Biological Evaluation of Compounds

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 IonWorks^TM 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

(updated by Mat, original file still attached below. Reason for update: units provided in AZ assay were micromolar, as an IC50. These have been converted into the more standard pIC50, which is -log of the IC50 when expressed in Molar and is unitless)

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

(updated by Mat, original file still attached below)

Excel Data
(updated by Mat, original file still attached below)

Preliminary Conclusions

Initial examination of the new data suggests that amides/amines show problematic hERG activity whereas ethers are tolerated. However, it is also possible that the Para OCHF₂ ether is the problem. One of the original two data points (for MMV669844) shows that a compound lacking both is still active in the assay, so the answer is more subtle. There is little correlation between CLogP and hERG activity, but clogp is an approximation to actual solubility.

Future Work

The team need to synthesise some amides containing the para-nitrile aromatic group (or pyridyl) on the 'right-hand-side' in order to determine whether amides containing different aryl groups show hERG activity. Despite these data, only compounds possessing lower LogP values should be synthesised in the next round to aid solubility, and this will provide more data for the hERG/logP correlation. Following the next two rounds of synthesis and evaluation, more compounds will be evaluated in this assay.

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 IonWorks^TMHT. Journal of Pharmacological and Toxicological Methods 54: 189–199 (10.1016/j.vascn.2006.02.003)

 Post originally authored by Alice E Williamson