Deubiquitinases as Drug Targets in Neglected Tropical Diseases Global Challenges Research Fund Jeremy Mottram York Biology Tony Wilkinson Chemistry Ana Paula Lima Federal University of Rio de Janeiro, Brazil Julio Martin Glaxo-Smith-Kline, Spain Boris Rodenko UbiQ, Netherlands Drugs for Negelected Diseases Initiative (DNDi) Switzerland

NMTs from Protozoan Parasites and their Human Host H2N Protein substrate Gly N H + CoA-SH Multiple substrates Leishmania Human Brannigan et al., J. Mol. Biol. 396, 985 (2010); Brannigan et al., IUCr J. 1, 250 (2014) Frearson et al., Nature 464, 728 (2010) Hutton et al., J. Med. Chem. 57, 8664 (2014) Thinon et al., Nat Commun 5, 4919 (2014)

Ubiquitin and Ubiquitination Isopeptide bond between Lysine on Substrate Protein and C-terminal Glycine of Ub Subsequent isopeptide linkages between Lysines on proximal Ub and C-term of distal Ub Komander & Rape The Ubiquitin Code Annu Rev Biochem 81, 203 (2012)

Polyubiquitin Lys63 tetra-Ub Lys48 tetra-Ub 3HM3; Datta et al., J. Mol. Biol. 392, 1117 (2009) Lys48 tetra-Ub 2O6V; Eddins et al., J. Mol. Biol. 367, 204 (2007)

Ubiquitin Ligases, Ubiquitin Binding Proteins and Deubiquitinases Substrate Substrate Ub Deubiquitinase (DUB) Ub Ub Downstream Effects Mediated by proteins with Ubiquitin Binding Domains Protein Degradation – poly Ub(63) Regulation of Gene Expression – poly Ub(48)

Parasite deubiquitinases DUB – diUb Complex JC Mottram Laboratory 19 DUBs discovered in Leishmania using activity-based probes LmDUB1 shown to be essential – also essential in Trypanosoma brucei 1.7 million compounds screened by GSK against TbDUB1 using rhodamine-labelled Ub based fluorescence assay Qualified hit-list of 14 compounds Proximal Ub Distal Ub 5LRV; Mevissen et al., Nature (2016)

Plan Further genetic and chemical validation of DUBs as drug targets Crystallise and determine crystal structure of LmDUB1 as platform for inhibitor development Improve chemical properties of inhibitors – potency, selectivity, DMPK Funding Outline Application made for MRC GCRF Foundation Award – unsuccessful Bid for Pump-Priming to UoY BBSRC GCRF – successful – Elena Blagova 4 months 50 % + Consumables + Affinity Reagent