s:** - Biochemical binding assays measuring PROTAC selectivity for APOE4 vs APOE3 - Mass spectrometry-based degradation kinetics in primary neurons -

SUMMARY

# s:** - Biochemical binding assays measuring PROTAC selectivity for APOE4 vs APOE3 - Mass spectrometry-based degradation kinetics in primary neurons - ## 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