VCU researchers design new receptor and enhance bioassay to advance drug discovery process

Virginia Commonwealth University chemists have created a new molecular receptor for a fluorescent dye used to track a drug candidate’s activity inside cell membranes.

These findings may help researchers more efficiently design, evaluate and test new drugs like antibiotics and therapeutics for genetic diseases, such as cystic fibrosis and Bartter’s syndrome, because they will know precisely where the drug is acting inside a cell.

According to the study in the Journal of the American Chemical Society, published online on September 27, researchers designed and synthesized a new cyclen-based receptor, and demonstrated its ability to strongly bind the fluorescent dye, pyranine, under near-physiological conditions. Furthermore, researchers were able to improve upon the currently applied membrane leakage assay used to evaluate specific properties of a developmental drug compound. Assays are used to help develop safer drugs by evaluating properties of absorption, distribution and metabolism.

“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, Ph.D., 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 said.

According to Sidorov, the 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.

The new receptor is ideal because it selectively binds to pyranine, he said. Pyranine does not penetrate the lipid membrane of the cell, and therefore could provide the basis for a membrane leakage assay.

“The receptor we have created requires an extremely low concentration for the dye to be bound,” said Sidorov. In current assays, researchers use high concentrations of probe DPX, a dicationic organic compound used to quench the fluorescence of pyranine. The 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,” he said.

“The therapeutic properties and side effects produced by the wide variety of drugs are tightly associated with their function in cell membranes,” Sidorov said. “Therefore, the 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 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.

This work was supported by grants from the National Science Foundation, VCU startup fund and Jeffress Memorial Trust.