We developed a high-throughput screen that takes advantage of the loading of fluorescent nucleotides onto GTPases and catalyzed by GEFs (see figure).  Compounds that inhibit nucleotide loading prevent increased fluorescence and the screen uses a single end-point measurement to identify potential inhibitors.  

Fluorescence-based exchange assay:

GTPase_exchange_assay

A.  The addition of a GEF is required to catalyze the loading of a fluorescent nucleotide (GTP*) onto a GTPase using purified proteins.  Bound GTP* is sequestered from solvent that otherwise quenches fluorescence.  B.  Real-time exchange assay.  Black diamonds indicate spontaneous loading of nucleotide onto Rac1.  GEF is added at arrow (red circles). 

Importantly, the KXTbio GTPase exchange assays works with numerous GTPases. Linearization of readouts further facilitates HTS application.

GTPasescreen

Excellent performance to identify GTPas inhibitors.

The assay is robust (Z`-score ~0.8), resistant to DMSO, and amenable to many GTPase/GEF pairs. 

Performance GTPase exchange assay

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