I am planning to see the effect of a small molecule inhibitor on bloodstream form Trypanosoma brucei. For this purpose, I will do growth assay of the parasites after treating them with different concentration of the drug. There is no report on use of this compound in parasites, so I was wondering:
1. What concentration of this compound should I start with? There is a report in other model organism, where the IC50 of this compound was 30micromolar, but I am not sure how to plan the experiment in parasites (micromolar or nanomolar range?)!
2. Is Alamar blue treatment a good strategy to check cell viability post drug treatment?
Any help will be appreciated!!
Hi Aditi,
30 micromolar sounds a bit on the higher side for parasites. I think it will be a good idea to start the concentration at nanomolar level and reach upto say 10 micromolar. Thanks!
Hi, Mukherjee
do not warry about the using experimental concentrations, you should do the pilot study before choosing the mean concentrations. As my knowledge, Tryparsamide, Pentamidine isethionate and Suramine … etc. usually used to treatment of trypanosomiasis. However, the below links could be useful for your work:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811327/
https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0005125
Best regards
Hi Ayan,
Thanks for the suggestions! I was looking at the literatures where others used small molecule inhibitors as drug target in Kinetoplastids and found different range of concentrations being used (even 33micromolar in a study). I also think its a good idea as you mentioned, to select a wide range of concentrations starting from nanomolar to micromolar range and see how the cells behave!!
Check out this paper Article Discovery of trypanocidal coumarins with dual inhibition of ...
Hi Aditi,
Assuming you have enough compound, I'd recommend carrying out a pilot EC50 analysis using 5 or 10-fold serial dilutions across a wide range of concentrations (from high micro to sub-nanomolar). You'll then be able to focus on a narrower range that includes the EC50.
Repeating the analysis using two-fold dilutions that encompass this range will give you an accurate estimate of the compound's EC50 versus cultured BSF T. brucei.
Details of the protocol and a couple of key refs can be found here:
https://blogs.lshtm.ac.uk/alsfordlab/protocols/ec50-analysis/
Good luck with your experiments.
Sam.