Back to Basics: Label-Free Technologies for Small Molecule Screening

Abstract

Small molecule high-throughput screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

“…impedance of layers of cells in culture caused by receptor-mediated changes in cell mass redistribution (Verdonk et al, 2006;McGuinness, 2007;Peters et al, 2007;Shiau et al, 2008;Peters and Scott, 2009). In general, whole-cell responses are the result of the integration of numerous pathway activations and as such are ideal for detecting bias in signaling.…”

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

“…An additional advantage that RF-MS/MS enjoys is that it requires either no or very minimal changes in sample preparation as compared to LC-MS/MS analysis, making it easy to be incorporated into existing automated in vitro assays. [19] To date this technology has been successfully used to support several high-throughput screening assays, [20,21] cytochrome P450 (CYP) inhibition assays, [22,23] as well as a metabolic stability assay, [24] and in this manuscript we describe the development of a sensitive, selective, reproducible and robust RF-MS/MS method for digoxin bioanalysis to support an in vitro P-gp inhibition screen.…”

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

“…It is a significant challenge to engineer a rapid injection system that uses small volumes, has low carry-over between injections, uses low flow rates, and is reliable. A rapid system that requires just 4 to 5 s per analysis and consumes 1 to 5 L of sample has been commercialized [10]; however, more common systems are considerably slower and require a few minutes per sample. For HTS, it is desirable to lower the volume of sample consumed, to reduce reagent costs, and further increase throughput.…”