Though pharma companies have already invested a ton in R&D for neurodegenerative diseases (ND), there aren’t any effective drugs for treatment. It’s because researchers face specific technical challenges when it comes to understanding the molecular mechanisms behind ND. If you’re facing these challenges too, learn how NanoTemper is empowering researchers to overcome them.
Protein misfolding is important in neurodegenerative diseases
Progressive neuronal death — a common feature for all neurodegenerative diseases like Parkinson’s (PD), Alzheimer’s (AD), and Huntington’s — leads to neurological impairment. Although these diseases display diverse clinical symptoms, they share the formation of misfolded protein aggregates. Understanding the mechanisms behind this process will help develop novel therapeutics and early detection methods.
Uncovering the molecular mechanisms
The formation of misfolded protein aggregates is a hallmark event in neurodegenerative diseases. The goal of many researchers is to determine the key players and how they interact and modulate the process. To do this, they measure molecular interactions and look at the effect of mutations on the stability and binding of key players.
Novel role for phosphorylated tau in amyloid-β toxicity
MST helped confirm that phosphorylated tau inhibits amyloid-β (Aβ) toxicity in early AD by measuring binding affinities of wild type and variant tau. The results correlated with those from pulldown assays, but required less sample and were truly quantitative providing calculated Kds.
A single point mutation affects the neuropathology of ataxia
The mechanisms behind Purkinje neurons (PN) loss seen in ataxias remain unknown. This study found that misfolding, proteotoxic stress, and eventual PN death is caused by the Elongator complex subunit 6 (Elp6). nanoDSF data confirmed the compromised stability of mutated Elp6, and MST helped correlate the decreased stability to a reduced tRNA binding capacity in vitro.
The role of zinc in the aggregation of TDP-43
Researchers investigated whether zinc-induced aggregation of TDP-43 took place by direct binding. Using label-free nanoDSF, they looked at TDP-43 thermostability in the presence of zinc ions and concluded that zinc binds to a specific domain in TDP-43 and decreases its thermostability.
Finding novel therapeutics
For researchers focusing on therapeutic agents — ones that inhibit and modulate protein misfolding pathways — it’s important to use the right biophysical tools to characterize their interactions with misfolded proteins.
Molecular interactions of monomeric and aggregated ⍺-synuclein
Understanding the interactions between α-synuclein aggregates and small molecules and other proteins can help to discover and develop therapeutics. MST overcame the challenge of characterizing ⍺-synuclein oligomeric structures, which often occur only at very low concentrations and have short lifetimes. The authors measured affinities of very challenging samples like supramolecular protein aggregates while using small amounts of sample.
The therapeutic potential of purine derivative drugs
Purine derivative drugs (PDD) are small molecules with therapeutic potential in the treatment of ND through various mechanisms including hyperphosphorylation of tau protein in Alzheimer’s disease (AD). MST was used to show that a PDD binds to prohibitin (PHB), a protein that may play a role in Parkinson’s disease and AD. The interaction rescued cognitive deficit, attenuated neuroinflammation, reduced the decline in neurogenesis, and restored synaptic function in aged mice.
At Celon Pharma, we work with target proteins that lack enzymatic activity, making screening for potential drug candidates very challenging. Monolith provides us a new approach that brings a fast and reliable solution for the screening of small molecule-protein and protein-protein interactions. The benefits are multiple: reduced costs due to low protein consumption, time saving for screening campaigns due to fast run times, and easy setup so everyone in the lab can use it.
Dr. Mikołaj Matłoka
Team Leader
Celon Pharma SA
Discovering effects on degradation pathways
All cells — including neurons — are constantly producing toxic misfolded proteins and the ubiquitin-proteasome pathway have evolved to tag, destruct and eliminate them. Molecules that enhance or enable these pathways in neurons are considered potential therapeutic candidates.
Interference of clearance pathway by Aβ peptide
Aβ accumulation role in AD progression suggests a malfunction in the proteasome ubiquitin system (UPS), but the mechanism is still poorly understood. MST was used in this study to measure the affinity of the interaction between Aβ40 and ubiquitin in solution. It also ruled out artifacts from SPR assays that could potentially result from the immobilization of Aβ40 to a solid substrate.
The role DJ-1 plays in regulating 20S proteasome activity
Overexpression of DJ-1 has been recently implicated in early-onset familial form of PD, but the exact mechanism is still unclear. This study demonstrates that DJ-1 binds and inhibits the activity of the 20S proteasome by physically binding to it, rescuing partially unfolded proteins from degradation. MST was used to measure the binding affinity of the 20S proteasome with DJ-1 wild type and a mutant.
Developing early diagnostic tools
Treatments at an early stage of ND are more effective because they’re started before irreversible changes in the brain have occurred. Neuroimaging techniques like positron emission tomography (PET) are useful for the early diagnosis of PD and AD. For this technique, new small molecule ligands specific for ND, such as tau PET are needed.
Interactions of small molecules with fibrillar protein aggregates
Fibrillar protein aggregates — AD and PD pathological hallmark — can be selectively detected in patient’s brains using PET and used to diagnose and study disease progression. The challenge is finding small molecules (radioligands) that bind to the protein fibrils and to measure their interactions with accuracy and sensitivity. In this proof of concept study, researchers show how MST was the technology able to measure the binding affinity of known small molecule to α-synuclein fibrils.
Using stem cells for in vitro models and drug discovery
The lack of physiologically relevant in vitro models is a major barrier to understanding the molecular mechanisms of AD. Technologies that use induced pluripotent stem cells (iPSC) from dementia patients offer a unique way to overcome this challenge. Under the right culture conditions, the iPSC can be differentiated into neurons and provide a physiologically relevant in vitro model. This strategy has been tested to model AD pathology, understand disease mechanisms, and assist in drug discovery. Ask how NanoTemper tools are used when combined with this approach.
Finding new roles for CRISPR technology
Growing evidence points to a genetic component in the generation of misfolded proteins. It makes sense that researchers have turned to gene-editing tools like CRISPR to generate ND-specific animal models and screen for potential drugs. CRISPR — with its efficiently edit sequence-specific genes — could offer a new form of treatment. Inquire how NanoTemper tools are used when combined with this approach.