A chromatin-bound kinase, ERK8, protects genomic integrity by inhibiting HDM2-mediated degradation of the DNA clamp PCNA

ERK8 prevents genome instability by blocking the association of the HDM2 E3 ubiquitin ligase with the genome-protecting protein PCNA

Chemical tools to probe the function of TRIM33

Epigenetic modifications, including chemical modification of DNA and post-translational modifications (PTMs) to histone proteins, are important in regulating gene expression. Patterns of PTMs recruit specific recognition modules, including bromodomains, which recognise acetylated lysine residues. Bromodomains play a central role in epigenetic gene regulation and, as aberrant access to genetic information is responsible for many diseases, they are considered attractive therapeutic targets. TRIM33 belongs to the C-VI subfamily of TRIM proteins, possessing a C-terminal chromatin binding unit, which comprises a bromodomain adjacent to a PHD finger. In the PARP-dependent DNA damage repair pathway, TRIM33 is responsible for the timely dissociation of chromatin-remodelling enzyme ALC1 from chromatin. Regulation of ALC1 at chromatin is important as prolonged chromatin relaxation leaves DNA susceptible to further damage. It is hypothesised that TRIM33 bromodomain ligands will prevent TRIM33 binding to chromatin, resulting in the prolonged association of ALC1 at chromatin, inefficient repair of single-strand breaks, and further DNA damage. This dissertation describes the development of chemical tools to validate TRIM33 as a therapeutic target. Bromodomain ligands and PROTACs, which enable the role of the bromodomain to be disconnected from the function of the whole protein, were designed and synthesised. Previous work in the group had identified benzimidazolone 23 as a TRIM33 bromodomain ligand (TRIM33β IC50 = 11.9 μM) (Figure 1). The optimisation of the selectivity and affinity of 23 through iterative cycles of design, synthesis and testing is detailed. Compounds were first evaluated using an AlphaScreen™ assay, and hits were validated in waterLOGSY and ITC experiments. Initial work demonstrated the importance of the benzylic amine substituent for imparting selectivity over the closely related TRIM24 bromodomain. The selectivity results from interactions with a glutamic acid residue (E981) in TRIM33, which is switched to an alanine residue (A923) in TRIM24. Compound 90 (TRIM33β IC50 = 13.7 μM) was developed as a selective ligand, which retains affinity for TRIM33, and was subsequently used as the basis for ligand design (Figure 1). The region ‘above’ the acetyl-lysine binding pocket was explored by extending off the headgroup of 90. The amine chain and methylation state of the benzimidazolone core were also investigated. Despite an extensive investigation focused on benzimidazolone analogues, a relatively flat SAR was observed, with most ligands displaying low micromolar affinity. A structural water molecule was identified as preventing the amine chain residing in the ZA channel. Subsequent ligand design focused on a bidentate interaction of the amine chain with E981 which avoided displacing the tightly bound ZA channel water molecule. This approach resulted in 161 (TRIM33β IC50 = 4.83 μM), which has increased affinity for the TRIM33 bromodomain, potentially due to the phenyl ring enforcing a favourable conformation for a bidentate interaction with E981 (Figure 1). The knowledge gained from ligand optimisation was used to facilitate PROTAC design. The core structure of the bromodomain ligands was incorporated as the TRIM33β bromodomain binding moiety, and tethered via an appropriate attachment point to a ligand for the E3 ligase cereblon. A range of linker lengths and linking strategies were used to maximise the chance of achieving potent degradation of TRIM33. Some of the potential TRIM33 PROTACs were found to interfere with the AlphaScreen™ assay therefore an alternative assay is required to evaluate their binding to TRIM33. Together the bromodomain ligands and PROTACs can be used to gain a deeper understanding of the physiological and pathological roles of TRIM33 and its bromodomain.</p

Broadly tunable continuous-wave diode-pumped Cr4+:YAG laser

NRC publication: Ye

SPIE

NRC publication: Ye

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

Project Extension for Community Health Outcomes (ECHO) Autism: A Successful Model to Increase Capacity in Community-Based Care

Individuals with autism spectrum disorder (ASD) struggle to access high-quality health care due to the shortage of trained providers. ECHO (Extension for Community Healthcare Outcomes) Autism is a unique educational program that allows ASD experts to provide knowledge and skills to professionals in local communities to deliver evidence-based care to children with ASD and their families. The model teaches clinicians how to screen and diagnose ASD, as well as manage common co-occurring medical and mental health issues. ECHO Autism is particularly useful for addressing the complex needs of children with ASD and reducing disparities often present in rural and underserved communities. The model can be disseminated globally due to its flexibility in accommodating local and regional differences in social norms and constructs. This article provides an overview of the format of the ECHO Autism model, data supporting the model&rsquo;s efficacy, and discusses future research directions

Controlling intramolecular interactions in the design of selective, high-affinity, ligands for the CREBBP Bromodomain

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise ten domains through which they interact with >400 proteins, making them important transcriptional coactivators, and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable binding of acetylated lysine residues from histones, and a number of other important proteins, including p53, p73, E2F and GATA1. Here we report work to develop a high affinity, small molecule, ligand for the CREBBP and EP300 bromodomains [(−)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc, and a reduction in H3K18 and H3K27 acetylation. In hypoxia (<0.1% O2), inhibition of the CREBBP/EP300 bromodomain results in enhanced stabilization of HIF-1α

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)