High-Affinity, Structure-Validated and Selective Macrocyclic Peptide Tools for Chemical Biology Studies of Huntingtin

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Huntington's disease (HD) is a fatal neurodegenerative disorder.
• It is caused by a CAG repeat expansion in the Huntingtin (HTT) gene.
• Currently, no disease-modifying therapies are available for HD, and the HTT protein's function remains unclear due to a lack of selective chemical tools.

Purpose of the Study:

• To identify and characterize novel chemical tools targeting the HTT protein.
• To investigate the molecular function of HTT and its interactions.
• To develop potential therapeutic strategies for Huntington's disease.

Main Methods:

• Identification and characterization of macrocyclic peptide binders.
• In vitro affinity assays.
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) for interface analysis.
• Chemoproteomics to confirm selective binding in cell extracts.
• Analysis of HTT-HAP40 complex formation across different cell lines and HTT variants.

Main Results:

• Novel macrocyclic peptide binders targeting HTT were identified and characterized.
• These binders exhibit low-nanomolar affinity in vitro.
• The binders engage distinct HTT and HTT-HAP40 interfaces.
• Chemoproteomics confirmed selective binding to HTT in wildtype but not HTT-null cell lines.
• HAP40 consistently co-purified with HTT across various cell lines and HTT variants, indicating the prevalence of the HTT-HAP40 complex.

Conclusions:

• Macrocyclic peptide binders represent promising chemical tools for studying HTT.
• These binders can selectively target HTT and its complexes.
• The findings provide new insights into the HTT-HAP40 complex and its relevance in Huntington's disease.
• This work lays the foundation for developing novel therapeutic approaches for HD.