The broad inhibitor of HDACs, TSA, has been shown to influence eNOS expression in HUVECs

performance Ni sepharose column to remove the TEV and the tag. The Ni column was washed with 10 CV of order TMS buffer A. Both wash and flow through from nickel were collected and injected onto a HiLoad 26/10 Superdex column 200 that was pre-equilibrated in buffer C. The column was eluted in 1.5CV of buffer C. Light Scattering Experiments Proteins Light scattering experiments were conducted using an Agilent 1100 series HPLC coupled with a Dawn model EOS multiangle light scattering photometer and an Optilab Rex refractive index detector. Protein samples were heated at 56uC for 80 min before injecting 100 ml on a Wyatt 30 S guard column followed in series by a Wyatt 30 S column. Experiments were carried out in 50 mM HEPES, 50 mM NaCl, 10% glycerol and either 4 mM dithiothreitol or 4 mM TCEP pH 8.0. Size exclusion chromatography was carried out at a flow rate of 0.5 mL/min at room temperature with a run time of 40 min. The experimental data was analyzed using Astra software. S5a Ubiquitination HTRF assay FL FLAG-Parkin was thermally treated by incubating at 56uC for 30 min. The thermally treated FL FLAGParkin stock was stored at 280 C. 5 15930314 ml of thermally treated or non-treated FLAG-Parkin diluted in assay buffer was added to wells of a 384-well non-binding plate. 5 ml of a premix of 15 nM E1, 300 nM E2 UbcH7, 1600 nM Ub, 20 nM Ub-Eu K, 200 nM biotinylated-S5a, and 1 mM Mg-ATP in assay buffer was added to each well. The reaction was allowed to proceed for 120 min at 30uC. 10 ml of stop-detection mix was added to a final concentration of 75 nM streptavidin XL665 conjugate, 12 mM EDTA in buffer containing 100 mM Na2HPO4 pH 7.0, 300 mM KF, and 0.1% BSA. The 20 ml reaction mixture was incubated for 60 min at room temperature. HTRF read using a LJL Analyst plate reader at excitation 320 nm and emission 665 nm & 615 nm. UblD1 5.4 UbcH72 4.7 8.1 7.1 2 Fragment Library Protein/Ligand FL -FLAG-Parkin RT 4 Ubq3 82 n/a 96 FL4-FLAG-Parkin 56uC 7.4 R0RBR Domain RT 1 6 Ubiquitin-like domain of Parkin; Ubiquitin conjugating enzyme E2; Ubiquitin; 4 Full-length. From the in-house screening collection of 25,000 fragments, a subset of 5260 compounds was chosen based on the following criteria: CNS lead-likeness: The compounds selected had low molecular weight, few rings and rotatable bonds, consistent with properties of historic leads that were optimized to drugs. In addition, the compounds had low cLogP and low toplogical polar surface area to enhance their potential for high oral bioavailability and CNS penetration Chemical diversity: We calculated a Parkin SPR Fragment Screening A Response Units 100 B 100 50 Rmax 50 0 0 200 400 600 0 50 100 Number of Fragments Number of Fragments Unity 2D fingerprint for each compound with Sybyl 8.0. Each compound selected was no closer than a Tanimoto similarity of 0.85 to any other selection. Solubility: Each compound had solubility.100 mM by light scattering assay. The distributions of physical chemical properties were calculated from the software package ACD/PhysChem Batch. For the 12419798 Negative Control Test Set 38 compounds were chosen from our in-house library of lead-optimized hits and drugs on the market based on the same criteria as for the fragment library. Surface Plasmon Resonance Experiments Fragment screenings and binding level screens of the negative control test set were performed on a GE Healthcare Biacore 4000 instrument. Briefly, the carboxyl groups of the sensor surface were activated by injection of a solution containi