At the Forefront of Functional binders generation against membrane - - PowerPoint PPT Presentation

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Ion Channel and GPCR binders At the Forefront of

Functional binders generation against membrane proteins

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Theranyx activities

Functional binders pipeline Ion Channel Structures Expression- Purification Membrane receptor Binders (VHHs and Cytobodies) Membrane Preparations Purified Receptors Cell lines

SERVICES RESEARCH CATALOGUE (GPRCs and Ion Channels)

Nanobodies

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Membrane Proteins at Theranyx

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hGlycine a1

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hGlycine a2

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hGlycine a3

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hGlycine a1b

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hGlycine a2b

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hHTR3A mGlycine a1

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mHTR3A

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hHTR3AB

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mHTR3AB

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hZacN

Ion Channels

10e0 10e1 10e2 10e3 10e4 aStrepTag Chromeo488 (GRN-HLog) 270 203 135 68 Count

M1 M2

10e0 10e1 10e2 10e3 10e4 aStrepTag Chromeo488 (GRN-HLog) 270 203 135 68 Count Plot3: Gated by: Gate 1

97%

10e0 10e1 10e2 10e3 10e4 aStrepTag Chromeo488 (GRN-HLog) 530 398 265 133 Count

M1 M2

10e0 10e1 10e2 10e3 10e4 aStrepTag Chromeo488 (GRN-HLog) 530 398 265 133 Count Plot3: Gated by: Gate 1

99.2%

300 ORF ; 150 cell lines-

Confocal imaging FACS analyses

GPCR

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Purified Membrane Receptors

• 83 GPCR/IC (m,h,r)-

Achievements i.e. : Validated applications:

Strengths:

• Highly expressing systems
• Good adaptation in large scales
• Purified receptors are close to their native properties

SPR Immunization Molecule screening by display approaches Electrophysiology X-ray structure EM

Serotonin 5HT3A h/m Serotonin 5HT3AB h/m Glycine GLRA1 h/m Glycine a1b h Glycine a2b h Gaba GABRB3 h/m Alpha7-5HT3Am h/m Alpha9-5HT3Am h/r Vasopressin V1B h Glucagon GCGR h Endothelin ET-A h Sphingosine SPR3 h Purinergic P2Y1 h Melatonin MT1A h Chemokine CXCR4 h Serotonin 5HT1B h

Ion Channel receptors GPCRs

Thermophoreses

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VHH Generation

Scientific American 293, 78 - 83 (2005)

• W. Wayt Gibbs

Target GC Immunization VHH dvp library VHH selection VHH expr/charact.

1 week 6 weeks 3 weeks 2 weeks 2-4 weeks

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VHHs offer many advantages

• High affinity in monovalent form
• Easy to make bi-specific fusion
• Straight forward expression in E-Coli
• Easy to label (fluorescent protein in fusion)
• Better shaped to bind cavities than classical antibody increase

there potential functional properties

• Higher specificity than small molecules…

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Good for Membrane Receptor modulation:

Potential membrane dye Inhibition dose response VHH confocal Immuno staining Patch clamp inhibition assay SPR Kd determination at 8 nM EM

5HT3R Channel blockers (patent pending)

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Good for Nature publication:

5HT3R Xray structure in complexe with a VHH

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Good for Nobel award

Brian Kobilka 2012 Nobel Prize in Chemistry

Beta2 Adrenergic receptor structure in complexe with trimeric G protein stabilised by a VHH

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Good for our drug discovery pipeline

More than 150 membrane receptor stable cell lines ready to purify available More than 20 membrane receptors purified and injected to Llama since 2014 VHHs for 12 targets

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Good for you?

Purified receptor Camel VHH Xray Structure 133 cell lines Binding based screening

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Collaboration

• Dr Hugues Nury

IBS Grenoble Xray Structure

• Pr. Horst Vogel

EPFL Lausanne Xray Structure

• Dr. Xiao Dan Li

PSI Zurich EM Structure

• Dr. Olof Karlsson

GE Uppsala SPR

• Vincent Dive

CEA Saclay Yeast Display

Many thanks to: