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25th September 2014 @ 10:00

Six "Near Neighbour" compounds in OSM Series 1 were synthesised by an undergraduate cohort at Lawrence University supervised by Stefan Debbert. The compounds have been evaluated for potency by Julie Clark in Kip Guy's lab. The results are summarised below and may be found in full in the attached spreadsheet. Julie said "I ran 2 independent experiments with a data analysis for each experiment, then I re-ran the data analysis for both runs together (combined analysis)." The data will be folded into the first OSM paper that is being written.

Debbert Guy Data

Two of the compounds (OSM-S-37 and OSM-S-111) have been evaluated for potency before. The new numbers matche very well in one case, and not so well in the other. One compound (OSM-A-2 note that the "A" stands for Appleton, where the compounds were made) appears to be unstable, given the difference in the values obtained between the two measurements. Two of the other compounds (OSM-A-1 and OSM-A-4) display low activity. One new compound of the six (OSM-A-3) displays good potency, in fact the best of the set.

At the time of writing, this series is parked, but can be pursued further by anyone. Compounds are potent and synthetically accessible.

(A note from the OSM team to the students who made these compounds - way to go, guys)

 

(This entry authored by Mat)

Codes:

OSM-S-111, SJ000849514-1, 

OSM-A-2, SJ000849515-1, 

OSM-A-1, SJ000849516-1, 

OSM-A-4, SJ000849517-1, 

OSM-S-37, SJ000849518-1, 

OSM-A-3, SJ000849519-1, 

Attached Files
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 -p(IC50) and LogP vs -log10(IC50)

Excel Data

hERG data September 2014.xlsx

Raw Data

*MHT to post

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)

 Post originally authored by Alice E Williamson

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