Discovery and Validation of Small Molecule Inhibitors of the p38α/MK2 Protein-Protein Interaction for Alzheimer’s Disease Open Access

Abstract

Alzheimer's disease (AD) is the most common cause of dementia, and chronic neuroinflammation is one of the core AD hallmarks. Extensive in vitro, in vivo, and clinical studies have established two protein kinases – p38α and MK2 – as the key drivers of neuroinflammation in AD. In response to stress stimuli, p38α phosphorylates and activates MAPK-activated protein kinase 2 (MK2), which regulates inflammatory cytokines, including tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and IL-6. While many p38α and MK2 inhibitors have advanced to clinical trials, none have been approved as a drug. This lack of success is mainly attributed to i) poor selectivity of the inhibitors against other kinases and ii) other substrates affected by the inhibition of p38α kinase activity. These limitations led to a broad range of adverse effects and often opposite therapeutic responses. The failure of p38α and MK2 kinase inhibitors in clinical trials highlight the urgent need for novel approaches to regulate p38α-MK2-dependent neuroinflammation. To address this unmet medical demand, we propose a novel approach to control the p38α pathway by selectively targeting p38α-MK2 protein-protein interaction (PPI) rather than their individual kinase activity. Through computational structural analysis, we identified high-affinity binding pockets on the p38α-MK2 interface, which allowed us to design short inhibitory peptides and conduct virtual screening of small molecule p38α-MK2 PPI inhibitors. The validation of computationally discovered hits in TR-FRET and pulldown assays confirmed their p38α-MK2 PPI inhibitory activity at low micromolar range in vitro and in endogenous conditions. The identified compounds also disrupted the p38α-MK2 PPI in human microglial cells, suppressing oxidative stress-induced MK2 phosphorylation. To discover new potent p38α-MK2 PPI inhibitors with improved pharmacological profiles, we have further optimized and miniaturized the TR-FRET assay for ultra-HTS 1,536-well plate formats. Early TR-FRET validation of selected primary hits exhibited enhanced inhibitory potency and revealed potential inducers of the complex. Together, our results demonstrate druggability of the p38α- MK2 PPI, provide the first chemical tools to disrupt this pathogenic complex, and establish new, robust, and integrative uHTS/computational platforms to discover small molecule p38α-MK2 PPI inhibitors to facilitate therapeutic discovery in Alzheimer’s disease. 

Table of Contents

Table of Contents

1. INTRODUCTION..................................................................................................................... 1

1.1 Neuroinflammation is a hallmark of Alzheimer’s disease........................................... 1

1.2 The p38α-MK2 signaling axis regulates pathogenic neuroinflammation..................... 3

1.3 Previous attempts at targeting p38α or MK2 have been challenging........................... 7

1.4 Inhibition of protein-protein interactions is a promising therapeutic strategy............ 10

 

2. METHODS.............................................................................................................................. 13

2.1 Molecular Modeling and Docking............................................................................. 13

2.2 Cell Cultures........................................................................................................... 13

2.3 Transfection and Lysis............................................................................................. 13

2.4 Fluorescence Polarization........................................................................................ 14

2.5 Dose-Response Time-Resolved Fluorescence Resonance Energy Transfer.................... 14

2.6 GST-Pulldown......................................................................................................... 15

2.7 Co-Immunoprecipitation......................................................................................... 15

2.8 Western Blot............................................................................................................ 15

2.9 Biolayer Interferometry............................................................................................ 16

2.10 Reverse-Transcription Quantitative Polymerase Chain Reaction................................ 17

2.11 TR-FRET Ultra-High-Throughput-Screening............................................................ 19

 

3. RESULTS................................................................................................................................. 20

3.1 Preliminary computational structural analysis revealed high affinity binding pockets... 20

3.2 MK2-derived peptides bind to and inhibit the p38α-MK2 interaction........................... 21

3.3 Virtual screening revealed the first p38α-MK2 PPI small molecule inhibitors............... 22

3.4 Hit compounds were validated through chemical biology techniques........................... 23

3.5 p38α-MK2 PPI inhibitors decrease pro-inflammatory cytokine levels in microglia........ 27

3.6 Ultra-High-Throughput-Screening revealed more p38α-MK2 PPI modulators............... 29

 

4. DISCUSSION........................................................................................................................... 32

4.1 The p38α-MK2 PPI is a druggable target................................................................... 32

4.2 Discovery of small molecule p38α-MK2 PPI inhibitors showed promising potency........ 33

4.3 TR-FRET uHTS hits demonstrate enhanced inhibitory properties................................ 34

 

5. CONCLUSION......................................................................................................................... 36

 

6. REFERENCES.......................................................................................................................... 37

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Health Sciences, Pharmacology Biology, Neuroscience
Keyword	Protein-protein interactions High-throughput screening Pharmacology Neuroinflammation Alzheimer's Disease Chemical biology MK2 p38
Committee Chair / Thesis Advisor	Ivanov, Andrey, Emory University
Committee Members	Cole, Megan, Emory University Zhang, Qiang, Emory University

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