Fluorescent screening, fluorescence-based

The discovery of inhibitors of fibrillogenesis has been hampered by the complexity of the folding pathway, the presence of mixed populations of aggregates, and the lack of sensitive markers of each oligomeric species.40 Screening assays based on thioflavin-S fluorescence or Congo-red absorbance have been used to identify inhibitors of aggregation of synthetic Af peptide. The reported compounds suffer from relatively low potency (effective at <10 pM) and "flat" SARs." 43... 

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

Next to the detection of enzyme inhibition, ESI-MS can also be used to monitor protein-ligand interaction, employing an assay format similar to fluorescence-based receptor assays. Using a similar continuous-flow analytical screening system as shown in Fig. 5.2, a competitive assay can be set up using ESI-MS to measure the interaction of the analyte(s) with an affinity protein such as an antibody, receptor or enzyme [28]. Figure 5.10 shows the equilibrium reactions that form the basis of the assay concept. In a first step, the sample was injected into a con-... 

Figure 3.11 Left Fluorescence-based RBDCC screen. One pool of resin (pool 7) from the screen exhibits significant fluorescence, and as such a small-molecule ligand for the target DNA. Right Selected homodisulfide resin-bound dynamic combinatorial library member that selectively binds DNA Sequence 2.

Unfortunately, none of these catalysts displayed practical levels of selectivity in the KR of aryl aUcyl yec-alcohols. Miller therefore embarked in the design of a third generation catalyst that could enable the KR of a larger number of substrates. In this context, he developed an elegant fluorescence-based activity assay which allowed rapid screening of a large number of structurally unique catalysts. This protocol based on proton-activated fluorescence led to the identification of octapeptide 52 as a highly selective catalyst for the KR of aryl alkyl. yec-alcohols but also alkyl yec-alcohols... 

Robertson, B. and Knox, R.J. (2004) A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells. Journal of Biomolecular Screening, 9, 671-677. 

DeLange RJ, Glazer AN. Phycoerythrin fluorescence-based assay for peroxy radicals a screen for biologically relevant protective agents. Analytical Biochemistry. 1989 177(2) 300-306. 

Zou L, Harkey MR, Henderson GL. Effects of intrinsic fluorescence and quenching on fluorescence-based screening of natural products. Phytomedicine... 

The advantage of fluorescence-based assays is their high sensitivity. It is therefore perhaps surprising that few such systems have been developed for evaluating the enantioselectivity of enzyme-catalyzed reactions. Fluorescence as a detection method is used in an enzyme-coupled assay [26] (see Section 9.3.4.3) and in the capillary array electrophoresis [25] (see Section 9.3.6.5). If several substrates need to be screened simultaneously, fluorescence-based substrate arrays as enzyme fingerprinting tools can be used, although enantioselectivity still needs to be addressed [26e],... 

Williams GJ, Thorson JS (2008) A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution. Nat Protoc 3 357-362... 

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays [56, 57] or fluorescence-based assays [58-61], Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer [62]. Their technique involves a DNA template that forms a stable hairpin structure labeled at two positions ... 

Fluorescent-based flux or membrane potential assays provide superior throughput compared to traditional electrophysiology-based screening technologies. For fluorescent-based membrane potential assays, however, the response to increasing channel activity can be non-linear due to the inherent non-linear relationship between channel activity and membrane potential (Fig. 4A). This prevents an accurate readout of efficacy and reduces the reso-... 

Figure 11.13 Synthetic route to the Pt-Rh-O-Ir-Ru discrete catalyst library L9 and fluorescence-based screening strategy based on 11.8 to discover the novel catalysts 11.9 and 11.10.

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