One of the best ways to identify the target of a compound is to use genetics. If we alter how much of the protein ‘target’ is made by the parasite, we should alter the effect of the drug on the parasite. For example, if we increase the amount of target protein made by the parasite, it will take more compound than usual to kill the parasite. We call increasing the amount of protein in a cell ‘overexpression’. We use genome-wide, high-throughput genetics to overexpress every protein made by the parasite. Then we grow these overexpressing parasites with our compound. Only parasites that continue to overexpress the ‘target’ of the compound will survive. We can figure out what proteins are being overexpressed using DNA sequencing. DNA sequencing usually produces a short list of possible targets. Using bioinformatics or follow up experiments we can identify the exact target of the compound. Although it sounds a bit complicated, this strategy is extremely robust and fast.
Read the papers using this technique.
(1) Clinical and veterinary trypanocidal benzoxaboroles target CPSF3. Wall RJ, Rico E, Lukac I, Zuccotto F, Elg S, Gilbert IH, Freund Y, Alley MRK, Field MC, Wyllie S, Horn D. Proc Natl Acad Sci U S A. 2018;115: 9616-9621.
(2) Pharmacological validation of N-myristoyltransferase as a drug target in Leishmania donovani. Corpas-Lopez V et al, ACS Infectious Disease. 2019, 5, 1, 111-122.
Another genetic strategy we use to identify the targets of compounds is just the opposite- we decrease protein expression. If we decrease the amount of target protein made by the parasite, it will take less compound than usual to kill the parasite. In T. brucei we can use genome-wide, high-throughput genetics to decrease the expression of every protein made by the parasite. This strategy is called RIT-Seq and was developed by the Horn Lab (see ‘How does gene expression affect parasite success?’). T. brucei have the ability to specifically stop production of specific proteins via a system called ‘RNAi’ or RNA interference. By high-jacking this system (which is natural to these parasites), we can artificially ‘interfere’ with the construction of proteins. We use RIT-Seq to stop production of every single parasite protein. Then we grow these under-expressing parasites with our compound. Only parasites that under-express the ‘target’ of the compound will not survive. We can figure out what proteins are being under-expressed using DNA sequencing.
These genetic tools for over or under expressing proteins are so robust for kinetoplastid parasites that we are now working to expand their utility for other parasites. Stay tuned!
After we have used genetic tools to identify possible targets of compounds, we use genetic tools to validate that these predictions are in fact the target. We do this by specifically modulating the expression of the predicted target protein. We can use methods describe above, or alternatively we can use CRISPR to engineer specific mutations of these target proteins, being developed in David Horn’s Lab for T. brucei and Mattie Pawlowic’s Lab for Cryptosporidium.