A Novel Reporter System for Molecular Imaging and High‐Throughput Screening of Anticancer Drugs

Abstract

Apoptosis is irreversible programmed cell death characterized by a cellular cascade activation of caspase-3, which subsequently degrades proteins and other components of cells with a motif sequence. Here we report a novel reporter system to detect apoptosis, growth arrest and cell death based on controlled and self-amplified protein degradation. The key element of the reporter system is an apoptotic sensor chimerical protein which consists of three components, the procaspase 3, ubiquitin (Ub), and a strong consensus sequence of N-degron. Between each of these units, there is a DEVD (Asp-Glu-Val-Asp) sequence, which acts as the cleavage target of caspase-3. This non-conventional signal loss approach is much more sensitive than other native methods based on signal gain. The superior sensitivity is demonstrated by its effective application in 386-well HTS with lower drug concentrations and short incubation time. The HTS selection process using this reporter system is very simple and economic. The simplicity eliminates potential errors introduced by multiple steps; there is no need for any substrate. Furthermore the cells in the assay need not be disrupted and the morphology of the cells can provide additional information on mechanisms; the intact cells after HTS can also be used for other analytic analysis. This system thus has a potentially important role in the discovery and development of new anticancer drugs, it also appears to be very versatile, can be used both in vitro and in vivo, with different linked reporter genes, it can be used for a variety of imaging applications.

High Throughput Screening for Colorectal Cancer Specific Compounds

“…Another is Nisoldipine, a calcium channel blocker of the dihydropyridine class, used to treat high blood pressure by relaxing blood vessels. This compound was also selected out when we screened the NIH clinical library [8]; the potential application of this class of molecules as anti-tumor drugs may merit further investigation in the future.…”

“…HTS assays were performed based on a reporter system we developed [8] to detect cellular apoptosis, growth arrest and cell death based on controlled and self-amplified protein degradation. The key component of the reporter system is an apoptotic sensor as a part of a chimerical fused fluorescent protein, consisting of a peptide sequence DEVD (Asp-Glu-Val-Asp)-linked procaspase 3, ubiquitin (Ub), and a strong consensus sequence of N-degron.…”

“…Prior to HTS assays, C6 and DLD1 cells were stably transfected with the reporter system as previously described [8]. The transfected C6 (1000) or DLD1 (500) cells were seeded in multiple 386-well plates for concurrent and comparative drug selection in 30μl of complete growth medium and allowed to settle overnight.…”

A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

“…To maintain the capacity to minimize the amount of enzyme required for screening multiple substrates, a high‐throughput screening platform capable of dealing with very small sample volumes was explored for rapid individual enzyme/substrate screening. High‐throughput (HT) screening platforms have been widely applied in drug discovery and protein crystallization screens, but have seen less use for enzyme characterization. A challenge in doing such screening is that the platform would need to be able to handle small volumes accurately, to subject the enzymes to low enough shear forces to maintain activity, and to avoid cross‐contamination between samples—an especially important trait when screening catalytic components.…”

Degron Protease Blockade Sensor to Image Epigenetic Histone Protein Methylation in Cells and Living Animals

“…In addition, N-terminal degrons have been used to design sensors for imaging apoptosis, cell death, and cell growth arrest. 16 …”