A major focus in drug discovery involves the identification of compounds that reduce or modulate biological processes by altering the activity of cellular receptors or signal transduction components such as kinases. Located in the cell membrane, the cytoplasm, or the cell nucleus, receptors are capable of highly specific binding to biological ligands. Upon the binding of a receptor to a ligand, signal transduction processes, including phosphorylation cascades mediated by kinases, regulate various biological processes required for cell growth and function.
For decades, heterogeneous filter-binding analyses have been employed, particularly under more demanding assay conditions (e.g., using unpurified tissue homogenates, cell membrane fragments, or cellular extracts), to study receptor binding and kinase activity.
Radiometric filter-binding assays are a proven methodology that directly measure either the extent of ligand binding to a wide range of receptors or, in the case of kinase screening, phosphorylation of target proteins and peptide sequences. Radiometric assays are less susceptible to artifacts generated by naturally fluorescent or quenching compounds (such as aromatic compounds). Filter-binding assays typically require minimal set-up and are fully automatable. The protocols are well documented in the peer reviewed literature.
Radiometric filter-binding assays have been successfully performed on 96-well platforms, but increasingly this format has fallen short of meeting the throughput and cost targets of secondary screening laboratories. This reality has fueled the development of higher throughput platforms that perform adequate library screening in a timely and cost-effective manner.
Millipore's (www.millipore.com) MultiScreenHTS 384-well filter plate (Figure 1) fulfills these needs in two different ways: by quadrupling the throughput of filter-based assays and by significantly reducing reagent consumption and costs. These benefits have been achieved without sacrificing assay sensitivity, robustness, or precision. The reduced filter-binding surface area and smaller assay volumes improve well-to-well reproducibility and reduce nonspecific binding. Furthermore, assays can be performed directly in the filter plate, which decreases the number of manipulations, reduces radioactive solid waste, and eliminates the need for a separate incubation plate.