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21st September 2014 @ 00:15

Background

A selection of series 4 compounds were sent for evaluation again the hERG ion channel using a  "medium-throughput electrophysiology-based hERG assay using IonWorksTM HT" at AstraZeneca.

The compounds were selected from compounds synthesised at The University of Sydney (biological data here) and also those inherited from MMV following online discussion (see GitHub Issue #211).

 

Results

 

[image]

 

Graphical Representation of ClogP vs -log10(IC50) and LogP vs -log10(IC50)


General Assay Principle

"The hERG-expressing Chinese hamster ovary K1 (CHO) cells described by Persson, Carlsson, Duker, and Jacobson (2005) were grown to semi-confluence at 37 °C in a humidified environment (5% CO2) in F-12 Ham medium containing L-glutamine, 10% foetal calf serum (FCS) and 0.6 mg/ml hygromycin (all Sigma- Aldrich). Prior to use, the monolayer was washed using a pre- warmed (37 °C) 3 ml aliquot of Versene 1:5000 (Invitrogen). After aspiration of this solution the flask was incubated at 37 °C in an incubator with a further 2 ml of Versene 1:5000 for a period of 6 min. Cells were then detached from the bottom of the flask by gentle tapping and 10 ml of Dulbecco's phosphate-buffered saline containing calcium (0.9 mM) and magnesium (0.5 mM) (PBS; Invitrogen) was then added to the flask and aspirated into a 15 ml centrifuge tube prior to centrifugation (50×g, for 4 min). The resulting supernatant was discarded and the pellet gently re- suspended in 3 ml of PBS. A 0.5 ml aliquot of cell suspension was removed and the number of viable cells (based on trypan blue exclusion) was determined in an automated reader (Cedex; Innovatis) so that the cell re-suspension volume could be adjusted with PBS to give the desired final cell concentration. It is the cell concentration at this point in the assay that is quoted when referring to this parameter. CHO-Kv1.5 cells, which were used to adjust the voltage offset on IonWorksTM HT, were maintained and prepared for use in the same way."

Reference

Bridgland-Taylor MH, Hargreaves AC, Easter A, Orme A, Henthorn DC, Ding M, Davis AM, Small BG, Heapy CG, Abi-Gerges N, Persson F, Jacobson I, Sullivan M, Albertson N, Hammond TG, Sullivan E, Valentin J-P,  Pollard CE (2006) Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorksTMHT. Journal of Pharmacological and Toxicological Methods 54: 189–199 (10.1016/j.vascn.2006.02.003)

 

Attached Files
hERG Results Aug 2014 AEW.cdx
hERG compounds for MMV numbers.pdf
hERG Results Aug 2014 AEW.png
hERG for MMV numbers.png
hERG compounds for MMV numbers.xlsx