ABSTRACT
We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We report the investigation of various subunits in this series and advanced studies on compound 8. Compound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral efficacy in a rat lung infection model.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Topoisomerases, Type II/chemistry , Dioxanes/chemistry , Dioxanes/pharmacology , Naphthyridines/chemistry , Naphthyridines/pharmacology , Piperidines/chemistry , Topoisomerase II Inhibitors/chemistry , Animals , Anti-Bacterial Agents/therapeutic use , DNA Topoisomerases, Type II/metabolism , Dioxanes/therapeutic use , Disease Models, Animal , Dogs , Haplorhini , Humans , Lung Diseases/drug therapy , Microbial Sensitivity Tests , Naphthyridines/therapeutic use , Piperidines/pharmacology , Piperidines/therapeutic use , Rats , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacology , Topoisomerase II Inhibitors/therapeutic useABSTRACT
As part of our wider efforts to exploit novel mode of action antibacterials, we have discovered a series of cyclohexyl-amide compounds that has good Gram positive and Gram negative potency. The mechanism of action is via inhibition of bacterial topoisomerases II and IV. We have investigated various subunits in this series and report advanced studies on compound 7 which demonstrates good PK and in vivo efficacy properties.
Subject(s)
Amides/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Topoisomerases, Type II/chemistry , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Topoisomerase II Inhibitors/chemistry , Amides/chemical synthesis , Amides/pharmacokinetics , Animals , Anti-Bacterial Agents/chemical synthesis , Binding Sites , Computer Simulation , DNA Topoisomerases, Type II/metabolism , Dogs , Haplorhini , Humans , Microbial Sensitivity Tests , Protein Structure, Tertiary , Rats , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemical synthesis , Topoisomerase II Inhibitors/pharmacokineticsABSTRACT
The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.
Subject(s)
Cell Line/cytology , Drug Discovery/methods , Electrophysiology/methods , Ion Channels , Electrophysiology/instrumentation , Humans , Tissue Array AnalysisABSTRACT
Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.
