Product Spotlight: XY-69

Posted on December 03, 2021

Xy 69 For Final Website Blog

We are excited to announce the addition of XY-69 into our product catalog!

The Need for Novel Assays for Phospholipase C Isozymes

XY-69 is a capable and sensible alternative to radioactive phosphatidylinositol 4,5-bisphosphate (PIP2) typically used in conventional phospholipase C (PLC) assays. To understand the importance of this product, we must first look at why a more robust and practical PLC assay method is necessary. As a little background, PLC catalyzes the conversion of membrane bound PIP2 into two secondary messengers. These secondary messengers are involved in a variety of cellular functions and thus, PLCs regulate key functions such as proliferation, differentiation, migration, and nerve conductance.

PLCs are grouped into six subfamilies: β, δ, γ, ζ, and η. The subfamilies of PLCs are autoinhibited by diverse mechanisms specific to each. Therefore, developing a robust and practical PLC assay is important to understand how PLCs are regulated to control normal cellular and disease state processes.

Past methods for assaying PLC activity included (1) using a radiolabeled PIP2 substrate to assay the purified PLC lipase activity, (2) measuring cellular PLC activity from the production of radiolabeled inositol phosphates, and (3) using cell-permeable dyes that increase fluorescence after binding with Ca2+. Besides the added complication of using radiolabeled substances, these methods do not allow for the direct measurement of PLC activity. So, to bypass these problems, researchers at the University of North Carolina at Chapel Hill developed a biosensor capable of reporting real-time activation of PLCs. XY-69 is readily partitioned into membranes and specifically and efficiently hydrolyzed to produce a robust fluorescent signal that is suitable for monitoring PLCs.

Practical Applications of XY-69 in Drug Discovery Screening

Their research continued towards the development of a high-throughput assay capable of capturing both orthosteric and allosteric inhibitors. The newly developed assay focused on PLC-γ isozymes which have several regulatory domains unlike other PLC subfamilies. Aberrant regulation of PCL-γ has been found in diseases such as cancer and Alzheimer’s disease. As an example, PLC-γ1 gain-of-function mutations are seen in over one-third of T cell lymphoma patients. The therapeutic relevance of the PLC-γ isozyme and the importance of discovering selective inhibitors is undeniable.

After proving that XY-69 is capable of monitoring PLC activity at the membrane level in real time, the research indicated that XY-69 could also report the activation of PLC-γ1 upon tyrosine phosphorylation or mutations. With this information they sought to develop a high-throughput assay using XY-69 liposomes to identify allosteric inhibitors of PLC-γ1. The assay was validated by a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC-1280). The results of this screening were proven to be reproducible and had a Z’-factor of <0.7. Of the screened molecules in the LOPAC-1280, 12 compounds were hits, and most of them had not been identified by any of the other PLC activity assays previously developed. The results of this study show the potential of the newly developed liposome-based assay in identifying new allosteric inhibitors of the PLC-γ isozyme for use in drug discovery research.

You can read the full scope of this research by reading the articles listed below! And don’t forget to check out XY-69 and our other new products by clicking the HERE!

A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes (2018)

A High-Throughput Assay to Identify Allosteric Inhibitors of thePLC‑γ Isozymes Operating at Membranes (2020)

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes (2021)