Project Details
Description
Leishmaniasis threatens millions of people in the tropics and sub-tropics with >1.5 million new cases reported each year and ~50k deaths. Current treatments are difficult to administer, toxic and prone to emerging drug resistance, articulating an urgent unmet need for new drugs. We have identified a parasite enzyme (deubiquitinase, DUB1) with excellent characteristics as a drug target. We propose here to produce and purify DUB1, grow crystals of the enzyme so that we can determine its structure by X-ray diffraction methods. This will provide an atomic level image of the enzyme and a platform for inhibitor discovery and design.
Layman's description
Leishmaniasis threatens millions of people in the tropics and sub-tropics with >1.5 million new cases reported each year and ~50k deaths. Current treatments are difficult to administer, toxic and prone to emerging drug resistance, articulating an urgent unmet need for new drugs. We have identified a parasite enzyme (deubiquitinase, DUB1) with excellent characteristics as a drug target. We propose here to produce and purify DUB1, grow crystals of the enzyme so that we can determine its structure by X-ray diffraction methods. This will provide an atomic level image of the enzyme and a platform for inhibitor discovery and design.
Key findings
Using the award, we have made progress in characterising LmDUB1. Specifically, we have (1) expressed the 65 kDa LmDUB1 in good yield using a baculovirus-based expression system established by Andreas Damianou. (2) developed an effective protocol for purification of the intact protein to ~95 % homogeneity as judged by Coomassie staining of polyacrylamide gels. (3) concentrated this protein to ~ 8 mg.ml-1 without precipitation and initiated crystallisation trials. An obstacle to crystallisation and structural study however is the susceptibility of this protein to proteolysis during storage at 4˚C. Thus (4) we are targeting smaller enzyme fragments. Both trypsin and chymotrypsin generate a ‘stable’ 43 kDa fragment which we deduce to be the result of N-terminal truncation by peptide fingerprinting mass spectrometry.
Next Steps - The funds have allowed a collaboration between Jeremy Mottram and Tony Wilkinson to be established and this collaboration has been extended and incorporated into ‘A Global Network for Neglected Tropical Diseases’ centred on Durham and York with partners in Argentina, Brazil, India, Pakistan and Uruguay which has attracted substantial support from RCUK. JCM is a CoI and AJW is a collaborator on this award.
Next Steps - The funds have allowed a collaboration between Jeremy Mottram and Tony Wilkinson to be established and this collaboration has been extended and incorporated into ‘A Global Network for Neglected Tropical Diseases’ centred on Durham and York with partners in Argentina, Brazil, India, Pakistan and Uruguay which has attracted substantial support from RCUK. JCM is a CoI and AJW is a collaborator on this award.
| Short title | Deubiquitinase Inhibitors |
|---|---|
| Acronym | DUBInhib |
| Status | Finished |
| Effective start/end date | 1/10/16 → 30/09/17 |
Funding
- BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL)