High-throughput screening and drug development for neurodegenerative diseases
Neurodegenerative diseases, NDDs, by affecting memory, cognition, behavior, sensory and motor functions, severely impact the daily lives of millions of patients.
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As mentioned in a previous article "Compound Screening Guide! " , Using automated rapid testing of thousands to millions of samples, HTS can verify biological activity at the model organism, cell, or molecular level, screening for small molecular compounds, chemical mixtures, natural products, oligonucleotides, and antibodies with known structures.
After reading the previous content, many students may have already gained some understanding of the high-throughput screening protocol. However, there may still be some confusion about its application in specific experiments. In order to provide a deeper understanding of high-throughput screening, in this issue, let's join MCE to study some instances of high-throughput screening in neurodegenerative disease research, starting from the pathological hallmarks of NDDs!
The hallmarks of neurodegenerative diseases
According to a review "Hallmarks of neurodegenerative diseases" published by Wilson DM 3rd in Cell, NDDs have 8 hallmarks: pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy metabolism, DNA and RNA defects, inflammation, and neuronal cell death [1]. In fact, these hallmarks also serve as crucial starting points in the development of drugs related to NDDs. This article mainly introduces three of the pathological markers of NDDs.
Pathological protein aggregation
The aggregation of pathological proteins is a key pathological hallmark of NDDs, playing an important role in the classification, diagnosis, and drug development of NDDs. For example, following the onset of Parkinson’s Disease (PD), abnormal deposition of alpha-synuclein (α-syn) in the brain forms Lewy bodies. In Alzheimer's Disease (AD), deposition of β-amyloid protein (Aβ) leads to amyloid plaques and phosphorylation of the tubulin-associated unit (Tau) protein results in neurofibrillary tangles. Next, let's take the high-throughput screening test for Tau protein inhibitors as an example to feel more intuitively.
Application: Screening for Tau Protein Inhibitors
Compared with traditional drug screening, Dehdashti SJ et al used Homogeneous Time-Resolved Fluorescence (HTRF) and AlphaLISA experiment to perform fast and efficient high-throughput screening. Eventually, Dehdashti SJ's team screened out four 4 lead compounds from 1,280 substances that could inhibit the level of Tau protein in SH-SY5Y cells, which significantly accelerating the screening speed for Tau protein inhibitors in NDDs[2].
Aberrant Proteostasis
Accumulation of toxic protein aggregates in a variety of neurodegenerative diseases is associated with defects in the Autophagy-Lysosome Pathway (ALP, one of the pathways that maintain protein homeostasis in eukaryotic cells). TFEB, the master regulator of lysosomal function, can improve disease phenotypes in a variety of neurodegenerative disease models.
Application: Screening for Kinase Regulators of TFEB and TFE3
Carling PJ's team using high-throughput screening to discover kinase regulators of TFEB and TFE3 with neuroprotective effects from the kinase inhibitor library. Through related mechanistic research, the authors found that quinazoline analogs upregulate the transcription of CLEAR genes, clear Huntingtin (HTT) aggregates, thereby regulating lysosomal function to exert neuroprotective effects[3].
During the initial screening, the author treated human induced pluripotent stem cells (hiPSCs) with compounds (1 μM), and used a luminescent assay kit to detect changes in the levels of adenylate kinase (AK) (Figure 3 B, C). They further assessed the lysosomal activity using DQ? Red BSA reagent (Figure 3 D, E). Based on the AK content and lysosomal activity results, compounds that could induce cytotoxicity or mitochondrial toxicity were eliminated.
In the secondary screening, the author used immunocytochemistry (ICC) to determine the effect of compounds (0.01-2 μM) on the nuclear entry of TFEB and TFE3 (Figure 3 F,G), screening out 74 compounds that significantly promoted the nuclear entry of TFEB or TFE3.
Altered Energy Metabolism
Neurons are a type of active cell that has high energy demands. Disruptions in energy metabolism can significantly affect the normal function of neurons, leading to the onset of NDDs. Nicotinamide Adenine Dinucleotide (NAD) is an important coenzyme in the process of energy metabolism, and having sufficient and stable levels of NAD is a major factor for maintaining neuronal health.
Application: Screening for Small Molecule Activators Targeting NAMPT
Focusing on the hot scientific topic of NAD metabolism regulation in neurodegenerative diseases, Hong Yao used targeted high-throughput screening and drug design based on target structure to screen the activator of NAMPT(Nicotinamide Phosphoribosyl Transferase, a key rate-limiting enzyme in NAD synthesis). Experimental results showed that NAT (NAMPT activator) could exert a neuroprotective effect by promoting the biosynthesis of NAD[4].
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First, the author screened several small molecules (10 μM) that could stimulate NAMPT vitro recombinant enzyme experiments. From these, the author selected NAT for further study, which strongly stimulated NAMPT activity but had no activity in Nicotinamide Nucleotide Adenylyltransferase (NMNAT) assay tests. Then, in order to improve the pharmacological properties of NAT, the authors modified and optimized the structure of NAT, and synthesized 81 NAT derivatives. Finally, the author validated the mechanism of action of the NAT at both the cellular and animal levels, finding that NAT-5r effectively increased intracellular NAD levels and induced subsequent metabolic and transcriptional reprogramming processes, thereby exerting a neuroprotective effect.
Summary
This time, starting from the pathological features of NDDs, Little M listed three instances where high-throughput screening technology has been applied in drug screening of neurodegenerative diseases. This article vividly illustrates the application of high-throughput screening technology from theory to practical operation.
MCE Compound Library
1,800+ compounds with neuroprotective activity or compounds targeting major neurodegenerative disease targets (such as Amyloid-β, Dopamine Receptor, COMT, etc.).
1,100+ compounds with anti-Parkinson's activity and compounds targeting major Parkinson's disease targets (such as Dopamine Receptor, LRRK2, 5-HT Receptor, etc.).
2,200+ small molecule compounds related to neural signaling, mainly targeting G Protein-Coupled Receptors (GPCRs) and Notch signaling. These are useful tools for studying neuron regulation and neurological diseases.
900+ compounds with potential neuroprotective effects, mainly targeting major targets in neuroprotective pathways such as calcium ion channels, sodium ion channels, adenosine A1 receptors, etc.
1,600+ neurotransmitter receptor related compounds are effective tools for drug screening for neurological diseases.
700+ has been clearly reported as a small molecule compound that can cross the blood-brain barrier and is an effective tool for developing drugs for brain diseases such as brain tumors, mental disorders and neurodegenerative diseases.
200+ 5-hydroxytryptamine (5-HT) receptor inhibitors and agonists can be used in the development of a variety of psychotropic drugs.