January 2018 Newsletter

It is a matter of bicarbonate

Oscar Moran – Istituto di Biofisica, CNR, Genova, Italy

Cystic fibrosis (CF) is a genetic disease that affects several organs. The malfunction or lack of the protein CFTR severely reduces the an­ion transport in the apical membranes of epi­thelia, that form the lumen of internal organs, like bronchi, intestine, sweet glands and pan­creas ducts. One of the systems most affect­ed by the disease are the airways, because of the loss of control of the liquid in the luminal surface of the bronchi (ASL). The modification of ASL causes the thickening of the mucus, resulting in the reduction of the ciliary motil­ity, which is fundamental to “washing away” the bacteria that are present in the breathing air. This condition leads to continuous bacte­rial infections, including those due to atypical bacteria, which, becoming chronic, conduce to the destruction of the lung parenchyma.

Almost 30 years after the identification of the gene encoding the CFTR, the exact mecha­nism of the loss of ASL regulation is still con­troversial. The study of the physiology of ionic transport in the bronchial epithelium, which includes more than twenty different trans­port mechanisms among channels, carriers and pumps, has given rise to two opposing hypotheses. The “high salt” hypothesis, pro­posed by Welsh group of the University of Iowa, postulates that the CFTR defect causes an elevated concentration of sodium and chlo­ride in the airway surface liquid (ASL) which inhibits the native bactericidal activity. On the other hand, the “low volume” hypothesis, sup­ported by the Boucher group of the University of North Carolina, proposes that the volume of the ASL is severely decreased as a result of abnormally increased sodium absorption, which causes cilia to collapse and halt mucus clearance. In a critical analysis of published data, Marquette and Luckie1 concluded that neither hypothesis is correct, as suggested by in vivo studies conducted on human or porcine. These studies strongly suggest that in CF disease there is not an increased salt concentration in the ASL, nor the depth of the airway surface liquid is lowered or depleted.

An alternative hypothesis suggests that the cause underlying CF lung disease is the dif­ference in the extracellular pH. The refocus­ing on the pH of the ASL is a direct conse­quence of the original finding that CFTR not only transports chloride ions but bicarbonate ones as well. Thus, a lower ASL pH, caused by a reduced bicarbonate secretion, could be the potential mechanism that causes lung infections and pulmonary complications asso­ciated with CF. On the other hand, increased levels of bicarbonate in the ASL of CF subjects could increase pH, with a dramatic reduction of mucus thickness.

One advantage of the anion carriers being developed in the project TAT-CF, the aniono­phores, is their ability to transport bicarbon­ate. Therefore, these small molecules are potentially capable not only to substitute the chloride reabsorption in the apical membrane, but also to promote the bicarbonate secretion, with consequent beneficial effects if directly applied to the bronchial epithelia of CF pa­tients. The TAT-CF consortium is now carrying out in vitro experiments on model CF-epithelia to measure the magnitude of the pH change on the ASL due to the application of aniono­phores and to reveal consequent changes in the viscoelastic properties of the mucus. We are confident that these experiments will indi­cate us the best conditions to assay the carri­ers in animal models to approach step by step the therapy of CF.

Reporting period of TAT-CF project successfully completed

TAT-CF is a 3 years project and is organ­ized in 8 Work Packages (WP). The first period of the TAT-CF project, which cor­responds to the first 18 months (from 01/01/2016 to 30/06/2017), has been successfully completed and reported to the European Commission. Most of the planned tasks, deliverables and milestones have been achieved. Overall the results of project are most satisfactory and bode well for the next period, in which TAT-CF project will tackle challenging tasks and objectives to reach the proposed goals.

During the first period of the project, UBU and AVIDIN have synthesized more than 150 compounds within WP1, based on three different pharmacophores structures identified in WP4. The organic substances synthesized were screened in WP2 by measuring their ability to transport anions. The transport assays were designed to measure the concentration changes of the halide -iodide or chloride- as well as the bicarbonate-chloride interchange in unila­mellar vesicles and in cells. On the other hand, a correlation of transporter activity of anionophores and cytotoxicity was found in most of the cases within WP3. Thus, new molecules have been synthesized to balance toxicity and enhanced membrane permeability. Finally, six compounds have been selected as the most promising after a first screening. A complete characteriza­tion of these compounds is being carried out and new and optimized analogs of the compounds are being synthesized.

These results will be completed in the fol­lowing months with data regarding the impact on percilliary fluid volume/viscos­ity/pH/chloride concentration as a result of the application of non-toxic doses of anionophores (WP3). These assays will be mainly performed in artificial epithelia mon­olayer grown using both wt and KO Calu-3 cells expressing the YPF Iodide sensor and testing the 6 lead compounds. In addition, the functionality of lead compounds will be also tested in organoids and in vivo mod­els (WP5). All these activities will allow to select the final compounds and innovative and effective drug delivery systems will be developed in the last stages of the project.

On the other hand, deliverables and mile­stones are also indicators of the project progress. All deliverables and milestones established for the first reporting period of the project have been completed and submitted to the EC at the time they were planned, with the exception of MS5 and MS17.

DELIVERABLES 1st RP (M1 – M18)

Del N °Deliverable nameWPLeader beneficiaryTypeDissemin.
Level
DateSubmission date
D7.1Project websiteWP7BIOPRAXISWebsites, patents filling, etc.PUM0329/03/2016
D7.2Dissemination plan
WP7BIOPRAXISReportCOM0630/06/2016
D2.1pH-insensitive Cl-sensor assayWP2IGGReportPUM0729/07/2016
D2.2Bicarbonate
sensor assay
WP2IGGReportCOM1130/11/2016
D4.1Pharmacophore reportWP4AVIDINReportCOM1223/12/2016
D5.1iPS cells from F508del patientsWP5CSICReportPUM1223/12/2016
D8.1Project progress
(report 12 months)
WP8UBUReportCOM1223/12/2016
D3.1Calu-3 mutant CFTR F508del and G551D cellsWP3CNRReportPUM1428/02/2017
D4.2Lead-like
molecules
WP4AVIDINReportCOM1628/04/2017
D2.3Hits transporting Cl- and HCO3-WP2IGGReportCOM1828/06/2017
D4.3SAR ReportWP4AVIDINReportCOM1828/06/2017
D5.2iPS cells from G551D patientsWP5CSICReportPUM1828/06/2017
D5.3Lung epithelial cells from F508del patientsWP5CSICReportPUM1828/06/2017
D7.3Exploitation planWP7BIOPRAXISReportCOM1830/06/2017
D9.1POPD – Requirement Nº6WP9UBUReportCOM1828/06/2017
D9.2H – Requirement Nº2WP9UBUReportCOM1828/06/2017
D9.3A – Requirement Nº1WP9UBUReportCOM1803/07/2017
D9.4HCT – Requirement Nº3WP9UBUReportCOM1830/06/2017

MILESTONES 1st RP (M1 – M18)

Mil. N °TitleWPLeaderDateAchieved date
MS3Non-ratiometric chloride sensorWP2IGGM630/06/2016
MS27Dissemination planWP1,WP2,WP3,WP4, WP5,WP6,WP7,WP8BIOPRAXISM730/06/2016
MS4Ratiometric chloride sensorWP2IGGM823/07/2016
MS12Definition of 3 pharmacophoresWP1,WP4AVIDIN30/08/2016
MS5Ratiometric bicarbonate FRET sensorWP2IGGM1001/04/2017
MS6Bicarbonate CEPAC FRET sensorWP2IGGM1130/11/2016
MS1Anionophores synthesizedWP1,WP2,WP5UBUM1230/12/2016
MS7Identification of lead anionophoresWP2IGGM1828/06/2017
MS14SAR ReportWP2, WP4AVIDINM1828/06/2017
MS17CFTR iPS cells collectionWP5CSICM18Partially achieved
MS28Exploitation planWP1,WP2,WP3,WP4, WP5,WP6,WP7,WP8BIOPRAXISM1830/06/2017

WP2: Compound activity screening (IGG)

Cystic fibrosis is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial cells in the airways, intestine, pancreas and other organs. Defective chloride and bi­carbonate secretion, arising from CFTR mutations, causes a multi-organ disease. In the airways, the impaired ion transport results in a thick mucus, dehydratation of the pericilliar region and bacterial infections. The chronic persistence of bacteria triggers a severe in­flammatory response that causes progressive loss of respiratory function (Fig1).

TAT-CF aims to identify small compounds, named anionophores, able to restore the ion transport activity across the CF epithelial cells and therefore to correct the properties of the fluid composition of the airway surface liquid.

During WP1 a novel libraries of anionophores, based on different structures, have been synthesized and successively considered for their biological evaluation.

The strategy conducted in WP2 has allowed to test compounds for their ability to transport anions with different methods (Fig2).

The screening has been done initially in phospholipid liposome mod­el to identify active compounds. Successively, we have moved to cell models and procedure 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.

Finally, a correlation of transporter activity and cytotoxicity has al­lowed to select six lead anionophores showing high efficacy, potency and low cytotoxicity.

Right now, we are ready to proceed in WP3 with pharmacological and biophysical properties of the selected lead compounds. In this part of the project we will also evaluate the effect of the induced anion transport in the pericilliar fluid properties of bronchial epithelial cells, that is one of the main task for the CF therapy.

Figure 1: CF epithelium disease caused by CFTR loss of function.

Next project meeting and Workshop in Mallorca


Cystic fibrosis is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial cells in the airways, intestine, pancreas and other organs. Defective chloride and bi­carbonate secretion, arising from CFTR mutations, causes a multi-organ disease. In the airways, the impaired ion transport results in a thick mucus, dehydratation of the pericilliar region and bacterial infections. The chronic persistence of bacteria triggers a severe in­flammatory response that causes progressive loss of respiratory function (Fig1).

TAT-CF aims to identify small compounds, named anionophores, able to restore the ion transport activity across the CF epithelial cells and therefore to correct the properties of the fluid composition of the airway surface liquid.

During WP1 a novel libraries of anionophores, based on different structures, have been synthesized and successively considered for their biological evaluation.

The strategy conducted in WP2 has allowed to test compounds for their ability to transport anions with different methods (Fig2).

The screening has been done initially in phospholipid liposome mod­el to identify active compounds. Successively, we have moved to cell models and procedure 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.

Finally, a correlation of transporter activity and cytotoxicity has al­lowed to select six lead anionophores showing high efficacy, potency and low cytotoxicity.

Right now, we are ready to proceed in WP3 with pharmacological and biophysical properties of the selected lead compounds. In this part of the project we will also evaluate the effect of the induced anion transport in the pericilliar fluid properties of bronchial epithelial cells, that is one of the main task for the CF therapy.

Next project meeting and Workshop in Mallorca

Several TAT:CF partners attended the Seminary in Mainz on the 21st and 22nd of September, in order to advance in the knowledge of this key Topic (organoids as 3D model for diseases), and also to show relevant advances from our project. UBU, CSIC and SIG presented several talks and posters at this relevant Seminary.