Correction for light absorption in fluorescence studies of protein-ligand interactions

Birdsall, B.; King, Roy W.; Wheeler, Miriam R.; Lewis, Charles A.; Goode, Scott R.; Dunlap, R. Bruce; Roberts, Gordon C. K.

doi:10.1016/0003-2697(83)90020-9

Cited by 242 publications

(127 citation statements)

References 15 publications

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“…where IF obs is the measured fluorescence, IF is the fluorescence after IFE correction, and A ex and A em are the absorbances at the excitation and emission wavelengths, respectively [35]. The compounds tested in this study absorbed at both 280 and 340 nm so that, in principle, IFE could have contributed to the signal change observed.…”

Section: Discussionmentioning

confidence: 96%

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Bodenreider

Beer

Keller

et al. 2009

Analytical Biochemistry

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In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC (50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions. b s t r a c tIn drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC 50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other proteinligand interactions. Ó 2009 Elsevier Inc. All rights reserved.High-throughput screening (HTS) 2 is a major method in drug discovery for new small lead molecules. In high-throughput enzyme inhibition assays, more than a million compounds are usua...

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Section: Discussionmentioning

confidence: 96%

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Bodenreider

Beer

Keller

et al. 2009

Analytical Biochemistry

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“…The sensitised fluorescence was measured at 480 nm. All data were corrected for inner-filter effects according to Birdsall et al (1983).…”

Section: Methodsmentioning

confidence: 99%

Determination of a Binding Site for a Non‐Substrate Ligand in Mammalian Cytosolic Glutathione S‐Transferases by means of Fluorescence‐Resonance Energy Transfer

Sluis‐Cremer

Naidoo

Kaplan

et al. 1996

European Journal of Biochemistry

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To determine the location of the non-substrate-ligand-binding region in mammalian glutathione Stransferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-a glutathione S-transferase, and in a human Trp284Phe mutant class-n glutathione S-transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class-a and class-n enzymes, respectively. Since glutathione S-transferases bind one non-substrate ligandprotein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis Mol. Biol. 246,. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.Keywords: glutathione S-transferase; energy transfer; non-substrate ligand; 8-anilinonaphthalene 1 -sulfonate.Cytosolic glutathione S-transferases (GST) a superfamily of heterodimeric and homodimeric proteins, catalyse the nucleophilic addition of the thiol of reduced glutathione (y-glutamylcysteinylglycine) to electrophilic centres in a wide variety of hydrophobic electrophiles, including alkyl and aryl halides, epoxides, quinones and activated alkenes (Armstrong, 1991). Although they can be grouped into five species-independent gene classes ( a, p, z, r~ and O), GST share a common protein fold (Dirr et al., 1994a). Three-dimensional structures of isoenzymes from the a (Sinning et al., 1993;Cameron et al., 1995), p (Ji et al., 1992Raghunathan et al., 1994), n (Reinemer et al., 1992; Dim et al., 1994b; Garcia-Saez et al., 1994), r~ (Ji et al., 1995) and B (Wilce et al., 1995; Reinemer et al., 1996) classes have been determined, and this abundance of structural information has provided much insight into the protein-architectural relationships between the different gene classes. Each glutathione Stransferase subunit is folded into two distinct structural domains. Domain 1 has an papappa folding topology, in which its conserved core provides the principal framework for the active site. Domain 2 has an all-a-type core structure. There are two structurally independent active siteddimer, each of which consists of a glutathione-binding site (G-site) and an electrophilic-hydrophobic-substrate-binding site (H-site).

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“…Small aliquots of known concentration of compound Ref I, dissolved in DMSO, were added to the protein solution and each time the fluorescence intensity was measured. These fluorescence intensities, corrected for ligand adsorption, 23 were plotted against the total ligand concentration and fitted with a 4-parameter sigmoidal function. The KD value was obtained applying the law of mass action.…”

Section: Fluorescence Quenching Experimentsmentioning

confidence: 99%

Fragment-Based Screening Using Surface Plasmon Resonance Technology

et al. 2009

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Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 °C at a fragment concentration of 200 µM in the presence of 2% DMSO. Applying the filter cascade, affinity-selectivity-competition (competition with reference compounds and cross-competition with fragments), 80 compounds show up as positive screening hits. Competition experiments between fragments show that they bind to different parts of the active site. Of 36 fragments cocrystallized for X-ray studies, 12 could be located in the active site of the protein. These results validate the authors' library and demonstrate that the application of SPR technology as a filter in fragment screening can be achieved successfully. (Journal of Biomolecular Screening. 2009:337-349)

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Correction for light absorption in fluorescence studies of protein-ligand interactions

Cited by 242 publications

References 15 publications

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Determination of a Binding Site for a Non‐Substrate Ligand in Mammalian Cytosolic Glutathione S‐Transferases by means of Fluorescence‐Resonance Energy Transfer

Fragment-Based Screening Using Surface Plasmon Resonance Technology

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