Ubiquitin conjugating system in Leishmania parasites

This project aims to identify players in ubiquitin conjugating cascades in the parasitic protozoan Leishmania and to validate their tractability as drug targets. Experiments will be performed in Leishmania mexicana, which causes cutaneous leishmaniasis and is easily cultured and genetically manipulated. The project further intends to investigate whether inhibitors can be developed, which are specific for leishmanial variants of ubiquitin conjugating enzymes over those present in the mammalian host cell. The project will be undertaken in collaboration with UbiQ, a Dutch biotech company that specialises in ubiquitin related reagents, peptides and assays. UbiQ is a spin-out of the Netherlands Cancer Institute in Amsterdam and has a wide range of technologies for target ID/validation and drug discovery.
The objectives of the project are (1) To identify novel ubiquitin-system targets in Leishmania using chemical proteomics (2) To validate potential targets using genetic manipulation such as CRISPR-Cas9 and inducible DiCre. These technologies will allow an assessment of essentiality in vivo in an animal model of infection, as well as in vitro to determine the role of essential ubiquitin conjugating enzymes in the biology of the parasite (3) To generate recombinant labelled fusion variants of leishmanial ubiquitinating enzymes in a heterologous expression system for drug screening purposes and raising antibodies for target localisation studies (4) To test the efficacy and selectivity of UbiQ's inhibitors of ubiquitinating enzymes on recombinant leishmanial ubiquitinating enzymes, on Leishmania cells and host cells in vitro and potentially in in vivo models of leishmaniasis.