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

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

Oct 18, 2023 594 likes •830 views

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

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SLIDE 1

Professor Kevin Pfleger

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

Profiling novel pharmacology of GPCR complexes using Receptor-HIT

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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

  • utcome of legal proceedings and the effectiveness of patent protection.
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Receptor-HIT: Receptor Heteromer Investigation Technology

Jaeger, Armstrong, Hill and Pfleger (2014) Frontiers in Endocrinology 5: 26

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SLIDE 4

Receptor-HIT: Receptor Heteromer Investigation Technology

Receptor A Receptor A Receptor B Receptor B

Jaeger, Armstrong, Hill and Pfleger (2014) Frontiers in Endocrinology 5: 26

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SLIDE 5

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

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SLIDE 6

Our publications describing Receptor-HIT

Reviews/Book Chapters:

Ayoub and Pfleger (2010) Current Opinion in Pharmacology 10:44-52 Mustafa et al. (2010) Drug Discovery Today: Technologies 23:1767-1776 Mustafa and Pfleger (2011) Journal of Laboratory Automation 16:285-291 Johnstone and Pfleger (2012) Frontiers in Endocrinology 3:101 Jaeger et al. (2014) Frontiers in Endocrinology 5:26 Johnstone and Pfleger (2015) in G-Protein- Coupled Receptors in Drug Discovery: Methods and Protocols 191-204 Gomes et al. (2016) Annual Reviews in Pharmacology and Toxicology 56:403-425

Original Articles:

See, Seeber et al. (2011) Assay and Drug Development Technologies 9:21-30 Porrello et al. (2011) Cellular Signalling 23:1767-1776 Mustafa, See et al. (2012) Journal of Biological Chemistry 287:12952-12965 Ayoub et al. (2013) PLoS One 8:e64672 Watts et al. (2013) British Journal of Pharmacology 168:1662-1674 Ayoub, Zhang et al. (2015) PLoS One 10:e0119803 White et al. (2017) Scientific Reports 7:3187 O’Brien et al. (2018) Biochemical Pharmacology 158: 232-242 Pickering et al. (2019) Journal of Clinical Investigation 129: 406-421

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SLIDE 7

Receptor-HIT with ⍺1aAR-CXCR2 heteromer

CXCR2-Rluc8 1A-AR

-Arrestin2-Venus Venus Rluc8

Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965 NE or CXCL8 CXCR2/Rluc8 + β-arresin2/Venus

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SLIDE 8

Receptor-HIT with ⍺1aAR-CXCR2 heteromer

Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965 Seeber and Pfleger, unpublished data:

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SLIDE 9

Constitutive complex

Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965 Constitutive complex that is not dynamically regulated by ligand Despite both CXCR2 and V2R appearing to be in close proximity to α1AAR, only CXCR2 appears to alter α1AAR pharmacology

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SLIDE 10

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

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SLIDE 11

Key role of CXCR2 protomer!

Transactivation mechanism within the ⍺1aAR-CXCR2 heteromer

Mustafa, See, Seeber, Armstrong, White, Ventura, Ayoub, Pfleger Journal of Biological Chemistry (2012) 287:12952-12965

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SLIDE 12

Receptor-HIT: modulation of CCR2-Gi coupling by AT1 receptor

Ayoub, Zhang, Kelly, See, Johnstone, McCall, Williams, Kelly, Pfleger (2015) PLoS One 10:e0119803

DMX-200

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SLIDE 13

Receptor-HIT with endogenous CXCR4 tagged with Nluc using CRISPR

White, Vanyai, See, Johnstone and Pfleger (2017) Scientific Reports 7:3187

  • nCXCR4/Nluc + exV1bR
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Receptor-HIT ligand binding: AT1 receptor heteromerisation with β2AR

BRET between BODIPY- propranolol and Nluc-AT1 indicates heteromerisation with β2AR.

50 100 150 200

  • 0.05

0.00 0.05 0.10 0.15 [BODIPY-propranolol] nM Specific Binding (BODIPY-propranolol- induced BRET) Nluc-b2AR

Nluc B-Prop BRET Prop

β

2AR

Kd = 3.64 ± 0.40 nM

50 100 150 200

  • 0.005

0.000 0.005 0.010 0.015 [BODIPY-propranolol] nM Specific Binding (BODIPY-propranolol- induced BRET) Nluc-AT1 + b2AR

Nluc

β

2AR

B-Prop BRET Prop AT1

Kd = 7.71 ± 5.50 nM

Johnstone and Pfleger, unpublished observations

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SLIDE 15

See, Shepherd and Pfleger unpublished observations

DMX-700

Receptor-HIT: CXCR2-Gi coupling induced by AT1 receptor activation with AngII

10 20 30 40 0.2 0.4 0.6

Time (min) Ligand induced BRET

CXCR2/Rluc8 + Venus/mGsi + AT1 receptor

CXCL8 AngII Both 100nM CXCL8 1μM AngII

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SLIDE 16

See, Shepherd and Pfleger unpublished

  • bservations

DMX-700

Receptor-HIT: CXCR2 inhibitors acting on CXCR2/Rluc8 + Venus/mGsi + AT1 receptor

10nM CXCL8 100nM AngII 10μM Inhibitor

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

vehicle

Time (min) Ligand-induced BRET veh > veh veh > CXCL8 veh > AngII veh > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

CXCR2 inhibitor 1

Time (min) Ligand-induced BRET CXCR2 Inhibitor 1 > CXCL8+AngII CXCR2 Inhibitor 1 > AngII CXCR2 Inhibitor 1 > CXCL8 CXCR2 Inhibitor 1 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

CXCR2 inhibitor 2

Time (min) Ligand-induced BRET CXCR2 Inhibitor 2 > CXCL8+AngII CXCR2 Inhibitor 2 > AngII CXCR2 Inhibitor 2 > CXCL8 CXCR2 Inhibitor 2 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

CXCR2 Inhibitor 3

Time (min) Ligand-induced BRET CXCR2 Inhibitor 3 > CXCL8+AngII CXCR2 Inhibitor 3 > AngII CXCR2 Inhibitor 3 > CXCL8 CXCR2 Inhibitor 3 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

CXCR2 Inhibitor 4

Time (min) Ligand-induced BRET CXCR2 Inhibitor 4 > CXCL8+AngII CXCR2 Inhibitor 4 > AngII CXCR2 Inhibitor 4 > CXCL8 CXCR2 Inhibitor 4 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

CXCR2 Inhibitor 5

Time (min) Ligand-induced BRET CXCR2 Inhibitor 5 > CXCL8+AngII CXCR2 Inhibitor 5 > AngII CXCR2 Inhibitor 5 > CXCL8 CXCR2 Inhibitor 5 > veh

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SLIDE 17

Receptor-HIT: AT1R and CXCR2 inhibitors acting

  • n CXCR2/Rluc8 + Venus/mGsi + AT1 receptor
  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

Angiotensin receptor blocker (ARB)

Time (min) Ligand-induced BRET ARB > veh ARB > CXCL8 ARB > AngII ARB > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

ARB + CXCR2 Inhibitor 1

Time (min) Ligand-induced BRET ARB+CXCR2 Inhibitor 1 > CXCL8+AngII ARB+CXCR2 Inhibitor 1 > AngII ARB+CXCR2 Inhibitor 1 > CXCL8 ARB+CXCR2 Inhibitor 1 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

ARB + CXCR2 Inhibitor 2

Time (min) Ligand-induced BRET ARB+CXCR2 Inhibitor 2 > CXCL8+AngII ARB+CXCR2 Inhibitor 2 > AngII ARB+CXCR2 Inhibitor 2 > CXCL8 ARB+CXCR2 Inhibitor 2 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

ARB + CXCR2 Inhibitor 3

Time (min) Ligand-induced BRET ARB+CXCR2 Inhibitor 3 > CXCL8+AngII ARB+CXCR2 Inhibitor 3 > AngII ARB+CXCR2 Inhibitor 3 > CXCL8 ARB+CXCR2 Inhibitor 3 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

ARB + CXCR2 Inhibitor 4

Time (min) Ligand-induced BRET ARB+CXCR2 Inhibitor 4 > CXCL8+AngII ARB+CXCR2 Inhibitor 4 > AngII ARB+CXCR2 Inhibitor 4 > CXCL8 ARB+CXCR2 Inhibitor 4 > veh

  • 20
  • 10

10 20 30 40 50

  • 0.1

0.0 0.1 0.2 0.3 0.4

ARB + CXCR2 Inhibitor 5

Time (min) Ligand-induced BRET ARB+CXCR2 Inhibitor 5 > CXCL8+AngII ARB+CXCR2 Inhibitor 5 > AngII ARB+CXCR2 Inhibitor 5 > CXCL8 ARB+CXCR2 Inhibitor 5 > veh

See, Shepherd and Pfleger unpublished

  • bservations

DMX-700

Similar data with 8 different ARBs

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SLIDE 18

See, Shepherd and Pfleger unpublished

  • bservations

Receptor-HIT localisation: AT1 receptor/Rluc8 + Venus-tagged localisation marker +/- CXCR2

  • 20
  • 10

10 20 30 40 50 60

  • 1.5
  • 1.0
  • 0.5

0.0 0.5

Time (min) Ligand-induced BRET

AT1R/Rluc8 + Venus/Kras

CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 1.5
  • 1.0
  • 0.5

0.0 0.5

Time (min) Ligand-induced BRET

AT1R/Rluc8 + CXCR2 + Venus/Kras

CXCL8 AngII Both

DMX-700

  • 20
  • 10

10 20 30 40 50 60

  • 0.1

0.0 0.1 0.2 0.3 0.4 0.5 0.6

AT1R/Rluc8 + Venus/Rab5

Time (min) Ligand-induced BRET CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.1

0.0 0.1 0.2 0.3 0.4 0.5 0.6

AT1R/Rluc8 + CXCR2 + Venus/Rab5

Time (min) Ligand-induced BRET CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.05

0.00 0.05 0.10

AT1R/Rluc8 + Venus/Rab8

Time (min) Ligand-induced BRET CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.05

0.00 0.05 0.10

AT1R/Rluc8 + CXCR2 + Venus/Rab8

Time (min) Ligand-induced BRET CXCL8 AngII Both

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SLIDE 19

See, Shepherd and Pfleger unpublished

  • bservations

DMX-700

Receptor-HIT localisation: CXCR2/Rluc8 + Venus- tagged localisation marker +/- AT1 receptor

  • 20
  • 10

10 20 30 40 50 60

  • 1.0
  • 0.5

0.0 0.5

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + Venus/Kras

CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 1.0
  • 0.5

0.0 0.5

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Kras

CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.05

0.00 0.05 0.10 0.15 0.20 0.25

CXCR2/Rluc8 + Venus/Rab5

Time (min) Ligand-induced BRET CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.05

0.00 0.05 0.10 0.15 0.20 0.25

CXCR2/Rluc8 + AT1R + Venus/Rab5

Time (min) Ligand-induced BRET CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + Venus/Rab8

CXCL8 AngII Both

  • 20
  • 10

10 20 30 40 50 60

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

CXCL8 AngII Both

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SLIDE 20

See, Shepherd and Pfleger unpublished

  • bservations
  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Kras

veh > veh veh > AngII veh > CXCL8 veh > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Kras

ARB2 > AngII ARB3 > AngII ARB1 > AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

CXCR2/Rluc8 + AT1R + Venus/Kras

Time (min) Ligand-induced BRET ARB1 > CXCL8+AngII ARB2 > CXCL8+AngII ARB3 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

CXCR2/Rluc8 + AT1R + Venus/Kras

Time (min) Ligand-induced BRET CXCR2 Inhibitor 1 > AngII CXCR2 Inhibitor 2 > AngII CXCR2 Inhibitor 3 > AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

CXCR2/Rluc8 + AT1R + Venus/Kras

Time (min) Ligand-induced BRET CXCR2 Inhibitor 1 > CXCL8+AngII CXCR2 Inhibitor 2 > CXCL8+AngII CXCR2 Inhibitor 3 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

CXCR2/Rluc8 + AT1R + Venus/Kras

Time (min) Ligand-induced BRET ARB3+CXCR2 Inhibitor 3 > CXCL8+AngII ARB2+CXCR2 Inhibitor 3 > CXCL8+AngII ARB1+CXCR2 Inhibitor 3 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

CXCR2/Rluc8 + AT1R + Venus/Kras

Time (min) Ligand-induced BRET ARB3+CXCR2 Inhibitor 2 > CXCL8+AngII ARB2+CXCR2 Inhibitor 2 > CXCL8+AngII ARB1+CXCR2 Inhibitor 2 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.6
  • 0.4
  • 0.2

0.0 0.2 0.4

CXCR2/Rluc8 + AT1R + Venus/Kras

Time (min) Ligand-induced BRET ARB1+CXCR2 Inhibitor 1 > CXCL8+AngII ARB2+CXCR2 Inhibitor 1 > CXCL8+AngII ARB3+CXCR2 Inhibitor 1 > CXCL8+AngII

Receptor-HIT localisation: CXCR2/Rluc8 + Venus/Kras plasma membrane marker + AT1R

DMX-700

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SLIDE 21

DMX-700

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

veh > veh veh > AngII veh > CXCL8 veh > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

ARB2 > AngII ARB3 > AngII ARB1 > AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

ARB1 > CXCL8+AngII ARB2 > CXCL8+AngII ARB3 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

CXCR2 Inhibitor 1 > AngII CXCR2 Inhibitor 2 > AngII CXCR2 Inhibitor 3 > AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

CXCR2 Inhibitor 1 > CXCL8+AngII CXCR2 Inhibitor 2 > CXCL8+AngII CXCR2 Inhibitor 3 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

ARB3+CXCR2 Inhibitor 3 > CXCL8+AngII ARB2+CXCR2 Inhibitor 3 > CXCL8+AngII ARB1+CXCR2 Inhibitor 3 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

ARB3+CXCR2 Inhibitor 2 > CXCL8+AngII ARB2+CXCR2 Inhibitor 2 > CXCL8+AngII ARB1+CXCR2 Inhibitor 2 > CXCL8+AngII

  • 20
  • 10

10 20 30 40 50

  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08 0.10

Time (min) Ligand-induced BRET

CXCR2/Rluc8 + AT1R + Venus/Rab8

ARB1+CXCR2 Inhibitor 1 > CXCL8+AngII ARB2+CXCR2 Inhibitor 1 > CXCL8+AngII ARB3+CXCR2 Inhibitor 1 > CXCL8+AngII

Receptor-HIT localisation: CXCR2/Rluc8 + Venus/Rab8 trafficking to plasma membrane marker + AT1R

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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

Academic Collaborators:

Leigh Stoddart, Amanda Wheal, Joëlle Goulding, Stephen Hill and Laura Kilpatrick: Nottingham Anatoly Tiulpakov and Ivan Dedov: Endocrinology Research Centre, Moscow Nathan Pavlos, Audrey Chan, Julian Heng and Hannah Vanyai: University of Western Australia

Industry Collaborators:

Matthew Robers, Thomas Machleidt and Keith Wood (Promega), James Balmer and John Abbenante (BMG Labtech), James Williams, Liddy McCall, Nina Webster and Robert Shepherd (Dimerix)

Funding: ARC, NHMRC, Dimerix Limited, Promega, BMG Labtech, University of Nottingham, Australian Government

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