RPIB - RECHERCHES PARTENARIALES ET INNOVATION BIOMEDICALE

Drug discovery from venoms using miniaturized artificial bilayers – VenomPicoScreen

Drug discovery from venoms

Drug discovery from venoms using miniaturized artificial bilayers

Identification of new drug candidates acting on ion channels and receptors from venom peptides

The endpoint of the VenomPicoScreen project is the identification of new drug candidates acting on ion channels and receptors from venom peptides. To achieve this goal, we propose to develop a membrane protein platform for the screening of nanoliter volumes of venoms, enabling assays based on a single spider, the tiny volume of natural venom sources being the bottleneck in these investigations. This platform will bring together 5 state-of-the-art technologies: 1) DIB for 'Droplet interface bilayers' to create artificial lipid bilayers from tiny 0.3 µl droplets; 2) In-droplet expression of membrane proteins from cDNA by incorporation within droplets of in-vitro transcription/translation mix; 3) EWOD for 'Electrowetting on dielectric' to remotely manipulate multiple droplets and form lipid bilayers; 4) ICCR for 'Ion-channel coupled receptors' to transduce ligand binding to a single receptor into a detectable electrical signal. 5) Handling of picomoles of venoms and their constituent peptides. Including experts in venoms, microfluidics, electronics, electrophysiology, and protein engineering, the consortium possesses hands-on experience in each of the technologies involved. <br />The platform will integrate on a single chip the microfluidics and electronics necessary for generating droplets, forming bilayers, and recording transbilayer currents. Tests will be directed towards proteins for which few drugs are available: initially, voltage-dependent potassium channels, and, at a later stage, selected GPCRs. Positive outcome, in addition to the advent of a new venom screening method, will be judged by the identification of venom peptides with modulatory activity on these targets.

The project is divided in 9 tasks. There are many interactions between tasks that will be dealt with by task 0, dedicated to project management. The project will start from different angles in order to prepare the missions needed for the pico-screening. In tasks 2 and 4, potassium channels will first be screened using known peptides, then using venoms fractions isolated in task 5. The K+ channels will be embedded in DIB made first manually and then automatically using 2 droplets. This will leave time to prepare the engineered GPCR/K channel chimera targets in task 3 and the complex 13-reservoirs chip in task 1. After 2 years, the relevance of the GPCRs and K channel targets will be re-evaluated on the basis of preliminary tests in order to start the picoscreen (task 6) from the best angle. The peptide drug leads will be synthesised and characterised in task 7. During the project task 8 will take care of the intellectual property of the potential drug candidates, the engineered targets as well as the innovative picoscreen plateform.

Both Kv1.1 and Kv1.5 channels were expressed by bacterial cell free synthesis. 1mg of protein was obtained per 2 mL synthesis, which is a good yield for a membrane protein. The insertion of both proteins in liposomes was optimized. Their activity at the bilayer interface of two 300 nL droplets was recorded in vitro.
The automation of the droplet interface bilayer concept was realized on an electronic biochip. The droplets are moving using electro-wetting.
The Hi-Clamp (Multichannel systems) robot was acquired by the project. It will be used to screen venom peptides for their interaction with GPCR receptors. These binding will be measured using the electric activity of the Kir 6.2 channel fused to this GPCR (the resulted protein is called ICCR for 'Ion-channel coupled receptors').
Several fractions of venoms have been realized by the partners Smartox et CEA Saclay (80 venoms, in particular from spiders and Dendroaspis angusticeps).

Because the discovery of biomolecules is such a challenging issue in drug discovery, innovative ideas have to be strategically set and planned, particularly when the source of biomaterials is limited. Our approach will offer new insights as to what is needed to perform screening starting from nanoliter volumes of venoms. We will target members of the potassium channel family which have so far no known usable modulators. In addition, as an alternative to binding assays or cellular assays, we will follow a single-molecule approach to screen GPCRs using the ion-channel coupled receptor concept pioneered by a partner within the consortium. Both targets, K+ channels and GPCRs are promising in term of paving a way towards anti-inflammatory and anti-cancerous drugs and patent applications will be filed for each discovered peptide lead. An industrial partner (VenomeTech) is included in the consortium and has interest in taking the peptide leads of this project for further investigations. The results will be presented in conferences related to microsystems, and venom screening and published in peer-reviewed international journals.

Niescierowicz K, Caro L, Cherezov V, Vivaudou M, Moreau CJ. Functional Assay for T4 Lysozyme-Engineered G Protein-Coupled Receptors with an Ion Channel Reporter. Structure. 2013 Nov

The endpoint of the VenomPicoScreen project is the identification of new candidate drugs acting on ion channels and receptors from venom peptides. To achieve this goal, we propose to develop a membrane protein platform for the screening of nanoliter volumes of venoms, enabling assays based on a single spider, the tiny volume of natural venom sources being the bottleneck in these investigations. This platform will bring together 4 state-of-the-art technologies: 1) DIB for 'Droplet interface bilayers' to create artificial lipid bilayers from tiny 0.3 µL droplets; 2) In-droplet expression of membrane proteins from cDNA by incorporation within droplets of in-vitro transcription/translation mix; 3) EWOD for 'Electrowetting on dielectric' to remotely manipulate multiple droplets and form lipid bilayers; 4) ICCR for 'Ion-channel coupled receptors' to transduce ligand binding to a single receptor into a detectable electrical signal. Including experts in venoms, microfluidics, electronics, electrophysiology, and protein engineering, the consortium possesses hands-on experience in each of the technologies involved. The platform will integrate on a single chip the microfluidics and electronics necessary for generating droplets, forming bilayers, and recording transbilayer currents. Tests will be directed towards proteins for which few drugs are available: initially, voltage-dependent potassium channels, and, at a later stage, selected G protein coupled receptors. Positive outcome, in addition to the advent of a new venom screening method, will be judged by the identification of venom peptides with modulatory activity on these targets.

Project coordinator

Madame Schaack Beatrice (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES) – beatrice.schaack@ibs.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

VT VENOMETECH
LEGI CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
SMARTOX SMARTOX
CEA COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - CENTRE D'ETUDES NUCLEAIRES SACLAY
IBS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

Help of the ANR 932,263 euros
Beginning and duration of the scientific project: February 2012 - 48 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter