The MoA group was established 4 years ago to underpin the drug-discovery programmes within WCAIR and also works with multiple academic groups and key stakeholders in NTD drug discovery. The group is focused on determining the MoA and/or molecular targets of compounds that are phenotypically-active against parasites. In the course of our studies, we have developed an integrated drug target deconvolution strategy, employing a matrix of established and new methodologies encompassing high-throughput genetics, cell biology and chemical proteomics (Figure 1). This multi-disciplinary approach to drug target deconvolution has proven extremely effective, with the molecular targets of 19 compound series (including 4 pre-clinical candidates) identified to date. Information emanating from these MoA studies has been invaluable in guiding anti-kinetoplastid drug discovery programmes. For example, novel drug targets have been exploited using structure-based approaches and new target-based screens, compounds that act via mechanisms unsuitable for drug development have been de-prioritised, and diverse chemical series have been identified that inhibit a particularly promiscuous molecular target. In addition, we have used our MoA toolkit for unbiased confirmation of on-target activity for compounds developed in target-focussed programmes. Owing to our success, we have recently received funding from the Bill and Melinda Gates Foundation and Medicines for Malaria Venture to support MoA studies in Plasmodium. In our new programme of research we propose to capitalise on experience gained in recent years and to substantially expand our multi-disciplinary approach to drug target deconvolution into new disease areas.
Figure 1. Overexpression library workflow.KD, knock-down; OE, over-expression; WGS, whole genome sequencing; FACS, fluorescence activated cell sorting; CETSA, cellular thermal shift assay; SILAC, stable isotope labelling by amino acids in cell culture.
Innovation in drug target deconvolution
To support our multi-disciplinary approach to drug target deconvolution the MoA group has established and implemented several novel methodologies.
High-throughput genetics – We have developed genome-wide overexpression libraries in all three kinetoplastid parasites (Trypanosoma brucei, Leishmania donovani and T. cruzi). These libraries are based on the principal that overexpression of a drug target may confer a selective advantage to parasites under drug selection (Figure 2). Thus, our genome-wide overexpression libraries, in combination with Next Generation Sequencing, now enable us to carry out unbiased and massive parallel screens of the parasite proteomes for the targets of drugs. We are also in the process of developing an analogous overexpression library in Plasmodium knowlesi.
Figure 2. Overexpression library workflow.
Cell biology and biochemistry – As a direct result of our MoA studies, we have identified a number of novel and high value drug targets that are exploitable for future drug discovery. In these cases, we have created complementary cell-based tools to facilitate our subsequent drug discovery efforts.
Chemical proteomics – Chemical proteomics has proved a powerful route for the identification of drug targets in our studies. We use compounds immobilised on magnetic beads to directly pull-down proteins from parasite cell extracts. To improve the discrimination of proteins that bind specifically/non-specifically to beads, we carry out these pulldown studies in conjunction with Stable Isotope Labelling by Amino acids in Cell culture (SILAC) quantitative proteomics methodology (Figure 3). Proteins specifically binding to drug beads are subsequently identified by mass spectrometry.
Figure 3. Pulldown strategy.
In cases where immobilisation compromises the ability of the compound to bind to its target(s), or if there is limited / no structure-activity relationship (SAR) data, we employ thermal proteome profiling (TPP) assays (Figure 4). The MoA group was the first to establish this innovative approach for use in the kinetoplastids. TPP is an unbiased, powerful label-free proteomics approach based on the principle that binding of a drug to its protein target may significantly increase the thermal stability of that protein.
Figure 4. TPP workflow.