VCU Researchers Design New Receptor And Enhance Bioassay To Advance Drug Discovery Process

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

Accordingto the study in the Journal of the American Chemical Society, publishedonline on September 27, researchers designed and synthesized a newcyclen-based receptor, and demonstrated its ability to strongly bindthe fluorescent dye, pyranine, under near-physiological conditions.Furthermore, researchers were able to improve upon the currentlyapplied membrane leakage assay used to evaluate specific properties ofa developmental drug compound. Assays are used to help develop saferdrugs by evaluating properties of absorption, distribution andmetabolism.

“There is a growing need for the development ofassays to rapidly assess the activity of developmental drug compoundsunder near-physiological conditions,” said Vladimir Sidorov, Ph.D., aprofessor of organic chemistry at VCU and lead investigator of thisstudy. “Therefore, we wanted to improve on the existing membraneleakage assay.

“The high affinity of this receptor to pyranine,its impermeability to the lipid bilayer membrane and fast kinetics ofbinding were used as a basis for the new membrane-leakage assay,” hesaid.

According to Sidorov, the membrane leakage assay iscompatible with a second type of assay that monitors the ionophoreticactivity of the drug candidate in the cell model. Ionophoretic activityis the ability of compound to transport ions across biologicalmembranes. Using the assays together allows researchers to distinguishbetween selective ion transport and formation of large pores perturbingthe integrity of cell membranes within a single set of experiments.

Thenew receptor is ideal because it selectively binds to pyranine, hesaid. Pyranine does not penetrate the lipid membrane of the cell, andtherefore could provide the basis for a membrane leakage assay.

“Thereceptor we have created requires an extremely low concentration forthe dye to be bound,” said Sidorov. In current assays, researchers usehigh concentrations of probe DPX, a dicationic organic compound used toquench the fluorescence of pyranine. The affinity and specificity ofDPX to pyranine is low.

“The problem with using highconcentrations of a probe is that it becomes difficult to detect wherethe activity is actually occurring and difficult to determine theimpact it may have on the membrane or cell itself,” he said.

“Thetherapeutic properties and side effects produced by the wide variety ofdrugs are tightly associated with their function in cell membranes,”Sidorov said. “Therefore, the methods allowing accurate assessment ofthese membrane functions have crucial importance for the development ofsafer and more efficient drugs.”

The assay described in thisstudy allows researchers to assess the mechanism of ion transport,which can detect potential therapeutics against cystic fibrosis andBartter’s syndrome. Both are inherited genetic diseases associated withthe malfunction of natural proteins transporting chloride anions acrosscell membranes. The synthetic compounds capable of such transport canfunction in place of compromised proteins and therefore, one day, treatthe diseases. Sidorov and his colleagues are also currentlyinvestigating the development of such Cl- transporters.

Bartter'ssyndrome causes the kidneys to excrete excessive amounts ofelectrolytes such as potassium, sodium and chloride, resulting inelectrolyte abnormalities. Two potential outcomes of Bartter’s syndromeare 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.