Reporter-Based Assays

1 Chloramphenicol Acetyl Transferase, Secreted Placental Alkaline Phosphatase, P-Galactosidase 

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Walker SL, Ariga J, Mathias JR, Coothankandaswamy V, Xie X, Distel M, Koster RW, Parsons MJ, Bhalla KN, Saxena MT, Mumm JS (2012) Automated reporter quantification in vivo high-throughput screening method for reporter-based assays in zebrafish. PLoS One, 7, e29916. 

Cell based assays for NRs range from reporter gene assays to in vivo recruitment assays. The most reported of these is the GAL4 reporter assay. This assay takes advantage of the fact that the GAL4 response element of yeast does not exist in mammalian systems. 

In another 2009 patent application, a series of 2-pyrazolopyrimidinones were reported to be PHD inhibitors [37]. Although most of the compounds exemplified in this application lacked a carboxylic acid moiety (vide supra), compound 45 was reported as having an IC50 value of < 10 nM in a FRET assay, although no cell-based assay data were disclosed. 

The best combination turned out to be L5 /A9 (ee = 90% at room temperature and 99% at —78 °C with benzaldehydes and ee = 92-99% with other aldehydes).87 Further improvements were reported later.89 Although only a few dozen reactions were monitored by a JASCO-CD-995 instrument, the CD-based assay is amenable to high-throughput screening of enantioselective catalysts. The chemistry itself lends itself ideally to combinatorial asymmetric metal catalysis, since the principle of asymmetric activation is turning out to be very powerful.89... 

Recent reports have emerged of several TRPV1 antagonists possessing a biaryl amide (14-16), urea (17), or urea isostere (18-20) scaffolds. Bicyclic derivatives 14-16 block capsaicin- or pH-stimulated calcium influx in FLIPR-based assays... 

The number of multiplexed particles-based assays reported is manifold and summarized in several reviews [85, 86, 97, 99], Bead sets for various nucleic acid or protein assays are commercially available that are fully optimized for clinical diagnostics and research purposes. 

It is apparent that signal amplification provides increased sensitivity over direct labeling. This is especially true for fluorescent-based assays. One of the most sensitive signal detection technologies is the immunoRCA (Schweitzer et al., 2000). Rolling circle amplificahon (RCA) is combined with antibody detection. RCA involves the amplification of circularized oligonuceotide probes under isothermal conditions by DNA polymerase (Lizardi et al., 1998). With immunoRCA, the 5 primer is attached to the reporter antibody. Initiation of the amplification starts when circular DNA template binds to the attached primer. 

Crystal strnctnres of a DIS construct bound to aminoglycosides have been pnblished. A flnorescence-based assay for evalnating ligand binding to DIS has been reported. 

S. W. (2002) A cell-based reporter gene assay for determining induction of CYP3A4 in a high-volume system. Journal of Pharmacology and Experimental Therapeutics, 303, 412-423. 

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