ABSTRACT
Small molecule screening, the systematic encounter of biology space with chemical space, has provoked the emergence of a whole industry that recreates itself by constant iterative improvements to this process. The authors describe an approach to tackle the problem for one of the most time-consuming steps in the execution of a screening campaign, namely, the reformatting of high-throughput screening test compounds from master plates to daughter assay plates used in the execution of the screen. Through an engineered storage procedure, they prepare plates ahead of the screening process with the respective compounds in a ready-to-use format. They show the biological inertness of the method and how it facilitates efficient recovery of compound activity. This uncoupling of normally interconnected processes provides time and compound savings, avoids repeated freeze-thaw cycles of compound solutions, and removes the problems associated with the DMSO sensitivity of certain assays types.
Subject(s)
Chemistry, Pharmaceutical/methods , Drug Evaluation, Preclinical/methods , Automation , Chromatography, Liquid , Combinatorial Chemistry Techniques , Drug Stability , Drug Storage/methods , Inhibitory Concentration 50 , Mass Spectrometry , Models, Chemical , Molecular Weight , Nanotechnology , Pharmaceutical Preparations , Solubility , Specimen Handling , Temperature , Time FactorsABSTRACT
Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Enterococcus/drug effects , Enzyme Inhibitors/chemical synthesis , Methionine-tRNA Ligase/antagonists & inhibitors , Quinolones/chemical synthesis , Staphylococcus/drug effects , Abscess/drug therapy , Abscess/microbiology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Quinolones/chemistry , Quinolones/pharmacology , Rats , Rats, Sprague-Dawley , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Structure-Activity RelationshipABSTRACT
A series of 1-(biphenylmethylamidoalkyl)-pyrimidones has been designed as nanomolar inhibitors of recombinant lipoprotein-associated phospholipase A(2) with high potency in whole human plasma. 5-(Pyrazolylmethyl) derivative 16 and 5-(methoxypyrimidinylmethyl) derivative 27 demonstrated excellent pharmacodynamic profiles which correlated well with their pharmacokinetic effects.
Subject(s)
Phospholipases A/antagonists & inhibitors , Pyrimidinones/pharmacokinetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Administration, Oral , Animals , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Inhibitory Concentration 50 , Kinetics , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Rabbits , Structure-Activity RelationshipABSTRACT
This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.
