ABSTRACT
Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates.
Subject(s)
Biological Assay , High-Throughput Screening Assays , Kv1.3 Potassium Channel/antagonists & inhibitors , Membrane Potentials/physiology , Potassium Channel Blockers/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Electrophysiology , Fluorometry , Inhibitory Concentration 50 , Patch-Clamp Techniques , Sodium Azide/pharmacologyABSTRACT
Piperidine, pyrrolidine, and azetidine sulfonamides were examined as linkers in designing novel human beta(3) adrenergic receptor (beta(3)-AR) agonists. The azetidine derivative 37, and piperidine derivatives 7, 8, and 13 were found to be potent beta(3)-AR agonists and have good selectivity against beta(1)- and beta(2)-AR.
Subject(s)
Adrenergic beta-3 Receptor Agonists , Adrenergic beta-Agonists/chemical synthesis , Adrenergic beta-Agonists/pharmacology , Sulfonamides/chemistry , Animals , Azetidines/chemical synthesis , Azetidines/pharmacology , CHO Cells , Cricetinae , Cyclization , Drug Design , Humans , Hydrogen Bonding , Molecular Conformation , Piperidines/chemical synthesis , Piperidines/pharmacology , Pyrrolidines/chemical synthesis , Pyrrolidines/pharmacologyABSTRACT
The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue degradation. In our preceding paper, we have reported on a series of novel and orally active N-hydroxy-alpha-phenylsulfonylacetamide derivatives. However, these compounds had two drawbacks (moderate selectivity and chirality issues). To circumvent these two problems, a series of novel and orally active N-substituted 4-benzenesulfonylpiperidine-4-carboxylic acid hydroxyamide derivatives have been synthesized. The present paper deals with the synthesis and SAR of these compounds. Among the several compounds synthesized, derivative 55 turned out to be a potent, selective, and an orally active MMP inhibitor in the clinically relevant advanced rabbit osteoarthritis model. Detailed pharmacokinetics and metabolism data are described.
