Biological Evaluation of Compounds

Data from Sandra Duffy, Griffith University Received April 25th 2012

Four compounds from the arylpyrrole series tested in a late stage anti-gametocyte imaging assay.

Relevant compounds:

Note - mistake in picture ZHY 6 was sent, which is OSM-S-38, not OSM-S-37 as shown in the figure.

Method: The assay involves the use of a Pfs16-GFP transgenic NF54 Plasmodium falciparum strain kindly provided by Dr David Fidock (Columbia University). Compound is added to 384 well imaging plates containing 20,000 highly synchronous late stage IV gametocytes. The plates are incubated for 72 hours in standard incubation conditions (5%CO2, 5%O2, 37oC, 5% humidity). After incubation the viability marker MitoTracker Red CM-H 2 XRos is added to the wells. The plates are then imaged on the OPERA confocal high-throughput imaging system. The images obtained are then analysed using an algorithm for determining the number of viable gametocytes still remaining in the compound treated wells based on viability marker fluorescent intensity and the morphology of the GFP expressing parasite. The number of gametocytes per well in this assay is the same number of parasites used in the asexual imaging assay for 3D7 and K1. Results: PMY10-2 has no significant activity whilst PMY14-1 has activity comparable to that in the asexual assay. (MHT: Others?)

Raw data:

Caveat: It has to be noted that the compounds have been in storage for a while and the plates defrosted several times for screening. The inactive compound could have lost asexual activity at the same time due to some instability. This was not tested.

Assay performed by Essen Bioscience 17th April 2012

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:


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



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.

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

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

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:

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