Pathogenic alterations in NOTCH3 cause CADASIL, an accelerated and currently untreatable form of cerebrovascular disease. CADASIL mutant NOTCH3, which frequently harbors abnormalities in EGF repeat cysteine number, adopt disulfide dependent abnormal conformations. To seek potential strategies to mitigate the impact of CADASIL mutations on NOTCH3, we investigated whether cysteine-targeting compounds may affect pathological NOTCH3. LSL-NOTCH3, a split luciferase assay that discriminates between benign and pathogenic NOTCH3 conformations, was used to quantify the capacity of 21 small molecule compounds to restore NOTCH3 reporter activity. We assayed the activity of each of the small molecules on 16 different pathogenic mutants distributed over three different regions of NOTCH3. Ten of 21 compounds had statistically significant effects on at least one mutant NOTCH3. Five of 21 compounds had mitigating effects on a majority of mutant NOTCH3 in three regions of the protein, with disulfiram and auranofin targeting the most favorable range of mutants. These findings support the concept that cysteine-targeting can potentially mitigate the effects of a broad range of NOTCH3 pathogenic mutants and provide an impetus to investigate whether cysteine-targeting strategies affect disease-relevant phenotypes in EGF repeat-related human disorders.
Cartee et al. (2026) studied this question.