Enzymology Is a Competitive Business
When Eduard Sergienko, Ph.D., was looking to set up appropriate assays to identify modulators of tissue-nonspecific alkaline phosphate (TNAP), he realized that buffers such as diethanolamine (DEA), traditionally used in alkaline phosphatase (AP) assays, can actually participate as a substrate in the reaction, re-directing it toward transphosphorylation.
This could pose a problem since a high concentration of DEA routinely employed for boosting the assay sensitivity would saturate the reaction, making it nearly impossible to find competitive inhibitors.
“The physiological significance of the transphosphorylation reaction and the identity of its alcohol substrate are still unknown,” the director of assay development at Conrad Prebys Center for Chemical Genomics (CPCCG) at Sanford-Burnham Medical Research Institute said. “We wanted to find compounds that would inhibit different potential enzyme activities and thus wanted to have compounds with various mechanisms of action.”
Dr. Sergienko and his colleagues adapted CDP-star, a chemiluminescence substrate widely used for detection of AP in blotting, allowing them to eliminate DEA if they so chose. The assay was sensitive enough, and with a large enough dynamic range, to screen at low enzyme concentration and with the substrate at Km. “One can emphasize a certain mechanism of action by playing with the concentration of substrate relative to the Km level,” he explained.
They screened TNAP against the Molecular Library Screening Center Network (MLSCN) collection containing 64,394 compounds and found three major categories of inhibitor structural scaffolds along with some minor categories and singletons. One of these scaffolds contained the first competitive inhibitors for TNAP, and for APs in general.
Only four of the 55 hits identified by the high-throughput luminescent assay screen could be confirmed in a colorimetric assay performed with high DEA concentration—giving evidence that it is perhaps directly competing for the enzyme’s binding site.
To support TNAP lead-optimization efforts at CPCCG, Dr. Sergienko and his team have gone on to develop a biomarker assay for AP activity within blood plasma that can be run at physiological pH using small-volume aliquots of minimally diluted plasma samples—making it amenable to high-throughput methodology.
This approach allowed testing of structure-activity relationship compounds and predicting their efficacy in vivo. In addition, the biomarker assay allows monitoring activity of TNAP in the samples taken from animals after dosing them with TNAP inhibitors in pharmacokinetic studies, providing pharmacodynamic information for animal models relevant to TNAP.