Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function

Development of novel nucleic acid technologies for tackling neurological diseases

Nucleic acid technologies such as antisense oligonucleotides (AOs) and DNAzymes can bind specifically to target messenger RNA and modulate gene expression by different mechanisms of actions. Recent approval of Nusinersen (Spinraza), by the United States Food and Drug Administration for the treatment of spinal muscular atrophy has demonstrated the potential of nucleic acid technologies in treatment of neuromuscular diseases. This thesis explores the potential of AOs and DNAzymes for tackling neurological diseases, particularly multiple sclerosis and Alzheimer’s disease. Chapter 1 provides a broad overview of the various nucleic acid technologies including the importance of chemical modifications and delivery of the nucleic acid molecules for clinical applications. Chapter 2 focused on developing DNAzymes targeting integrin subunit alpha 4 (ITGA4), a validated therapeutic target in multiple sclerosis. A DNAzyme candidate, RNV143, was identified to efficiently cleave exon 9 of ITGA4 RNA. This chapter also briefly explored the use of chemical modifications for nuclease resistance. Towards this, the DNAzyme, RNV143 was chemically modified and further evaluated for its nuclease resistance and cleavage activity. The focus of Chapter 3 was to develop DNAzyme and splice modulating AOs for tackling Alzheimer’s Disease by targeting amyloid precursor protein (APP), beta-site amyloid precursor protein cleaving enzyme (BACE1), and microtubule-associated protein tau (MAPT). Splice modulating AOs targeting APP, BACE1, and MAPT were developed and evaluated at the RNA and protein levels. DNAzymes targeting MAPT were also developed and its efficacy was evaluated in vitro. The results presented here highlight the scope of DNAzymes and splice modulating AOs for tackling multiple sclerosis and Alzheimer’s disease

Systematic evaluation of 2′-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro

Abstract Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2′-Fluoro (2′-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2′-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2′-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2′-O-methyl (2′-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2′-F nucleotides induced efficient exon-23 skipping, with LNA/2′-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2′-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2′-OMe/2′-F chimeras. Overall, our findings certainly expand the scope of constructing 2′-F modified AOs in splice modulation by incorporating 2′-OMe and LNA modifications

Development of cell-specific aptamers: recent advances and insight into the selection procedures

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures

Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro

We have developed a novel functional nucleic acid aptamer to amyloid- peptide 1-40 (A(1-40)) and investigated its potential to detect A peptide fragments in neuropathologically confirmed Alzheimer brain hippocampus tissues samples. Our results demonstrate that the aptamer candidate RNV95 could detect tetrameric/pentameric low-molecular-weight A aggregates in autopsy hippocampal tissue from two neuropathologically confirmed Alzheimer disease cases. Although these are preliminary observations, detailed investigations are under way. This is the first demonstration of aptamer-A binding in Alzheimer brain tissues