Table 1.

Summary of results from DAST, FB-28 and FB-71 in computational docking, ubiquitin vinyl sulfone assay and MTT in human breast cancer cells.

OB compound	Free energy of docking to UCHL5 (kcal/mol)	Inhibition of UCHL5 in breast cancer cells	Free energy of docking to USP14 (kcal/mol)	Inhibition of USP14 in breast cancer cells	MTT IC50 values (μM in MBA-MD-468 cells)	MTT IC50 values (μM in MCF7 cells)
DAST	−5.6	+	−5.7	+	28	10
FB-28	−6.5	++	−6.3	+	12	9
FB-71	−7.8	+++	−8.3	+	10	9

The free energy values of the reactions between UCHL5 and DAST, FB-28 and FB-71 were −5.6, −6.5, and −7.8 kcal/mol, respectively, and reactions between USP14 and DAST, FB-28, and FB-71 were similar at −5.7, −6.3, and −8.3 kcal/mol, respectively. The levels of inhibition of UCHL5 or USP14 activities by these OBs in breast cancer cells were based on the data of Figure 3, in the order of +++ > ++ > +. In MDA-MB468 cells, IC₅₀ values of DAST, FB-28 and FB-71 were 28, 12, and 10 μM, respectively, at 72 h. In MCF7 cells, IC₅₀ values of DAST, FB-28 and FB-71 were 10, 9, and 9 μM, respectively at 72 h. DAST, 4,4’-Diamino-2,2’-stilbenedisulfonic acid; DUB, deubiquitinating enzyme; FB-28, Fluorescent Brightener 28; FB-71, Fluorescent Brightener 71.