Characterization and structure-activity relationships of indenoisoquinoline-derived topoisomerase I inhibitors in unsilencing the dormant Ube3a gene associated with Angelman syndrome.

Background: Angelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A ( UBE3A ), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of Ube3a in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal UBE3A in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics.

Methods: We tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of Ube3a in mouse neurons. Primary cortical neurons were isolated from embryonic day 14.5 (E14.5) mice with a Ube3a-YFP fluorescent tag on the paternal allele ( Ube3a m+/pYFP mice) or mice that lack the maternal Ube3a allele and hence model AS ( Ube3a m-/p+ mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay.

Results: All 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal Ube3a . Several compounds exhibited favorable paternal Ube3a unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated Emax (maximum response of unsilencing paternal Ube3a ) and EC50 (half maximal effective concentration).

Conclusions: We provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal Ube3a unsilencers. All 13 tested compounds were effective at unsilencing paternal Ube3a , although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of Ube3a unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed.