KXTbio is built on more than two decades of research on the biological mechanisms of GPCR and GTPase signaling pathways from the University of North Carolina at Chapel Hill. Based on this experience, KXTbio focuses on developing and applying new technologies to assay signaling cascades controlled by GTPases, including heterotrimeric G proteins. Currently, we have two drug screening platforms.

The first one relies on monitoring the loading of a fluorescent derivative of guanine nucleotides onto the GTPase under study. Various fluorescent derivatives modified on either the sugar hydroxyls or the 5’ terminal phosphate are applicable in this assay and the quantum yield of fluorescence typically increases 2-3 fold upon binding of these compounds to the GTPases. We have used this format to monitor the activation of diverse, purified GTPases of the Ras superfamily and heterotrimeric G proteins. We have developed an assay for use in ultra-high-throughput (e.g., 1536 well plates) screens to identify small molecules that inhibit the direct activation of Ras-related GTPase by guanine nucleotide exchange factors.

The second platform takes advantage of a proprietary reagent to monitor the activity of PLCs directly activated by diverse inputs, including several heterotrimeric G proteins and small GTPases. PLCs normally hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers, inositol trisphosphate and diacylglycerol.

WH-15 (renamed PLCglow) was specifically designed and synthesized to monitor this catalytic activity and mammalian PLCs hydrolyze PIP2 and WH-15 with equal efficiency. The hydrolysis of WH-15 produces a highly fluorescent product that accurately reports the phospholipase activity of purified mammalian PLCs as well as active PLCs in cell lysates. WH-15 is not hydrolyzed by other phospholipases (i.e., PLA2, PLD1) and is stable in cell lysates lacking active PLCs.