WP1. Chemistry (UBU): This WP will be responsible for the synthesis of the new compounds. WP1 is lead by UBU, having an important expertise in the synthesis and design of anionophores. WP1 is responsible for synthesize all compounds to be tested in this project. Moreover WP1 will use feedback from WP2-6 to refine and prepare lead compounds. As mentioned in the introduction suitable anionophores capable of inducing anion transmembrane permeation in living cells are already available. This will allow a smooth start of most of WP tasks at the beginning of the project. Therefore there will be suitable candidates to be tested from the start of the project. The continuous feedback from other partners involved in the testing of the substances (WP2-6) will allow identification and optimization of hits and lead compounds. As a result of TAT-CF the number of available anionophores and our understanding of anionophore properties, mechanisms of action and design will increase dramatically.
WP2. Compound activity screening (IGG): The primary screening of the anion transport activity of these compounds will be the main objective of WP2, lead by IGG. Several methods will be used. First the transmembrane transport activity will be screened in model phospholipid liposmes. Active compounds will be then tested on Fisher Rat Thyroid (FRT) cells using a fluorescent assay based on the different sensitivity to halides of the Yellow Fluorescent Protein (YFP). TAT-CF aims to develop new methods to explore the transmembrane transport activity. These methods will be useful not only for this project but for the wider scientific community. Thus a CHO bicarbonate sensor cell line and a cell based assay for measuring bicarbonate transport activity of compounds will be developed within this project. In addition a pH insensitive chloride sensor CHO cell line and a cell based assay will be developed Finally, a secondary screening of hit analogues using different techniques will be carried out.
WP3. Cell biophysics and physiology (IBF): WP3 will be lead by IBF. This work package is focus in the biophysical and physiological characterization of the anionophores and the assessment of the physiological impact of the anionophore-induced anion transport on normal and cystic fibrosis bronchial cell models. Up to date very little is known about the fundamentals of transmembrane anion transport. WP3 will asses several aspects of anionophore activity which are crucial for further development of these compounds as drugs. Electrophysiological characterization of compounds will be obtained using voltage clamp in frog oocytes and patch clamp in mammalian cells. It includes a detailed functional analysis (transport efficiency, ionic composition dependence, pH dependence, temperature effects, voltage dependence), pharmacological analysis, and cell distribution of compounds by biochemical methods. Of particular relevance is the effect in the bronchial epithelial cell, which is among the most affected in the CF pathology. Therefore it is of key importance to validate the previous observations investigating the action of anionophores in preparation of primary bronchial epithelial cells from normal subjects and CF patients in culture. CFTR wild type and mutant Calu-3 sensor cell lines developed in task 3.2 (SIZ) will be grown on filters and the transport of anionophores, as well as the incorporation of the anionophores in the cells will be assessed. Further synergism of anionophores with CFTR correctors and potentiators or interference with the actions of these compounds will be evaluated. Another most relevant point is to study the periciliary fluid. CF affects the properties of the periciliary fluid, modifying the visco-elastic properties of the mucus. The resultant sticky mucus is responsible for recurrent pulmonary infections in CF patients. The improvement of trans-epithelial transport by anionophores is expected to cause also an increase of airway hydration and a consequent improvement of the properties of CF mucus. Calu-3 CFTR mutant and wt cell lines stably transfected with genetically encoded chloride and/or bicarbonate sensors will be used for evaluating the effect of anionophore compounds on anion and fluid transport through the apical membrane (SIZ). We will also analyse the effect of anionophores on a mucus secreting intestinal co-culture model consisting of goblet and Caco-2 cells, which is very relevant if these substances should be orally administered (B:F).
WP4. Medicinal Chemistry (AVD): Structure-activity relationship studies and evaluation of the drugability of the compounds along with in silico optimization of selected compounds will result in pharmacophore identification. This WP4 will be lead by AVD the partner with expertise in chemioinformatics.. These studies regarding the drugability of substances will result in optimized clinical candidates for preclinical studies in WP5. Detailed quantitative structure-activity relationship (QSAR) analyses will shed light on the relationships between chemical structure and pharmacological activity for a series of lead-like compounds. Using chemioinformatics optimisation we propose novel synthetic modifications on lead compounds for WP1 in order to improve their drugability.
WP5. Preclinical studies (CSIC): WP 5 will be lead by CSIC. This WP5 will focus on the more advances stages of the preclinical development of anionophores. The efficacy of lead compounds in animal models will be determined In vivo. ADME-tox, bioavailability, biodistribution and PK/PD will be assessed in animal studies. The potential antimicrobial activity of these compounds will also be determined. A number of cation selective ionophores are regularly used as antibiotics. It is expected that anion-selective ionophores will also work similarly. A dual beneficial effect would enhance the therapeutic potential of this approach. iPS cell lines obtained from different individuals with diverse CFTR mutations and genotypes will be used to test the efficacy of lead anionophores irrespective of the cause of the loss of CFTR function. Intestinal organoids derived from iPS cells from CFTR patients with different CFTR mutations and diverse genetic backgrounds, to test the physiological properties of the iononophores produced in WP1 and WP2 and validated in WP3 .
WP6. Drug delivery and formulation (B:F): This WP will be responsible for determining the optimal formulation of the lead compound. Different alternatives based on nanoformulation will be developed and evaluated including lipid (pulmonary) and polymer (oral) based nanoparticles, to address pulmonary delivery as primary route and oral route as a secondary object, in order to deliver the new compounds also to the gastro-intestinal tract. As final pharmaceutical form, two different potential products will be developed: one will be based in pulmonary delivery, as a dry powder formulation or as a nebulized one (Praxis). The second one will be intended for oral administration, as a tablet or a liquid-filled capsule (B:F). Scalability of the production processes will be assessed and designed, in order to find any potential drawbacks, which may compromise the future translation of the innovative drugs based on anionophores.
WP7. Dissemination and exploitation (PRAX): All partners will contribute to this WP, leaded by one of our industrial partners. This WP will be responsible for the strategy to be followed in order to protect the intellectual property susceptible of commercial exploitation. This should be made compatible with open access dissemination of the data generated as well as maximizing the visibility of the work carried out through the outreach activities planned. A Dissemination and communication Plan will guide TAT-CF measurements to maximize the visibility and awareness of TAT-CF results. Likewise, the Exploitation Plan will be used to manage the different exploitable results of the project.
WP8. Coordination and management (UBU): The objectives of this WP are to manage the project in a fair and transparent manner, ensuring that the project deliverables are met in an integrated and timely fashion within the agreed budget and to enable integration of work packages and ensure effective communication between members of the research consortium. WP0 will be lead by UBU and dedicated to management and coordination of the project.