Leveraging Biased Signaling in Addiction Therapeutics (PDF)

Leveraging biased signaling in the search for addiction therapeutics Lauren M. Slosky, Ph.D. Department of Pharmacology University of Minnesota [email protected] January 30, 2024 PHCL4001:Complementing Signal Transduction Lecture II NTSR1 G protein and β-arrestin signaling may mediate distinct physiol...

Leveraging biased signaling in the search for addiction therapeutics Lauren M. Slosky, Ph.D. Department of Pharmacology University of Minnesota [email protected] January 30, 2024 PHCL4001:Complementing Signal Transduction Lecture II NTSR1 G protein and β-arrestin signaling may mediate distinct physiological effects NT NTSR1 Gαq GTP β-Arrestin Attenuation of addictionassociated behaviors Hypothermia Hypotension Motor impairment β-arrestin biased NTSR1 ligands may be preferable to unbiased agonists for the treatment of chemical addictions NT Biased allosteric modulator SBI-553 NTSR1 Gαq GTP Hypothermia Hypotension Motor impairment β-Arrestin Attenuation of addictionassociated behaviors β-arrestin biased NTSR1 ligands may be preferable to unbiased agonists for the treatment of chemical addictions NT SBI-553 NTSR1 Gαq GTP Hypothermia Hypotension Motor impairment β-Arrestin Attenuation of addictionassociated behaviors Addiction is a Brain Disorder Adapted from: National Institute on Alcohol Abuse and Alcoholism. Draft strategic plan for research 2017–2021. Drug Addiction is a Growing Health Concern 107,000 2021 Medications for the Treatment of Addiction 12000 Publications 10000 8000 Buprenorphine 6000 4000 Naltrexone Methadone 2000 0 1960 Use Disorder Opioid Cocaine Amphetamine 1 0 0 1980 0 0 0 2000 1 0 0 2020 1 0 0 FDA-approved treatments 3 0 0 Addiction is a Brain Disorder Adapted from: National Institute on Alcohol Abuse and Alcoholism. Draft strategic plan for research 2017–2021. Addiction and Mesolimbic Dopamine Striatum NAc Midbrain VTA DA Addiction and Mesolimbic Dopamine NAc ✓ ✓ ✓ ✓ ✓ ✓ Alcohol Nicotine Opioids THC Cocaine VTA Methamphetamine DA Addiction and Mesolimbic Dopamine NAc ✓ ✓ ✓ ✓ ✓ ✓ Alcohol Nicotine Opioids THC Cocaine VTA Methamphetamine DA Addiction and Mesolimbic Dopamine NAc ✓ ✓ ✓ ✓ ✓ Diverse Roles for Dopamine Reward Movement Mood Motivation Executive function VTA DA Neurotensin (NT) Modulates Dopamine Signaling NAc VTA DA Neurotensin (NT) Modulates Dopamine Signaling NAc VTA DA NT Woodworth et al. (2018) Neuropeptides Hanson et al. (2012) Neuroscience Studler et al. (1988) Neuropeptides Neurotensin (NT) Modulates Dopamine Signaling Glu Frontal cortex VTA NAc DA NT Neurotensin receptor 1 (NTSR1) As reviewed: Torruella-Suárez & Mcelligot (2020) Neuropharmacology Ferraro et al. (2016) Journal of Psychopharmacology Binder et al. (2001) Pharmacological Reviews Neurotensin (NT) Modulates Dopamine Signaling Glu NAc Frontal cortex D2 VTA DA NT Neurotensin receptor 1 (NTSR1) As reviewed: Torruella-Suárez & Mcelligot (2020) Neuropharmacology Ferraro et al. (2016) Journal of Psychopharmacology Binder et al. (2001) Pharmacological Reviews NT Antagonizes D2 Signaling via Action at NTSR1 NAc D2 VTA DA NT Neurotensin receptor 1 (NTR1) Thibault et al. (2011) J Biol Chem Jomphe et al. (2006) Eur J Neurosci Jiang et al. (1994) J. Physiol. (Lond.) Li et al. (1995) J. Neural Transm. Gen. Sect. NTSR1 NTSR1 is a Promising Therapeutic Target Psychostimulants NTSR1 Peptide Agonists Hyperlocomotion Hypolocomotion Neurotoxicity Neuroprotective Psychotic episodes Antipsychotic Cognitive deficits Cognitive enhancers Skoog et al. (1986) Neuropharmacology 25, 777-82. Torup et al. (2003) Neurosci Lett 351, 173-6. Feifel et al. (1999) J Pharmacol Exp Ther 288, 710-3. Grimond-Billa et al. (2008) J Psychopharmacol 22, 290-9. Models of GPCR Activation Rajagopal et al. (2010) Nature Reviews Drug Discovery 9: 373-386. Models of GPCR Activation NTSR1 NT Gαq PLC, IP Ca2+ mobilization Rajagopal et al. (2010) Nature Reviews Drug Discovery 9: 373-386. Models of GPCR Activation Rajagopal et al. (2010) Nature Reviews Drug Discovery 9: 373-386. Screen for NTSR1 β-arrestin Agonists β-arrestin2 GFP Development of Small Molecule NTSR1 β-arrestin Agonists β-arrestin translocation screen 400,000 compounds Pinkerton et al. (2019) J Med Chem Barak et al. (2016) ACS Chem Biol Peddibhotla et al. (2013) ACS Med Chem Lett Screen developed by Lawrence Barak Structural modifications aimed at improving solubility 200-300 hits 100s of variants with newly identified chemical backbone 10-15 hits share common chemical backbone ML314 SBI-553 Current lead compound 40 (pmol/mg protein) Specific [3H]NT Binding SBI-553 is an Allosteric Ligand 30 20 10 M SBI-553 1 M SBI-553 100 nM SBI-553 10 nM SBI-553 Vehicle Bmax 10 0 Kd 0.001 0.01 0.1 1 [3H]NT (nM) 10 100 Slosky et al. (2020) Cell 181(6):1364-1379. NTSR1 Signaling NT Gq Pathway Gq activation IP3 generation Ca2+ mobilization NTSR1 GRK2 Gα q GTP β-Arrestin β-Arrestin Pathway GRK recruitment NTSR1 phosphorylation β-arrestin2 recruitment NTSR1 internalization NTSR1 Signaling NT Gq Pathway Gq activation IP3 generation Ca2+ mobilization NTSR1 GRK2 Gα q GTP β-Arrestin β-Arrestin Pathway GRK recruitment NTSR1 phosphorylation β-arrestin2 recruitment NTSR1 internalization NT is a Balanced Agonist NT NTSR1 Gα q NT 0.1 0.0 -10 Slosky et al. (2020) Cell 181(6):1364-1379. -8 -6 -4 Log [Compound], M (Objects per Area) 0.2 β-Arrestin -Arrestin Translocation (Net Luminescence) Ca2+ Mobilization GTP 600 400 NT 200 0 -10 -8 -6 -4 Log [Compound], M SBI-553 is a β-arrestin Biased Agonist SBI-553 NTSR1 Gα q NT 0.1 SBI-553 0.0 -10 Slosky et al. (2020) Cell 181(6):1364-1379. -8 -6 -4 Log [Compound], M (Objects per Area) 0.2 β-Arrestin -Arrestin Translocation (Net Luminescence) Ca2+ Mobilization GTP 600 400 NT SBI-553 200 0 -10 -8 -6 -4 Log [Compound], M SBI-553 Confers β-arrestin Bias to NT NT SBI-553 NTSR1 Gα q 0.2 0.1 NT + SBI-553 0.0 -10 Slosky et al. (2020) Cell 181(6):1364-1379. -8 -6 -4 Log [Compound], M 1500 (Objects per Area) (Net Luminescence) Ca2+ Mobilization NT + Vehicle 0.3 β-Arrestin -Arrestin Translocation GTP 1000 NT + SBI-553 500 NT + Vehicle 0 -10 -8 -6 Log [NT], M -4 Biased Allosteric Modulators: A New Class of GPCR Ligands Agonist Inactive Conformations Inactive Conformations BAM Multiple Active Conformations Subset of Active Conformations + Biased Allosteric Modulator (BAM) Multiple signaling outcomes Multiple, distinct physiological effects a b c Subset of signaling outcomes a b c Subset of distinct physiological effects Slosky et al. (2021) Trends in Pharmacological Sciences 42(4):283-299. Structural (Cryo-EM) Basis of Biased Signaling Now Understood 3 Krumm et al. 2023 Biochemistry, 62, 1233-1248 Duan et al. 2023 Nature (Authors include Slosky [Minnesota], Pinkerton [SBP], Barak [Duke] and cryoEM Team [UNC Chapel Hill]) Epub August 2, dio: 10.1038/s41586-023-06395-9 NT SBI-553 Does SBI-553 reduce stimulantassociated behaviors? Slosky et al. (2020) Cell 181(6):1364-1379. Psychostimulant-Induced Hyperlocomotion Methamphetamine 400 30 60 90 Time (min) 120 150 1000 500 0 0 30 60 90 Time (min) 120 150 Saline + Vehicle 1500 Cocaine + Vehicle Distance Traveled 800 (cm / 5 min) Distance Traveled (cm / 5 min) Distance Traveled 1500 Saline (cm / 5 min) Meth + Vehicle 1200 0 0 Cocaine 1000 500 0 0 30 60 90 120 150 Time (min) Slosky et al. (2020) Cell 181(6):1364-1379. Psychostimulant-Induced Hyperlocomotion Methamphetamine * * 30 * * 60 90 T i m e ( m in ) 120 150 500 * * * * 0 0 30 * * 60 90 T i m e ( m in ) 120 150 ( c m / 5 m in ) ** * * * 400 1000 S a lin e + V e h ic le S a lin e + S B I 1500 C o c a in e + V e h ic le C o c a in e + S B I- 5 5 3 ( c m / 5 m in ) 800 1500 D is ta n c e T r a v e le d ( c m / 5 m in ) D is ta n c e T r a v e le d M e th + S B I-5 5 3 Saline D is ta n c e T r a v e le d M e th + V e h ic le 1200 0 0 Cocaine 1000 500 0 0 30 60 90 120 150 T i m e ( m in ) Slosky et al. (2020) Cell 181(6):1364-1379. SBI-553 Attenuates Stimulant-Induced Locomotion via a β-Arrestin2-Dependent Mechanism Methamphetamine * 800 *** * * 400 * * 0 0 30 * * 60 90 (cm / 5 min) 1500 V e h ic le S B I- 5 5 3 Distance Traveled ( c m / 5 m in ) C57BL/6J β-arrestin2 D is ta n c e T r a v e le d 1200 Cocaine Vehicle SBI-553 * 1000 500 ** * ** * 0 0 120 150 30 Slosky et al. (2020) Cell 181(6):1364-1379. ns 800 400 0 0 30 60 90 T i m e ( m in ) 120 150 1500 (cm / 5 min) ( c m / 5 m in ) D is ta n c e T r a v e le d β-arrestin2 KO β-arrestin2 KO V e h ic le S B I- 5 5 3 Distance Traveled T i m e ( m in ) 1200 60 90 120 150 Time (min) Vehicle ns SBI-553 1000 500 0 0 30 60 90 120 150 Time (min) SBI-553 Attenuates Stimulant-Induced Locomotion via a β-Arrestin2-Dependent Mechanism Striatum NAc D2 Midbrain VTA DA NT Neurotensin receptor 1 (NTSR1) Slosky et al. (2020) Cell 181(6):1364-1379. SBI-553 Attenuates Cocaine Self-Administration *** 10 *** 40 30 20 10 0 0 Vehicle SBI-553 No treatment 150 Lever Accuracy 20 *** 50 Vehicle SBI-553 No treatment (% Active Lever Responses) 30 Latency to First Response (min) Cocaine Reinforcements (#) *** n.s. 100 50 0 Vehicle SBI-553 No treatment Slosky et al. (2020) Cell 181(6):1364-1379. SBI-553 Attenuates Cocaine Self-Administration SBI-553 attenuates psychostimulant-associated behaviors 10 *** 40 30 20 10 0 0 Vehicle SBI-553 No treatment 150 Lever Accuracy 20 *** 50 Vehicle SBI-553 No treatment (% Active Lever Responses) 30 *** Latency to First Response (min) Cocaine Reinforcements (#) *** n.s. 100 50 0 Vehicle SBI-553 No treatment Slosky et al. (2020) Cell 181(6):1364-1379. SBI-553 Attenuates Cocaine Self-Administration Does SBI-553 produce other effects characteristic of peptide NTSR1 ligands? 10 *** 40 30 20 10 0 0 Vehicle SBI-553 No treatment 150 Lever Accuracy 20 *** 50 Vehicle SBI-553 No treatment (% Active Lever Responses) 30 *** Latency to First Response (min) Cocaine Reinforcements (#) *** n.s. 100 50 0 Vehicle SBI-553 No treatment Slosky et al. (2020) Cell 181(6):1364-1379. Unbiased NTSR1 Ligand β-arrestin Biased NTSR1 Ligand PD149163 Gαq GTP SBI-553 NTSR1 NTSR1 Gαq β-Arrestin Drug addiction-associated behaviors Side effects: Core body temperature Blood pressure Motor coordination impairment β-Arrestin GTP Drug addiction-associated behaviors Side effects: ? PD149163 = NT peptide analogue with improved metabolic stability SBI-553 Does Not Induce Hypothermia Vehicle PD149163 (0.03 mg/kg) PD146193 (0.1 mg/kg) PD146193 (0.3 mg/kg) PD146193 (1 mg/kg) 35 * *** *** *** *** 30 *** *** 25 0 *** *** *** *** 60 120 180 240 300 Time (min) 40 Body Temp (C) Body Temp (C) 40 Vehicle SBI-553 (2 mg/kg) SBI-553 (6 mg/kg) SBI-553 (12 mg/kg) SBI-553 (30 mg/kg) 35 30 25 0 60 120 180 240 300 Time (min) Slosky et al. (2020) Cell 181(6):1364-1379. 80 Vehicle PD149163 (0.03 mg/kg) PD149163 (0.1 mg/kg) PD149163 (0.3 mg/kg) PD149163 (1 mg/kg) *** 60 40 20 0 *** *** Latency to Grasp Wire (s) Latency to Grasp Wire (s) SBI-553 Does Not Impair Performance in the Wire Hang Test 80 Vehicle SBI-553 (2 mg/kg) SBI-553 (6 mg/kg) SBI-553 (12 mg/kg) SBI-553 (30 mg/kg) 60 40 20 0 Slosky et al. (2020) Cell 181(6):1364-1379. SBI-553 Does Not Induce Hypotension 0 Dennis Abraham and Lan Mao Duke Cardiovascular Physiology Core -10 -20 * 45 30 15 60 ** -30 0 ( mmHG) Systolic Blood Pressure Vehicle SBI-553 (12 mg/kg) PD149163 (0.3 mg/kg) Time (min) Slosky et al. (2020) Cell 181(6):1364-1379. NTSR1 G protein and β-arrestin signaling may mediate distinct physiological effects PD149163 NTSR1 Gαq GTP β-Arrestin Attenuation of addictionassociated behaviors Hypothermia Hypotension Motor impairment β-arrestin biased NTSR1 ligands may be preferable to unbiased agonists for the treatment of chemical addictions PD149163 SBI-553 NTSR1 Gαq GTP Hypothermia Hypotension Motor impairment β-Arrestin Attenuation of addictionassociated behaviors PD149163-Induced Hypothermia and Inactivity Vehicle + Vehicle PD149163 + Vehicle PD149163 + SBI-553 (10 mg/kg) PD149163 + SBI-553 (30 mg/kg) PD149163 + SBI-553 (100 mg/kg) 40 * 30 * *** * *** *** 30*** * * *** *** ****** *** *** *** 25 25 0 -60 0 60 120 180 240 300 60 120 180 240Time 300 (min) 1200 (Counts) 35 35 Horizontal Activity Body Temp (C) Body Temp (C) 40 1500 900 600 300 0 0 10 20 30 Time (min) 40 50 Slosky et al. (2020) Cell 181(6):1364-1379. Oral SBI-553 Pretreatment Attenuates PD149163-Induced Hypothermia and Inactivity Vehicle + Vehicle PD149163 + Vehicle PD149163 + SBI-553 (10 mg/kg) PD149163 + SBI-553 (30 mg/kg) PD149163 + SBI-553 (100 mg/kg) 40 * 30 *** * *** *** 30*** * * * *** *** *** * * * *** *** ****** *** *** *** 25 25 0 -60 0 60 120 180 240 300 60 120 180 240Time 300 (min) 1200 (Counts) 35 35 Horizontal Activity Body Temp (C) Body Temp (C) 40 1500 900 600 300 0 0 10 20 30 Time (min) 40 50 Slosky et al. (2020) Cell 181(6):1364-1379. NTSR1 G protein and β-arrestin signaling may mediate distinct physiological effects NT NTSR1 Gαq GTP Hypothermia Hypotension Motor impairment β-Arrestin Attenuation of addictionassociated behaviors β-arrestin biased NTSR1 ligands may be preferable to unbiased agonists for the treatment of chemical addictions NT SBI-553 NTSR1 Gαq GTP Hypothermia Hypotension Motor impairment β-Arrestin Attenuation of addictionassociated behaviors Acknowledgements Slosky Laboratory Noah Foster Abigail Alwin Campbell Krusemark Madelyn Moore Kaeli Edwards Kelsey Person Crystal Lemchi Briana Jimenez Lavina Iskander Nancy Xiong (LSSURP alum) Sanford Burnham Prebys Medical Discovery Institute Dr. Anthony Pinkerton Dr. Michael Jackson Dr. Steven Olsen Dr. Colin Loweth CROs Volochem Seran WuXi AppTec Pharmaron Caron Laboratory Dr. Marc Caron Dr. Lawrence Barak Dr. Yushi Bai Dr. Krisztian Toth Lauren Rochelle Caroline Ray Dr. Rahul Chandrasekhar Xiu Qin Zhang Wendy Roberts Dr. Peter Nicholls Andrea Pires Nicholas Clark Namratha Atluri Dr. Yang Zhou Dr. Josh Gross Fiona Porkka University of North Carolina at Chapel Hill Dr. Bryan Roth Dr. Brian Krumm Duke Mouse Behavioral Core Dr. William Wetsel Duke Center for In vivo Microscopy Dr. Alexandra Badea UNC-CH BRIC Dr. Hong Yuan Funding UG3 DA050316 (Jackson) P30 DA029925 (Caron) K99/R00 DA048970 (Slosky) F32 DA043931 (Slosky) University of Minnesota Foundation (Slosky) R33 DA038019 (Pinkerton) W81XWH-22-1-0266-PR211151 (Barak/Ji) P30 DA048742 (Thomas) Questions? Contact: [email protected] @LaurenSlosky Justice & Dhillon (2016) Disease Models & Mechanisms 9:101-103.