In the past, we have developed an assay for inhibitors for carbonic anhydrases (CA). Human carbonic anhydrases (hCA) are a family of enzymes (15 isoenzymes in mammalian cells localized in various tissues and cell compartments) that catalyze the conversion of CO2 and water to HCO3- and back thereby contributing to regulation of pH and CO2 levels. Isoenzymes hCA IX and hCA XII are maintaining acidic pH in solid tumors thereby contributing to unfavorable prognosis. A selective hCA IX and hCA XII inhibitors would be a boon to treatment of many cancers, and a significant effort toward the synthesis of such small-molecule inhibitors has been expended. Numerous potential inhibitors must be tested for all CA isoenzymes because we need drugs that inhibit the cancer associated CAs (hCA IX and hCA XII), but not the other CAs. This is an immense challenge because all hCAs have very similar structure. While we do not develop new drugs in our laboratory, we have developed a fluorescence-based assay based on a competitive mechanism whereas our probe is bound by the enzyme (fluorescence is quenched) and upon addition of competitive inhibitor the fluorophore (probe) is released, and its fluorescence is regenerated. The figure shows the structure of hCA (A), the structure of the probe (B) as well as one of the inhibitors and the docking of the probe in the binding pocket of the enzyme (C). We can clearly see how the aromatic surface of the probe preferentially interacts with the hydrophobic amino acids (shown by the red color).
While the probe is bound within the enzyme, its fluorescence is quenched (D). However, upon addition of the inhibitor, the probe is released and becomes brightly fluorescent, which is visible by naked eye (E). A titration by the inhibitor allows for calculation of the binding constant (Kassoc) (F). Numerous potential inhibitors can be tested and compared (G) to identify promising leads. Leads that inhibit the cancer-associated hCAs but not the other isoenzymes.
Medicinal chemists must generate large numbers of potential inhibitors that have to be tested using all pertinent isoenzymes. This requires a rapid and reliable assay capable of addressing Kassoc in the range of 105 - 1010 M-1. Reference: Chem 2017, 2, 271-282.
Recently, we focused on determination of conversion of ATP into ADP+Pi or into AMP+PPi. This is because numerous biological processes perform these reactions and we feel that our receptors and sensor can address these needs. Recently, during the Covid-19, a need for running millions PCR was realized. But the traditional DNA stains such as SYBR Green I are not always reliable due to variable fluorescence to different dsDNA sequences, sensitivity to additives, and reacts to loops/palindromes in ssDNA. Hybridization probes utilizing dual labeling such as TaqMan (gold standard in Covid-19 diagnosis) are sensitive to storage conditions, and expensive to the point that it cannot be available for large populations in economically developing countries. We have embarked on research to develop fluorescence-based sensors that selectively and reversibly bind PPi (product of PCR) in the presence of large concentration of dNRPs. Furthermore, the sensor cannot interact with the primers, DNA polymerase regardless of the origin (TaqDNA, PfuDNA, etc.) and must utilize the same wavelengths of dyes to be compatible with all the current PCR instruments. The idea of Zn(II) based receptors combined with fluorophores is not entirely new (e.g., Chem. Commun. 2016, 52, 8463-8466), but the problem is that most of the sensors interfere with the polymerase function! We are currently testing the first sensors, and the comparison with SYBR Green is favorable.
