Not all parasites are amenable to genetic approaches, therefore we need other methods to identify the target of compounds. First, we isolate the total protein from parasites and then allow the total collection of proteins to interact with the compound. We use physical force to break the parasites open and release all the proteins inside, this produces what we call a ‘lysate’.

We can use the lysate in two different experiments to identify their protein targets. One approach is to immobilise the compound, usually on a magnetic bead. The compound is free to interact with all the proteins in the lysate. After enough time, the compound will eventually bump into the target protein and bind. Using a magnet, we can physically pull the compound (via the magnetic bead) out of the lysate with its target protein attached. We call this a ‘pull down’ as we physically separate the compound and its target from the entire lysate. Using mass spectrometry, we can identify which protein is bound to the compound. We do this experiment in combination with ‘Stable Isotope Labeling by Amino acids in Cell Culture’ or SILAC to discriminate between real targets and proteins that bind non-specifically to the compound.

Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages. Urbaniak MD, Guther ML, Ferguson MA. PLoS One. 2012; 7: e36619.

Sometimes by adding the magnetic bead, we disrupt the ability of the compound to interact with its target protein. In this case we create a lysate and use a ‘Thermal Proteome Profiling’ approach to identify the protein target of the compound. This innovative approach is based on the idea that when a compound binds it target protein, that protein becomes stable at higher temperatures. We incubate the lysate with the compound and split this into several identical test tubes. Then we expose each tube to a different temperature. We use labels called ‘TMTs’ or Tandem Mass Tags to effectively colour-code each tube so we know what temperature it was exposed to. We then combine the samples and analyse them using a mass spectrometer. We can decode which proteins came from which tube based on the colour-coding and predict the identity of the target protein.

Pharmacological validation of N-myristoyltransferase as a drug target in Leishmania donovani. Corpas-Lopez V et al, ACS Infectious Disease. 2019, 51111-122.

Because it is often straightforward to produce a lysate from any parasite, these chemical proteomics approaches described here are a great starting point to identify targets in difficult-to-work with parasites and pathogens.