It is our desire to better understand the regulation of GPCRs, heterotrimeric G proteins and GTPases at the molecular level with the ultimate goal of using this information to design therapies to correct abnormal signaling mediated by these proteins and thereby treat associated pathologies.

Current major initiatives of KXTbio include:

PLCglow: A fluorescence sensor for simple, fast & real-time monitoring of mammalian phospholipase C isozymes

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Phospholipase C (PLC) isozymes catalyze the hydrolysis of the minor phospholipid, phosphatidylinositol 4,5-bisphophate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. These two second messengers act in concert to increase intracellular calcium concentrations and activate protein kinase C leading to the regulation of numerous cellular processes, including fertilization, division, differentiation, and chemotaxis.  As such, the critical role of PLC isozymes in various signaling processes has been appreciated since the 1980’s.  Yet surprisingly, very little is known about how these proteins are regulated at the molecular level.  In collaboration with the laboratory of Dr. Ken Harden (Pharmacology, UNC Chapel Hill) we have determined a series of crystal structures of PLC-? isozymes and are using this information along with complementary biochemical and cellular studies to understand the regulation of PLC isozymes at atomic resolution and within the context of various signaling cascades.  This work is part of a long-term and extensive collaboration with Dr. Harden that also includes similar studies of other PLC isozymes (e.g. PLC-?, -? and –?) and has led to our recent description of a general framework for understanding the regulated auto-inhibition of the entire PLC family.

The PLCglow sensor does away with conventional radioactive assays to study mammalian PLCs.  PLCglow is useful for all PLC isozymes; it will not report on other lipases; and it can be used with purified proteins or cell extracts. PLCglow is ideal for cost-effective screens of compound collections during drug discovery projects that target PLCs. 

 

Identifying hits that inhibit activation of Rac1 by P-Rex1

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The Ras superfamily of small GTPases primarily function as molecular switches: GDP-bound forms are “off” while GTP-bound forms are “on” and directly activate diverse downstream effectors.  Cycling between these two states is tightly regulated and its misregulation contributes to numerous human diseases, including cancer and immunological disorders.  For example, mutated forms of Ras that are constitutively active are frequent oncogenes.

The Rho subfamily of small GTPases are major regulators of actin dynamics and gene expression. These proteins are expressed in all eukaryotes and are needed for myriad cell processes, including neuronal pathfinding and chemotaxis as well as proliferation and division.  The best studied members of this group include RhoA, Rac1, and Cdc42.  The overexpression of Rho GTPases is often associated with cancer and recent genome-wide screens identified mutated Rac1 that could no longer hydrolyze GTP as a major driver of melanoma. 

KXTbio has developed proprietary assays to monitor the cycling of guanine nucleotides onto GTPases.  One of these assays is currently being used by GlaxoSmithKline to screen its ~2 million compound collection for inhibitors of Rac1 with the ultimate goal of treating melanomas harboring mutated Rac1.  Related formats are useful for monitoring other GTPases as well as heterotrimeric GTPases. 

Launching a novel high through-put screen for direct GPCR monitoring

Details not disclosed

Details not disclosed.