Chemical tools to probe the role of bromodomains in the parasite Trypanosoma cruzi

Chagas disease is a chronic infection caused by the parasite Trypanosoma cruzi (T. cruzi). The development of the parasite in the human heart muscle or digestive tract causes severe damage, resulting in organ failure. The current treatments, benznidazole and nifurtimox, have a limited efficacy, show significant side effects, and increasing resistance. Post-translational modifications (PTMs) play a crucial role in the life cycle of protozoan parasites. Bromodomains (BDs) are readers of lysine acetylation, one of the PTMs found on histone tails. The function of BDs remains uncharacterised in T. cruzi. The life cycle of the parasite is complex and is thought to rely heavily on epigenetic control and therefore our main interest is to validate the T. cruzi BDs as therapeutic targets. Optimisation of expression and purification protocols of the protein BD-containing factors (BDFs) in E. coli (Chapter 1) was followed by the development of a waterLOGSY- based assay to screen acetyl-lysine mimic containing compounds (Figure 0.1). The binding of the hits was further characterised by 1H NMR ligand-observed protein titration to obtain KD. Excitingly, the human BD and kinase ligand, BI-2536 (8), was shown to bind to TcBDF3 with a KD value between 25 and 55 μM. The binding was confirmed to be 14.6±3.0 μM (n=3) using ITC. BI-2536 (8) is to date the highest affinity ligand identified and was chosen as a lead structure for further development (Chapter 2). Based on this initial work, eight functional probes were designed and synthesised to enable the development of higher-throughput assays for T. cruzi BDs (Chapter 3). The 19F NMR reporter molecules, photo-activatable diazirines, and fluorescent probes were assessed against TcBDF3 to establish novel assays (Chapter 4). Firstly, the fluorinated probes were used as 19F NMR reporter molecules to observe their binding and displacement to and from TcBDF3. Secondly, a protein LCMS assay was established to characterise binding and subsequent cross-linking of the diazirine probes. Finally, fluorescent probes were validated by fluorescence polarisation. Satisfactorily, one probe binds to TcBDF3, with a KD value of 3 μM. Displacement of the fluorescent probe by the parent compound BI-2536 (8) results in a dose-dependent response, however, a full curve and an IC50 value could not be obtained due to assay interference at high ligand concentrations. Further validation is required, using higher affinity ligands to show that the fluorescence polarisation displacement can generate IC50 values. With GSK collaborators, 556 diazirines of the GSK PhaBits library were screened against TcBDF3 and 5-1, identifying one and six new binding fragments, respectively. Additionally, a DNA-encoded library of ca. 1.9 trillion encodings were screened against TcBDF2 and 5- 1, giving 82 and 134 hit clusters, respectively, to be investigated for binding. The low hit rates for both screening campaigns highlights that the T. cruzi BDs are challenging targets. In this work we report the first production of soluble T. cruzi BD proteins and their study using NMR assays such as waterLOGSY and NMR titration. Critically, this initial work led to the development of functional probes to provide a tool box to study TcBDF3 at higher throughput with diverse and orthogonal assays. The novel biophysical assays established here can be used to screen efficiently for binding compounds and opens up the possibility of rapid SAR. Identified ligands will enable validation of the bromodomains of T. cruzi as targets for Chagas disease for the first time. This will have a wide range of implications in the use of epigenetics targets for treatments of parasites more broadly.</p

Fragment-based identification of ligands for bromodomain-containing factor 3 of Trypanosoma cruzi

The Trypanosoma cruzi (T. cruzi) parasite is the cause of Chagas disease, a neglected disease endemic in South America. The life cycle of the T. cruzi parasite is complex and includes transitions between distinct life stages. This change in phenotype (without a change in genotype) could be controlled by epigenetic regulation, and might involve the bromodomain-containing factors 1–5 (TcBDF1–5). However, little is known about the function of the TcBDF1–5. Here we describe a fragment-based approach to identify ligands for T. cruzi bromodomain-containing factor 3 (TcBDF3). We expressed a soluble construct of TcBDF3 in E. coli, and used this to develop a range of biophysical assays for this protein. Fragment screening identified 12 compounds that bind to the TcBDF3 bromodomain. On the basis of this screen, we developed functional ligands containing a fluorescence or 19F reporter group, and a photo-crosslinking probe for TcBDF3. These tool compounds will be invaluable in future studies on the function of TcBDF3 and will provide insight into the biology of T. cruzi

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½ = 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½ = 388 min)

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½ = 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½ = 388 min)