Kinase-Focused Target Identification and Structure Activity Relationship Exploration for a Novel Angelman Syndrome Therapeutic.

About This Grant

Summary Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of the maternal UBE3A allele, leading to intellectual disability, ataxia, microcephaly, and seizures. Only maternal UBE3A is expressed in mature neurons, as the paternal transcript is silenced by a long noncoding antisense RNA (UBE3A-ATS). This unique biology offers an unprecedented therapeutic opportunity of restoring UBE3A expression by activating the intact but silenced paternal allele, an approach being vetted in clinical trials using antisense oligonucleotides (ASOs) to downregulate UBE3A-ATS and, hence, unsilence paternal UBE3A. Despite promising clinical data, ASOs face limitations including uneven biodistribution, risk of hydrocephaly, and invasive delivery. In contrast, blood brain barrier (BBB)-penetrant small molecules offer even brain biodistribution, reduced invasiveness, and greater patient accessibility. Our lab recently identified that the BBB- penetrant small molecule (S)-PHA533533 potently unsilences paternal UBE3A/Ube3a in AS patient-derived neurons and mouse primary neurons. (S)-PHA533533 was designed to be a cyclin-dependent kinase 2 and 5 (CDK2 and CDK5) inhibitor; however, these kinases are not responsible for the unsilencing mechanism of action (MoA). Here, I aim to identify the MoA by which (S)-PHA533533 unsilences paternal Ube3a, allowing rational drug design away from CDK inhibition and toward enhanced on-target potency, improving therapeutic index for pediatric administration and advancing clinical candidate selection. Toward this goal, my lab developed a novel dual Ube3a reporter mouse that harbors a targeted knock-in following Ube3a containing NanoLuciferase for high throughput screening and nuclear-tagged superfolder GFP that enables nuclear sorting and rapid biodistribution assessments. Using this mouse model, I will explore the mechanism by which (S)-PHA533533 unsilences paternal Ube3a and will profile the structure-activity relationship of (S)-PHA533533 analogs, synthesized by our medicinal chemist collaborators. I will investigate the MoA through targeted ASO knockdown of known chemical interactors, a CRISPR whole kinome knockout screen, and a kinase chemogenetic inhibitor small library screen. I also aim to identify chemical modifications of (S)-PHA533533 that increase its potency and decrease its cytotoxicity, hence maximizing its therapeutic index. These experiments hold promise to develop a first-in-class, brain penetrant small molecule therapy for AS, ultimately improving quality of life for affected individuals and their caretakers.