For detection of GTP binding to small GTPases we used a flow cytometry-based assay 14
. This method was found to be very sensitive, and allowed us to measure a dose response of GTP binding to individual GST-tagged small GTPases even though different conditions were required for optimal activity of individual GTPases. BODIPY-GTPγNH was identified as a better substrate for some of the small GTPases such as RhoA wt, Rac1wt, Cdc42, while others (Rab proteins14
and Ras) preferred binding to BODIPY-FL-GTP. Mg2+
ions were crucial for Rho enzyme activity measurement, while Rab proteins and H-Ras more effectively bound fluorescent GTP in the presence of EDTA. Activity of H-Ras was completely inhibited in the presence of 0.01% dodecyl maltoside in the assay, while this detergent had negligible effect on GTP binding by other GTPases. Based on these preliminary results, we chose specific conditions, which though not optimal for all GTPases, allowed us to simultaneously measure GTP binding of several small GTPases. Experiments comparing GTP-binding to individual GTPases in multiplex or single-plex assay format were identical, therefore a multi-plex flow cytometric assay was used for HTS of a MLSCN library. Approximately 200,000 compounds were screened for identification of potential activators and inhibitors of GTP binding to six different small GTPases. This approach allowed us to collect and analyze more than 1.2 million data points in the primary screen. A total of 100–500 positive compounds for each member of multiplex were identified in the primary screen (the results from this multi-plex screen were published on PubChem AID 757, 758, 759, 760, 761, 764). False positives such as autofluorescent compounds and compounds affecting GST-protein binding to GSH-beads were eliminated, and ~1200 compounds were selected and tested in secondary dose-response assays of which 18 % of the compounds judged to be active in the primary screen were confirmed in dose response analysis. Here we identified Rho family selective (MLS000532223) and Cdc42 specific (MLS000573151) inhibitors and characterized the cellular activity of MLS000532223 in detail. The pull-down assay of active Rac1 showed that MLS000532223 completely inhibited EGF induced Rac1 activation in Swiss 3T3 cells. Live cell imaging and confocal microscopy studies demonstrated the inhibition of actin reorganization and cell morphology changes downstream of Rho family GTPase activation.
Inhibitors of Rho family GTPases are of significant interest as targets for drug therapy. Hyperactivated small GTPases are implicated in 30% of all human cancers and are particularly prevalent in myeloid leukemia, pancreatic, lung and colon carcinomas.4, 5
Rho family GTPases have been found central to the control of gene regulation, cell proliferation and cell migration.2
Thus, it is not surprising that based on overexpression or hyperactivation they are increasingly associated with cancer cell metastasis and invasion, as well as uncontrolled proliferation and loss of growth control 31
. Based on the inhibitory activity of MLS000532223 on Rac1, Rac2 and Cdc42 in hematopoietic cell types, it will be of interest to further develop this probe for possible use to control the growth of leukemic cells.
Kinetic experiments revealed that MLS000532223 did not affect the Kd of GTP binding and since its activity was not reversible (data not shown), suggesting that it is not a competitive inhibitor for the GTP binding site. The observed incomplete inhibition of GTP binding could represent an allosteric binding site, limited molecular solubility, or protein heterogeneity which has not been resolved by the experiments performed to date.
Comparison of MLS000532223 against two other Rac1 specific inhibitors that have been developed, NSC23766 and EHT1864, reveals the importance of biological context. 22, 30
NSC23766 was developed using rational drug design and found to specifically inhibit guanine nucleotide exchange and Rac1 activation without affecting Cdc42 or RhoA both in vitro and in in cell-based assays.22
At the doses (50 μM) used on NIH 3T3 cells, NSC23766 proved toxic to RBL-2H3 cells and at lower doses (10 μM) it failed to inhibit the activated FcεR1-induced cell spreading and lamellopodia formation. EHT1864, identified as an inhibitor of amyloid precursor processing, interacts with Rac1 and related isoforms. Although EHT1864 blocked lamellipodia formation in NIH 3T3 fibroblasts following platelet derived growth factor stimulation30
, as shown here it did not block activated FcεR1-induced cell spreading and ruffling in RBL-2H3 cells. Together the data suggest that MLS000532223 is an effective Rho GTPase inhibitor for hematopoietic cells and that cell-type specific inhibition of Rho family GTPases may be possible and a worthwhile therapeutic strategy.
In sum, our data indicate that multi-plex bead-based assay could successfully be used via flow cytometry for HTS of libraries. This is a low-cost, time saving and highly efficient method for the simultaneous measurements of the activity for the number of proteins. Moreover, it enabled the analysis, comparison and detection of the specific activators/inhibitors of individual GTPases in a systematic and previously unparalleled manner. The identified small molecules provide a new chemical platform for the rationale design of selective inhibitors of key small G protein members that could represent a boon for understanding the biology and pharmacology of small GTPases.