My research will focus on the discovery of novel drug-like inhibitors of Pin1 with reactive groups that can form a covalent, yet reversible bond with the amino acid cysteine in proteins. The peptidyl-prolyl isomerase Pin1 is a cysteine-containing enzyme that specifically recognizes phosphorylated serine or threonine next to proline residues and catalyzes the rotation of the amide bond. The protein is highly conserved across species and is a key regulatory enzyme of mitosis in the cell cycle. Abnormal activity of the enzyme has been strongly linked to many types of cancer. One cysteine residue in the protein is located adjacent to the active site and thus is a promising target for a new cysteine-reactive drug. I will use a two-pronged approach to discover a new inhibitor. The first will capitalize on a known inhibitor to discover a cysteine-reactive peptidomimetic. The second will use a strategy known as fragment-based drug discovery to identify non-reactive leads that can be developed into cysteine-reactive inhibitors. The reactive groups will be selected such that related examples can be found in approved pharmaceuticals to ensure their potential biological compatibility. To detect the interactions, I will use two methods;one will be a fluorescence anisotropy assay using a fluorescently-labeled peptide as the known ligand. The second will use surface plasmon resonance, a technique that measures the change in mass at a surface, to observe the association of small molecules with a protein that has been immobilized on the surface. The binding orientation of promising hits will be determined by x-ray crystallography. Molecules that interact specifically with the binding site will be elaborated by chemical synthesis for the discovery of a potent and selective cysteine-reactive inhibitor. Finally, I will study the role of Pini in mitosis using the inhibitor I discover and high-resolution and fluorescence microscopy to observe chromosomes and other structural elements of cells. Methods are available to arrest cells at specific stages of mitosis, allowing for a step-by-step analysis of Pin1 activity. Comparing the phenotypes resulting from of Pin1 inhibition and normal cells will provide exciting new results on the function of Pin1 in cellular regulation.
A potent, drug-like inhibitor will aid in the validation of Pini as a target for cancer treatment, as the protein is over-expressed in many cancers, including prostate and breast cancer. The study of Pini in mitosis will lead to a better understanding of the regulatory function of the enzyme, this important cell cycle stage, and how Pin1 activity contributes to normal and disease phenotypes.
|Bradshaw, J Michael; McFarland, Jesse M; Paavilainen, Ville O et al. (2015) Prolonged and tunable residence time using reversible covalent kinase inhibitors. Nat Chem Biol 11:525-31|
|Serafimova, Iana M; Pufall, Miles A; Krishnan, Shyam et al. (2012) Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat Chem Biol 8:471-6|