Investigating bromodomain proteins as targets for antileishmanial drug discovery

Leishmania is a kinetoplastid parasite and the causative agent of the neglected tropical disease, leishmaniasis. Despite the high disease burden, current treatments for leishmaniasis show toxic side effects and growing resistance, so new antileishmanials are urgently needed. Bromodomain inhibitors have shown huge potential for the treatment of other diseases, and may also present an attractive drug target in Leishmania. Five bromodomain factor (BDF) proteins are found in Leishmania which are essential for promastigote survival. Bromodomains are small, epigenetic reader domains which recognise acetylated lysine residues on histone tails and regulate gene expression. The aim of this work was to validate Leishmania bromodomain proteins as targets for the development of new antileishmanials. The binding of ligands to recombinant Leishmania donovani BDF bromodomains was investigated using biophysical methods including thermal shift assays, fluorescence polarisation assays and NMR. Exploring the peptide recognition profile of the tandem bromodomain protein, Leishmania donovani BDF5 (LdBDF5) revealed binding sites in acetylated peptides derived from the tails of histones H2B and H4. Screening a library of 15 commercially available human bromodomain inhibitors against LdBDF proteins identified three binding compounds, including SGC-CBP30 which bound to LdBDF5 in biophysical assays and also displayed significant cytotoxicity against Leishmania promastigotes in cell viability assays. Subsequent work sought to identify novel inhibitors of BDF5. Collaborative high-throughput screens and SAR-optimisation efforts yielded a series of hit compounds. Using orthogonal biophysical assays, binding interactions were characterised and a smaller cohort of promising lead compounds selected. Gratifyingly, these compounds were phenotypically active against promastigotes, with EC50 values comparable to miltefosine, a leading antileishmanial drug. These findings validate Leishmania BDF5 as a viable drug target and describe the first steps taken towards the generation of novel antileishmanial compounds to tackle the global burden of this neglected tropical disease.