Patch clamp assays

The two most frequently studied compounds with T-type calcium channel antagonist properties are ethosuximide 1 and mibefradil 3. However, the modest potency of ethosuximide ( 200 pM) [48] and the poor selectivity of mibefradil [49] make these compounds suboptimal tools for the investigation of these channels. Guided by a pharmacophore model [50], several analogs of 3 were prepared. Compound 4 represents the most potent compound identified (IC50 8 nM, patch-clamp assay) with good selectivity over the L-type calcium channel [51], Compound 4 showed a modest brain-to-plasma ratio (0.25) after oral dosing to rats at 50 mg/kg. However, no in vivo efficacy assay results have been reported with this compound. 

The benzhydryl substituent is common to many reported N-type calcium channel blockers. A recently reported series derived from the neuroepileptic flunarizine (5), which exhibits N-type calcium channel potency in a whole-cell patch-clamp assay (IC50 0.08 iM) as well as L-type potency... 

However, when compound 15 was tested for h E RG K+ channel inhibition, a basic cardiovascular safety indicator related to the QT prolongation syndrome [32], and linked to ventricular fibrillation and sudden death [33], we were unpleasantly surprised. Although the lead compound 1 exhibited very weak binding to the hERG K+ channel (IC50 > 5000 nM), the optimized compound 15 was afairly potent hERG blocker with an inhibition concentration IC50 of 300 nM (in patch-clamp assay). 

Redfern et al. studied the occurrence of QT prolongation and the lethal arrhythmia torsades de pointes (see Chapter 16) in marketed drugs from the perspective of the therapeutic index [50]. This group proposed a therapeutic index of 30-fold, calculated from the free Cmax at the therapeutic dose and the in vitro potency in the manual hERG patch clamp assay. This level, or even higher, seems to be generally followed by the industry. 

Owing to the pioneering efforts of C. Kung, the best characterized mechanosensitive channel is the prokaryotic MscL, the mechanosensitive channel of large conductance. MscL was originally identified, isolated, and characterized by Kung and co-workers using a biochemical approach in combination with a patch-clamp assay to isolate an intrinsically stretch activated channel from E. coli membranes (Sukharev et al.,... 

Herg blockade is mesmerising the pharmaceutical industry, not just due to the implications for drug discovery, but also because the difficulty is understanding the interplay between displacement of dofetilide, patch clamp assays, dog telemetry and the occurrence of toursade des pointes in humans. Several groups have developed... 

HERG ("Human-ether-a-go-go related gene) at low concentrations. Conformationally constrained isoquinuclidine analogs of BRL-32872 were also prepared to study the effects of molecular flexibility towards class III and class IV anti-arrhythmic properties [48]. Both standard microelectrode and patch clamp assays indicated that while both epimers displayed a pharmacological profile similar to BRL-32872, epimer 52b was more effective than 52a. 

Note 4 Manual patch clamp assay is a gold standard for functional analysis of ion channels and their interactions with the drugs. Major limitation of this assay includes low through put, need of high amount of compound for testing, and requirement of highly skilled technician. The assay can be used in the late phase of drug discovery for lead optimization. 

Hierachical PLS N= 1312 molecules data generated in house with lonWorks HT patch clamping assay. N = 7520 additional molecules tested over time in the same assay. Used SELMA, DRONE, VOLSURF, fragment descriptors training set mean RMSE = 0.40, test set RMSE 0.49, within experimental error of assay (0.5 log units). 166... 

Each of these techniques can give quite different hERG IC50 values for the same compound. Eor example, terfenadine s IC50 has been measured at 30 nM in the patch-clamp assay, 4.8 pM in the rubidium flux assay, and 500 nM in a binding assay. Needless to say, numbers from different assays, different cells, or different labs for that matter, can t be directly compared. 

Amuzescu B, Scheel O, Knott T (2014). Novel automated patch-clamp assays on stem cell-derived cardiomyocytes will they standardize in vitro pharmacology and arrhythmia research J Phys Chem Biophys 4 4. 

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