Covalent Inhibition of Essential Deubiquitinases of Leishmania

Background: The ubiquitin proteasome system is an important mechanism for protein degradation and cell signaling. Ubiquitin is coupled to target proteins through the sequential action of E1, E2 and E3 conjugating enzymes. Meanwhile, deubiquitinases (DUBs) remove ubiquitin from target proteins and breakdown polyubiquitin chains. Four of the 20 identified DUB genes in L. mexicana are essential and the encoded DUBs are cysteine proteases belonging to the ubiquitin-specific protease and ubiquitin-carboxyl terminal hydrolase families. Here we propose to screen a library of cysteine reactive fragments to identify those that form covalent adducts with the DUBs.
Previous collaborative work between York and our industrial collaborator has demonstrated the promise of this approach targeting bromodomain-containing proteins with a panel of 240 fragments with sulfonyl fluoride warheads. These reacted with binding site tyrosine residues and displayed selectivity between targets.

Objectives:
Screen a library of covalent fragments against the four essential DUBs of L. mexicana using mass spectrometry and activity assays
Crystallise DUB:fragment complexes to guide the development of higher potency inhibitors
Develop a method to measure on-target engagement in parasites using an LC-MSMS based quantitative assay
Use inhibitors as chemical tools to probe the downstream biological impact of DUB inhibition and identify substrate proteins and interactors

Novelty: Leishmania belong to the kinetoplastida - a deeply branched group of eukaryotes whose molecular biology differs from that of model organisms and the human host. Specific inhibitors of DUBs have the potential to serve as chemical tools to elucidate pathways of regulation in these organisms.

Timeliness: The essential components of the ubiquitination and deubiquitination system of L. mexicana have been established. Meanwhile, the Chemical Biology group of our industrial collaborator has been developing a suite of reactive fragment platforms for the efficient identification of covalent inhibitors and these have been applied successfully to bromodomain-containing proteins from Leishmania.

Experimental Approach: We have constructs expressing three of the target DUBs and the first goal will be to develop a construct expressing the fourth. The student will purify the four recombinant enzymes for screening against a covalent fragment library. This will involve parallel incubation of the DUBs with the fragments followed by assay by intact protein LCMS to identify specific mass shifts. Fragments of interest based on their reactivity and selectivity will be analysed by LC-MSMS of tryptic digests of the adducts to identify the site of covalent attachment. A round of hit expansion will be performed to explore the SAR of emerging hits and improve potency. Binding of these fragments to the DUBs will be characterised in biophysical assays and through crystallisation and structure determination. Their capacity to inhibit DUB activity can be measured in fluorimetric assays with ubiquitin-fluorophore conjugates.

The capacity of identified covalent fragments to engage with the selected DUB inside living Leishmania parasites will be assessed. These measurements can be made quantitative by spiking the resulting parasite lysates with a mass-labelled mixture of the free and covalent inhibitor complexed DUB.