Researcher's new receptor advances drug discovery process
that is set to advance the drug discovery process for diseases such
as cystic fibrosis and Bartter's syndrome, tracking a drug's
activity inside the cell.
The new cyclen-based receptor, and its ability to strongly bind to the fluorescent dye, pyranine, under near-physiological conditions, brings renewed hope to discovering new antibiotics and therapeutics for these genetic diseases which suffer from a lack of an effective treatment.
According to the study in the Journal of the American Chemical Society, published online on September 27, researchers set out to improve upon the currently applied membrane leakage assay used to evaluate specific properties of a developmental drug compound.
According to one of the researchers the new receptor is ideal because it selectively binds to pyranine. Pyranine does not penetrate the lipid membrane of the cell, and therefore could provide the basis for a new type of membrane leakage assay.
In addition, the new membrane leakage assay is compatible with a second type of assay that monitors the ionophoretic activity of the drug candidate in the cell model.
Ionophoretic activity is the ability of compound to transport ions across biological membranes.
Using the assays together allows researchers to distinguish between selective ion transport and formation of large pores perturbing the integrity of cell membranes within a single set of experiments.
"There is a growing need for the development of assays to rapidly assess the activity of developmental drug compounds under near-physiological conditions," said Vladimir Sidorov, a professor of organic chemistry at VCU and lead investigator of this study.
"Therefore, we wanted to improve on the existing membrane leakage assay. The high affinity of this receptor to pyranine, its impermeability to the lipid bilayer membrane and fast kinetics of binding were used as a basis for the new membrane-leakage assay," he added.
The receptor created requires an extremely low concentration for the dye to be bound.
In current assays, researchers use high concentrations of probe DPX, a dicationic organic compound used to quench the fluorescence of pyranine. The difficulty is affinity and specificity of DPX to pyranine is low.
"The problem with using high concentrations of a probe is that it becomes difficult to detect where the activity is actually occurring and difficult to determine the impact it may have on the membrane or cell itself," Sidorov said.
"The therapeutic properties and side effects produced by the wide variety of drugs are tightly associated with their function in cell membranes," he added.
The significance of this research means that methods allowing accurate assessment of these membrane functions have crucial importance for the development of safer and more efficient drugs.
The assay described in this study allows researchers to assess the mechanism of ion transport, which can detect potential therapeutics against cystic fibrosis and Bartter's syndrome.
Both are inherited genetic diseases associated with the malfunction of natural proteins transporting chloride anions across cell membranes.
The synthetic compounds capable of such transport can function in place of compromised proteins and could therefore, one day, treat the diseases.
Sidorov and his colleagues are also currently investigating the development of such Cl- transporters.
Bartter's syndrome causes the kidneys to excrete excessive amounts of electrolytes such as potassium, sodium and chloride, resulting in electrolyte abnormalities.
Two potential outcomes of Bartter's syndrome are kidney failure and inner-ear defects resulting in deafness.
