Development of small molecule binders targeting toxic CAG-repeat RNA in Huntington's Disease

Abstract

Huntington’s disease is an inherited neurodegenerative disorder caused by an expansion mutation in the CAG-repeat tract of the huntingtin gene. Expression of the gene results in a toxic CAG-repeat RNA transcript that underlies pathogenesis of the disease. A bisamidinium compound DB213 has been identified as a small molecule inhibitor for CAG-repeat RNA, which exhibits neuroprotective effects in affected cells and animals. Detailed binding studies have revealed the molecular binding model of DB213 with CAG-repeat RNA. Based on the studies with DB213, small molecule binders are rationally designed to target CAG-repeat RNA for therapeutic and biomedical applications in the treatment of Huntington’s disease.

In Chapter 1, an introduction on the chemistry of RNA as a relevant drug target in medicine is given. The molecular pathogenesis of Huntington’s disease and recent therapeutic efforts in its treatment are also discussed. Strategies of targeting the toxic CAG-repeat RNA in Huntington’s disease with small molecule binders are highlighted, which is concluded with the recent discovery of DB213 as an inhibitor of the RNA.

In Chapter 2, a unique binding of DB213 at the CAG-repeat major groove via specific non-covalent interactions was first revealed by NMR, and selectivity studies have demonstrated that the bisamidinium pharmacophore of DB213 is essential to its recognition of CAG-repeat RNA. Eleven bisamidinium small molecule binders are rationally designed and synthesized based on non-covalent interactions with CAG-repeat RNA. ITC binding studies show that π-π stacking and hydrogen bonding have relatively minor contribution to RNA binding. Two bisamidinium cyclic binders, DBc5BR and DBc5BS, display higher binding affinities of Kd = 0.62 μM and 0.76 μM respectively towards CAG-repeat RNA.

In Chapter 3, azide-alkyne click chemistry is utilized in the rational design of dimeric RNA binders based on the DB213 pharmacophore. Four bisamidinium dimeric binders are synthesized with different lengths of the linker. ITC binding studies suggest that the dimeric binders are more potent than DB213 in the inhibition of CAG-repeat RNA, in which DBPr2132 and DBHx2132 exhibit nanomolar affinities of Kd = 7 nM. Further cell studies have demonstrated the in vivo potency of DBEt2132 and DBPr2132 in the inhibition of RNA toxicity in neuronal cells.

In Chapter 4, DB213-fluorophore conjugates are rationally designed as small molecule fluorescent probes for CAG-repeat RNA. Environment-sensitive dyes and aggregation-induced emission (AIE) fluorophores are linked to the bisamidinium binder via azide-alkyne click chemistry. The fluorescent compounds display remarkable environment-responsive fluorescence toward solvent polarity, ionic strength, and pH in fluorescent studies. The AIE compound DBTPE is identified as a selective fluorescent probe with a 19-fold fluorescent turn-on towards CAG-repeat RNA and 3-fold selectivity against canonically-paired RNA.

In Chapter 5, encapsulation of DB213 by macrocyclic hosts is investigated as an alternative therapeutic approach. Complexation of DB213 with cucurbiturils and cyclodextrins is studied by NMR. A 1:1 complex is formed between DB213 and cucurbit[7]uril with a binding constant of Ka = 10-5 M-1, which results from an encapsulation of the DB213 bisamidinium ring within the macrocycle cavity.