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20th April 2012 @ 21:22
Assay performed by Essen Bioscience 17th April 2012

Relevant compounds:
Relevant Compounds


Methods overview
Compounds were tested for inhibition of the human ether a go-go related gene (hERG) K+ channel using IonWorks patch clamp electrophysiology. 8-Point concentration-response curves were generated using 3-fold serial dilutions from the maximum final assay concentration shown in this spreadsheet.

Image of results:

Snapshot of table

(legend: Compound (ID, number in assay), pIC50 (value, 95% CI), IC50 in micromolar(value, 95% CI), Curve profile (slope, [max] in micromolar, cell count, QC, notes). For full explanations, see notes in spreadsheet linked above)

Graphs of outputs:

OSM-S-35 (ZYH 3):
Response Curve ZYH

OSM-S-5 (Original GSK, PMY 10):
GSK hERG

Quinidine:
Response Curve Quinidine


In each graph the log of molar concentration of test compound is plotted against the effect of the compound on the hERG current, expressed as a percentage of the pre-compound signal. In this experiment an average of 9±3% signal decay was observed in the time- and vehicle (DMSO) control. Data are normalised for this vehicle-response such that a value of 100% = no drug effect. The smaller grey symbols represent data from individual cells. The larger blue points show the mean data at each test concentration. The line of best fit (4 parameter logistic equation) is generated from the individual cell data points. Outlier data points (boxed symbols) were manually excluded from the curve fit. No more than two per curve were excluded, other than where compounds were visibly out of solution.

Details of method
Electrophysiological recordings were made from a Chinese Hamster Lung cell line stably expressing the full length hERG channel. Single cell ionic currents were measured in the perforated patch clamp configuration (100 μg ml-1) amphoterocin) at room temperature (21-230C) using an IonWorks Quattro instrument. The internal solution contained (mM): 140 KCl, 1 MgCl2, 1 EGTA, 20 HEPES and was buffered to pH 7.3. The external solution contained (mM): 138 NaCl, 2.7 KCl, 0.9 CaCl2, 0.5 MgCl2, 8 Na2HPO4, 1.5 KH2PO4 also buffered to pH7.3. Cells were clamped at a holding potential of -70mV for 30s and then stepped to +40mV for 1s. This was followed by a hyperpolarising step of 1s to -30mV to evoke the hERG tail current. This sequence was repeated 5 times at a frequency of 0.25Hz. Currents were measured from the tail step at the 5th pulse, and referenced to the holding current. Compounds were then incubated for 6-7 minutes prior to a second measurement of the hERG signal using an identical pulse train.

Electric cycle method


The following QC conditions were applied:

(1) Individual cells with any of the following properties were excluded from subsequent analysis: (1) seal resistances <50MOhms (2) hERG currents <150pA (3) seal resistances that changed by >50% during the experiment

(2) A minimum of 17 cells were required for each pIC50 curve fits

(3) pIC50 curve fits with a 95% confidence limit of > ±0.5 log were failed

(4) Entire assay plates in which the pIC50 of the standard compound (quinidine) was outside of the normal range [5.6-6.3] were failed.
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19th April 2012 @ 04:51

Data obtained for compounds synthesised by Soumya Bhattacharyya and Sanjay Batra.

Conclusions: 1) 3-fluoro "near neighbour" derivative is much less active than closely related compounds described here. 2) The IC50 for PMY 10-6 broadly matches the results obtained by other labs, meaning this is a good control to use in conjunction with chloroquine.

Data for (3-fluorophenyl) pyrrole derivative SBBH-1-9 (or isomer) Synthesis of (3E)-3-((1-(3-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-1-phenyl-5-(phenylimino)pyrrolidin-2-one or it’s isomer (SBBH-1-9)

(Original data posted to G+ (with wrong name) - screenshot attached)

3D7:

Sample Code, IC50 (microM), CC50 (microM), SI SBBH-1-9: 3.2, 92.4, 28.87 Chloroquine: 0.0063, 125.85, 19976.2

K1 strain:

Sample Code, IC50 (microM,) CC50 (microM), SI SBBH-1-9: >5.0, 92.4, 18.48 Chloroquine: 0.235, 125.85, 535.53

Original 3-F data

Control compounds sent from Sydney was PMY 10-6 (TCMDC-123812) Synthesis of TCMDC-123812 via acid chloride (PMY 10-6), which was evaluated (13/04/2012) vs.chloroquine. Data attached

Controls

and results pasted here:

For 3D7: PMY 10-6 IC50 0.581 microM, CC50 (cytotoxicity) nd, SI (selectivity index) - CQ IC50 0.0083 microM, CC50 125.85, SI 15162.7

For K1: PMY 10-6 IC50 0.641, CC50 nd, SI - CQ IC50 0.252, CC50 125.85, SI 499

Method used for cytotoxicity assay:

cytotox assay SOP

In more recent correspondence in late 2014, assay protocol was updated by Sanjay Batra:

Text S Assay Protocol Batra New.docx
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