State of the art - Ion channels represent about 10% of current therapeutic targets. There are about a hundred pharmacological modulators of ion channels approved for clinical trials. In the past ten years, the explosion of the “omics” data has led to a level of complexity that none of the classical screening approaches can handle. It is clear that the discovery of promising compounds targeting ion channels needs new tools and test innovative assay formats to account for this diversity. From the development by Cisbio Bioassay of proprietary technology, HTRF®, a highly sensitive, robust technology for the detection of molecular interactions has led to the emergence of new screening assays, which are widely used by the pharmaceutical industry during the high-throughput screening stage of drug development (35). Recently, Cisbio Bioassays introduced a new type of cell-based assay called Tag-lite®. Tag-lite® is a new cellular platform for cell surface proteins study and screening which combines HTRF® and SNAP-tag technologies (36,37). Tag-lite® is the fruit of a very close collaboration and tight scientific interactions with IGF in Montpellier through an established collaborative lab unit within IGF.
Aims – One of the key goals of the ICST project is to funnel newly acquired knowledge (protein networks identified in TASK 1.2 and validated in TASK 2.1) toward the development of new therapeutic strategies. However, high-throughput screening assays for ion channels rely on the use of fluorescent ion indicators that strictly report pore activity (i.e., ion currents and electrical membrane potential). These assays often lack sensitivity or are poorly appropriate in the case of voltage-dependent ion channels. In addition these assays cannot be developed for channels with background activity or for which gating mechanisms are poorly characterized. Hence, the first necessary step toward drug development will be to implement original screening assays relying on emerging technologies. One way to achieve this goal is to implement new FRET-based technologies to ion channel functions and regulation.
Previous results and projects - Classical high-throughput screening assays are, in most cases, inappropriate for ion channels. An alternative strategy is to assess conformational modifications of the protein that occur during activation. This can now be achieved using time-resolved fluorescence energy transfer technology (TR-FRET). This approach has initially been developed for GPCR through a collaboration between Cisbio Bioassay and IGF, which led to a series of innovations in GPCR screening assays using TR-FRET and Tag-Lite®. These include, analysis of ligand binding (38,39), cell surface protein-protein interactions (40,41), allosteric modulation of GPCR (42). The collaborative program between Cisbio Bioassays and IGF is funded by a grant from FUI (Fonds Unique Interministériel) to develop these approaches to other membrane proteins, particularly ion channels. A work program has been initiated to get a proof of concept that TagLite® technology is suitable for screening ion channel function and regulation. As part of this program, P2X receptors have been chosen due to the strong interactions between the FR team and the Cisbio-IGF collaborative team.
In addition, different partners of the ICST consortium have developed their own research project.
• The FR team has shown that interaction of P2X2 receptors with the intracellular calcium sensor Vilip can be monitored by FRET. Importantly, these results demonstrated that this approach is sensitive enough to follow the dynamic of the interaction (8). These results indicate that FRET technology is suitable to assess transient protein-protein interaction and is compatible with high throughput screening format. This group has also provided evidence that P2X receptor stoichiometry can be resolved using a combination of BRET and Bimolecular fluorescence complementation (submitted for publication).
• The EB team is investigating interactions of Cav channels with other proteins. This group has pioneered the development of tag-Cav channels (including fluorescent tag channels) that were instrumental in analyzing Cav interacting protein complex (8). This group is now developing TR-Fret assays to analyze Cav channel interaction with GPCR. In addition, EB has successfully developed a ligand-based virtual screening assay for Cav channels (43).
• The NP team is developing Fret-based assays to analyze the TRPM8 signaling complex, which will be developed in medium to high throughput assays.
Added value of the LabEx - The strength of the consortium comes from the tight links existing between Cisbio Bioassays and IGF, from the expertise of the Screening Interactome facility at the IGF. This pharmacology facility (www.arpege.cnrs.fr) offers state-of-the-art screening equipment for the determination of the composition and dynamics of membrane protein complexes and intracellular signaling pathways. The platform is suited to perform assays using the Resonance Energy Transfer method, BRET, FRET, TR-FRET and HTRF. Cisbio has a strong interest in further developing HTRF® and TagLite® screening assay for ion channels. Within the consortium, the association of Cisbio Bioassays with the different academic partners will promote the development of new screening assays by sharing and combining respective expertise.
State-of-the-art - Exome sequencing is becoming a current strategy to discover disease-associated gene coding variants and mutations. Mutations in ion channel genes are known to cause a broad range of channelopathies (44,45). Sporadic channel variants are also emerging as candidates for risk in complex traits such as psychiatric disorders, hypertension or cancer. Capture methods will be designed to enrich the material to be sequenced in ion channel genes to cover with high accuracy this particular set of targets. This technology will be achieved at the IPMC genomic platform and will benefit from the France Génomique network for sequence analysis.
The finding by the group of Noebels (45), that even deleterious ion channel mutations confer uncertain risk in epilepsy to the individual depending on the other variants with which they are combined, adds a further level of complexity to the analysis of the results of exome sequencing studies. The high level of expertise of the consortium in the field of ion channels should therefore be an asset to correctly evaluate how discovered genetic variants contribute to the studied pathology in terms of risk, severity and mechanisms underlying the disease.
Projects - 1) Profiling the transportome in prostate carcinogenesis (NP). The aim is to describe a set of markers allowing not only the detection of clinically evident prostate cancer, but also early disease before the occurrence of symptoms. Systematic screening of transport gene mutations in tumoral and healthy samples will be performed using targeted resequencing. The NP group will focus on deep sequencing from TRP, ORA and Cav gene families. Other channel gene variants discovered will be shared with the consortium. 2) Searching for channel variants in thyroid adenomas (JB, MM/SBL). Mutations in the KCNJ5 K+ channel gene are present and probably causative for one third of the very frequent aldosterone-producing adrenal adenomas found in approximately 1 in 30 patients with essential hypertension (44). Thyroid adenomas are very similar to adrenal adenomas. Both are very frequent, are generally not malignant and produce abnormally high level of hormones. Moreover, both TSH-producing thyrocytes and aldosterone-producing glomerulosa cells use the membrane potential as control for hormone secretion. Somatic or familial channel gene mutations are likely to be associated or even responsible for thyroid adenoma formation. We will screen for channel gene variations/mutations in adenoma and healthy samples available at the thyroid biobank set up at Nice University Hospital ((46). JB team will focus on K+ channels variants. 3) Discovery of modifier genes in episodic diseases (JB). Many genetic diseases are episodic and highly variable in terms of frequency or severity of crisis. This is the case for life-threatening idiopathic ventricular arrhythmias where carriers of the same channel mutation often show a spectrum of clinical phenotypes, including the absence of disease, even within the same pedigree. The functional impact of additional mutation(s) can exacerbate or mask the excitability disorder. Therefore, the personalized channel SNP pathogenicity in such disease must be defined in the context of all other channel subunits present. Cohorts of patients with Long QT Syndrome are available in French University Hospitals. The project will be extended to cohorts of patients with atrial fibrillation. The same approach will be applied for discovery of regulatory genes involved in the phenotypic variation in congenital dysfunction of heat automaticity. MM/SBL team has access to a large cohort of patients with congenital dysfunction of automaticity due to Cav1.3 loss-of-function, as well as healthy individuals with abnormally high resting heart rate. Modifier genes will be shared among the consortium for characterizing their action on ion channels.
Added value of the LabEx - The strength of the consortium, combining almost all current advances in ion channel studies, will be of invaluable importance in the analysis allowing the extraction of predictive phenotypic information on network behavior from ion channel SNP profiles.
State-of-the-art - An important issue of innovative medicine is the development of nanotechnologies for drug delivery and non-invasive diagnostic procedures. Quantum dots (QDs) are colloidal nanocrystalline semiconductors with unique optical and electrical properties. These tiny light-emitting particles, on the nanometer scale, are emerging as a new class of fluorescent probes for in vivo biomolecular and cellular imaging. Fluorescence recovery after photobleaching (FRAP) and quantum dot tracking have been used to examine the mechanisms underlying Kv2.1 potassium channels-containing surface domains in both HEK cells and cultured hippocampal neurons. The results clearly indicate that channels within a surface cluster are mobile (47). Using dual-color labeling with quantum dots, it was possible to detect single hIK1 channel molecules within the plasma membrane (48,49). From the above report and others, we anticipate that the technology of QDs can be easily applied to other channels (TRP, ASIC, P2X). Nanoparticle drug delivery systems: The ability to use nanotechnology to alter the characteristics of a drug to increase solubility, decrease degradation during circulation, and concentrate the drug at the desired site of action promises to increase efficacy while decreasing unwanted side effects. This huge potential has spurred much funding and research aimed toward the development of various nanoparticle drug delivery systems (50,51).
Aims - Our objective is to synthesize and functionalize nano-objects of different nature (semiconducting, oxide, metallic and lipidic) for imaging, delivery (small organic molecules such as drugs, siRNA, DNA, etc), light controlled activation of cells and photodynamic therapy applications.
Ongoing and future projects - 1) Synthesis of quantum dots. The preparation of QDs with high quantum yields and good photostability, which does not blink will be achieved through established methods. We will also use a new approach, consisting of encapsulating organic dyes in lipidic nanocapsules. The QDs will be used to study the mobility of ion channels at high temporal and spatial resolution. We will combine the QDs technology with confocal or super resolution microscopy systems such as SIM (Structured Illumination Microscopy) to monitor single channel mobility. 2) Encapsulation of small organic molecules and siRNA. Lipidic nanocapsules, known as drug carriers, will be used for the encapsulation of small organic molecules such as drugs (channel blockers), pre-designed siRNA or photosensitizers for photodynamic therapy. The technique is now well established in the IRI (Institute for Interdisciplinary research), Lille. Nanocapsule size and composition will be optimized to reach high loading efficiency and controlled release of the bioactive agent. 3) Large-scale synthesis of functional nanoparticles. One challenging issue in the use of nanoobjects is their production at a large scale (gram range). During this project, we will develop a novel and relatively inexpensive technique for the preparation of nanoparticles of controlled diameter through planetary ball milling. In this technique, the nanoparticles are composed of only biocompatible molecules and the loading is achieved by simply mixing all the components. The system will allow for controlled, protected and targeted release and delivery of any type of active agents including antigen and therapeutic proteins.
Added value of the LabEx - Application of nanotechnologies to biomedical problems are increasingly catching the attention of scientists and clinicians. The development of nanoparticules for imaging, delivery, light controlled activation of cells and photodynamic therapy applications will allow creating new therapeutic tools for different pathologies studied within the consortium, such as epilepsy, pain, cancer, Parkinson’s disease, Alzheimer’s disease, diabetes. A close interaction with different members of the consortium is expected from the beginning of the project to its end.
State-of-the-art - For most ion channels, pharmacological characterization started with the discovery of specific high-affinity toxins. Many toxins, in turn, became lead compounds for the development of clinically used drugs (52).
Aims - The consortium proposes to develop new pharmacological tools against ion channels of interest (Cav1.3, HCN1, ASICs, K2Ps, Orai, SK channels, P2X receptors, TRPV1, TRPV6, TRPM8, Kv1.3, Nav1.6, Nav1.7, Nav1.9) through the screening and the characterization of novel animal toxin blockers from venom libraries.
Previous results of the consortium - The groups of EL, MM, EB and MDW [both by its academic contribution and by creating a start-up company devoted to toxins] in the consortium have a strong expertise in the field of ion channels and animal toxins and have developed an international leadership in the identification and characterization of pharmacologically interesting toxins (53-58).
Added value of the LabEx - A collection of venoms is already available in the different institutes of the consortium (IPMC, IGF, GIN) and will be complemented from private companies/suppliers as well as via ad-hoc collaborations with academic labs. The ICST consortium will also take advantage of novel experimental approaches (59) currently developed by MV at IBS that permit the screening of nanoliter volumes of compounds using artificial bilayers (60) coupled to cell-free channel protein synthesis (61), which is particularly well adapted for tiny volumes of natural venom sources. Collaboration with CBS in Montpellier and privileged access to IBS in Grenoble via the consortium will permit the exploration of the three-dimensional structure of the new toxins.
The powerful combination of venoms containing hundreds of distinct, active molecules with a set of pertinent ion channel targets present in the ICST consortium will favor the discovery of new, bioactive molecules that will be of interest for understanding the role of these channels, but also, for some of them, for further development toward disease treatment (epilepsy, pain, cancer, Parkinson disease, Alzheimer’s disease, diabetes, autoimmune disorders or cardiovascular disorders) through industrial partnerships and startup companies linked to the consortium, like Theralpha (www.theralpha.com) and Smartox Biotechnologies (www.smartox-biotech.com). Patent applications will be filed on new active peptide toxins showing in vivo effects and potential therapeutic interest. Selected toxins could then be exploited as free peptides or as tethered toxins delivered with methods that allow specific cell and temporal expression to target particular tissues or regions (in vivo biodistribution of toxins of interest can be performed by Smartox Biotechnology).