Advancing Small Molecule Therapies for Myotonic Dystrophy Type 1

Author

Sawyer M. HicksFollow

ORCID

0000-0003-4041-8105

Date of Award

Fall 2024

Language

English

Embargo Period

11-25-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Program

Biology

First Advisor

John A. Berglund

Committee Members

Paolo E. Forni, Melinda Larsen, Frank Middleton

Keywords

DM1, small molecule, drug discovery, therapeutics, bioinformatics

Subject Categories

Animals | Disease Modeling | Heterocyclic Compounds | Musculoskeletal Diseases | Nucleic Acids, Nucleotides, and Nucleosides | Organic Chemicals | Other Chemicals and Drugs | Polycyclic Compounds

Abstract

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by the expression of expanded CUG (CUG^exp) repeat RNA from the myotonic dystrophy protein kinase (DMPK) gene. This CUG^exp repeat RNA’s gain-of-function mechanism leads to the sequestration of muscleblind-like (MBNL) proteins, resulting in widespread alternative splicing dysregulation across tissues. Despite significant research, there are currently no approved therapeutics targeting the underlying causes of DM1. This dissertation explores the development and optimization of a novel class of small molecules, Modified Polycyclic Compounds (MPCs), designed to address splicing dysregulation in DM1. MPCs were designed from previously published works identifying diamidines as small molecules with potential for therapeutic efficacy. A structure activity relationship strategy was applied while developing MPC01 to MPC10, where MPC03 and MPC04 were identified as compounds capable of rescuing dysregulated alternative splicing at nanomolar concentrations in patient-derived fibroblast and myotube cell models. Minimal to no toxicity was observed at concentrations which returned splicing towards control levels. Global effects of MPC04 were assessed through bulk RNA-sequencing of two patient-derived myotube cell lines. We observed the rescue of thousands of skipped-exon events and of dysregulated gene expression after MPC04 treatment in both cell lines. MPC03 and MPC04 were also tested in the HSA^LR mouse model, which contains ~220 CTG repeats and recapitulates core features of DM1 in skeletal muscle tissue. After 5 days of treatment, MPC03 and MPC04 decreased the expression of the CUG^exp transgene and partially rescued dysregulated splicing in tibialis anterior skeletal muscle. Next, we explored how different functional groups attached to the end-cap regions of the MPC core structure improved or nullified activity in patient derived cell models. We identified four promising compounds, MPC11, MPC17, MCP20, and MPC21, capable of rescuing splicing to greater degrees than MPC04 at higher treatment concentrations. Based on the activities of MPCs in human cell models with minimal cell viability effects and the reduction of CUG^exp RNA expression in human DM1 patient-derived cell models and a transgenic mouse model, MPCs are a novel and potentially effective class of small molecules to treat patients with DM1.

Another aspect of this body of work addressed tissue specific differences in the HSA^LR mouse model. We performed an analysis of publicly available RNA-seq data from the HSA^LR mouse model and enhanced our understanding of alternative splicing dysregulation in the model and assessed the effectiveness of various treatments. We observed distinct tissue-specific differences in CUG^exp RNA levels among gastrocnemius, quadriceps, and tibialis anterior skeletal muscles, which aligned with the severity of dysregulated splicing. A core set of 95 splicing events consistently exhibited dysregulation, serving as a comparative reference point for evaluating the splicing rescue achieved by seven therapeutic interventions. Some therapeutics, like Pip6a-PMO and long-term treatment of EMIQ rescued many of the commonly dysregulated splicing events with minimal off-target effects. This study contributes to a broader understanding of the HSA^LR model’s role in DM1 research and its potential for preclinical drug development.

In summary, the research presented in this dissertation identifies a new class of small molecule therapeutics for DM1 and establishes a structure-function relationship to splicing rescue. Novel modifications to the end-cap region of the MPC class of molecules improved the degree of splicing rescue. Key insights into the HSA^LR model and comparisons of different potential therapeutic molecules were made and reported to the DM1 research community to support preclinical drug development efforts. Future studies are expected to determine beneficial modifications for MPCs to move further along the pre-clinical pathway and hopefully into the clinic for DM1.

License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Hicks, Sawyer M., "Advancing Small Molecule Therapies for Myotonic Dystrophy Type 1" (2024). Electronic Theses & Dissertations (2024 - present). 72.
https://scholarsarchive.library.albany.edu/etd/72