Biomedical subjects
Publications and source records attributed to A J Pope.
Carbocyclic analogues of the microbial metabolite SB-219383 have been synthesised and evaluated as inhibitors of bacterial tyrosyl tRNA synthetase. One compound showed highly potent and selective nanomolar inhibition.
Novel inhibitors of bacterial tyrosyl tRNA synthetase have been synthesised in which the cyclic hydroxylamine moiety of SB-219383 is replaced by C-pyranosyl derivatives. Potent and selective inhibition of bacterial tyrosyl tRNA synthetase was obtained.
Novel pyranosyl analogues of SB-219383 have been synthesised to elucidate the structure-activity relationships around the pyran ring. Analogues with highly potent stereoselective and bacterioselective inhibition of bacterial tyrosyl tRNA synthetase have been identified. A major reduction in the overall polarity of the molecule can be tolerated without loss of the nanomolar level of inhibition.
Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.
In order to accommodate the predicted increase in screening required of successful pharmaceutical companies, miniaturized, high-speed HTS formats are necessary. Much emphasis has been placed on sensitive fluorescence techniques, but some systems, particularly enzymes interconverting small substrates, are likely to be refractory to such approaches. We show here that simple absorbance-based assays can be miniaturized to 10-microl volumes in 1536-well microplates compatible with the requirements for ultra-high throughput screening. We demonstrate that, with low-cost hardware, assay performance is wholly predictable from the 2-fold decrease in pathlength for fully filled 1536-well plates compared to 96- and 384-well microplates. A number of enzyme systems are shown to work in this high-density format, and the inhibition parameters determined are comparable with those in standard assay formats. We also demonstrate the utility of kinetics measurements in miniaturized format with improvements in assay quality and the ability to extract detailed mechanistic information about inhibitors.
G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.
DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [(3)H]-dNTPs onto the growing primer 3' end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [(3)H]-rNTPs or by subsequent polymerase extension with [(3)H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [(33)P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.
We have developed homogeneous miniaturized assays to measure ligand binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants of fluorescently labeled interleukin-8 (IL-8) and a low-molecular-weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1 microl without compromising their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand-binding assay performed in such low volumes. The resulting savings in reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.
This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd approximately 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.
A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.
Synthetic analogues of the microbial metabolite SB-219383 have been synthesised with defined stereochemistry. Densely functionalised hydroxylamine containing amino acids were prepared by the addition of a glycine anion equivalent to sugar-derived cyclic nitrones. One of four stereoisomeric dipeptides incorporating these novel amino acids was found to be a potent and selective inhibitor of bacterial tyrosyl tRNA synthetase, suggesting analogous stereochemistry of the natural product.
Aminoalkyl adenylates and aminoacyl sulfamates derived from arginine, histidine and threonine, have been prepared and tested as inhibitors of their cognate Staphylococcus aureus aminoacyl tRNA synthetases. The arginyl derivatives were both potent nanomolar inhibitors of the Class I arginyl tRNA synthetase whereas for the Class II histidyl and threonyl tRNA synthetases, the acyl sulfamates were potent inhibitors but the adenylates had very little affinity.
This paper describes the design and characterization of novel inhibitors of IleRS, whose binding affinity approaches the tightest reported for noncovalent inhibition. Compounds were designed from a binding model for the natural product pseudomonic acid-A (PS-A) together with a detailed understanding of the reaction cycle of IleRS and characterization of the mode of binding of the reaction intermediate IleAMP. The interactions of the compounds with IleRS were characterized by inhibition of aminoacylation of tRNA or PP(i)/ATP exchange at supersaturating substrate concentration and by transient kinetics and calorimetry methods. A detailed understanding of the interaction of a comprehensive series of compounds with IleRS allowed the identification of key features and hence the design of exquisitely potent inhibitors. Predictions based on these results have been recently supported by a docking model based on the crystal structure of IleRS with PS-A [Silvian, L. F., Wang J. M., and Steitz T. A. (1999) Science 285 1074-1077].
We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.
New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cell-based assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.
SB-219383 is a naturally occurring antibiotic, which acts by inhibition of tyrosyl tRNA synthetase. Semi-synthetic derivatives of SB-219383 were prepared with the objective of elucidating the key features required for inhibition of tyrosyl tRNA synthetase in order to improve the antibacterial activity. Some ester and amide derivatives as well as monocyclic analogues exhibited sub-nanomolar inhibitory activity against tyrosyl tRNA synthetase.
A novel, potent and selective inhibitor of bacterial tyrosyl tRNA synthetase, designated SB-219383 has been isolated from Micromonospora sp. NCIMB 40684. The fermentation, isolation and some properties are described, whilst the structure determination is described in the succeeding paper). SB-219383 showed competitive, inhibitory activity against a Staphylococcus tyrosyl tRNA synthetase, with an IC50 of <1 nM, and exhibited weak in vitro activity against some Streptococcus sp.
Molecular modelling and synthetic studies have been carried out on tyrosinyl adenylate and analogues to probe the interactions seen in the active site of the X-ray crystal structure of tyrosyl tRNA synthetase from Bacillus stearothermophilus, and to search for new inhibitors of this enzyme. Micromolar and sub-micromolar inhibitors of tyrosyl tRNA synthetases from both B. stearothermophilus and Staphylococcus aureus have been synthesised. The importance of the adenine ring to the binding of tyrosinyl adenylate to the enzyme, and the importance of water-mediated hydrogen bonding interactions, have been highlighted. The inhibition data has been further supported by homology modelling with the S. aureus enzyme, and by ligand docking studies.