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22nd September 2014 @ 05:02

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

 

 

*[To be edited by Paul Willis] Graphical Representation of ClogP vs -log10(IC50) and LogP vs -log10(IC50)

Excel Data


Preliminary Conclusions

Initial examination of the compounds might suggest that amides/amines show problematic hERG activity whereas ethers are tolerated. However, it is also possible that the Para OCHF2 ether is the problem. 

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. In general, only compounds possessing lower LogP values should be synthesised in the next round in order to reduce the likelihood of hERG interactions. 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 IonWorksTMHT. Journal of Pharmacological and Toxicological Methods 54: 189–199 (10.1016/j.vascn.2006.02.003)

 

Attached Files
21st September 2014 @ 01:15

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
4th September 2014 @ 07:21

A further set of Series 4 Triazolopyrazine compounds have been sent to have their efficacy evaluated against Plasmodium falciparum in-vitro at Syngene.

The compounds are ethers and amides. Compounds are intended by default to have cLogP < 3.5. Those that do not are either older compounds already made, or are evaluating something specific, e.g. the prototypical ether compound OSM-S-260 or OSM-S-259 which assesses potency of benzylic ethers. Results will be posted in due course.

 

OSM Compounds Sep Syngene.png

General assay principle:
"This protocol assesses compound efficacy against Plasmodium falciparum in-vitro. This assay is using [3H]-hypoxanthine incorporation or DNA labeling by SYBR Green as a markers of parasite growth.
This procedure is designed for use with culture adapted P. falciparum strains or clones only. On one 96-well plate typically 03 drugs are tested in duplicate. Standard strains: Plasmodium falciparum, NF54 (sensitive to all known drugs), Plasmodium falciparum, K1 (chloroquine and pyrimethamine resistant). The assay can be performed in dose response mode (12 concentrations in duplicate, 24 data points) which allows determining IC50, or in single concentration mode (one concentration in triplicate, 3 data points) which allows determining the percentage of growth inhibition.
For more information, see Desjardins et al. (Antimicrob. Agents Chemother., 16(6), 710, 1979)."

 

(Post originally authored by Alice Williamson, tweaked by Mat)

Attached Files
27th August 2014 @ 01:56

The following compounds have been evaluated by Kiaran Kirk and Adele Lehane in their ion regulation assay, and will now be evaluated vs ATP4-resistant mutants in Kiaran's lab. The same samples are being sent to David Fidock's lab at Columbia to testing vs his ATP4 resistant mutants, for comparison. Compounds being shipped 28th August 2014. Results will be posted to this lab notebook when obtained.

 

Kirk and Fidock ATP4 Resistance Assay

Attached Files
14th August 2014 @ 22:58

An assay was performed on several Series 1 compounds in July 2013 by GSK to determine activity vs P. falciparum Dihydroorotate dehydrogenase (DHODH). Assay carried out by María José Lafuente. Reports/Data attached.

Compounds tested:

PMY 12-5 OSM-S-8 Preparation of OSM-S-8

PMY 27-2 OSM-S-16 Preparation of OSM-S-16

PMY 31-5 OSM-S-19 Preparation of OSM-S-19

PMY 34-1 OSM-S-21 Preparation of OSM-S-21

ZYH 3-1 OSM-S-35 Preparation of OSM-S-35

ZYH 5-1 OSM-S-37 Preparation of OSM-S-37

ZYH 6-1/6-2 OSM-S-38 Preparation of OSM-S-38

ZYH 7-2 OSM-S-39 Preparation of OSM-S-39

ZYH 10-2 A OSM-S-42 Preparation of OSM-S-42

ZYH 10-2 B OSM-S-43 Preparation of OSM-S-43

ZYH 12-1/12-2 OSM-S-45 Preparation of OSM-S-45

ZYH 15-1 OSM-S-48 Preparation of OSM-S-48
ZYH 16-1 OSM-S-49 Preparation of OSM-S-49
ZYH 17-1 OSM-S-50 Preparation of OSM-S-50
ZYH 18-1 OSM-S-51 Preparation of OSM-S-51
ZYH 19-1 OSM-S-52 Preparation of OSM-S-52
ZYH 22-3 OSM-S-54 Preparation of OSM-S-54
ZYH 23-1 OSM-S-55 Preparation of OSM-S-55

PMY12-1 OSM-S-7 Preparation of OSM-S-7
PMY 2-4 OSM-S-2 Preparation of OSM-S-2
PMY 6-1 OSM-S-3 Preparation of OSM-S-3
PMY 8-2 OSM-S-4 Preparation of OSM-S-4
PMY 11-2 (TCMDC-123794) OSM-S-6 Preparation of OSM-S-6

PMY 10-2 (TCMDC-123812) OSM-S-5 Preparation of OSM-S-5

PMY 14-1 OSM-S-9 Preparation of OSM-S-9

Result: No activity

 

(This post authored by Mat Todd)

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Attached Files