Project Grant R21AG081962

DEVELOPING HELICAL PEPTIDE ANTAGONISTS OF THE GROWTH HORMONE RECEPTOR - PROJECT SUMMARY GROWTH HORMONE (GH) IS A SINGLE-CHAIN POLYPEPTIDE THAT ACTS AS A KEY STIMULATOR OF CELL GROWTH, PROLIFERATION AND METABOLISM IN MAMMALS. GH ACTS TO PROMOTE LONGITUDINAL GROWTH AND PROPER ORGAN DEVELOPMENT DURING CHILD- HOOD AND STIMULATES PRODUCTION OF INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) IN THE LIVER AND OTHER TISSUES THROUGHOUT LIFE. NUMEROUS STUDIES HAVE INDICATED THAT REDUCED GH ACTIVITY IN VIVO RESULTS IN HEALTHY AGING AND INCREASED LIFESPAN; IN FACT, THE LONGEST-LIVED LABORATORY MOUSE RESULTS FROM GLOBAL DISRUPTION OF THE GH RECEPTOR (GHR) GENE. NOTABLY, SUCH GHR-/- MICE HAVE DEMONSTRATED ENHANCED RESISTANCE TO GH-MEDIATED DISORDERS THAT CONTRIBUTE TO UNHEALTHY AGING, INCLUDING DIABETIC END ORGAN DAMAGE AND CERTAIN TYPES OF CANCER. THIS CONNECTION BETWEEN REDUCED GH ACTION AND EXTENDED LIFESPAN HAS LED TO THE HYPOTHESIS THAT INHIBITING GH ACTION MAY DELAY THE ONSET OF AGE-RELATED MORBIDITIES. PEPTIDES REPRESENT AN ATTRACTIVE CLASS OF MOLECULE TO SERVE AS THERAPEUTIC LEADS BECAUSE THEY CAN BE DESIGNED TO MIMIC THE VARIABLE STRUCTURES AND SEQUENCES OF PROTEIN INTERACTION DOMAINS. MOREOVER, PEPTIDES ARE SEQUENCE SPECIFIC AND SYNTHETICALLY TRACTABLE, ALLOWING THEM TO CIRCUMVENT MANY OF THE PRODUCTION PROBLEMS ASSOCIATED WITH PROTEIN-BASED DRUGS. OUR GROUP RECENTLY IDENTIFIED A NOVEL PEPTIDE-BASED GHR ANTAGONIST (TERMED SH1) THAT MITIGATES GH-MEDIATED SIGNALING IN CULTURED CELL LINES. S1H WAS DESIGNED AS A DIRECT SEQUENCE MIMIC OF A SMALL HELICAL REGION (RESIDUES 36-51) OF GH THAT INTERACTS WITH THE GHR. STRUCTURE ACTIVITY RELATIONSHIPS OF S1H SHOWED A STRONG CORRELATION BETWEEN PEPTIDE HELICITY AND GHR ANTAGONISM, LEADING US TO HYPOTHESIZE THAT HELICAL PROPENSITY IS REQUIRED FOR BIOLOGICAL ACTIVITY. WE NOW SEEK TO TEST THIS HYPOTHESIS BY DEVELOPING S1H DERIVATIVES THAT FOLD INTO STABLE HELICAL STRUCTURES AND USE THEM TO INHIBIT GH-MEDIATED SIGNALING IN VITRO AND IN VIVO. IN AIM 1 OF THIS PROPOSAL, WE WILL SYNTHESIZE TWO SEPARATE CLASSES OF STRUCTURED S1H DERIVATIVE. THE FIRST CLASS WILL BE GENERATED BY INSTALLING OLEFINIC SIDE CHAINS INTO THE WILD-TYPE S1H SEQUENCE SO THE PEPTIDES CAN BE 'STAPLED' INTO A-HELICAL STRUCTURES. THE SECOND CLASS WILL BE DEVELOPED BY TRANSPOSING S1H RESIDUES ONTO THE A-HELIX OF SCYLLATOXIN, A SMALL PROTEIN THAT FOLDS INTO A STABLE A/B MOTIF. THE RESULTING PEPTIDES WILL THEN BE USED IN A SERIES OF DIRECT BINDING AND ELECTROPHORETIC MOBILITY SHIFT ASSAYS AGAINST RECOMBINANT GHR. IN AIM 2, WE WILL INVESTIGATE THE ABILITY OF OUR STRUCTURED S1H DERIVATIVES TO INHIBIT GH SIGNALING IN CELLS THAT OVEREXPRESS THE GHR AND IN VIVO MOUSE MODELS OF AGING. FIRST, WE WILL EMPLOY A CELL-BASED SURROGATE ASSAY TO DETERMINE WHETHER STRUCTURED S1H DERIVATIVES CAN INHIBIT GH-MEDIATED PHOSPHORYLATION OF DOWNSTREAM TRANSCRIPTION FACTORS, SUCH AS STAT5. NEXT, WE WILL ASSESS HOW STRUCTURED S1H DERIVATIVES AFFECT THE SERUM LEVELS OF IGF-1 AND IGF-BP3, AND BODY COMPOSITION OF C57BL6 MICE. DATA GENERATED FROM THIS PROPOSAL WILL SERVE AS A FOUNDATION FOR FUTURE STUDIES THAT EXPLORE THE MOLECULAR MECHANISMS THROUGH WHICH S1H (AND ITS STRUCTURED DERIVATIVES) AFFECTS GH-MEDIATED SIGNALING AND WILL IDENTIFY POTENTIAL LEAD COMPOUNDS TO BE USED AS EMERGING THERAPEUTICS DESIGNED TO MITIGATE GH-MEDIATED DISORDERS THAT CONTRIBUTE TO UNHEALTHY AGING.