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1.
Assay Drug Dev Technol ; 16(7): 372-383, 2018 10.
Article in English | MEDLINE | ID: mdl-30307314

ABSTRACT

Fluorescence assay technologies are commonly used in high-throughput screening because of their sensitivity and ease of use. Different technologies have their characteristics and the rationale for choosing one over the other can differ between projects because of factors such as availability of reagents, assay performance, and cost. Another important factor to consider is the assay susceptibility to artifacts, which is almost as important as the ability of the assay to pick up active compounds. Spending time and money on false positives or missing the opportunity to build chemistry around false negatives is something that every drug project tries to avoid. We used a BET family Bromodomain, BRD4(1), to explore the outcome of a screening campaign using three fluorescent assay technologies as primary assays. A diverse 7,038 compound set was screened in fluorescence lifetime, fluorescence polarization, and homogeneous time-resolved fluorescence to look at primary hit rates, compound overlap, and hit confirmation rates. The results show a difference between the fluorescence assay technologies with three separate hit lists and some overlap. The confirmed hits from each assay were further evaluated for translation into cells (NanoBRET™). Most of the actives confirmed in cells originated from compounds that overlapped between the assays. In addition, a well-annotated set of compounds with undesirable mechanism of inhibition was screened against BRD4(1) to compare the ability to discriminate true hits from artifact compounds. The results indicate a difference between the assays in their ability to generate false positives and negatives.


Subject(s)
Fluorescence , Nuclear Proteins/analysis , Transcription Factors/analysis , Cell Cycle Proteins , Fluorescence Polarization , Fluorescent Dyes/analysis , High-Throughput Screening Assays , Humans , Surface Plasmon Resonance
2.
Chembiochem ; 12(5): 802-10, 2011 Mar 21.
Article in English | MEDLINE | ID: mdl-21337480

ABSTRACT

Cyclophilin A (CypA) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A (CsA). Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template. Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands. Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans.


Subject(s)
Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans/drug effects , Cyclophilin A/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Cyclophilin A/metabolism , Cyclosporine , Drug Design , Ligands , Models, Molecular , Protein Binding , Spectrometry, Mass, Electrospray Ionization
3.
J Antimicrob Chemother ; 65(12): 2566-73, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20861142

ABSTRACT

OBJECTIVES: We sought to identify and characterize new inhibitors of MurA and MurZ, which are enzymes involved in the early stages of bacterial peptidoglycan synthesis. METHODS: A library of ∼650 000 compounds was screened for inhibitors of Escherichia coli MurA in an endpoint assay measuring release of inorganic phosphate from phosphoenolpyruvate. Hits were validated by determining the concentrations required for 50% inhibition (IC(50)) of MurA from E. coli and MurA/MurZ from Staphylococcus aureus. The mode of action of selected inhibitors was explored by examining the reversibility of MurA inhibition, the binding of a radiolabelled inhibitor to MurA proteins and through docking studies. Inhibitors were further characterized by determining their antibacterial activity against E. coli and S. aureus. RESULTS: Benzothioxalone derivatives were identified that inhibited MurA from E. coli and MurA/MurZ from S. aureus with IC(50) values between 0.25 and 51 µM. Several inhibitors also exhibited activity against S. aureus with MICs in the range 4-128 mg/L. Inhibition of MurA was irreversible and a radiolabelled inhibitor from this compound class displayed stoichiometric binding to the enzyme, which was displaced by dithiothreitol. Binding was undetectable with a C115D mutant MurA protein. CONCLUSIONS: The results suggest a mode of action for the benzothioxalones that involves the formation of a disulfide bond with MurA/MurZ, via attack from an active site cysteine on the thioxalone ring carbonyl group, followed by ring opening to yield an S-acylated protein. The proposed covalent mode of action may prove useful in the design of new antibacterial agents.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors , Escherichia coli/drug effects , Lactones , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , High-Throughput Screening Assays , Humans , Lactones/chemical synthesis , Lactones/chemistry , Lactones/pharmacology , Microbial Sensitivity Tests , Peptidoglycan/biosynthesis , Staphylococcus aureus/enzymology , Structure-Activity Relationship
4.
Chem Commun (Camb) ; 46(37): 6929-31, 2010 Oct 07.
Article in English | MEDLINE | ID: mdl-20733984

ABSTRACT

A novel long lifetime fluorescence reporter based on 9-aminoacridine was designed, the lifetime of which can be modulated in a defined manner when in proximity to a tryptophan residue enabling fluorescence lifetime based biochemical assays to be configured.


Subject(s)
Aminacrine/chemistry , Biological Assay/methods , Fluorescence , Peptides/chemistry , Molecular Structure , Peptides/chemical synthesis
5.
Anal Biochem ; 402(1): 54-64, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20230774

ABSTRACT

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the "gold standard" radiometric kinase assays with respect to Z' values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.


Subject(s)
High-Throughput Screening Assays/methods , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Chelating Agents/metabolism , Fluorescence , Fluorescent Dyes/chemistry , Iron/metabolism , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Substrate Specificity
6.
Mol Microbiol ; 72(2): 335-43, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19298367

ABSTRACT

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Bacterial Proteins/metabolism , Peptidoglycan/biosynthesis , Staphylococcus aureus/enzymology , Alkyl and Aryl Transferases/genetics , Bacterial Proteins/genetics , Enzyme Inhibitors/pharmacology , Fosfomycin/pharmacology , Genes, Bacterial , Models, Molecular , Promoter Regions, Genetic , Protein Structure, Tertiary , Sequence Deletion , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics
7.
Bioorg Med Chem Lett ; 18(5): 1730-4, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-18261901

ABSTRACT

Several 2-aminotetralones were identified as novel inhibitors of the bacterial enzymes MurA and MurZ. A number of these inhibitors demonstrated antibacterial activity against Staphylococcus aureus and Escherichia coli with MICs in the range 8-128 microg/ml. Based on structure-activity relationships we propose that the alpha-aminoketone functionality is responsible for the inhibitory activity and evidence is provided to support a covalent mode of action involving the C115 thiol group of MurA/MurZ.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Tetralones/chemistry , Tetralones/pharmacology , Binding Sites , Escherichia coli/drug effects , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Staphylococcus aureus/drug effects , Structure-Activity Relationship
8.
Antimicrob Agents Chemother ; 52(5): 1737-42, 2008 May.
Article in English | MEDLINE | ID: mdl-18180348

ABSTRACT

Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPS(A) phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.


Subject(s)
Acetamides/pharmacology , Oxazolidinones/pharmacology , Peptidyl Transferases/genetics , Point Mutation , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Diterpenes/pharmacology , Drug Resistance, Multiple, Bacterial , Linezolid , Models, Molecular , Peptidyl Transferases/chemistry , Peptidyl Transferases/metabolism , Polymerase Chain Reaction , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Analysis, DNA , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics
9.
Biochem Biophys Res Commun ; 363(4): 1013-9, 2007 Nov 30.
Article in English | MEDLINE | ID: mdl-17927958

ABSTRACT

Cyclophilins, which are found in all cellular compartments and with diverse biological roles, are now drug targets for a number of diseases including HIV infection, malaria and ischaemia. We used the database-mining program LIDAEUS and in silico screening to discover the dimedone family of inhibitors which show a conserved 'ball and socket' binding mode with a dimethyl group in the hydrophobic binding pocket of human cyclophilin A (CypA) mimicking a key interaction of the natural inhibitor cyclosporin A (CsA). The most potent derivative binds CypA with a K(d) of 11.2+/-9.2 microM and an IC50 for activity against Caenorhabditis elegans (C. elegans) of 190 microM compared to 28 microM for CsA. These dimedone analogues provide a new scaffold for the synthesis of families of peptidomimetic molecules with potential activity against HIV, malaria, and helminth parasite infections.


Subject(s)
Caenorhabditis elegans/drug effects , Caenorhabditis elegans/enzymology , Cyclohexanones/chemistry , Cyclohexanones/pharmacology , Cyclophilin A/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Animals , Crystallography, X-Ray , Cyclohexanones/chemical synthesis , Cyclohexanones/classification , Cyclophilin A/chemistry , Cyclophilin A/metabolism , Cyclosporine/chemistry , Cyclosporine/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/classification , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Structure , Phenotype , Protein Binding , Structure-Activity Relationship
10.
J Am Chem Soc ; 128(7): 2224-5, 2006 Feb 22.
Article in English | MEDLINE | ID: mdl-16478171

ABSTRACT

Deracemization of racemic chiral tertiary amines has been achieved by combination of an enantioselective amine oxidase, obtained through directed evolution, and ammonia borane in a one-pot process.


Subject(s)
Amines/chemical synthesis , Monoamine Oxidase/chemistry , Amination , Aspergillus niger/enzymology , Monoamine Oxidase/metabolism , Stereoisomerism
11.
Anal Biochem ; 345(2): 214-26, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-16102717

ABSTRACT

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.


Subject(s)
Biosensing Techniques/methods , Cyclophilin A/antagonists & inhibitors , Cyclophilin A/analysis , Enzyme Inhibitors/analysis , Surface Plasmon Resonance , Binding, Competitive , Cyclophilin A/genetics , Cyclophilin A/isolation & purification , Cyclophilin A/metabolism , Cyclosporine/analysis , Cyclosporine/metabolism , Enzyme Inhibitors/chemistry , Enzyme Stability , Enzymes, Immobilized , Fluorescence , Histidine/chemistry , Humans , Kinetics , Ligands , Models, Molecular , Molecular Weight , Nitrilotriacetic Acid/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solutions , Static Electricity , Time Factors , Titrimetry
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