatly accelerating the discovery of therapeutic peptide leads. The Candidate,
Dr. Jeffery Tharp’s long-term career goal is to establish an independent research program that uses genetic
code expansion and phage display to develop antiviral peptides for the diagnosis, treatment, and prevention of
infectious diseases. Herein we propose a detailed five-year Career Development Plan supervised by mentors
Drs. Dieter Söll and Craig Wilen, and a team of subject-matter experts. This plan will augment previous training
and address key training gaps to prepare Dr. Tharp for accomplishing his long-term career goal.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Acceleration><Address><Adoption><Affinity><Amines><Amino Acids><Amino Acyl T RNA Synthetases><Amino Acyl-tRNA Ligases><Amino Acyl-tRNA Synthetases><Aminoacyl Transfer RNA Synthetase><Aminoacyl-tRNA Synthetase><Antibodies><Antiviral Agents><Antiviral Drugs><Antivirals><Bacteriophage M13><Bacteriophages><Binding><COVID-19 virus><COVID19 virus><Cell Body><Cells><Chemicals><CoV emergence><CoV-2><CoV2><Coliphage M13><Communicable Diseases><Coronaviridae><Coronaviridae Infections><Coronavirus><Coronavirus Infections><Coupled><Cyclic Peptides><Cyclicity><Cyclization><Cysteine><Development><Development Plans><Diagnosis><Diagnostic><Directed Molecular Evolution><Disease><Disorder><Drug Targeting><Engineering><Enterobacteria phage M13><Evolution><Genetic Code><Goals><HCoV><Half-Cystine><Human><In Vitro><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><L-Cysteine><Laboratories><Learning Skill><Libraries><Ligands><M13 Phage><MERS corona virus><MERS coronavirus><MERS virus><MERS-CoV><Mediating><Membrane Fusion><Mentors><Metabolic Glycosylation><Methods><Middle East Respiratory Syndrome Corona Virus><Middle East Respiratory Syndrome Coronavirus><Middle East Respiratory Syndrome Virus><Middle East Respiratory Syndrome-CoV><Middle East Respiratory coronavirus><Middle Eastern Respiratory Syndrome Corona virus><Middle Eastern Respiratory Syndrome Coronavirus><Middle Eastern Respiratory Syndrome Virus><Middle Eastern Respiratory Syndrome-CoV><Modeling><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Peptide Conformation><Peptide Library><Peptide-based drug><Peptides><Periodicity><Phage Display><Phages><Phase><Prevention><Property><Proteins><Reaction><Research><Respiratory Disease><Respiratory System Disease><Respiratory System Disorder><Rhythmicity><Ribosomes><Route><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-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><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 related corona virus 2><Side><Surface><System><Techniques><Technology><Therapeutic><Training><Transfer RNA><Transfer RNA Synthetase><Translations><Triplet Codon-Amino Acid Adaptor><Tyr-tRNA Ligase><Tyrosine-tRNA Ligase><Tyrosine-transfer RNA Ligase><Tyrosyl T RNA Synthetase><Tyrosyl-tRNA Ligase><Tyrosyl-tRNA Synthetase><Viral><Viral Activity><Viral Function><Viral Physiology><Virus><Work><Wuhan coronavirus><amine><aminoacid><aminoacid tRNA ligase><anti-viral agents><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><antiviral compound><antiviral medication><antiviral therapeutic><bacterial virus><career><career development><combinatorial><conformation><conformational><conformational state><conformationally><conformations><corona virus><corona virus emergence><coronavirus disease 2019 virus><coronavirus disease-19 virus><coronavirus emergence><developmental><directed evolution><emergent CoV><emergent corona virus><emergent coronavirus><emerging CoV><emerging corona virus><emerging coronavirus><functional group><future pandemic><glycosylation><hCoV19><human CoV><human corona virus><human coronavirus><in vivo><inhibitor><nCoV><nCoV2><new CoV><new corona virus><new coronavirus><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><next generation therapeutics><next pandemic><novel><novel CoV><novel corona virus><novel coronavirus><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><peptide drug><prevent><preventing><programs><protein protein interaction><scaffold><scaffolding><screening><screenings><severe acute respiratory syndrome-CoV><small molecule><tRNA><tRNA Synthetase><tech development><technology development><therapeutic peptide><thioether><tool><transfer Ribonucleic acids><translation>