December 2018 Newsletter

Tat-cf: Final achievement and reflections

Cystic Fibrosis (CF) is caused by malfunction of CFTR, a transmembrane protein expressed in the apical membranes of epithelial tissues. Current therapeutic treatments targeting CF basic molecular defect aim to correct trafficking or folding defects preventing CFTR reaching the plasma membrane or to potentiate the activity of CFTR. There are over 2000 mutations described in the CFTR gene and these approaches are mutation specific, thus benefiting only cohorts of patients sharing the same functional defect. TAT-CF project seeks to develop molecules that mimic CFTR protein function, facilitating the permeation of anions such as chloride and bicarbonate through the membrane. By replacing CFTR function independently from the protein our approach is mutation agnostic and in principle could benefit all CF patients.

The main project innovations were to design and synthesize the appropriate compounds in the first instance. This was a challenging task and more than 200 compounds were evaluated by the different partners involved in our consortia. We needed to find the right balance between the molecular properties, transport activity, and toxicity of the candidates to select the appropriate leads for our project. In addition, we have created new research tools for cystic fibrosis such as induced pluripotent stem cell (iPSC) lines from cystic fibrosis patients and new assays to evaluate intracellular concentrations of anions.

Our most important achievement is the demonstration of the viability of this therapeutic approach. The best in vitro preclinical model of CF disease are synthetic epithelium derived from primary human bronchial epithelial cells. We have proved that our compounds can correct CF epithelia function to normal values in terms of fluid reabsorption and mucus viscosity, two key parameters in the pathophysiology of CF patients.

We have produced and studied several cell lines obtained from both patients with different mutations and through gene editing. This means that we have been able to test our compounds in cells and organoids having different functional defects as well as models in which CFTR expression was completely absent. Moreover we have tested TAT-CF compounds in combination with approved CF drugs and observed additive effects, which could also be envisaged to be a promising approach for the treatment of different mutations.

Due to the promising results obtained within the TAT-CF project, our idea is to continue these investigations. At this stage, drug development requires substantial investments to complete the preclinical assessment and proceed to clinical phases of development. A patent application has been recently submitted and we are looking for new sources of funding, both public and private, that allow us conducting the next steps for the development of the product. Although this is challenging in the context of a rare disease yet we are confident that it is possible.

Tat cf: main technical achievements

Each Technical Work Package (WPs 1 to 6) has obtained synergistic, relevant results, in order to achieve the main goals of the project. In brief, this are the main results for the mentioned WPs:

  • WP1. Chemistry

    This WP focused on the synthesis of new compounds capable of facilitating transmembrane transport of anions (anionophores). Both UBU and AVIDIN were the partners mainly involved in this WP, having a well-established expertise in the synthesis and characterization of novel compounds. Based in the extensive background on small molecule anion transporters synthesis and design the first candidates were produced from the beginning of the project and over the lifetime of the project UBU and AVIDIN have synthesised around 200 novel compounds, based on different potential anionophores structures. The designs are based in small molecules having convergent hydrogen bonding pockets in which the anions are stabilized. The supramolecular complex anionophores-anion is thus able to diffuse across the lipid membrane releasing the anion in the other side of the membrane leaflet. Overall this process mimics the function of the transmembrane protein facilitating the translocation of the anion (chloride and/or bicarbonate) across the phospholipid bilayer. The continuous feedback from other partners involved in the screening of the molecules (WP2-6) has allowed identification of 30 compounds (Milestone MS13) subjected to subsequent selection that returned seven lead candidates for further development. The seven lead compounds have been selected based on their transport activities in different cell lines as well as their toxicological profile.

  • WP3. Cell Biophysics and Physiology

    The central objective of this workpackage has been the characterization of the transport mechanism of anionophores, identifying those that have a reduced toxicity, so that they can be applied to cells or model systems of bronchial epithelium. Ultimately, it has been proven that the application of anionophores on the apical side of the epithelium induces a reduction in the viscosity of the mucus, probably related to the transport of anions. The workpackage obtained six major results:
    We determined the in vitro toxicity of all synthesized anionophores, extending the studies to the induction of apoptosis for the leading compound. It was useful to determine the limit concentration that can be used without damaging the cells.The biophysical characterisation of the anionophores, done in artificial membranes, yielded a functional model of reversible uniporters. Interestingly, for the prodigiosines and tambjamines, the compound must be ionised to work, depending on the pH. The anionophores have the same transport properties on cells, and efficacies similar or higher than the CFTR can be obtained at no-toxic concentrations.
    Noteworthy, anionophores does not interfere with the function of CFTR, nor to the CFTR modulators, and their anion transport are additive. At high concentration, anionophores are distributed on all cellular membranes. Conversely, at the low concentrations used in our experiments, localization is limited to the plasma membrane, and was not detectable in other intracellular membranes. When anionophores are applied to the apical side of CF-human bronchial epithelium, there is a reduction of the water reabsorption to levels similar to those of the non-CF epithelium. Anionophore-driven chloride and bicarbonate transport in the apical membranes of epithelium lead to a modification of the pericilliar fluid, resulting in a decrease of the mucus viscosity in CF-bronchial epithelium.
    Regarding sensors, two Chinese Hamster Ovary (CHO) sensor cell lines detecting chloride transport and bicarbonate transport activity of anionophores were developed. SIG characterized several compounds using the developed chloride sensor cell as well as the bicarbonate sensor cell. The chloride transport activity was compared with the bicarbonate transport activity for the 6 lead compounds MM3, MM34, EH130, RQ363, ACF516 and ACF820.

  • WP5. Preclinical Studies

    We have obtained relevant preclinical information for the selected leads, including in vitro and in vivo efficacy and toxicology data. Innovative preclinical models, like lung epithelial cells from p.F508del iPS cells and organoids have been developed, helping to obtain more significant efficacy models. On a second step, acute and repeated dose toxicity, in oral and pulmonary delivery have been studied for the more relevant compounds in rodents, showing promising, low toxicity results. A pilot, limited study has been performed using knockout mice harboring CF mutation.

  • WP2. Compound Activity Screening

    During WP2 a novel libraries of anionophores were considered for their biological evaluation.This phase has allowed to test compounds for their ability to transport anions with different methods.
    The screening has been done initially in phospholipid liposome model to identify active compounds. Successively, we have moved to cell models and procedures with increasing complexity, such as fluorescent and bicarbonate biosensor based assay, in order to measure the efficacy in transporting chloride and bicarbonate. The toxicity properties of the anionophores have also determined as a relevant point to consider to optimize analogs with improved features.
    On the basis of the data obtained, we have been able to select six lead anionophores showing high capacity of transport iodide, chloride and bicarbonate, potency and low cytotoxicity.
    In order to provide compounds with a better balance toxicity and improved drugability, leads’ analogs synthesized in WP1 have been validated for their activity as transporters.

  • WP4. Medicinal Chemistry

    Based on already described ionophore molecules and drug-like compounds identified in WP2, three pharmacophore structures were identified and the defined basic structures serve the basis for generating additional new and diverse scaffolds by using scaffold hopping. Pharmacophores were also used for hit identification and lead optimization. Three pharmacophore structures were identified: tambjamine-based molecules, 1,2,3-triazole-based molecules and thiourea-quinoline-based molecules (Milestone MS12). Based on these core structures, 30 lead-like molecules were selected taking into account their transport properties and covering different substituents (hydrogen bond donors, hydrogen bond acceptors, hydrophobic groups) in order to generate molecular diversity. From all the synthesised compounds, and thanks to the continuous feedback from other partners involved in the testing of the substances, we have been able to identify six compounds as the lead ones. Continuous information on selected chemical structures and their cellular in vitro activity has enabled us to perform a detailed SAR analysis. For all three selected scaffolds structure activity relationship rules could be obtained. This information has driven the optimization of lead compounds.

  • WP6. Drug Delivery and Formulation

    We have performed characterization of the substances in order to determine their drugability profiles. This has included stability, solubility, at different conditions. We have also developed different formualtions, including nanoformulations in lipid matrices in order to obtain final formulations for oral and pulmonary delivery, including nanoparticles freeze drying. The formulations have been successfully tested in animal models (oral and pulmonary delivery).

TAT-CF dissemination, exploitation and management

WP7 and WP 8 have contributed to a right dissemination, communication and exploitation strategy and to the overall coordination and management of the project. Some highlights are the filling of a PCT patent, the final conference and the continuous meetings and workshops, including the final meeting in Burgos to successfully close the project.

TAT-CF final project meeting in Burgos (Spain)

The last TAT-CF project meeting took place the first week of December in Burgos. The University of Burgos organized a 3-days meeting, including the TAT-CF workshop (Monday, 3 December), the final project conference (Tuesday, 4 December) and the meeting of the Steering Committee (Wednesday, 5 December). A summary of the activities performed throughout the entire project was presented in the workshop and a project self-evaluation was done by the consortium during the Steering Committee session.

TAT-CF final conference

TAT-CF organized its final conference “Research and New Treatments in Cystic Fibrosis”, in the context of the last project meeting. This event took place in the National Reference Centre for People with Rare Diseases (CREER), on 4th December in Burgos. This conference was aimed at associations and patients, as well as professionals in the sector and the objectives were to present the main results obtained within the TAT-CF project and establish a debate on the therapeutic advances that are currently being made in the field of Cystic Fibrosis. Around 40 people attended to this event in person, in which 7 presentations were made by renowned experts in the area. Additionally, the event was broadcast live to allow a wider participation and the videos are hosted in YouTube in the following link:
https://www.youtube.com/playlist?list=PLNdLQDwGFm9GxI2vYqGeczJVUMDPEiLye.

Overall the conference was very successful and the availability of the material produced in the internet will enhance the communication potential of this activity well beyond the lifetime of our project.