We then assessed the effects of these compounds on the three proteases activities associated with the Perifosine side effects proteasome, using fluorogenic compounds in HeLa cells extracts. All compounds that increased GFP expression in GFPu 1 cells inhibited chymotrypsin and caspase like activities of the proteasome, the chymotrypsin like activity being generally the most affected. In addition, lactacystin and curcumin inhibited trypsin like activity. These findings indicate that the compound 5929407 is a novel proteasome inhibitor that preferentially inhibits the chymotrypsin like activity of the proteasome. Most chemicals that inhibit the FA pathway inhibit homologous recombination Since the FA pathway is required for efficient DNA double strand break repair by HR, we tested whether the identified compounds affect HR efficiency in human cells using the DR GFP reporter system.
In this assay, GFP expression reflects the occurrence of an HR repair event. compounds that disrupt HR repair will decrease GFP expression. Twenty four Inhibitors,Modulators,Libraries hours after transfection of a HA tagged I Sce1 encoding plasmid, U2OS DR GFP cells were exposed to the identified FA pathway inhibitors for 24 hours. The concentration used for each drug was optimized Inhibitors,Modulators,Libraries to induce minimal decrease in cell viability, and did not significantly affect HA tagged I Sce1 expression, with the exception of MG132, UCN 01, compounds 5195423 and 7012246. The HA positive population was analyzed for GFP expression. In the absence of drug, 9. 5 0. 9% of the HA positive cells expressed GFP. With the exception of SB218078, HNMPA 3 and TPEN, all FA pathway inhibitors significantly decreased HR.
No significant differences in the cell cycle distribution were observed under these conditions, except for UCN 01, Inhibitors,Modulators,Libraries 17 AAG, wortmannin, HNMPA 3 and compounds 5929407 and 5315179. BRCA1 and RAD51 are required for efficient HR and are known to interact Inhibitors,Modulators,Libraries with FANCD2. We therefore tested whether the FA pathway inhibitors block FANCD2, BRCA1 and RAD51 foci formation upon DNA damage in U2OS DR GFP cells. To do so, we used drug treatments identical to those used during the DR GFP assay, consisting in longer exposure to lower concentrations of chemicals than the initial screen and confirmation experiments. Under such conditions, most of the drugs still significantly inhibited IR induced foci formation of FANCD2 and RAD51, without significantly modify ing cell cycle distribution.
The drugs that failed to significantly inhibit FANCD2 foci formation under these conditions demonstrated significant inhibition Inhibitors,Modulators,Libraries at higher dosage, consistently with the initial screen. IR induced monoubiquitination of FANCD2 was in most cases moderately inhibited or unaffected, with the exception of CA 074 Me, which strongly inhibited it. IR induced foci formation of BRCA1 selleck chem was also mildly affected or unaffected by the compounds.