Scratch n’ Screen for Inhibitors of Cell Migration
2005; Elsevier BV; Volume: 12; Issue: 3 Linguagem: Inglês
10.1016/j.chembiol.2005.03.001
ISSN1879-1301
Autores Tópico(s)14-3-3 protein interactions
ResumoYarrow et al. have identified a small molecule inhibitor of cell migration, 3-(4-pyridyl)indole (“Rockout”), that targets Rho-kinase via a novel screening method using a scratch wound healing assay adapted to a high-throughput format and automated microscopy. Yarrow et al. have identified a small molecule inhibitor of cell migration, 3-(4-pyridyl)indole (“Rockout”), that targets Rho-kinase via a novel screening method using a scratch wound healing assay adapted to a high-throughput format and automated microscopy. Small molecule inhibitors are extremely useful for dissecting complex cellular events that require the coordination and regulation of many proteins. By inhibiting a specific component of a biochemical network, one can deduce its function in the process. A popular approach has been to use pure proteins and screen for inhibitors in activity assays. However, this limits the target to known elements with known activities. A more difficult and potentially fruitful approach is to design a “phenotypic screen” that assays the entire cellular process. In this issue of Chemistry & Biology, Yarrow and colleagues have designed an innovative, imaging-based screen for inhibitors of cell migration and have discovered the compound “Rockout,” which targets the cytoskeletal regulator Rho-kinase [1Yarrow J.C. Totsukawa G. Charras G. Mitchison T. Chem. Biol. 2005; 12 (this issue): 385-395Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar]. Cell migration is attractive as a target for phenotypic small molecule screens because it relies on the integration of several processes and because it is essential in many cell types. Wound healing, tissue formation, immune responses, angiogenesis, and tumor cell metastasis all depend on cell motility [2Chodniewicz D. Klemke R.L. Exp. Cell Res. 2004; 301: 31-37Crossref PubMed Scopus (55) Google Scholar]. The process generally begins with an extracellular stimulus that triggers a signal transduction cascade leading to polarized migration of the cell, which requires the reorganization of actin filaments and microtubules, polarization of the secretory apparatus, membrane protrusion, and dynamic adhesion to the substrate [2Chodniewicz D. Klemke R.L. Exp. Cell Res. 2004; 301: 31-37Crossref PubMed Scopus (55) Google Scholar, 3Fukata M. Nakagawa M. Kaibuchi K. Curr. Opin. Cell Biol. 2003; 15: 590-597Crossref PubMed Scopus (402) Google Scholar]. Although a variety of stimuli can trigger migration, many aspects of the intracellular pathways that contribute to cell polarization and motility are conserved in multiple cell types and species [3Fukata M. Nakagawa M. Kaibuchi K. Curr. Opin. Cell Biol. 2003; 15: 590-597Crossref PubMed Scopus (402) Google Scholar, 4Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (710) Google Scholar, 5Van Haastert P.J. Devreotes P.N. Nat. Rev. Mol. Cell Biol. 2004; 5: 626-634Crossref PubMed Scopus (548) Google Scholar]. For example, Rho family GTPases—namely Rho, Rac, and Cdc42—have conserved functions in linking extracellular signals to rearrangements of both actin microfilaments and microtubules [3Fukata M. Nakagawa M. Kaibuchi K. Curr. Opin. Cell Biol. 2003; 15: 590-597Crossref PubMed Scopus (402) Google Scholar, 4Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (710) Google Scholar]. By activating downstream effectors, these GTPases regulate processes that range from microtubule/actin interactions at the leading edge of a migrating fibroblast to polarizing actin structures in budding yeast [4Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (710) Google Scholar, 6Pruyne D. Bretscher A. J. Cell Sci. 2000; 113: 365-375Crossref PubMed Google Scholar]. A major goal is to understand how these Rho GTPases function to regulate polarity in diverse cellular processes. Small molecule inhibitors have been used to study the migration of living cells, and the most readily available compounds directly disrupt actin microfilaments or microtubules [7Wakatsuki T. Schwab B. Thompson N.C. Elson E.L. J. Cell Sci. 2001; 114: 1025-1036Crossref PubMed Google Scholar, 8Kielbassa K. Schmitz C. Gerke V. Exp. Cell Res. 1998; 243: 129-141Crossref PubMed Scopus (44) Google Scholar]. Treating migrating cells with such compounds can address the requirements of microtubules or microfilaments in motile processes; however, these experiments exclude information about upstream regulation. Consequently, efforts have been made to identify small molecule inhibitors that target regulatory proteins using phenotype-based assays [9Saitoh M. Naka M. Hidaka H. Biochem. Biophys. Res. Commun. 1986; 140: 280-287Crossref PubMed Scopus (84) Google Scholar, 10Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. et al.Nature. 1997; 389: 990-994Crossref PubMed Scopus (2542) Google Scholar, 11Peterson J.R. Lokey R.S. Mitchison T.J. Kirschner M.W. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10624-10629Crossref PubMed Scopus (82) Google Scholar, 12Mc Henry K.T. Ankala S.V. Ghosh A.K. Fenteany G. Chembiochem. 2002; 3: 1105-1111Crossref PubMed Scopus (11) Google Scholar]. These approaches have yielded inhibitors of factors such as N-WASP, which activates the Arp2/3 complex and promotes actin polymerization [11Peterson J.R. Lokey R.S. Mitchison T.J. Kirschner M.W. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10624-10629Crossref PubMed Scopus (82) Google Scholar]; Rho-kinase, which is activated by the Rho GTPase and whose targets include cytoskeletal components [10Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. et al.Nature. 1997; 389: 990-994Crossref PubMed Scopus (2542) Google Scholar]; and myosin light chain kinase, which regulates myosin activity [9Saitoh M. Naka M. Hidaka H. Biochem. Biophys. Res. Commun. 1986; 140: 280-287Crossref PubMed Scopus (84) Google Scholar]. Because cell migration is so complex, a comprehensive understanding would be greatly facilitated by the discovery of additional, cell-permeable compounds that target other regulators, such as the Rho GTPases themselves, additional downstream actin and microtubule effectors, and factors that mediate interactions between these two cytoskeletal elements [4Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (710) Google Scholar]. In an unbiased approach to identify novel cell migration inhibitors, Yarrow and colleagues performed a visual screen based on classical wound healing assays performed in high-throughput format [12Mc Henry K.T. Ankala S.V. Ghosh A.K. Fenteany G. Chembiochem. 2002; 3: 1105-1111Crossref PubMed Scopus (11) Google Scholar, 13Wong M.K. Gotlieb A.I. J. Cell Biol. 1988; 107: 1777-1783Crossref PubMed Scopus (70) Google Scholar] and identified a new Rho-kinase inhibitor, Rockout [1Yarrow J.C. Totsukawa G. Charras G. Mitchison T. Chem. Biol. 2005; 12 (this issue): 385-395Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 14Yarrow J.C. Perlman Z.E. Westwood N.J. Mitchison T.J. BMC Biotechnol. 2004; 4: 21Crossref PubMed Scopus (313) Google Scholar]. In the screen, BS-C-1 cell monolayers were grown in 384-well tissue culture plates. The monolayers in each well were wounded with a pin transfer device normally used for transferring small volumes of liquid to 96-well plates. Pins were placed in the wells and then moved laterally to introduce a wound of uniform size and shape to each monolayer before the addition of small molecules. A library of 16,000 small molecules was screened. Cell monolayers were allowed a 7-hr recovery period before processing for microscopy (Figure 1) [14Yarrow J.C. Perlman Z.E. Westwood N.J. Mitchison T.J. BMC Biotechnol. 2004; 4: 21Crossref PubMed Scopus (313) Google Scholar]. An image of each well was captured using automated microscopy and compounds affecting wound healing were grouped according to phenotype. Compounds that were selected for further analysis decreased cell migration, caused aberrant morphology, increased the mitotic index, or caused a combination of these phenotypes. Compounds that caused gross disruptions of the cell monolayer were interpreted to be toxic and were not pursued. A set of seven secondary assays was then performed to test the selected compounds for their effects on the actin cytoskeleton as well as other processes. Rockout was identified as a nontoxic molecule that changed the morphology and dynamics of cells at the wound margin. In secondary assays, Rockout inhibited cell blebbing and the formation of stress fibers and focal adhesions. Importantly, Rockout did not disrupt actin filaments directly. Together these results suggest that Rockout disrupts the Rho pathway. Additional experiments showed that Rockout does not act on Rho itself, but rather inhibits the activity of Rho-kinase. Although Rockout is less potent than the pre-existing Rho-kinase inhibitor Y-27632 [10Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. et al.Nature. 1997; 389: 990-994Crossref PubMed Scopus (2542) Google Scholar], it provides a new scaffold that could be optimized to develop more potent and specific inhibitors of Rho-kinase. Rho-kinase is of interest as a small molecule target because it is activated by Rho and because it phosphorylates cytoskeletal proteins, including myosin light chain, which contributes directly to stress fiber formation [15Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (948) Google Scholar]. Other substrates of Rho-kinase include factors that function in cytokinesis and membrane ruffling [16Amano M. Fukata Y. Kaibuchi K. Exp. Cell Res. 2000; 261: 44-51Crossref PubMed Scopus (453) Google Scholar]. Like a similar screen performed previously [12Mc Henry K.T. Ankala S.V. Ghosh A.K. Fenteany G. Chembiochem. 2002; 3: 1105-1111Crossref PubMed Scopus (11) Google Scholar], the strategy used in this study has several advantages. For one, the readout is the direct visualization of a complex phenomenon (wound healing) whose proper completion relies on several fundamental mechanisms (signal transduction, cytoskeletal reorganization, etc). This makes possible the identification of compounds that affect regulators of multiple pathways that contribute to cell migration. In addition, the screen automatically selects for small molecules that are cell-permeable. Although the current study focused on finding compounds that target actin-associated proteins, one can imagine using the same screening method to find compounds that target other processes that are important for cell migration. For example, in standard wound healing assays, the Golgi apparatus orients toward the site of polarized growth in response to the introduction of a scratch wound, and this orientation can be quantified [17Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1210) Google Scholar]. One might discover interesting inhibitors in a screen for small molecules that disrupt Golgi orientation in this assay. The authors of this study were able to identify a target of Rockout using a combination of careful experimentation and inference. However, target identification is an inherent challenge in phenotypic small molecule screens and it is often necessary to use other methods to identify the target or targets of a compound. For example, affinity chromatography methods can be used for target identification by coupling the compound of interest to a matrix to retrieve target proteins from cellular extracts [18Wignall S.M. Gray N.S. Chang Y.T. Juarez L. Jacob R. Burlingame A. Schultz P.G. Heald R. Chem. Biol. 2004; 11: 135-146Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Another approach, known as the small molecule yeast-three-hybrid system, detects compound-protein interactions in living yeast cells [19Baker K. Sengupta D. Salazar-Jimenez G. Cornish V.W. Anal. Biochem. 2003; 315: 134-137Crossref PubMed Scopus (45) Google Scholar]. Here, the interaction between the compound and its target protein activates the expression of a reporter gene. Although such approaches have been used successfully, a major challenge remains to develop improved methods for identifying targets in phenotypic screens. In summary, the study by Yarrow and colleagues demonstrates the use of a high-throughput screening approach that is based on a wound healing assay. This work has set the stage for the identification of novel inhibitors as well as potentially novel targets. Depending on how the microscopy assay is applied, it could be used to screen for compounds that affect diverse migration-related pathways associated with wound healing. We are eagerly waiting to see what crawls out next.
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