The pursuit of GTPase inhibitors has been an enduring effort due to the important roles that this class of proteins plays in cell physiology and the strong indications they have in cancers, immune diseases, and neurological disorders (53
). Specifically, Cdc42 has been implicated in ovarian cancer (16
), breast cancer (57
), hepatitis C virus-related liver disease (58
), and mental retardation (59
). However, there has been limited success in GTPase inhibitor discovery. Prior Cdc42 inhibitors either lack selectivity by acting on multiple GTPases or only target particular protein-protein interactions (19
). The function of Cdc42 is controlled by multiple upstream regulators and downstream effectors (41
). Blocking one set of interactions may be useful in some scenarios. However, the protein could still remain active by interacting with other partners and allow undesirable bypasses. Moreover, sequestering a shared GDI would affect the function of other GTPases. We report here the characterization of a selective small molecule inhibitor for Cdc42 and a second active analog. Numerous other structurally related analogs were tested and found inactive (supplemental Fig. S1
), confirming specificity. The compounds inhibit Cdc42 while sparing Rac and Rho that are in the same GTPase family and have high sequence and structure homology to Cdc42. Furthermore, using a real-time fluorescence assay, CID2950007 was shown to be an allosteric inhibitor that binds the guanine nucleotide-associated Cdc42 and induces ligand dissociation. Supplemental Fig. S2
shows predicted docking of the compounds. In collaboration with Dr. Roger Goody (Max-Planck Institute of Physical Chemistry, Dortmund, Germany), crystallization trials were conducted and led to the conclusion that the synthesis of derivatives that can be covalently cross-linked to the GTPase will be required. It also remains to be determined how the compound affects RhoV, RhoU, and RhoJ GTPases that are often expressed in a cell type-specific manner and are classified as Cdc42-like GTPases (61
BODIPY® FL GTP has been widely used in research with guanine nucleotide-binding proteins because of its high sensitivity and the minimal interference from the fluorescent label (63
). As compared with radioisotope-labeled nucleotide analogs, it is environmentally friendly and convenient to use. The fact that CID2950007 was discovered in a fluorescence-based screening campaign using BODIPY® FL GTP (21
) and in the present study validated in multiple biochemical and cellular assays again proved the effectiveness of BODIPY®-labeled guanine nucleotide in GTPase studies.
Many currently available GTPase inhibitors target protein-protein interaction interfaces. Dbs is a GEF for both Cdc42 and RhoA. Whether CID2950007 can affect Dbs-facilitated guanine nucleotide exchange on Cdc42 was tested with minimal effect detected (supplemental Fig. S3
). Because the compound can displace both GDP and GTP and is inhibitory in cell-based assays, the compound could act independent of any effect on GEF activity or interaction, as supported by the docking studies (supplemental Fig. S2
). In this model, consistent with the proposed allosteric mechanism, the compound is found to bind to an allosteric pocket adjacent to the nucleotide binding site to promote nucleotide dissociation and thus would not interfere with GEF binding to the switch regions (supplemental Fig. S2
). However, small molecules may block a selective subset of protein-protein interactions as exemplified by NSC23766, which blocks Rac1 interaction with the Tiam and Trio GEFs, but not with Vav (64
). Therefore, it is still too early to conclude whether CID2950007 could affect the interaction between Cdc42 and its other specific GEFs besides being able to block the guanine nucleotide binding to the GTPase.
Previously, the function of Cdc42 in mammalian cells has been studied using its dominant-negative or constitutively active mutants (65
). However, these mutants can impede the functions of other GTPases with which Cdc42 shares upstream regulators and downstream effectors. For instance, a dominant-negative mutant can bind a shared GEF, whereas a constitutively active mutant may occupy a mutual effector, making them unavailable to other GTPases. Therefore, such an approach imposes limitations related to specificity, dosage, and clonal variability (68
). The use of a small molecule-selective inhibitor instead achieves selective inactivation of Cdc42 within a short time window and leaves analogous GTPases unaffected. This should greatly facilitate the functional assignment of Cdc42 and help to disentangle complex GTPase networks.
Due to its important roles in cytoskeleton organization, the activity of Cdc42 could be assessed in filopodia formation and cell migration assays. We observed that CID2950007 effectively inhibited Cdc42 in both assays, leading to less filopodia formation and ineffective cell migration. Contrary to our observation that Cdc42 promoted chemotaxis, which was inhibited by CID2950007, some early studies reported that Cdc42 negatively regulated cell migration (69
). This might be because the function of Cdc42 is cell type-specific (42
), or is likely due to the use of constitutively active Cdc42 mutants in those studies that may induce nonspecific effects and bias the conclusions. This again shows the value of small molecule-selective inhibitors.
Rac and Rho have been shown to be involved in the inside-out signaling pathways triggered by chemokines. The signaling results in α4β1-dependent up-regulation of the adhesion capacity of T-lymphocytes and leukocytes (50
). The involvement of Cdc42 in the same GTPase family remains unknown. In this study, the use of the small molecule Cdc42-selective inhibitor strongly suggests the participation of Cdc42. The briefness of the decrease of the fluorescence level, which is an indication of the VLA-4 activation state, indicates that redundant signaling pathways are capable of compensating the inhibited Cdc42 (70
). Moreover, our assay represents a breakthrough in the methodology to study the involvement of GTPases in integrin activation. Previous studies typically used either immunoprecipitation or radioactive labeling to isolate GTPases from the cell lysate and quantify nucleotide incorporation (50
). Such efforts are often time-consuming and involve complicated waste disposal. In addition, they would not reveal the concurrent changes happening in the cells. On the other hand, our assay could conveniently monitor the adhesion changes of the cells in real time by using flow cytometry and fluorescently labeled peptide.
In conclusion, we characterized a small molecule allosteric inhibitor that selectively inhibits nucleotide binding to the Cdc42 GTPase. The compound may find its applications in drug development aimed at Cdc42-related diseases. As illustrated by the examples, the compound can also help to unravel the complex signaling pathways that often involve multiple GTPases.