Profiling novel pharmacology of GPCR complexes Professor Kevin - PowerPoint PPT Presentation

Profiling novel pharmacology of GPCR complexes Professor Kevin Pfleger using Receptor-HIT Director Biomedical Innovation, The University of Western Australia and MTPConnect WA Life Sciences Innovation Hub Head of Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia Chief Scientific Advisor, Dimerix Limited

Forward-looking statements This presentation includes forward-looking statements that are subject to risks and uncertainties. Such statements involve known and unknown risks and important factors that may cause the actual results, performance or achievements of Dimerix to be materially different from the statements in this presentation. Actual results could differ materially depending on factors such as the availability of resources, the results of clinical studies, the timing and effects of regulatory actions, the strength of competition, the outcome of legal proceedings and the effectiveness of patent protection.

Receptor-HIT: Receptor Heteromer Investigation Technology Jaeger, Armstrong, Hill and Pfleger (2014) Frontiers in Endocrinology 5 : 26

Receptor-HIT: Receptor Heteromer Investigation Technology Jaeger, Armstrong, Hill and Pfleger (2014) Frontiers in Endocrinology 5 : 26 Receptor B Receptor B Receptor A Receptor A

Receptor-HIT: BRET configuration Pfleger and Eidne (2006) Nature Methods 3 :165-174 Pfleger et al. (2006) Nature Protocols 1 : 337-345 Ayoub and Pfleger (2010) Current Opinion in Pharmacology 10 :44-52

Our publications describing Receptor-HIT Original Articles: Reviews/Book Chapters: Ayoub and Pfleger (2010) Current Opinion in See, Seeber et al. (2011) Assay and Drug Development Technologies 9 :21-30 Pharmacology 10 :44-52 Porrello et al. (2011) Cellular Signalling 23 :1767-1776 Mustafa et al. (2010) Drug Discovery Today: Technologies 23 :1767-1776 Mustafa, See et al. (2012) Journal of Biological Chemistry 287 :12952-12965 Mustafa and Pfleger (2011) Journal of Laboratory Automation 16 :285-291 Ayoub et al. (2013) PLoS One 8 :e64672 Johnstone and Pfleger (2012) Frontiers in Watts et al. (2013) British Journal of Pharmacology Endocrinology 3 :101 168 :1662-1674 Jaeger et al. (2014) Frontiers in Endocrinology Ayoub, Zhang et al. (2015) PLoS One 10 :e0119803 5 :26 White et al. (2017) Scientific Reports 7 :3187 Johnstone and Pfleger (2015) in G-Protein- Coupled Receptors in Drug Discovery: O’Brien et al. (2018) Biochemical Pharmacology 158 : Methods and Protocols 191-204 232-242 Gomes et al. (2016) Annual Reviews in Pickering et al. (2019) Journal of Clinical Pharmacology and Toxicology 56 :403-425 Investigation 129 : 406-421

Receptor-HIT with ⍺ 1a AR-CXCR2 heteromer CXCR2/Rluc8 + β -arresin2/Venus NE or CXCL8  1A-AR  -Arrestin2-Venus CXCR2-Rluc8 Venus Rluc8 Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287 :12952-12965

Receptor-HIT with ⍺ 1a AR-CXCR2 heteromer Seeber and Pfleger , unpublished data : Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287 :12952-12965

Constitutive complex Constitutive complex that is not dynamically regulated by ligand Mustafa, See, Seeber, Armstrong, Despite both CXCR2 and V2R appearing to be in close proximity to α 1A AR, only CXCR2 appears to White, Ventura, Ayoub, Pfleger alter α 1A AR pharmacology Journal of Biological Chemistry (2012) 287 :12952-12965

Transactivation mechanism within the ⍺ 1a AR-CXCR2 heteromer CXCR2/Rluc8 + β -arresin2/Venus + ⍺ 1a-AR Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287 :12952-12965

Transactivation mechanism within the ⍺ 1a AR-CXCR2 heteromer Key role of CXCR2 protomer! Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287 :12952-12965

Receptor-HIT: modulation of CCR2-Gi coupling by AT 1 receptor DMX-200 Ayoub, Zhang, Kelly, See, Johnstone, McCall, Williams, Kelly, Pfleger (2015) PLoS One 10 :e0119803

Receptor-HIT with endogenous CXCR4 tagged with Nluc using CRISPR nCXCR4/Nluc + exV1bR White, Vanyai, See, Johnstone and Pfleger (2017) Scientific Reports 7:3187 �

Receptor-HIT ligand binding: AT 1 receptor heteromerisation with β 2 AR Nluc- b 2 AR BRET 0.15 (BODIPY-propranolol- Prop Specific Binding Nluc induced BRET) 0.10 B-Prop 0.05 BRET between β 2 AR 0.00 BODIPY- K d = 3.64 ± 0.40 nM -0.05 propranolol and 0 50 100 150 200 [BODIPY-propranolol] nM Nluc-AT 1 indicates Nluc-AT 1 + b 2 AR heteromerisation BRET 0.015 Prop with β 2 AR. (BODIPY-propranolol- Specific Binding induced BRET) 0.010 Nluc 0.005 B-Prop 0.000 AT 1 β 2 AR K d = 7.71 ± 5.50 nM Johnstone and Pfleger, -0.005 unpublished observations 0 50 100 150 200 [BODIPY-propranolol] nM

Receptor-HIT: CXCR2-Gi coupling induced by AT 1 receptor activation with AngII CXCR2/Rluc8 + Venus/mGsi + AT 1 receptor DMX-700 0.6 100nM CXCL8 Ligand induced BRET 1 μ M AngII 0.4 CXCL8 AngII 0.2 Both 10 20 30 40 See, Shepherd and Pfleger Time (min) unpublished observations

Receptor-HIT: CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT 1 receptor vehicle CXCR2 inhibitor 1 CXCR2 inhibitor 2 veh > veh veh > AngII 0.4 0.4 0.4 CXCR2 Inhibitor 1 > veh CXCR2 Inhibitor 2 > veh veh > CXCL8 veh > CXCL8+AngII CXCR2 Inhibitor 1 > CXCL8 CXCR2 Inhibitor 2 > CXCL8 Ligand-induced BRET 0.3 Ligand-induced BRET 0.3 Ligand-induced BRET 0.3 CXCR2 Inhibitor 1 > AngII CXCR2 Inhibitor 2 > AngII CXCR2 Inhibitor 1 > CXCL8+AngII CXCR2 Inhibitor 2 > CXCL8+AngII 0.2 0.2 DMX-700 0.2 0.1 0.1 0.1 0.0 0.0 0.0 10nM CXCL8 -0.1 -0.1 -0.1 100nM AngII -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 10 μ M Inhibitor Time (min) Time (min) Time (min) CXCR2 Inhibitor 3 CXCR2 Inhibitor 4 CXCR2 Inhibitor 5 0.4 0.4 0.4 CXCR2 Inhibitor 5 > veh CXCR2 Inhibitor 3 > veh CXCR2 Inhibitor 4 > veh CXCR2 Inhibitor 5 > CXCL8 CXCR2 Inhibitor 3 > CXCL8 CXCR2 Inhibitor 4 > CXCL8 Ligand-induced BRET 0.3 Ligand-induced BRET 0.3 Ligand-induced BRET 0.3 CXCR2 Inhibitor 5 > AngII CXCR2 Inhibitor 3 > AngII CXCR2 Inhibitor 4 > AngII CXCR2 Inhibitor 3 > CXCL8+AngII CXCR2 Inhibitor 4 > CXCL8+AngII CXCR2 Inhibitor 5 > CXCL8+AngII 0.2 0.2 0.2 0.1 0.1 0.1 See, Shepherd and 0.0 0.0 0.0 Pfleger unpublished observations -0.1 -0.1 -0.1 -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 Time (min) Time (min) Time (min)

Receptor-HIT: AT 1 R and CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT 1 receptor ARB + CXCR2 Inhibitor 1 ARB + CXCR2 Inhibitor 2 Angiotensin receptor blocker (ARB) 0.4 0.4 0.4 ARB+CXCR2 Inhibitor 1 > veh ARB+CXCR2 Inhibitor 2 > veh ARB > veh ARB > AngII ARB+CXCR2 Inhibitor 1 > CXCL8 ARB+CXCR2 Inhibitor 2 > CXCL8 ARB > CXCL8 ARB > CXCL8+AngII Ligand-induced BRET Ligand-induced BRET 0.3 Ligand-induced BRET 0.3 0.3 ARB+CXCR2 Inhibitor 1 > AngII ARB+CXCR2 Inhibitor 2 > AngII ARB+CXCR2 Inhibitor 1 > CXCL8+AngII ARB+CXCR2 Inhibitor 2 > CXCL8+AngII 0.2 0.2 0.2 DMX-700 0.1 0.1 0.1 0.0 0.0 0.0 Similar data with -0.1 -0.1 8 different ARBs -0.1 -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 Time (min) Time (min) Time (min) ARB + CXCR2 Inhibitor 3 ARB + CXCR2 Inhibitor 4 ARB + CXCR2 Inhibitor 5 0.4 0.4 0.4 ARB+CXCR2 Inhibitor 3 > veh ARB+CXCR2 Inhibitor 5 > veh ARB+CXCR2 Inhibitor 4 > veh ARB+CXCR2 Inhibitor 3 > CXCL8 ARB+CXCR2 Inhibitor 5 > CXCL8 ARB+CXCR2 Inhibitor 4 > CXCL8 Ligand-induced BRET Ligand-induced BRET 0.3 0.3 Ligand-induced BRET 0.3 ARB+CXCR2 Inhibitor 3 > AngII ARB+CXCR2 Inhibitor 5 > AngII ARB+CXCR2 Inhibitor 4 > AngII ARB+CXCR2 Inhibitor 3 > CXCL8+AngII ARB+CXCR2 Inhibitor 5 > CXCL8+AngII ARB+CXCR2 Inhibitor 4 > CXCL8+AngII 0.2 0.2 0.2 0.1 0.1 0.1 See, Shepherd and 0.0 0.0 0.0 Pfleger unpublished -0.1 -0.1 -0.1 observations -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 -20 -10 0 10 20 30 40 50 Time (min) Time (min) Time (min)

Receptor-HIT localisation: AT 1 receptor/Rluc8 + Venus-tagged localisation marker +/- CXCR2 AT1R/Rluc8 + Venus/Rab5 AT1R/Rluc8 + Venus/Kras AT1R/Rluc8 + Venus/Rab8 0.6 0.10 0.5 CXCL8 Ligand-induced BRET CXCL8 Ligand-induced BRET Ligand-induced BRET 0.5 AngII AngII 0.0 DMX-700 0.4 0.05 Both Both 0.3 -0.5 0.2 CXCL8 0.00 0.1 -1.0 AngII 0.0 Both -0.1 -1.5 -0.05 -20 -10 0 10 20 30 40 50 60 -20 -10 0 10 20 30 40 50 60 -20 -10 0 10 20 30 40 50 60 Time (min) Time (min) Time (min) AT1R/Rluc8 + CXCR2 + Venus/Rab5 AT1R/Rluc8 + CXCR2 + Venus/Kras AT1R/Rluc8 + CXCR2 + Venus/Rab8 0.6 0.5 0.10 CXCL8 Ligand-induced BRET CXCL8 Ligand-induced BRET Ligand-induced BRET 0.5 AngII AngII 0.0 0.4 Both 0.05 Both 0.3 -0.5 0.2 0.00 CXCL8 0.1 -1.0 See, Shepherd and AngII 0.0 Both Pfleger unpublished -0.1 -0.05 -1.5 -20 -10 0 10 20 30 40 50 60 -20 -10 0 10 20 30 40 50 60 observations -20 -10 0 10 20 30 40 50 60 Time (min) Time (min) Time (min)