Assay Method Information

Assay Name:  Biological Assay
Description:  Successful drug candidate compounds were subjected to numerous biochemical and cellular assays. Purified bacterial β-glucuronidases were challenged with compounds to determine inhibition properties in a standard, robust activity assay, utilizing p-nitrophenyl glucuronide (PNPG) as the enzymatic substrate. Reactions (n=3/inhibitor concentration) were conducted in a 96-well assay, consisting of PNPG substrate (12 concentrations between 25 μM and 5 mM), inhibitor solution (8 concentrations between 0.1 nM and 100 μM), and 5 nM enzyme. Preferred compounds exhibited potent IC50 values with values <1 μM.Additionally, preferred compounds exhibited negligible, if any, effect on purified mammalian β-glucuronidases, specifically, preferred compounds are >500-fold more selective and potent against purified bacterial enzymes. Purified bovine liver and human β-glucuronidase were dissolved in a reaction mixture containing 1 μM enzyme and 1 mM PNPG as substrate.
Affinity data for this assay
 

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