Publication: Smart-Engineered Small Molecule -Based Potential Therapeutic Development for Duchenne Muscular Dystrophy
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Date
2024-12-21
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Indian Institute of Technology, Jodhpur
Abstract
Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder characterized by progressive muscle degeneration and weakness, predominantly affecting males at a rate of approximately 1 in 3500 births. It arises from mutations in the dystrophin gene on the X chromosome, resulting in deficient or absent dystrophin protein. Current therapeutic strategies focus on specific mutations using exon skipping and stop codon read-through to restore dystrophin function, though limited to certain patient populations. Gene therapy approaches, employing viral vectors and gene editing like CRISPR/Cas9, aim to deliver functional dystrophin genes to muscle cells, yet clinical implementation remains under investigation. However, in the majority of cases, the current therapeutic paradigm for managing DMD primarily emphasizes on symptomatic treatment. Although these symptomatic and supportive treatments have shown limited success in improving the life expectancy of individuals with DMD, but there is no currently available treatment that can modify the course of the disease and treat the universal DMD population. Our research addresses these challenges with a multifaceted approach. Here, our overall goal is to design, develop and further validate small molecule and peptide based therapeutics targeting different key strategies to combat DMD and providing better life care support for global DMD population. This study presents a multi-faceted therapeutic approach addressing key challenges in DMD management, including utrophin modulation, antisense oligonucleotide (ASO) delivery, and the mitigation of secondary pathological hallmarks. Firstly, we designed and synthesized a library of seventy novel quinazoline- and quinoline-based small molecules as utrophin modulators. High-throughput In-cell ELISA screening identified SG-02 as the most promising candidate, exhibiting 2.7-fold upregulation of utrophin at a nanomolar concentration doses of 800 nM in a dose-dependent manner. Mechanistic studies performed identified SG-02 as a potent antagonist of the aryl hydrocarbon receptor (AhR). SG-02 exhibited high binding affinity to AhR, with a dissociation constant (Kd) of 41.68 nM, indicating strong target engagement. Furthermore, SG-02 enhanced myogenesis by upregulating myosin heavy chain (MyHC) expression, a key marker of muscle differentiation. These results highlight SG-02 as a promising therapeutic agent for DMD that targets utrophin upregulation through AhR antagonism, offering broad applicability to the DMD patient population. Second, to address the delivery limitations of antisense oligonucleotides (ASOs) for exon-skipping therapies, we developed a novel short non-cationic cell-penetrating peptide (CPP), ETWWK. This peptide was specifically designed by avoiding richness of positive charged amino acids to enable efficient conjugation with the negatively charged 2'-O-methyl phosphorothioate (2OMePS) ASO backbone using click chemistry. Functional studies demonstrated that the ETWWK-ASO conjugate successfully delivered ASO to the nuclear compartment, its intended site of action, achieving a 2-fold upregulation of dystrophin mRNA expression compared to untagged ASO. These findings mark a significant advancement in ASO delivery systems, addressing prior limitations such as low nuclear access, poor selectivity, and compromised cytotoxicity. Lastly, we focused on mitigating secondary pathological hallmarks of DMD progression by developing a novel therapeutic peptide, E.M.P-2. Designed with mitochondrial targeting and calcium-chelating properties, E.M.P-2 promoted myogenesis by upregulating key myogenic markers, myosin heavy chain (MyHC) and myogenic differentiation factor (MyoD), by more than 2-fold at a concentration of 150 nM. In addition, E.M.P-2 effectively mitigated fibrosis by downregulating collagen type I alpha 1 chain (COL1A1), a critical fibrosis marker, by 60%. The peptide also restored impaired calcium homeostasis within mitochondria and the cellular environment, a key factor in muscle regeneration. Furthermore, E.M.P-2 exhibited anti-inflammatory properties by significantly reducing IL-6 expression in DMD patient-derived cells, which display elevated baseline IL-6 levels. Overall, E.M.P-2 represents a first-in-class peptide therapeutic capable of addressing multiple pathological features of DMD, including fibrosis, impaired myogenesis, calcium dysregulation, and inflammation. This peptide holds the potential to replace corticosteroids, the current gold standard for DMD treatment, which are associated with significant long-term side effects. Together, these findings demonstrate a comprehensive therapeutic strategy for DMD, advancing the field through small-molecule utrophin modulators, innovative CPP-ASO conjugation systems, and peptide-based interventions targeting secondary disease hallmarks. My research endeavours provides a multifaceted therapeutic approach aimed at addressing the needs of the broader DMD population and providing cost-effective supportive care.
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Ghosh, Surajit (2019).Smart-Engineered Small Molecule -Based Potential Therapeutic Development for Duchenne Muscular Dystrophy (Doctor's thesis).Indian Institute of Technology, Jodhpur