Single‐bead, Single‐molecule, Single‐cell Fluorescence

Abstract

According to many current reports, the pharmaceutical business will hit a wall over the next few years. The generic competition is expected to wipe out a double-digit billion-dollar amount from top companies' annual sales between 2007 and 2012 (Wall Street Journal, online, December 6, 2007). The industry's science engine has stalled, new blockbusters are lacking, and patent expirations are a big problem. Also, the U.S. Food and Drug Administration is pulling back on approvals, requesting larger safety studies. Among the different approaches taken throughout the industry to improve productivity and to reduce the attrition rate of compounds in the drug discovery process, an extended application of quantitative biology and biophysical methods is ranked very high. Fluorescence spectroscopy and imaging represented the main detection technologies for assays and screening methods in recent years. Today, label-free detection methods, such as isothermal titration calorimetry, differential scanning calorimetry, tandem mass spectrometry (MS(n)), light scattering, or interferometry, start to provide viable alternative readouts for physicochemical characterization of leads and hit list triaging. However, the multidimensional nature of fluorescence along with its high sensitivity and single-molecule resolution remains an unparalleled source of molecular parameters to extract all different kinds of information on molecules and ligand-protein complexes in solution. Although fluorescence-based methods are currently applied throughout the different stages of the industrial drug discovery process, they are usually applied in an unconnected way. We have developed a fully integrated hit and lead discovery process combining bead-based synthesis and screening methods with confocal fluorescence microspectroscopy. The primary on-bead screening process provides fluorescent ligands that after a multistep characterization process ultimately leads to fully mechanistically characterized cellularly validated binders and inhibitors of target protein interactions. The unlabeled small-molecular inhibitors represent chemical starting points in drug discovery and target validation.

Clickable Long‐Wave “Mega‐Stokes” Fluorophores for Orthogonal Chemoselective Labeling of Cells

“…This reaction or its copper‐free, strain‐promoted alternative (SPAAC) has been shown to be quite versatile in terms of biological applications 6. 7 CuAAC also finds widespread applications in high‐throughput screening of libraries 8. The versatility of the combined use of copper‐catalyzed and Cu‐free click chemistries in sequential labeling methods was also demonstrated recently 9.…”

Clickable fluorophores for biological labeling—with or without copper

“…3 CuAAC also finds widespread applications in highthroughput screening of libraries. 4 In cases where copper toxicity can be an obstacle in the ligation process, a strain-promoted cycloaddition between cyclooctynes and azides can be used as an alternative as proposed by the Bertozzi and Boons groups. 5, 6 Very recently, we have presented our results on multiple labeling of biomolecules using a combination of copper free and copper mediated click chemistries in a sequential manner.…”

Facile Synthesis of a Next Generation Safety‐Catch Acid‐Labile Linker, SCAL‐2, Suitable for Solid‐Phase Synthesis, On‐Support Display and for Post‐Synthesis Tagging

“…Furthermore, in classical solid phase synthesis the final compound is often generated by the simultaneous detachment from solid support and deprotection of various side‐chain protecting groups. However, modern Chemical Biology research, which uses compounds as tools for understating biological systems increasingly utilizes post‐synthesis tagging methods . Also, the use of combinatorial libraries for on‐bead screening or similar display technologies requires that the final compounds can be fully deprotected while it still remains linked to the solid surface.…”