Mechanism-based assays

An ingenious whole-cell mechanism-based assay using an antisense fabF RNA was developed to increase the detection threshold to FabH/F inhibitors in S. aureus. A range of new natural products was thus... 

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

We have employed two different protocols for the chemical fractionation of GSE obtained from MegaNatural-AZ based on the amounts needed for bioactivity-based assays. Batches of GSE (50 g) were extracted in acetone/water (7 3) under N2 with mechanical agitation for 12 h. The acetone was removed on a rotary evaporator and the aqueous phase was freeze-dried to yield 48 g of tannin crude extract (TCE). TCE was further fractionated following two different methods. 

---