Competition binding screens

Fig. 1. Competition binding screens Bead/ligand compatibility test in early assay development. The beads PVT-WGA, Type A and Type B are based on polyvinyltoluene (PVT) not polyethyleneimine (PEI). PVT, polyvinyltoluene WGA, wheat germ agglutinin Ysi, yttrium silicate.

Fig. 2. Competition binding screens Examples of concentration-response curves for 125I-CCL1 binding to human CCR8. U87 cells expressing hCCR8 were incubated with trace amounts of 125I-CCL1 (lOOpM) and increasing concentrations of cold CCL1 (circles) or compound A (squares).

The development of maraviroc (21), much like other chemokine receptor antagonists, started with a high-throughput screen employing a competition binding assay and led to the hit compound UK-107,543. Chemical optimization of this compound led to the development candidate UK-427,857 during this optimization phase, a parallel characterization of the compounds was performed... 

Secondary assays depend on the project. Where the primary screen was a cell-based assay, the secondary assay may be a radioligand competition binding assay. In other cases, such as where the primary screen was a biochemical assay, the secondary assay may be a cellular assay, and may be functional or mechanistic. One of the issues that may arise at this stage is that compounds with reasonable activity in the primary assay may not show activity in the secondary assay. There can be a number of reasons for this, including insufficient potency, inability of the compound to get into cells, or a higher intracellular concentration of the natural ligand (e.g., ATP) if the inhibitor is a competitive inhibitor. It is often necessary at this stage to prepare additional compounds in the series to get compounds of sufficient potency and/or permeability so that cellular activity can be demonstrated. 

MS binding assays are also useful for library screening with subsequent hit identification. The concept is simple. First, a library is searched for active compounds in a competitive binding assay. If the result is positive (which is indicated by an increase of the marker signal), the target bound hit is liberated and identified. 

Dalvit, C., et al., High-throughput NMR-based screening with competition binding experiments. J Am Chem Soc, 2002,124, 7702-7709. 

Such an approach to the preliminary evaluation of photoaffinity reagents goes one step beyond the reversible binding experiments discussed earlier. It is a useful approach in that, like competitive binding experiments, radiolabeled ligand is not required and therefore many potential reagents may be screened. However, many pitfalls severely reduce its utility. 

Bunce NJ, Schneider UA, Brown MM, Logau RA, Millart LC (1995 a), Environ. Sci. Technol. 29 2595-2602. Screening assay for dioxin-like compounds based on competitive binding to the marine hepatic Ah recepor. 1. Assay development"... 

Figure 7 Depiction of competition-based screening. Low- to medium-affinity binders are displaced from their binding site by higher affinity binders. In this experiment, only the signals of these low- to medium-affinity binders (reporter ligands) are monitored...

Lu et u/. screened a prefiltered set of 11000 compounds from the NCI database with a pharmacophore model based on the crystal structure of murine double minute 2 (MDM2) oncoprotein in complex with the p53 tumor suppressor and several non-peptide small inhibitors. The remaining compounds were docked, scored, and 67 compounds were selected for testing, ten of which were active in a competitive binding assay. Similarly, Yu et used a Catalyst HipHop model combined with the molecular shape of an experimental X-ray... 

Fig. 15.2 Flow injection set-up for optical sensing and screening library members and competitive binding assay for fructose developed on-bead using fluorescent labeled glucose...

---