Targeting the p38/MK2 protein-protein interaction for therapeutic discovery in neurodegenerative diseases Restricted; Files Only
Hu, Maylynn (Spring 2024)
Published
Abstract
P38 kinases are essential components of nervous system signal transduction. Four known p38 isoforms (p38α, p38β, p38γ, and p38δ) serve non-redundant functions by binding various substrates, including transcription, signal transduction, protein folding, and cytoskeleton maintenance. The inhibition of p38 activity has emerged as a highly appealing therapeutic strategy in multiple neurological disorders, including Alzheimer’s Disease (AD). Although many p38 inhibitors have been developed, none of them have been approved as a drug due to a limited selectivity against p38 isoforms and other kinases. The failure of p38 inhibitors in clinical trials highlights the urgent need for new therapeutic approaches to p38 regulation. To address this unmet medical need, we have developed a novel approach to control p38 activity by selectively targeting p38 isoform-specific protein-protein interaction (PPI), rather than its kinase activity. The MAPK-activated protein kinase 2 (MK2), coded by MAPKAPK2, is one of the most clinically important p38 substrates. The p38 binding and phosphorylation of MK2 play a critical role in neurotoxicity and neuroinflammation in AD.
The discovery of potent p38/MK2 PPI inhibitors may open new avenues for p38-based therapeutic development. Our Time-Resolved Fluorescence Energy Transfer (TR-FRET) and affinity pulldown assays revealed that MK2 has a significantly higher binding affinity to p38α and p38β compared to p38γ and p38δ isoforms. Using computational structural analysis, we identified multiple contact sites on the p38/MK2 PPI interface that are critical for p38/MK2 interaction. The calculated druggability scores and contribution of p38 and MK2 residues to the MK2/p38 free binding energy allowed us to prioritize two pockets suitable for small molecule p38/MK2 disruptor binding. We have further optimized and miniaturized the TR-FRET assay for the high-throughput screening (HTS) 384- and ultra-HTS 1,536-well plate formats. We have shown that both p38α/MK2 and p38β /MK2 PPIs demonstrated strong TR-FRET signal with >20 signal/background ratio, which was stable for more than 48 hours and tolerated >10 % DMSO. The pilot uHTS of more than 10,000 structurally diverse pharmacologically active compounds, as well as a virtual screening done in parallel, followed by the dose-response confirmatory screen has revealed multiple low-micromolar inhibitors of p38/MK2 interaction. We selected one of the most promising compounds to further validate in orthogonal biochemical and biophysical assays. Using the in vitro and cellular thermal shift assays, we have determined the target engagement of this inhibitors with p38 and MK2.
Together, our studies have defined the p38/MK2 interaction as a druggable target for therapeutic discovery, provided a robust uHTS assay for screening small molecule p38/MK2 inhibitors, and revealed novel molecules to control this pathogenic axis and facilitate therapeutic discovery in Alzheimer’s other neurological diseases.
Table of Contents
Introduction
Alzheimer’s disease pathology
Neuroinflammation as an AD hallmark
Overview of p38 mitogen-activated kinases
The role of p38 in AD pathology
MK2 as a molecular regulator of neuroinflammation
Previous failures of p38 and MK2 inhibitors as therapeutic targets
Materials and Methods
Antibodies
Cell lines and culture conditions
Transfection
GST-pull down assay
Co-immunoprecipitation assay
Western blot
Time-Resolved Fluorescent Resonance Energy Transfer (TR-FRET) Assay Measurements
Miniaturization of the assay into a 1,536-Well uHTS Format
Pilot screening for potential p38-MK2 modulators through uHTS
Dose-response TR-FRET validation for compound hits
Structure preparation and analysis
MK2 complexes with MAPK11, 12, and 13
Molecular dynamics
Virtual screening
Thermal shift assay
Data analysis
Results
Exploring the biology of the p38/MK2 PPI
Computational analysis of the p38/MK2 complex
Virtual screening results
Ultra-high-throughput-screening TR-FRET assay results
Novel p38/MK2 PPI inhibitor disrupts the p38-MK2 complex
Discussion
Future Directions
Summary and conclusion
Bibliography
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