Screening Approaches to Identify Allosteric mDia1 Inhibitors

Similar to the approaches described for Pakl, chemical screens to identify allosteric inhibitors of mDia should be designed such that the chosen assays address both the inhibitory activity of a compound as well as the mechanism of action. One such approach would involve performing parallel in vitro actin polymerization assays (Cooper et al. l983): one with full-length mDial proteins, the other with a truncated mDial protein containing only the FH2-DAD domains. In the first screen, the ability of full-length mDial to stimulate actin polymerization in the presence of activator (GTP-bound Rho) and individual compounds would be assayed. A second screen would then be performed that would test the ability of the same compounds to inhibit the actin-nucleating activity of a truncated mDial protein containing only the catalytic FH2-DAD domains. This FH2-DAD fragment lacks the autoregula-tory domains required for autoinhibition, and as such displays high intrinsic catalytic activity (Li and Higgs 2003). Compounds that inhibit only the full-length protein, and not the isolated catalytic fragment, may target the autoregulatory domains of mDia1. In contrast, compounds that inhibit both the full-length and truncated mDia1 proteins are likely to act either non-specifically or by targeting the catalytic domains of the protein. This type of assay was used in the characterization of wiskostatin, where it was demonstrated that wiskostatin was unable to inhibit the catalytic VCA fragment of N-WASP, but significantly inhibited the catalytic activity of the full length N-WASP construct (see above and Peterson et al. 2004).

Another approach to assay compounds for their mechanism of action could be to directly monitor the interaction between recombinant protein fragments containing the autoregulatory DID and DAD domains in vitro using fluorescence polarization assays. These assays are commonly used in high throughput screens to identify compounds that disrupt protein-protein interactions (Owicki 2000; Burke et al. 2003); however, in this case the technique could be used to identify compounds that stabilize the autoinhibitory interactions of the DID and DAD. Fluorescence polarization assays have been used to investigate DID/DAD interactions (Lammers et al. 2005; Li and Higgs 2005; Rose et al. 2005; Wallar et al. 2006), and this assay has been used to demonstrate the mutually exclusive binding of Rho-GTP and a mDia1 construct containing the DID (Rose et al. 2005). In this report, an mDia fragment containing the DID was added to a DAD-containing fragment labeled with the fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA). The interaction between these two fragments resulted in a dramatic increase in the fuorescence polarization of the DAD construct, indicative of a binding interaction. Addition of activated RhoA, but not RhoA-GDP, reduced the fluorescence polarization back to basal levels, indicating a disruption of the DID/DAD interaction (Rose et al. 2005). This assay could be adapted to test the ability of individual compounds to stabilize the interaction between the DID and the DAD in the presence of active RhoA. Compounds that stabilize the interactions between the DID and fluorescently labeled DAD would result in increased fluorescence polarization of DID/DAD complexes that persist despite the addition of active RhoA. To further characterize those compounds that appear to stabilize the DID/DAD complex, a follow-up fluorescence polarization assay could be performed using a DID-containing fragment mutated from Ala to Asp at residue 256. This DID mutant is unable to interact with DAD-containing protein fragments (Otomo et al. 2005; Rose et al. 2005). Therefore, compounds that stabilize the interaction of mutated-DID/DAD interaction in this assay may be acting through a non-specific mechanism.

0 0

Post a comment