🧫 Experiment Protocol
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SUMMARY
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- Biochemical binding assays measuring PROTAC selectivity for APOE4 vs APOE3
- Mass spectrometry-based degradation kinetics in primary neurons
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## Background and Rationale
Proteolysis targeting chimeras (PROTACs) represent an innovative therapeutic strategy for selectively degrading disease-associated proteins in neurodegeneration. APOE4, the strongest genetic risk factor for Alzheimer's disease, differs from the protective APOE3 variant by only two amino acids, making selective targetin
METHODOLOGY NOTES
**Phase 1: PROTAC Synthesis and Characterization (Weeks 1-2)**
• Synthesize APOE4-selective PROTAC compounds using established linker chemistry
• Prepare radiolabeled versions with ¹⁸F or ¹¹C for BBB studies
• Validate compound purity >95% via HPLC-MS
• Confirm structural integrity using ¹H and ¹³C NMR spectroscopy
**Phase 2: Biochemical Binding Assays (Weeks 3-4)**
• Express and purify recombinant APOE3 and APOE4 proteins (n=3 batches each)
• Perform surface plasmon resonance (SPR) binding assays using Biacore 8K+
• Test PROTAC concentrations: 0.1-100 μM in triplicate
• Measure association/dissociation kinetics at 25°C in HBS-EP+ buffer
• Calculate KD values and selectivity ratios (KD-APOE3/KD-APOE4)
• Include negative controls with scrambled PROTAC sequences
**Phase 3: Primary Neuron Culture and Degradation Studies (Weeks 5-7)**
• Isolate primary cortical neurons from E18 C57BL/6 mouse embryos
• Culture 2×10⁶ cells per condition in neurobasal medium + B27 supplement
• Transfect neu