Dimerix Limited · ASX:DXB
Dimerix — ELRIG 2020
22 slides from Dimerix's conference presentation, 2020
Slide 1: Profiling novel pharmacology of GPCR complexes using Receptor-HIT
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 a
Slide 2: Forward-looking statements
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 fro
Slide 3: Receptor-HIT: Receptor Heteromer Investigation Technology
Receptor-HIT: Receptor Heteromer Investigation Technology Jaeger, Armstrong, Hill and Pfleger (2014) Frontiers in Endocrinology 5: 26
Slide 4: Receptor-HIT: Receptor Heteromer Investigation Technology
Receptor-HIT: Receptor Heteromer Investigation Technology Jaeger, Armstrong, Hill and Pfleger (2014) Frontiers in Endocrinology 5: 26 Receptor A Receptor B Receptor A Receptor B
Slide 5: Receptor-HIT: BRET configuration
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
Slide 6: Our publications describing Receptor-HIT
Our publications describing Receptor-HIT Original Articles: Reviews/Book Chapters: See, Seeber et al. (2011) Assay and Drug Ayoub and Pfleger (2010) Current Opinion in Development Technologies 9:21-30 Pharmacology 10:44-52 Porrello et al. (2011) Cellular Signalling 23:1767-1776 Mustafa et al. (2010)
Slide 7: Receptor-HIT with α1aAR-CXCR2 heteromer
Receptor-HIT with ⍺1aAR-CXCR2 heteromer CXCR2/Rluc8 + β-arresin2/Venus NE or CXCL8 CXCR2-Rluc8 1A-AR -Arrestin2-Venus Venus Rluc8 Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965
Slide 8: Receptor-HIT with α1aAR-CXCR2 heteromer
Receptor-HIT with ⍺1aAR-CXCR2 heteromer Seeber and Pfleger, unpublished data: Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965
Slide 9: Constitutive complex
Constitutive complex Constitutive complex that is not dynamically regulated by ligand Despite both CXCR2 and V2R appearing to be in Mustafa, See, Seeber, Armstrong, close proximity to α1AAR, only CXCR2 appears to White, Ventura, Ayoub, Pfleger alter α1AAR pharmacology Journal of Biological Chemistry
Slide 10: Transactivation mechanism within the α1aAR-CXCR2 heteromer
Transactivation mechanism within the ⍺1aAR-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
Slide 11: Transactivation mechanism within the α1aAR-CXCR2 heteromer
Transactivation mechanism within the ⍺1aAR-CXCR2 heteromer Key role of CXCR2 protomer! Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965
Slide 12: Receptor-HIT: modulation of CCR2-Gi coupling by AT1 receptor
Receptor-HIT: modulation of CCR2-Gi coupling by AT1 receptor DMX-200 Ayoub, Zhang, Kelly, See, Johnstone, McCall, Williams, Kelly, Pfleger (2015) PLoS One 10:e0119803
Slide 13: Receptor-HIT with endogenous CXCR4 tagged with Nluc using CRISPR
Receptor-HIT with endogenous CXCR4 tagged with Nluc using CRISPR nCXCR4/Nluc + exV1bR White, Vanyai, See, Johnstone and Pfleger (2017) Scientific Reports 7:3187
Slide 14: Ligand binding: AT1 receptor heteromerisation with β2AR
Receptor-HIT ligand binding: AT1 receptor heteromerisation with β2AR Nluc-b2AR BRET 0.15 (BODIPY-propranolol- Prop Specific Binding induced BRET) Nluc 0.10 B-Prop 0.05 BRET between β2AR 0.00 Kd = 3.64 ± 0.40 nM BODIPY- -0.05 0 50 100 150 200 propranolol and [BODIPY-propranolol] nM Nluc-AT1 indicates
Slide 15: CXCR2-Gi coupling induced by AT1 receptor activation with AngII
Receptor-HIT: CXCR2-Gi coupling induced by AT1 receptor activation with AngII CXCR2/Rluc8 + Venus/mGsi + AT1 receptor DMX-700 0.6 100nM CXCL8 Ligand induced BRET 1μM AngII 0.4 CXCL8 AngII 0.2 Both 10 20 30 40 See, Shepherd and Pfleger Time (min) unpublished observations
Slide 16: CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT1 receptor
Receptor-HIT: CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT1 receptor vehicle CXCR2 inhibitor 1 CXCR2 inhibitor 2 0.4 veh > veh veh > AngII 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-indu
Slide 17: AT1R and CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT1 receptor
Receptor-HIT: AT1R and CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT1 receptor Angiotensin receptor blocker (ARB) ARB + CXCR2 Inhibitor 1 ARB + CXCR2 Inhibitor 2 0.4 0.4 ARB+CXCR2 Inhibitor 1 > veh 0.4 ARB+CXCR2 Inhibitor 2 > veh ARB > veh ARB > AngII ARB > CXCL8 ARB > CXCL8+AngII ARB+CXC
Slide 18: AT1 receptor/Rluc8 + Venus-tagged localisation marker +/- CXCR2
Receptor-HIT localisation: AT1 receptor/Rluc8 + Venus-tagged localisation marker +/- CXCR2 AT1R/Rluc8 + Venus/Kras AT1R/Rluc8 + Venus/Rab5 AT1R/Rluc8 + Venus/Rab1 AT1R/Rluc8 AT1R/Rluc8 + Venus/Rab6 + Venus/Rab8 AT1R/Rluc8 + Venus/Rab4 AT1R/Rluc8 AT1R/Rluc8 + Venus/Rab7 + Venus/Rab9 0.5 0.6 0.15 0.06
Slide 19: CXCR2/Rluc8 + Venus-tagged localisation marker +/- AT1 receptor
Receptor-HIT localisation: CXCR2/Rluc8 + Venus- tagged localisation marker +/- AT1 receptor CXCR2/Rluc8 + Venus/Kras CXCR2/Rluc8 + Venus/Rab5 CXCR2/Rluc8 + Venus/Rab1 CXCR2/Rluc8 CXCR2/Rluc8 ++Venus/Rab8 CXCR2/Rluc8Venus/Rab6 + Venus/Rab4 CXCR2/Rluc8++Venus/Rab9 CXCR2/Rluc8 Venus/Rab7 0.5 0.25 0.08
Slide 20: CXCR2/Rluc8 + Venus/Kras plasma membrane marker + AT1R
Receptor-HIT localisation: CXCR2/Rluc8 + Venus/Kras plasma membrane marker + AT1R CXCR2/Rluc8 + AT1R + Venus/Kras CXCR2/Rluc8 + AT1R + Venus/Kras CXCR2/Rluc8 + AT1R + Venus/Kras CXCR2/Rluc8 + AT1R + Venus/Kras 0.4 0.4 0.4 0.4 ARB1 > CXCL8+AngII 0.2 0.2 0.2 ARB2 > CXCL8+AngII 0.2 Ligand-induced BRET
Slide 21: CXCR2/Rluc8 + Venus/Rab8 trafficking to plasma membrane marker + AT1R
Receptor-HIT localisation: CXCR2/Rluc8 + Venus/Rab8 trafficking to plasma membrane marker + AT1R CXCR2/Rluc8 + AT1R + Venus/Rab8 CXCR2/Rluc8 + AT1R + Venus/Rab8 CXCR2/Rluc8 + AT1R + Venus/Rab8 CXCR2/Rluc8 + AT1R + Venus/Rab8 0.10 veh > veh 0.10 ARB1 > AngII 0.10 ARB1 > CXCL8+AngII 0.10 veh > CXCL8 v
Slide 22: Acknowledgements
Acknowledgements Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research: Current: Ethan See Ruth Seeber Rekhati Abhayawardana Liz Johnstone Carl White Natasha Dale Selected Recent Past: Mohammed Akli Ayoub Matthew Dalrymple Werner Jaeger Sanam Mustafa Stephen Armstrong