Discovery and Validation of Novel LonP1 and Proteasome Inhibitor in IDH1-R132H Malignant Astrocytoma Models

Malignant astrocytomas are aggressive glioma tumors that have poor prognosis and limited treatments available following recurrence. These tumors are characterized by extensive hypoxia-induced, mitochondria-dependent changes such as glycolytic respiration, increased chymotrypsin-like (CT-L) proteasome activity, decreased apoptosis, and increased invasiveness. Mitochondrial Lon Peptidase 1 (LonP1) is an ATP-dependent protease directly upregulated by hypoxia-inducible factor 1 alpha (HIF-1α). Both LonP1 expression and CT-L proteasome activities are increased in gliomas and are associated with a high tumor grade and poor patient survival. Recently, dual LonP1 and CT-L inhibition has been found to exhibit synergy against multiple myeloma cancer lines. We now report that dual LonP1 and CT-L inhibition has synergistic toxicity in IDH mutant astrocytoma when compared to IDH wildtype glioma, due to increased reactive oxygen species (ROS) generation and autophagy. A novel small molecule, BT317, was derived from coumarinic compound 4 (CC4) using structure-activity modeling and was found to inhibit both LonP1 and CT-L proteasome activity and subsequently induce ROS accumulation and autophagy-dependent cell death in high-grade IDH1 mutated astrocytoma lines. In vitro, BT317 also had enhanced synergy with the commonly used chemotherapeutic temozolomide (TMZ), which blocked BT317-induced autophagy. This novel dual inhibitor was selective to the tumor microenvironment and demonstrated therapeutic efficacy both as a single agent and in combination TMZ in IDH mutant astrocytoma models. We show that BT317, a dual LonP1, and CT-L proteasome inhibitor exhibited promising anti-tumor activity and could be a promising candidate for clinical translation in the space of IDH mutant malignant astrocytoma therapeutics.