Relevance of an Allosteric mDia1 Inhibitor

A major challenge in the actin field is the difficulty of determining the contribution of different classes of actin filament nucleators to the generation of new actin filaments because of the presence of other abundant actin filament nucleating activities. A small-molecule inhibitor that is specific for the formin family would therefore be useful in discriminating the relative contribution of the formins in the context of the three classes of actin nucleators (Arp2/3 complex, Spire, and formins). However, sequence differences within the formin family suggest that a greater degree of inhibitor specificity may be possible. In contrast to the catalytic FH2 domain found in all members of the formin family, clear DID and DAD-like sequences are found only in three formin subfamilies: mammalian diaphanous (mDia) subfamily, formin-related gene in leukocytes (FRL) subfamily, and disheveled-associated activator of morphogenesis (DAAM) subfamily (Higgs and Peterson 2005). This suggests that small molecules that target the DID/DAD regulatory domains might exhibit formin subfamily specificity, which would aid in characterizing the role of these functionally related proteins. Furthermore, while core residues are conserved between the DID and DAD sequences across these three subfamilies, they differ significantly in overall DID and DAD sequence (Higgs and Peterson 2005), suggesting the possibility of identifying mDia-specific, FRL-specific, and DAAM-specific inhibitors. Finally, even within the mDia subfamily, limited differences in the sequence of the paralogues mDia1, mDia2, and mDia3 allow for isoform-specific small-molecule inhibitors that could be used to discriminate the distinct roles of mDia family members (Higgs and Peterson 2005).

A small-molecule inhibitor of mDial would offer additional advantages over conventional research tools used to study this protein. For example, it has been observed that deletion of the gene encoding mDia1 results in the up-regulation of mDia2 expression (Peng et al. 2003). A rapid-acting chemical inhibitor of mDial might allow for the study of loss of mDial function while avoiding the compensatory up-regulation of mDia2 expression, thereby facilitating the identification of iso-form-specific mDia functions. The temporal control afforded by the use of chemical inhibitors allows for inhibitor wash-out experiments, which may shed light on the role of mDial in the formation of such dynamic structures as filopodia (Peng et al. 2003; Higashida et al. 2004), the cytokinetic ring (Watanabe et al. 1997; Tominaga et al. 2000), and the mitotic spindle (Kato et al. 2001). Finally, chemical inhibition offers the advantage of knocking down protein function without the expression of dominant negative peptide fragments that may interact with endogenous binding partners (Watanabe et al. 1999). This advantage is especially relevant in the context of Rho GTPase effectors, where dominant negative constructs may bind to and titrate away the pool of activated Rho GTPases, clouding the interpretation of resultant phenotypes.

An allosteric mDial inhibitor may also serve as a useful tool in furthering our understanding of the molecular details of mDia regulation. For example, binding of Rho only partially relieves the autoinhibition of mDial actin nucleation in vitro (Li and Higgs 2003, 2005), which raises the possibility that additional, unidentified regulatory proteins are involved in mDia activation. A small-molecule inhibitor that stabilizes the cellular pool of mDial into an autoinhibited conformation may allow for the purification and identification of "dead end" mDial/activator complexes for subsequent analysis and identification of interacting proteins.

Finally, while there is as yet no direct link between mDia activity and the development of disease states, DRFs have been proposed as novel drug targets in cancer due to their role in regulating both the actin and microtubule cytoskeleton (Faix and Grosse 2006) and the importance of these cytoskeletal elements for mitosis and cytokinesis. mDial interacts with the tumor suppressor adenomatous polyposis coli (APC), and may serve as a scaffold for APC-mediated microtubule stabilization (Wen et al. 2004). In addition, mDial binds polycystin 2 (PKD2), a protein linked to the development of polycystic kidney disease (Rundle et al. 2004). Additional evidence for the involvement of these cytoskeletal regulators in the development of disease comes from the human DRFs, where mutations in the genes encoding hDial and hDia2 have been associated with non-syndromic deafness (Lynch et al. l997) and premature ovarian failure (Bione et al. l998). Given the role of DRFs in a number of important cellular processes, it is likely that aberrant DRF function will be implicated in additional human disease states. As such, small-molecule inhibitors of mDial could help define the role of these proteins in disease development.

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