STEROTRANS · Anion Transport by Steroid-Based Synthetic Channels

Final Activity Report Summary - STEROTRANS (Anion Transport by Steroid-Based Synthetic Channels)

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasian populations. CF is caused by loss of function of the cystic fibrosis trans-membrane conductance regulator (CFTR) chloride channel, a protein that regulates chloride concentration within the cells. Malfunction of CFTR disrupts salt and water movement across epithelia cells causing ducts and tubes to become blocked by thick, sticky mucus. Various strategies aim to restore salt and water transport to CF epithelia. These include: - replacing faulty CFTR with normal CFTR using gene therapy; - rescuing the expression and function of faulty CFTR using small molecules; - bypassing the loss of CFTR function using alternative Cl- transporters. Cholapods are amphiphilic anion-binding molecules derived from the natural steroid cholic acid. Some cholapods demonstrate Cl- transport from artificial liposomes. Unlike many other anion-binding molecules, they are electroneutral, and this makes them less likely to be toxic. There is a realistic hope that molecules from this family may promote anion transport across the cell membranes of CF patients, and thus alleviate the symptoms of the disease. Within this project, we have designed, synthesised and tested a series of cholapods. The design has been made according to our previous experience with similar compounds, focusing mainly on their capacity to bind anions. Also some computational modelling had been carried out to define the binding site of the cholapods for anions of different size. The synthesis of these molecules has been carried out at the School of Chemistry of Bristol, within the A.P. Davis laboratory. A classical multistep procedure has been used at the bench in order to obtain a reasonable amount of the desired cholapods. Then these compounds have been tested as potential anion transporters through artificial membranes. These tests have been carried out at the Department of Physiology and Pharmacology of Bristol using the planar lipid bilayer (PLB) technique. Basically this technique consists in the measurement of ion transport (current) through a thin lipid membrane separating two aqueous compartments containing a known composition of ions. A pure lipid membrane is impermeable to hydrophilic molecules such as anions, thus no transport could take place in a membrane free of transporters. The incorporation of cholapods within the membrane increases its permeability and reveals an ion transport through it. The tests have shown that an increase of cholapod concentration results in a current rise in a linear way. This suggests that the cholapods act as single molecules and not as aggregates (e.g. self-assembled channels). Also the membrane permeability tends to a limit as the anion concentration of the aqueous phases increase. This plateau signifies a saturation of the carrier activity which means that the current becomes only translocation rate-dependant. The cholapods have shown also a good selectivity for anion versus cations and certain selectivity between anions such as chloride, nitrate or bromide, the latter being the best transported ion. Cholapods are thus able to increase the anion permeability of artificial membranes. These results show that this transport is made via a carrier mechanism where the cholapods bind an anion at one side of the membrane, translocate through the membrane and then release the anion to the second aqueous phase. Although the anion selectivity needs to be improved toward chloride transport, we can speculate that such molecules might have applications in drug therapy for CF in the future. The PLB technique has been a very effective method in determining the mode of activity of the cholapods in artificial membrane and is a good technique to begin the study of potential drugs.

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