d at a single concentration, they were tested for binding to the target protein in dose response format, with a starting concentration of 50 mM followed by two-fold dilutions downward. A confirmed hit was defined as fragment binding in a concentration-dependent manner at equal or more than three concentrations. All single concentration hits were confirmed again for at least one concentration, and 36 fragments were confirmed to have a dose response. 15% of these fragments had weak affinities, with a calculated % Rmax below 10% at the concentrations tested. These fragments were therefore not further pursued. Concentrations higher than 50 mM were not tested to avoid loss of compound solubility, which could interfere with a good SPR signal. Another 38% of the confirmed hits showed ill-behavior at concentrations above 25 mM. 13 hits showed concentrationdependent binding and displayed sensorgrams without any sign of ill-behavior. The result was a 0.21% 118414-82-7 chemical information Overall hit rate as calculated from 17149874 the total number of fragments screened. These 13 confirmed hits and some of the single concentration hits with promising sensorgram shape were tested for binding to the R0RBR domain of Parkin by STD NMR. Overall, 16 fragments were confirmed as binders to Parkin by NMR. These comprised 12 of the confirmed hits and 4 of the single concentration hits. Taken together, good agreement between SPR and NMR binding data was achieved as 16 out of the 21 fragments tested by NMR bound to the Parkin R0RBR domain. The SPR screen used FLFLAG Parkin, while the NMR screen used the parkin R0RBR domain. It is, therefore, possible that the SPR hits that were negative in binding to R0RBR by NMR bind to a region outside of this domain. Parkin Fragment Screen Physical-Chemical Characteristics of SPR Fragment Hits To further characterize the SPR fragment hits, physicalchemical properties of the fragment library were plotted versus fragment hits by both SPR and NMR. The distributions of Parkin SPR Fragment Screening physical chemical properties for the fragment library are shown in The 10712926 transforming growth factor beta family of cytokines regulates a wide array of biological responses that are critical for proper development and homeostasis. Deregulation of TGFb-mediated responses contributes to the pathogenesis of diverse disease processes from pulmonary and renal fibrosis to cancer. A widely studied and key biological effect of TGFb is the inhibition of hematopoietic and epithelial cell proliferation, which has important consequences in cancer biology. Several types of carcinomas acquire resistance to TGFb-induced cell cycle arrest, leading to uncontrolled cell proliferation. TGFb ligands form heteromeric complexes with type I and II transmembrane TGFb receptors, which have intrinsic serine/ threonine kinase activities. The type II kinase transphosphorylates the type I receptor in a glycine-serine rich motif, thereby stimulating the type I kinase activity. The Smad family of intracellular signaling proteins is critical for transducing TGFb signals 
from the cell surface to the nucleus to regulate gene expression and consequent cellular processes. In particular, the TGFb-stimulated type I receptors associate and phosphorylate the receptor-regulated Smad proteins Smad2 and Smad3 on the C-terminal two serine residues in the SSXS motif. The phosphorylated RSmads then form a heteromeric complex with the common partner Smad4, and the R-Smad/Smad4 complex accumulates in the nucleus and bind