The Sonic hedgehog (Shh) pathway is involved in various forms of cancer, including brain, skin, pancreatic, ovarian, and gastrointestinal neoplasms. Drugs targeting this pathway hold great promise for oncology, but the current generation of Shh pathway inhibitors cause emergence of drug resistance. Most of the known compounds that inhibit the Shh pathway were discovered by high-throughput screening based on the expression of an artificial target luciferase construct. This method, however, has some major drawbacks and yields a large number of false hits. Here, we propose to develop a novel high-throughput screening method based on subcellular localization of Shh pathway components to an organelle called the primary cilium. Our method will not only be more robust than the currently used screening methods, but the high-content data generated in the course of screening can be used to probe molecular mechanisms of Shh signaling and ciliary function. We will use this newly developed high-throughput screening assay to discover new Shh inhibitors that will prevent cancer cells from evading treatment.
The discovery of new pharmaceuticals is accomplished by screening vast collections of chemical compounds for their ability to block specific biochemical events found only in disease states, such as cancer. Certain forms of cancer, such as a childhood brain tumor medulloblastoma, skin cancer, and tumors of the pancreas, colon, and ovaries, are characterized by increased movement of proteins to a tiny structure in the cell called the primary cilium. We will develop a method of automatic monitoring protein movement to the primary cilium, which will enable us to test hundreds of thousands of chemical molecules for their ability to block this transport phenomenon in hopes of discovering a potential new generation of anti-cancer drugs.
| Niewiadomski, Pawel; Rohatgi, Rajat (2015) Rapid Screening of Gli2/3 Mutants Using the Flp-In System. Methods Mol Biol 1322:125-30 |
| Ahrends, Robert; Niewiadomski, Pawel; Teruel, Mary N et al. (2015) Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry. Methods Mol Biol 1322:105-23 |
| Niewiadomski, Pawel; Rohatgi, Rajat (2015) Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR. Methods Mol Biol 1322:81-92 |
| Niewiadomski, Pawel; Kong, Jennifer H; Ahrends, Robert et al. (2014) Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. Cell Rep 6:168-181 |
| Lin, Yu-Chun; Niewiadomski, Pawel; Lin, Benjamin et al. (2013) Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier. Nat Chem Biol 9:437-43 |
