Uncovering the molecular target(s) of ORI-113, a potential brain-penetrant small molecule therapeutic for Huntington's disease.

ABSTRACT Huntington’s disease (HD) is an autosomal dominant, progressive and fatal neurodegenerative disease that effects 200,000 people worldwide. Despite the discovery of the causative gene 26 years ago, there is no cure for HD. The development of a therapy that slows, halts, or prevents disease would have a major impact on patients. HD is caused by the expansion of a CAG repeat in the huntingtin gene (HTT), resulting in an expanded stretch of glutamines in the huntingtin (HTT) protein. Normal huntingtin protein (wildtype or wtHTT) is essential throughout the brain and body to regulate cell physiology, synaptic transmission, neuroprotection, cell division, differentiation, gene transcription and the DNA damage response. In HD patients the expanded polyglutamine tract causes mutant HTT protein (mHTT) to fold abnormally, resulting in cleavage to generate toxic mHTT fragments. Lowering mHTT has reversed disease symptoms in animal models of HD and lowering of HTT RNA is showing promise in early clinical trials. However, these drug candidates are disadvantageous in that they (1) do not target or remove the toxic mHTT protein fragments that contribute to HD pathogenesis, (2) are not selective for mHTT, also removing essential wtHTT, (3) target specific portions of the brain and leave the rest of the body untreated, (4) require invasive surgery for delivery or (5) are not orally administered, limiting patient access. Therefore, an orally delivered, systemically distributed, brain-penetrant therapeutic that selectively eliminates toxic mHTT protein and reduces aggregates while sparing wtHTT to support normal physiology could offer an effective treatment for all HD patients. Origami Therapeutics has identified ORI-113, a first-in-class small molecule that selectively reduces soluble mHTT and insoluble mHTT aggregates in HD patient-derived cells and mouse models. Preliminary studies show ORI-113 decreased mHTT levels by 25-41% in cortex and striatum of YAC128 and R6/2 HD mice after only 2 weeks of treatment. The proposed research will identify the precise molecular target(s) of ORI-113 and elucidate its mechanism of action using three complementary proteomic techniques: 1) Cellular thermal shift assay to identify target proteins that physically interact with ORI-113, 2) Covalent protein painting to detect conformational changes in the proteome induced by mHTT and reversed by ORI-113, and 3) Assessment of post-translational modifications across the proteome including ubiquitination and phosphorylation after ORI-113 treatment. Defining the specific molecular target(s) and cellular pathways modulated by ORI-113 to achieve selective degradation of mHTT will critically enable lead optimization, biomarker development, target engagement strategies and predictive toxicology assessments. This knowledge will accelerate progression of ORI-113 as an exceptionally promising orally available disease-modifying therapy for HD patients.