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

Ion Channel and GPCR binders At the Forefront of Functional binders generation against membrane proteins

Theranyx activities SERVICES Expression- Binders (VHHs Purification and Cytobodies) Membrane receptor Cell lines CATALOGUE Membrane (GPRCs and Ion Channel Preparations RESEARCH Structures Ion Channels) Purified Functional binders Receptors pipeline Nanobodies

β € € € € € µ € β β € € € € € € € € € µ € β ρ € € € € € € € € € € ρ ρ € € € € α € € € € € € € € € € € ρ ρ € µ € µ € α € α € € € € € € € € € € € € € € € € € € € ρ µ € µ € µ € µ € α α α α β € € Membrane Proteins at Theranyx € € € € € € € € € € € € € € € € € € € € € € € 300 ORF ; 150 cell lines- µ € µ € µ € µ µ € € µ € µ € β α β α α β α α α Ion Channels € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € € µ € µ € µ € µ € µ € µ € µ € µ € µ € µ € µ € µ € mGlycine a 1 hGlycine a 2 hGlycine a 3 hGaba b 3 hGlycine a 1 mHTR3A hHTR3AB mHTR3AB hZacN hGlycine hGlycine hHTR3A α β α β α β α a 1 b a 2 b GPCR € € € € € € € € € € € € € € € € € € € € € € € € € µ € µ € µ € µ € µ € µ € µ € α β α β ρ € € € € € € € € € € € € € € € € € € µ € µ € µ € µ € α β Confocal imaging ρ € Plot3: Gated by: Gate 1 Plot3: Gated by: Gate 1 530 530 270 270 € € € € € € € 398 398 203 203 € € € € € € µ € Count Count Count Count 265 265 135 135 µ € 99.2% 97% M2 M2 133 133 M1 68 68 M1 ρ 0 0 0 0 10e0 10e0 10e1 10e1 10e2 10e2 10e3 10e3 10e4 10e4 10e0 10e0 10e1 10e1 10e2 10e2 10e3 10e3 10e4 10e4 aStrepTag Chromeo488 (GRN-HLog) aStrepTag Chromeo488 (GRN-HLog) aStrepTag Chromeo488 (GRN-HLog) aStrepTag Chromeo488 (GRN-HLog) € € FACS analyses € € € € € € α € € € € € € € µ € α € € € € € µ €

Purified Membrane Receptors -83 GPCR/IC (m,h,r)- Strengths: • Highly expressing systems • Good adaptation in large scales • Purified receptors are close to their native properties Achievements i.e. : Validated applications: Serotonin 5HT3A h/m Ion Channel receptors Serotonin 5HT3AB h/m Thermophoreses Glycine GLRA1 h/m Glycine a1b h Immunization Glycine a2b h SPR Gaba GABRB3 h/m Alpha7-5HT3Am h/m Molecule screening Alpha9-5HT3Am h/r by display approaches Vasopressin V1B h Glucagon GCGR h EM Endothelin ET-A h Electrophysiology GPCRs Sphingosine SPR3 h Purinergic P2Y1 h X-ray structure Melatonin MT1A h Chemokine CXCR4 h Serotonin 5HT1B h

VHH Generation Target GC Immunization VHH dvp library VHH selection VHH expr/charact. 1 week 6 3 2-4 weeks 2 weeks weeks weeks Scientific American 293, 78 - 83 (2005) W. Wayt Gibbs

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… •

Good for Membrane Receptor modulation: 5HT3R Channel blockers (patent pending) Patch clamp VHH confocal Potential membrane dye inhibition assay Immuno staining Inhibition dose response SPR Kd determination at 8 nM EM

Good for Nature publication: 5HT3R Xray structure in complexe with a VHH

Good for Nobel award Beta2 Adrenergic receptor structure in complexe with trimeric G protein stabilised by a VHH Brian Kobilka 2012 Nobel Prize in Chemistry

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

Good for you? 133 cell lines Purified receptor Binding based screening Xray Structure Camel VHH

Collaboration Many thanks to: • 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