Targeting LRRK2 for Parkinson’s Disease with the Transcreener® GDP GTPase Assay

GTPases act like switches and control a variety of cellular processes as they cycle between inactive (GDP bound) and active (GTP bound) conformations.  GTPases form a relatively large family of enzymes, all of which are characterized by the ability to hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP).

Many cellular processes are modulated by GTPases including actin cytoskeletal organization, cell growth, proliferation, differentiation, and survival. For these reasons, GTPases have become a target for many diseases including cancer, Alzheimer’s and Parkinson’s.

Small GTPases include the Ras, Rho, Rab, Arf, and Ran subgroups. Mutant K-Ras proteins are significant drivers of human cancers and may play a direct role in about one million cancer cases per year. Ras GTPases are important targets in drug discovery and development, as genetic alterations in Ras GTPase are very common in human cancers.

Rho family GTPases control cell growth, movement, and gene expression and are often misregulated in cancer pathways, especially cell migration and invasion. However, Rho GTPases are infrequently mutated and their tumorigenic functions are often mediated by overexpression of GEFs which positively regulate.

Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow the formation of the active, GTP-bound complex. Development of inhibitors that specifically disrupt GEF action on a target Rho GTPase may be a promising therapeutic strategy for blocking Rho-dependent oncogenesis.

Unlike GTP binding assays, the Transcreener method is based on multiple catalytic cycles for both the GEF and the GTPase with an unmodified GTP substrate making it is a more direct measure of GEF’s intrinsic functional activity.

Members of the regulator of G-protein signaling (RGS) superfamily have emerged as critical modulators of specific GPCR signal transduction pathways. Via their conserved RGS domain that confers GTPase-activating protein (GAP) activity, RGS proteins attenuate GPCR signaling by deactivating heterotrimeric G-protein alpha subunits. The steady-state GTPase activity of isolated Gα proteins is limited by GDP dissociation, so steady-state hydrolysis cannot be used to measure GAP activity in simple biochemical assays. To overcome this hurdle, mutated Gα proteins with decreased GTP hydrolysis rates and increased GDP dissociation rates are required. Mutated Gα proteins and RGS domains can then be coupled with the homogenous, fluorescence-based Transcreener GDP assay to demonstrate an HTS-compatible assay system for selective modulation of GPCRs.