WU250137S
S COPE OF WORK FOR THE RESEARCH PROJECT ENTITLED: EFFICACY AND SIGNALING MODULATION BY TARGETING THE SODIUM SITE AT MU OPIOID RECEPTOR COOPERATING INSTITUTION: BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY 455 BROADWAY DISCOVERY HALL REDWOOD CITY, CA 94063-3126 PRINCIPAL INVESTIGATOR: BRIAN KOBILKA, M.D. BACKGROUND MUSCLE ( MU ) OPIOID RECEPTOR (MOR) AGONISTS HAVE BEEN USED CLINICALLY FOR DECADES FOR THE TREATMENT OF MODERATE TO SEVERE PAIN. UNFORTUNATELY, USAGE OF PRESCRIPTION OPIATES ALONG WITH DIVERSIFICATION OF FENTANYL AND OTHER DESIGNER FENTANYLS HAS FUELED AN OPIOID EPIDEMIC WHICH CLAIMED 70,601 AMERICAN LIVES IN 2021 DUE TO OVERDOSE. THE SITUATION IS ALSO WORSENED BY THE FACT THAT THE ANTAGONIST NALOXONE (NARCAN) IS SHORT ACTING COMPARED TO FENTANYL, EFFECTIVELY UNABLE TO COUNTERACT OVERDOSES BEYOND 2 H. THUS, THERE IS AN URGENT NEED FOR SAFER OPIOID ANALGESICS AS WELL AS ANTAGONISTS WITH PROLONGED HALFLIVES. THE SODIUM (NA+) BINDING SITE IS CONSERVED ACROSS >300 CLASS A G-PROTEIN COUPLED RECEPTORS (GPCR S). NA+ HAS BEEN KNOWN TO BE A NEGATIVE ALLOSTERIC MODULATOR (NAM) IN THE MAJORITY OF CLASS A GPCR S. IN COLLABORATION WITH THE KOBILKA LABORATORY, THE MAJUMDAR LAB HAS RECENTLY REPORTED A RATIONAL STRUCTURE-BASED APPROACH TO TARGET THE SODIUM SITE AT MOR GENERATING BITOPIC LIGANDS BASED ON THE FENTANYL TEMPLATE. THIS PROJECT INTEGRATES CHEMISTRY, PHARMACOLOGY, AND STRUCTURAL BIOLOGY, PROPOSING TO UNDERSTAND THE ROLE OF THE SODIUM BINDING SITE IN MOR PHYSIOLOGY BY DEVELOPING NOVEL CHEMICAL PROBES WHICH EITHER LEAD TO GI/O/Z BIASED LIGANDS AND/OR ANTAGONISTS. THE KOBILKA LAB HAS EXTENSIVE EXPERIENCE IN THE USE OF BIOCHEMICAL, BIOPHYSICAL AND STRUCTURAL APPROACHES TO STUDY SIGNALING BY G PROTEIN COUPLED RECEPTORS. MORE RECENTLY, THEY HAVE USED CRYOELECTRON MICROSCOPY TO OBTAIN HIGH-RESOLUTION STRUCTURES OF GPCRS INCLUDING 8 STRUCTURES OF THE ÂΜOR (6 PUBLISHED, 2 UNPUBLISHED). TASKS THE KOBILKA LAB WILL BE RESPONSIBLE FOR THE STRUCTURAL STUDIES USING SINGLE- PARTICLE CRYO-ELECTRON MICROSCOPY OUTLINED IN AIM 2C AND THE MOLECULAR DYNAMICS SIMULATIONS OUTLINED IN AIM 2D. AIM 2. STRUCTURAL AND PHARMACOLOGICAL CHARACTERIZATION OF LEAD BITOPIC COMPOUNDS (MAJUMDAR AND KOBILKA LAB). ALL COMPOUNDS SYNTHESIZED IN AIM 1 WILL UNDERGO FUNCTIONAL CHARACTERIZATION FIRST USING THE CAMP ASSAY AND THEN Gα-SUBTYPE PROFILING USING TRUPATH AS WELL AS ARRESTIN SUBTYPE RECRUITMENT USING BRET BASED ASSAYS. GPCROME SCREENING AT 330 CNS/NON-CNS TARGETS WILL BE CARRIED OUT ON LEADS THROUGH THE PDSP. STRUCTURES OF COMPOUNDS WITH UNIQUE Gα-SUBTYPE BIAS AS WELL AS ANTAGONISTS WILL BE SOLVED. CRYOEM OF LEAD COMPOUNDS WILL BE USED TO GUIDE NEW GENERATION DESIGN, FACILITATE LIGAND OPTIMIZATION, AND PROVIDE STRUCTURAL INSIGHTS INTO THEIR DISTINCT SIGNALING, EFFICACY AND FUNCTION. BASIC ADME/PK CHARACTERIZATION WILL BE CARRIED OUT UNDER THIS AIM BEFORE COMPOUNDS ENTER ANIMAL STUDIES. FOR ANTAGONISTS AGAINST FENTANYL, PK ASSAYS WILL AIM TO OPTIMIZE FOR POTENT MOR ANTAGONISM WITH A LONGER DURATION OF ACTION COMPARED TO NALOXONE AIM 2C. CRYO-EM STRUCTURES OF LEADS (KOBILKA LAB). COMPOUNDS WITH FAVORABLE FUNCTIONAL SELECTIVITY PROFILES IN G-PROTEIN AND ARRESTIN SIGNALING ASSAYS AND/OR ARE ANTAGONISTS WILL BE EVALUATED USING CRYOEM MICROSCOPY. COMPOUNDS WITH APPROPRIATE ADME/PK PROPERTIES AS WELL AS A SAFER ANALGESIC PROFILE (IDENTIFIED THROUGH AIM 3) AND/OR ANTAGONISTS WITH LONGER DURATION OF ACTION THAN FENTANYL IN VIVO ARE ALSO OF HIGH INTEREST FOR CRYO-EM STUDIES. THE STRUCTURAL STUDIES WILL ALLOW US TO CORRELATE COMPOUND BINDING INTERACTIONS WITHIN THE ORTHOSTERIC AND NA+ SITES WITH THE IN VITRO SIGNALING OBSERVED AND ASSESS POTENTIAL RELATIONSHIPS BETWEEN THEM. SIMILARLY, ESTABLISHING A RELATIONSHIP BETWEEN STRUCTURES AND OBSERVED IN VIVO BEHAVIOR WITH OR WITHOUT CNS ADVERSE EFFECTS (IN AIM 3) WILL ALSO BE ATTEMPTED AS PART OF THIS SUBAIM. THE KOBILKA LAB HAS SOLVED CRYO-EM STRUCTURES OF MOR BITOPICS34 PRESENTED AS PRELIMINARY DATA IN THIS PROPOSAL AS WELL AS OTHER CB1 BITOPICS (DATA NOT SHOWN). IN ADDITION, THE LAB HAS USED CRYO-EM TO DETERMINE AN INACTIVE-STATE STRUCTURE OF MOR BOUND TO ANANTAGONIST AND A NAM, WHERE NB6 WAS USED FOR PARTICLE ORIENTATION (UNPUBLISHED),100 AS WELL AS AN ACTIVE-STATE STRUCTURE OF MOR BOUND TO AN AGONIST AND A PAM (FIG.11). WE ANTICIPATE SOLVING AT LEAST 8 STRUCTURES THROUGH THIS PROJECT, SUPPORTING OUR INTENT TO DRIVE SAR STUDIES BY CRYO-EM. WE WILL INITIALLY AIM TO SOLVE STRUCTURES OF AF73 BOUND TO GI3 AND POTENTIALLY RO88 BOUND TO MOR INACTIVE STATE. AIM 2D. COMPUTATIONAL MODELING. DOCKING AND MD SIMULATIONS ON LIGANDS WILL BE CARRIED OUT IN THIS AIM. THESE IN SILICO METHODS CAN BE USED TO SCREEN COMPOUNDS FOR POTENTIAL BINDING POSES AS WELL AS A QUICK TOOL FOR OPTIMIZATION FOR ENGAGING AMINO ACID RESIDUES IN THE NEAR VICINITY OF THE LIGAND AND/OR ENGAGING SURROUNDING WATERS IN THE POCKET. DOCKING OF COMPOUNDS OF INTEREST WILL BE CARRIED OUT USING AUTODOCK VINA101 OR BRISTOL UNIVERSITY DOCKING ENGINE (BUDE)102. MOLECULAR DYNAMICS SIMULATIONS (MDS) OF SOLVED CRYO-EM STRUCTURES WILL BE RUN USING AMBER103, THE STRUCTURES WILL BE INSERTED INTO A LIPID BILAYER AND MDS WILL BE GENERATED. BY GENERATING MDS, WE CAN EXPLORE THE INTERACTION BETWEEN LIGAND AND RECEPTOR IN A DYNAMIC ENVIRONMENT. THIS TECHNIQUE BUILDS ON THE INFORMATION GIVEN BY CRYOEM STRUCTURES AND WILL ENABLE US TO GIVE ACCURATE PREDICTIONS ON RESIDUE INTERACTIONS AND LIGAND STABILITY. IT WILL ALSO ALLOW FURTHER MODELING THAT MAY NOT BE INITIALLY HIGHLIGHTED IN CRYO-EM STRUCTURES SUCH AS THE DIFFUSION OF WATER MOLECULES INTO THE ORTHOSTERIC POCKET, HYDROGEN BONDS AND HELICAL MOVEMENTS. DELIVERABLES THROUGHOUT THE PROJECT, DR. KOBILKA WILL: Ï · COORDINATE MEETINGS AND SHARE INFORMATION WITH DR. MAJUMDAR. Ï · ENSURE THAT RESEARCH GOALS ARE MET IN A TIMELY MANNER WITH SCIENTIFIC INTEGRITY, THAT THE WORK IS DONE WITHIN BUDGETED AMOUNTS AND IN COMPLIANCE WITH THE UNIVERSITY AND FUNDING AGENCY REGULATIONS, AND THAT DATA ARE COLLECTED AND ANALYZED PROPERLY. Ï · ASSIST IN WRITING UP ALL ASPECTS OF THE DATA.
The Leland Stanford Junior University
Project Grant R01DA059978
$196.9k 10/11/24 WU250128S
SCOPE OF WORK FOR THE RESEARCH PROJECT ENTITLED: STRUCTURE-BASED DESIGN OF MU-OPIOID RECEPTOR AGONISTS COOPERATING INSTITUTION: UNIVERSITY OF FLORIDA DEPARTMENT OF PHARMACODYNAMICS 1345 CENTER DRIVE GAINESVILLE, FLORIDA 32610 PRINCIPAL INVESTIGATOR: JAY P. MCLAUGHLIN, PH.D. THE UNIVERSITY OF FLORIDA IS A NON-PROFIT PUBLIC UNIVERSITY AND RESEARCH INSTITUTION WITH HEADQUARTERS IN GAINESVILLE, FLORIDA. THE IN VIVO WORK WILL BE PERFORMED ON THE GAINESVILLE CAMPUS IN THE LABORATORY OF DR. JAY MCLAUGHLIN, WHO IS SERVING AS CONSORTIUM PI. MOST CLINICALLY-USED OPIOIDS ACT THROUGH MU OPIOID RECEPTORS (MOR) TO TREAT SEVERE PAIN, BUT WITH THE POTENTIAL FOR ABUSE AND RESPIRATORY DEPRESSION. AS EFFECTIVE ANALGESICS ARE ESSENTIAL, IT IS CRUCIAL TO IDENTIFY SAFER THERAPEUTICS TARGETING MOR WITH DIMINISHED SIDE EFFECTS. AN APPROACH AIMED AT ACHIEVING OPIOID FUNCTIONAL SELECTIVITY INVOLVES PARTIAL AGONISM AND Gα-PROTEIN ISOFORM BIAS AT MOR. EMERGING MODELING AND CELLULAR EVIDENCE SUGGESTS THE FEASIBILITY THAT BIAS OR EFFICACY TOWARDS A SPECIFIC G-PROTEIN SUBTYPE MAY PLAY A ROLE IN THE ADVERSE EFFECTS OF OPIOID AGONISTS. THIS PROJECT AIMS AT DEVELOPING OPIOID ANALOGS BITOPICALLY TARGETING THE MOR ORTHOSTERIC SITE AND THE NA+ BINDING SITES. PRELIMINARY RESULTS FROM THE MAJUMDAR AND MCLAUGHLIN LABORATORIES WITH LEAD BITOPIC LIGANDS, C5GUANO AND C6GUANO, SUPPORT THE FEASIBILITY OF DEVELOPING THESE ANALOGS TO PRODUCE ANALGESIA WITH FEWER CLINICAL LIABILITIES SUCH AS CONDITIONED PLACE PREFERENCE AND RESPIRATORY DEPRESSION CHARACTERISTIC OF CONVENTIONAL MOR AGONISTS SUCH AS MORPHINE. THIS PROJECT IS A JOINT PROJECT ACROSS MULTIPLE INSTITUTIONS, BUILDING ON EARLIER DATA. NOVEL LIGANDS OPTIMIZED FOR A BITOPIC MULTIFUNCTIONAL MOR ORTHOSTERIC AND ALLOSTERIC PHARMACOLOGICAL PROFILE WILL BE SYNTHESIZED IN THE LAB OF DR. SUSRUTA MAJUMDAR AT UHSP/WASHINGTON UNIVERSITY, AND CHARACTERIZED FOR THEIR IN VITRO PHARMACOLOGY (OPIOID SELECTIVITY AND AGONIST POTENCY IN OPIOID TRANSFECTED CELL LINES). PROMISING COMPOUNDS SO IDENTIFIED WILL THEN BE SCREENED AT THE UNIVERSITY OF FLORIDA BY DR. MCLAUGHLIN IN PRECLINICAL TRIALS WITH MICE TO EVALUATE AND CHARACTERIZE IN VIVO ACTIVITY AND ANTINOCICEPTIVE POTENCY, AND ASSESSMENT OF POTENTIAL LIABILITIES SUCH AS PHYSICAL DEPENDENCE, RESPIRATORY DEPRESSION OR POTENTIAL FOR SUBSTANCE ABUSE. THE ULTIMATE GOAL OF THIS INITIATIVE IS TO UTILIZE NEW INFORMATION FROM RECEPTOR STRUCTURES COUPLED WITH SYNTHESIS AND SCREENING OF NOVEL LIGANDS TO PRODUCE A VARIETY OF STRUCTURALLY UNIQUE LIGANDS WITH PARTIAL AGONISM, A BIAS FOR THE Gα-ISOFORM AND ANALGESIA WITH ATTENUATED SIDE-EFFECTS. DR. MCLAUGHLIN WILL BE THE CO-INVESTIGATOR OF THE PROJECT AT THE UNIVERSITY OF FLORIDA AND WILL HAVE OVERALL RESPONSIBILITY FOR THE SCIENTIFIC CONDUCT AND EXECUTION OF THE ADMINISTRATION OF COMPOUNDS, COLLECTION OF DATA AND ANALYSIS OF SAME FOR THE IN VIVO TESTING IN THE PROJECT. HE WILL ALSO BE RESPONSIBLE FOR PREPARATION OF REPORTS FOR PUBLICATION, SUPERVISING THE RESEARCH TECHNICIAN AND MONITORING THE ADMINISTRATIVE COMPONENTS OF THE STUDY AT THE UNIVERSITY OF FLORIDA. THROUGHOUT THE PROJECT, DR. MCLAUGHLIN WILL WORK IN COLLABORATION WITH DR. MAJUMDAR AT THE UNIVERSITY OF HEALTH SCIENCES AND PHARMACY (UHSP) IN ST. LOUIS AND ADDITIONAL COLLABORATORS, PARTICIPATING IN MEETINGS AND CONFERENCE CALLS TO DISCUSS PROGRESS AND ANALYZE DATA.
University Of Florida
Project Grant R01DA059978
$125.5k 10/10/24