Paediatric diffuse midline glioma (DMG) has the poorest prognosis of all childhood cancers due to a lack of effective treatments. Multiple clinical trials of traditional chemotherapy and existing targeted therapies have not improved patient survival outcomes. They are characterised by driver H3K27M mutations in the H3 histone which have been considered ‘undruggable’. An emerging chemoproteomic drug discovery strategy known as activity-based protein profiling (ABPP) can be utilised to discover covalent drugs, including compounds that target proteins deemed to be ‘undruggable’. However, this approach has not to date been employed in drug discovery for DMG. This study aims to identify new biological targets, including ‘undruggable’ proteins in DMG and utilise ABPP techniques to generate lead compounds that bind specifically to these targets.
To achieve this, this study will: (i) generate a map of druggable cysteines in DMG cell model proteomes by using cysteine-specific probes and mass spectrometry; (ii) validate the disease-relevance of potential targets based on their expression levels in DMG by conducting knockdown/knockout experiments in vitro; (iii) identify suitable drug-like molecules that bind to the validated targets; (iv) conduct in vitro and in vivo screening of these molecules and their derivative compounds to identify the most promising lead compounds for further development. Preliminary ABPP assays have identified a chemical probe, JNSY1, that covalently engages with cysteine residues on histone H3, including the ‘undruggable’ driver H3K27M mutant histone. Additionally, in silico docking simulations indicate that the binding of the chemical probe causes a conformational change in the N-terminus of histone H3 which is responsible for nucleosome structure and function. Given the importance of H3K27M as the critical driver mutation in DMG, we next assessed the cytotoxic activity of JNSY1 across a panel of DMG cell lines which includes non-mutant and H3K27M mutant cells. JNSY1 showed potent activity at low micromolar concentrations, with particular effect against H3.1 mutant cell lines over wildtype and others. Furthermore, a derivative compound of JNSY1 lacking the functional group required for forming covalent bonds is not cytotoxic in the same concentration ranges. This indicates that cytotoxicity caused by JNSY1 is due to its covalent interactions with its targets, such as histone H3.
Our initial findings suggest the discovery of a first-in-class histone-binding chemical probe. Future studies will investigate alternative binding sites of JNSY1 as well as its mechanism of action. Additionally, other probes will be analysed to identify other ligandable cysteine hotspots in the DMG proteome.