Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay.
Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small-molecule inhibitors. The authors describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different glutathione-S-transferase (GST)-tagged small GTPases were bound to glutathione beads, each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-guanosine triphosphate (GTP) binding was used as the readout. This novel multiplex assay allowed the authors to screen a library of almost 200,000 compounds and identify more than 1200 positive compounds, which were further verified by dose-response analyses, using 6- to 8-plex assays. After the elimination of false-positive and false-negative compounds, several small-molecule families with opposing effects on GTP binding activity were identified. The authors detail the characterization of MLS000532223, a general inhibitor that prevents GTP binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live-cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high-throughput screening via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.
Surviladze
Zurab
Z
University of New Mexico Center for Molecular Discovery, Albuquerque, NM 87131, USA. zsurviladze@salud.unm.edu
Waller
Anna
A
Wu
Yang
Y
Romero
Elsa
E
Edwards
Bruce S
BS
Wandinger-Ness
Angela
A
Sklar
Larry A
LA
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007073:Immunoglobulin E / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008863:Microspheres; D020559:Monomeric GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D000199:Actins; D004791:Enzyme Inhibitors; D008024:Ligands; D007073:Immunoglobulin E; D004815:Epidermal Growth Factor; D001619:beta-N-Acetylhexosaminidases; D020559:Monomeric GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins