Rho family GTPases belong to the Ras-like small GTPases superfamily and are intracellular signal transducers involved in diverse cell signal transduction processes from cell adhesion molecules, growth factor receptors, G-protein coupled receptors, and cytokines. It has become increasingly appreciated that Rho family members, RhoA and RhoC in particular, are often upregulated or hyperactivated in human diseases including cancer and inflammation, and these Rho GTPases can serve as useful targets in reversing pathologic conditions such as cancer cell proliferation and invasion (Faried et al., 2007
; Horiuchi et al., 2003
). Activation and signal transduction through Rho GTPase regulated pathways require cascades of protein-protein interactions, including enzymatic reactions of kinases and GTPases. However, Rho GTPases, like Ras, are considered undruggable by conventional means. This is in part because the small GTPases themselves are globular structures with limited surface structural areas suitable for high affinity binding by small molecules (Kristelly et al., 2004
). Thus, extensive efforts have previously been devoted to the development of inhibitors of the upstream regulators (e.g. farnesyl or gerynyl transferases (Sebti, et al., 2003
)), Rho GTPases themselves (e.g. by bacteria toxins (Genth et al., 2008
) or small molecules (Gao et al., 2004
; Onesto et al., 2008
)) and downstream effectors (e.g. ROCK and SRF (Narumiya et al., 2000
; Evelyn et al., 2010
), of the GTPase signaling pathways, but none have achieved clinical utility.
Activation of Rho GTPases by the Dbl family GEFs is known to be one major mechanism leading to the activated state of multiple Rho family members including RhoA, RhoC, Cdc42 and Rac1. Extensive structural and biochemical work has provided detailed information on how this class of GEFs recognizes a Rho GTPase substrate and catalyzes the GDP/GTP exchange. The studies have presented a possibility that small molecules designed for specifically targeting the GEF - Rho GTPase interaction could be obtained to affect the Rho GTPase activities, not unlike previously identified brefeldin A that can bind to and inhibit an ARF small GTPase GEF (Louis et al., 2003). Applying this rationale, we previously identified a lead Rac activation-specific inhibitor, NSC23766, that displays a micromolar binding affinity to Rac1 at the GEF binding site and is active for inhibiting GEF activation of Rac1(Gao, et al., 2004
). However, effort in enhancing the efficacy of this lead has proven difficult mostly due to the low affinity nature of the available targeting site on Rac1.
Here, we have identified a lead small molecule inhibitor that specifically targets RhoA and closely related RhoB and RhoC by structure-based rational design. This inhibitor, Rhosin, contains two aromatic chemical fragments that are tethered by a properly spaced linker, with each likely bound to a distinct, shallow groove of RhoA. In vitro it is able to specifically inhibit the RhoA-GEF interaction and RhoA activation by GEFs. In cells, Rhosin blocks RhoA activation and RhoA-mediated MLC phosphorylation, actin stress fiber formation and focal adhesion assembly without affecting the activity or signaling events of endogenous Cdc42 or Rac1. Moreover, this compound showed no cytotoxicity or non-specific effect on the constitutively active RhoA or ROCK mutant induced actomyosin changes. Finally, by suppressing RhoA or RhoC activity, Rhosin was active in decreasing mammosphere formation and inhibiting invasion of breast cancer cells, and promoting neurite outgrowth from neuronal cells. Thus, Rhosin constitutes a Rho-specific small molecule inhibitor that is useful in the study of the physiological role of Rho and for tackling Rho-mediated pathologies.
Cdc42 or Rac1 shares high homologies with RhoA in the region between switches I & II involved in Rhosin binding and GEF interaction. An analysis of the amino acid sequences and the 3D structure of the RhoA binding pocket indicates that Cdc42 contains a Phe56 instead of Trp58 in the critical site of Rhosin binding, whereas Rac1, although containing mostly conserved amino acid residues, has a minor structural conformation difference from RhoA (Worthylake et al., 2000
; Kristelly et al., 2004
). Likely due to similar reasons, the previously characterized Rac inhibitor, NSC23766, shows high specificity towards Rac1 (Gao et al., 2004
). The detailed mode of action by Rhosin in interaction with RhoA awaits further structural illustrations.
We have performed multiples assays in this study to test the target binding affinity (i.e. Kd), the IC50 for the inhibition of GEF-RhoA binding, and the EC50 of cellular activity, of G04. In the assay conditions, G04 displays a Kd of ~0.4 uM to RhoA, and a weaker efficacy in inhibiting the GEF binding to RhoA reaction or cellular RhoA-GTP formation (at over 10 uM). This is not unlike most kinase inhibitors that have a high binding affinity to targets but may require higher concentrations to be effective to inhibit a kinase-substrate interaction or a kinase reaction in cells. Although the lead inhibitors of RhoA identified in the current studies remain a distance from pharmaceutical applications, our studies put forward a demonstrated example that the GEF reaction of small GTPase activation is a valid site for rationalized targeting. The discovery of Rhosin, a lead inhibitor targeting RhoA that contains two aromatic chemical fragments tethered by a properly spaced linker, with each likely bound to a distinct, shallow groove of RhoA, adds to the previously characterized Rac GTPase-specific small molecule inhibitors that shared the principle of targeting the GEF recognition site of a Rho GTPase (Kristelly et al., 2004
). It further raises the possibility that design and search for double headed, low affinity binding structures tethered by a properly sized linker may be applicable to small GTPases or other difficult to target biological molecules.