Developing a preclinical-grade chemical into a potential therapy against malignant brain cancer

GB is the most common primary brain tumour in adults, with the current standard of care involving surgical resection with concurrent chemoradiotherapy. Despite current efforts exploring targeted therapies, immunotherapies and drug delivery systems to bypass the blood-brain barrier, clinical outcomes remain poor, underscoring the need for a novel therapeutic strategy.
GB cells exhibit metabolic adaptation, simultaneously upregulating glycolysis and OXPHOS, which presents a vulnerability. Previous work by Polson et al. (2018) identified the chaperone HSPD1 as a potential target given its role in regulating metabolism, and their small molecule KHS101 showing selective GB targeting.
This study identified and characterised 21 analogues of KHS101 for exploration of a new frontrunner compound and an additional analogue where KHS101 is conjugated to TPP to explore KHS101 ‘on-target’ effects with mitochondrial HSPD1 in patient-derived GB cells, including a non-cancerous control cell line.
An NADH-based HSPD1:HSPE1 refolding assay was utilised to demonstrate the compound's ability to affect HSPD1:HSPE1 activity. However, lacking sensitivity and sufficient consistency, an image-based multi-parametric assay was employed. This assay assessed six parameters measuring three phenotypes: autophagy and cytoplasmic degradation (metabolic dysfunction), vacuolised area of the cytoplasm and number of vacuoles (vacuolisation) and number of stress fibres and nuclear-to-cytoplasmic ratio (stress). Among the 21 KHS101 analogues, SCBT-21 emerged as the leading compound, demonstrating strong metabolic exhaustion and selectivity in GB and NP cells.
Further in vitro analysis revealed that SCBT-21 had a pronounced effect on cell viability of recurrent GB models over primary GB models, reflected in their IC₅₀ values. Additionally, SCBT-21-treated cell cycle indicator carrying GB cell models demonstrated a G1 cell cycle arrest in a small ‘resistant’ population of cells in GB models, while no such effect was observed in the NP model. In vivo studies using syngeneic mouse models and a pharmacokinetic (PK) study demonstrated SCBT-21 was able to penetrate the blood-brain barrier and accumulate in the brain.
To confirm KHS101 targets the mitochondria, a mitochondrial-targeted analogue (KHS101TPP) was synthesised to bypass the cytoplasm and cytoplasmic proteins (e.g., TACC3 and cytoplasmic HSPD1), showing enhanced potency across multiple assays, with the suggestion that KHS101 is targeting the mitochondria.
Overall, SCBT-21 represents an analogue with significant advancement over KHS101, with pronounced metabolic exhaustion and favourable pharmacokinetics in vivo. SCBT-21 lays the foundation for future optimisation, including SAR refinement and dosage studies towards an effective therapeutic agent against GB.