A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC-γ Isozymes Operating at Membranes

Abstract

The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC 1280 ) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.

The molecular basis of the dichotomous functionality of MAP4K4 in proliferation and cell motility control in cancer

“…PLCg activation is also induced by growth factor signaling, suggesting that targeted inhibition of PLCg could result in growth repression through Hippo pathway activation. Novel allosteric inhibitors with improved specificity and potency against PLCg are being developed (84), which may be explored in the future to drive RAP2-MAP4K4 mediated Hippo pathway activation and growth repression in growth factor activated tumors. A yeast two-hybrid screen for the identification of interactors of the 4.1, ezrin, radixin, moesin (FERM) domain of proline-rich tyrosine kinase 2 (PYK2) identified MAP4K4 as a binding partner (85).…”

“…PLCγ activation is also induced by growth factor signaling, suggesting that targeted inhibition of PLCγ could result in growth repression through Hippo pathway activation. Novel allosteric inhibitors with improved specificity and potency against PLCγ are being developed ( 84 ), which may be explored in the future to drive RAP2-MAP4K4 mediated Hippo pathway activation and growth repression in growth factor activated tumors.…”

The crystal and cryo-EM structures of PLCγ2 reveal dynamic inter-domain recognitions in autoinhibition

“…Lipid vesicles containing the fluorogenic substrate XY-69 was prepared as previously described 39,59 . Briefly, lipid vesicles were generated containing 0.46 µM XY-69, 57.6 µM phosphatidylinositol 4,5- bisphosphate (PIP2), and 230 µM phosphatidylethanolamine.…”

A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes

“…Activating mutations for both PLCγ1 and PLCγ2 are known, and ATP has been shown to be an inhibitor of both enzymes [24]. To further validate the C16CF3-coumarin micelle assay, we measured the reaction of the substrate with the PLCγ1-D1665H and PLCγ2-P522R activating mutants in the presence or absence of ATP.…”

“…XY-69 is incorporated into a liposome and is currently considered the best assay for monitoring PLC enzymatic activity, because the substrate is only processed when the PLC enzyme is in contact with the membrane, thus mimicking the cellular environment [23]. The liposomal XY-69 assay is therefore more suitable to screen for allosteric inhibitors and of course can identify orthosteric inhibitors as well [24]. Unfortunately, suitable liposomes are tedious to prepare in large quantities and often suffer from high batch-to-batch variability.…”