Volume 15, Issue 9, Pages 969-978 (September 2008) Identification of Chemical Inhibitors to Human Tissue Transglutaminase by Screening Existing Drug Libraries  Thung-S. Lai, Yusha Liu, Tim Tucker, Kurt R. Daniel, David C. Sane, Eric Toone, James R. Burke, Warren J. Strittmatter, Charles S. Greenberg  Chemistry & Biology  Volume 15, Issue 9, Pages 969-978 (September 2008) DOI: 10.1016/j.chembiol.2008.07.015 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Structures of ZM39923 and ZM449829 Chemistry & Biology 2008 15, 969-978DOI: (10.1016/j.chembiol.2008.07.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Ca+2-Dependent Inhibition of TGase/TGM2 by Chemical Inhibitors TGM2 was incubated in the absence (A) or presence (B) of 1 mM Ca+2 with inhibitors at concentration that inhibited >95% of TGase activity. The mixture was dialyzed in 41 of 50 mM HEPES (pH 7.5). The incubation mixtures were used to determine TGase activity using BP incorporation assay. The standard deviations were derived from triplicate experiments. Open bar: before dialysis; closed bar: after dialysis. Chemistry & Biology 2008 15, 969-978DOI: (10.1016/j.chembiol.2008.07.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Fluorescent GTP Binding Assay The fluorescence binding assay was performed in buffer B (50 mM Tris-Cl [pH 7.5], 2 mM DTT, and 1 mM EDTA) containing 400 nM of purified enzyme (TGM2) and 500 nM BODIPY FL-GTP. Buffer B, containing only BODIPY FL-GTP, was used as a reference; the results represented the difference in fluorescence upon binding to the enzymes. (A) Effects of 150 μM of GTP, GDP, GMP, ATP, ADP, and AMP on BODIPY FL-GTP binding. (B) Similar to (A) but with inhibitors at 160 μM in the presence (filled bar) or absence (open bar) of 1 mM Mg+2. (C) Different concentrations of inhibitors (reactive blue 2 and tyrphostin 47), GTP, and ATP were investigated for their effects on BODIPY FL-GTP binding. At each concentration, the binding of inhibitors, GTP, or ATP to BODIPY FL-GTP were used as background value to be subtracted from value obtained with TGM2 binding. There are slight quenching from reactive blue 2 and tyrphostin 47 at concentrations higher than 40 μM. Chemistry & Biology 2008 15, 969-978DOI: (10.1016/j.chembiol.2008.07.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Effects of Chemical Inhibitors in Rescuing Drosophila in MJD Model of Neurodegeneration Chemicals were investigated in these flies at 10 μM. Chemical stocks were diluted to final DMSO concentrations of ≤0.1%. Fly food was replenished every 2–3 days. The number of flies left in the tubes was counted every other day. Untreated flies (Q78) and flies that carried normal length of glutamine repeats (Q27) were used as controls. Each chemical was tested in at least three different tubes, and the survival rate of flies was calculated by dividing the number of flies that survived by the number of starting flies. The log rank p values compare treated groups and the control (untreated) group, as described in Methods. The standard deviations were derived from triplicate experiments. ZM: ZM39923; Cyst: cystamine; Me 3–4: Me-3,4 dephostatin; Q27: elav-Gal4/Q27; Q78: elav-Gal4-Q78. Chemistry & Biology 2008 15, 969-978DOI: (10.1016/j.chembiol.2008.07.015) Copyright © 2008 Elsevier Ltd Terms and Conditions