Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay

Area of Science:

• Cellular Biology
• Biochemistry
• Pharmacology

Background:

• Small GTPases are critical regulators of cellular processes.
• Dysregulation of small GTPases is implicated in various human diseases.
• Targeting small GTPases with small-molecule inhibitors is a promising therapeutic strategy.

Purpose of the Study:

• To develop and validate a multiplex, flow cytometry bead-based assay for high-throughput screening of small GTPase modulators.
• To identify novel small-molecule inhibitors and activators of small GTPases.
• To characterize the mechanism of action of identified compounds.

Main Methods:

• Development of a multiplex assay using glutathione-S-transferase (GST)-tagged small GTPases immobilized on differentially fluorescently labeled beads.
• Screening of a library of approximately 200,000 compounds using fluorescent-guanosine triphosphate (GTP) binding as the readout.
• Validation of hits through dose-response analyses, biochemical assays, cell-based assays, live-cell imaging, and confocal microscopy.

Main Results:

• The assay enabled the screening of nearly 200,000 compounds, identifying over 1200 initial hits.
• Subsequent validation identified several small-molecule families with opposing effects on GTP binding.
• Characterization of MLS000532223 revealed it as a general inhibitor of GTP binding, inducing actin reorganization and morphological changes consistent with Rho GTPase inhibition.

Conclusions:

• High-throughput flow cytometry screening is an effective strategy for identifying novel compounds targeting small GTPases.
• The developed assay facilitates the discovery of modulators for therapeutic intervention in GTPase-related diseases.
• The identified compounds, particularly MLS000532223, warrant further investigation for their therapeutic potential.