Bromodomain Factor 5 as a Target for Antileishmanial Drug Discovery

Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited, and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains a pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarization assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarization, and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC–CBP30, bromosporine, and I-BRD9; moreover, SGC–CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a possible drug target in Leishmania and provide a foundation for the future development of optimized antileishmanial compounds targeting this epigenetic reader protein.


Figure S1 .
Figure S1.(A) 17.5% SDS-PAGE analysis of purified recombinant His-tag-cleaved BD2 with an expected molecular mass of 14.5 kDa.(B) SEC-MALLS analysis of recombinant His-tagged BD2 with arrow indicating MALLS curve labelled with the associated estimated molecular mass (predicted molecular mass 16.6 kDa).

Figure S4 .
Figure S4.Thermal shift assay melting curves for LdBDF5 BD5T with (A) pan-acetylated H2B9-23K9 Ac K15 Ac K19 Ac K21 Ac and (B) unmodified H2B9-23 peptides at 400 µM.Curves are normalised for five parameter sigmoid equation model fitting for six replicate samples of protein with peptides (blue) alongside DMSO control samples (orange).Graphs produced using the online JTSA tool.

Figure S5 .
Figure S5.Structures of 15 human bromodomain inhibitor compounds and the family of human protein target(s) in brackets.Thermal shifts from TSA screen with the LdBDF5 recombinant proteins given below, where compound concentrations are given in brackets.

Table S3 .
His-tagged and His-tag cleaved LdBDF5 BD5T recombinant protein details; DNA sequence codon-optimised for E. coli.

Table S4 .
His-tagged and His-tag cleaved LdBDF2 recombinant protein details.

Table S5 .
LdBDF5 BD5.2 apo structure crystallographic data collection and refinement statistics.a Values in parentheses correspond to the outer resolution shell.R merge = ∑ hkl ∑ i |I i -<I> |/∑ hkl ∑ i <I> where I i is the intensity of the ith measurement of a reflection with indexes hkl and <I> is the statistically weighted average reflection intensity.d Rpim = ∑hkl[1/(n-1)] 1/2 ∑i|Ii -<I>|/∑hkl∑iI Rwork = ∑||F o | -|F c ||/∑|F o | where F o and F c are the observed and calculated structure factor amplitudes, respectively.Rfree is the R-factor calculated with 5% of the reflections chosen at random and omitted from refinement.Root-mean-square deviation of bond lengths and bond angles from ideal geometry.Percentage of residues in most-favoured/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot, according to PROCHECK.