8 uM total actin by high speed ultracentrifugation. Actin was first polymerized in vitro, and then G actin fractionated now from F actin by centrifugation at 150,000 X g for 1. 5 hours. Increasing Cofilin1 concentrations revealed an efficient effect of shifting actin from P to S fractions at 5 uM. To test the possibility that cucurbitacin E binds co valently to Cofilin1 as it does for actin, a range of cucurbitacin E concentrations at molar ratios up to 1 100 were incubated with purified Cofilin1 protein for 16 h and examined for their ability to induce a Cofilin1 mobility shift on 12% Bis Tris polyacrylamide gels. Although low cucurbitacin E concentrations had no obvious effect, reduced Cofilin1 mobility was clearly evident at 1 50 and 1 100 molar ratios.

Inhibitors,Modulators,Libraries Covalent modification of actin was similarly achieved with a 1 100 molar ratio of actin protein to cucurbitacin E. To determine whether Inhibitors,Modulators,Libraries similar effects were seen with additional cucurbitacin compounds, each of three cucurbitacin compounds was incubated at 1 100 molar Inhibitors,Modulators,Libraries ratio with Cofilin1 that had been dialysed to remove dithiothreitol. The mobility of Cofilin1 on 12% Bis Tris polyacrylamide gels was slowed by each cucurbitacin. consistent with stable modification of Cofilin1 by cucurbitacin compounds and increased mass. The inclusion of 5 mM DTT blocked the mobility shift induced by cucurbitacin compounds. However, there was no effect on the Cofilin1 mo bility shift if DTT was added after the incubation of cucurbitacin E with Cofilin1. These results in dicate that Cofilin1 was modified by cucurbitacin com pounds to produce Inhibitors,Modulators,Libraries stable modifications that could not be reversed by DTT.

The actin severing assay was used to determine whether cucurbitacin E inhibited Cofilin1 F actin severing activity. While DMSO vehicle control did not change the S P ratio, cucurbitacin E notably Inhibitors,Modulators,Libraries increased the pelleted F actin, consistent with a direct effect on inhibiting depolymerisation. The effect of Cofilin1 was to shift actin to the monomeric S fraction, which was unaffected by DMSO vehicle control. In contrast, treatment with cucurbitacin E inhibited the actin severing activity of Cofilin1, since the P fraction was significantly increased relative to DMSO treated Cofilin1 samples. Mass spectrometry revealed that each cucurbitacin treated sample had an increased mass co rresponding to the mass determined for Cofilin1 plus four times the mass of each cucur bitacin compound used.

Incubation with 10 mM DTT for 3 h resulted in virtually complete re action of 100 uM cucurbitacin E with no loss of mass in the product of the two reactants. To determine the exact binding sites of cucurbitacin E, the treated Cofilin1 was digested with trypsin, and the fragment sizes determined by MS. Fragmentation by MS MS of AG-014699 the pep tide containing Cys39 revealed that this was a site of cucurbitacin E conjugation.