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1.
J Biomol Screen ; 11(1): 75-81, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16361697

ABSTRACT

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappears with oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based on monitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.


Subject(s)
Drug Evaluation, Preclinical/methods , NADP/metabolism , NAD/metabolism , Biological Assay , Coloring Agents , Inhibitory Concentration 50 , Light , Phenothiazines/metabolism , Phosphopyruvate Hydratase/antagonists & inhibitors , Phosphopyruvate Hydratase/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/metabolism , Substrate Specificity , Time Factors
2.
J Biomol Screen ; 8(1): 19-33, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12854995

ABSTRACT

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence.


Subject(s)
Microscopy, Confocal , Alcohol Dehydrogenase/analysis , Alkaline Phosphatase/analysis , Antigens, Human Platelet/analysis , Antigens, Human Platelet/metabolism , Endopeptidases/analysis , Endopeptidases/metabolism , Fluorescence , Ligands , RNA/metabolism
3.
Eur J Med Chem ; 38(4): 351-6, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12750021

ABSTRACT

Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus aureus. Key elements were an alkyl membrane anchor, proline at P5 and lysine at P2. The proline at P5 induced a helical turn in the lipopeptide, as deduced from NMR studies, from P6 to P2 in membrane mimetic solvents. The substrate Decanoyl-LTPTAKAASKIDD-OH was cleaved by SpsB, as expected, between the P1 and P1' alanines with a k(cat)/K(m) of 2.3x10(6) M(-1)s(-1) at pH 8.5. Insertion of proline at P1' converted substrates to competitive inhibitors, whilst the incorporation of an alpha-ketoamide at the cleavage site transformed substrates to time dependent inhibitors of SpsB.


Subject(s)
Lipoproteins/chemical synthesis , Membrane Proteins , Oligopeptides/chemical synthesis , Serine Endopeptidases/metabolism , Staphylococcus aureus/enzymology , Amides/chemistry , Amides/pharmacology , Amino Acid Sequence , Binding Sites , Dose-Response Relationship, Drug , Enzyme Activation , Escherichia coli/enzymology , Escherichia coli/genetics , Hydrogen-Ion Concentration , Kinetics , Lipoproteins/chemistry , Lipoproteins/metabolism , Magnetic Resonance Spectroscopy , Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Conformation , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Serine Endopeptidases/drug effects , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Staphylococcus aureus/genetics , Substrate Specificity , Time Factors
4.
J Biomol Screen ; 7(6): 554-69, 2002 Dec.
Article in English | MEDLINE | ID: mdl-14599354

ABSTRACT

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.


Subject(s)
Microchemistry/instrumentation , Microchemistry/standards , Combinatorial Chemistry Techniques/instrumentation , Combinatorial Chemistry Techniques/standards , Data Interpretation, Statistical , Needles , Quality Control , Stainless Steel
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