Product Spotlight: XY-69

Posted on July 03, 2024


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Did you know that Avanti offers XY-69?

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 appreciate the importance of this product, it's essential to understand why a more robust and practical PLC assay method is necessary. PLC catalyzes the conversion of membrane-bound PIP2 into two secondary messengers. These messengers are involved in various cellular functions, allowing PLCs to regulate critical processes such as proliferation, differentiation, migration, and nerve conductance.

PLCs are grouped into six subfamilies: β, δ, γ, ζ, and η. Each subfamily is autoinhibited by mechanisms specific to them. Therefore, developing a robust and practical PLC assay is crucial to understand how PLCs regulate normal cellular processes and disease states.

Challenges with Previous Methods

Previous methods for assaying PLC activity included:

Using a radiolabeled PIP2 substrate to assay the purified PLC lipase activity.
Measuring cellular PLC activity from the production of radiolabeled inositol phosphates.
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. 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 led to 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 PLC-γ has been found in diseases such as cancer and Alzheimer’s disease. For 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 can monitor 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 developed 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 reproducible and had a Z’-factor of <0.7. Of the screened molecules in the LOPAC-1280, 12 compounds were hits, most of which had not been identified by any previous PLC activity assays. 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.

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You can read the full scope of this research by reading the articles listed below!

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)