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

Abstract

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene, with no disease-modifying therapies currently available. The precise molecular function of the HTT protein is unclear, and the lack of selective chemical tools has limited functional studies. We have identified and characterized macrocyclic peptide binders targeting HTT. These binders exhibit low-nanomolar affinity in vitro and engage distinct HTT and HTT-HAP40 interfaces, as revealed by hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy. Chemoproteomics confirmed selective binding in cell extracts from wildtype but not HTT-null cell lines. HAP40 consistently and stoichiometrically co-purified with HTT across cell lines, including with HTT variants containing different CAG repeat lengths, highlighting the broad presence of the HTT-HAP40 complex.

Keywords: HAP40; Huntingtin; Huntington’s disease; ligand discovery; peptide macrocycles.

Figure 1.. Macrocycle discovery and selection.

a, Structure diagram of HTT-HAP40. HTT contains the N17, polyglutamine (polyQ), and proline rich domain (PRD), as well as the globular N-HEAT (blue), Bridge (green), and C-HEAT (purple) regions. A large intrinsically disordered region (IDR – grey) interrupts the N-HEAT domain. The wildtype (WT) protein in this study is Q23 (3144 aa), and the mutant is Q54. HTT forms a stable noncovalent heterodimer with HAP40 (pastel green). An AlphaFold3 structure of full-length HTTQ23-HAP40 was used here model the full-length proteins. b, RaPID is an RNA-display technique coupled to FIT, facilitating the incorporation of nonstandard residues which enable macrocyclization. c, Spontaneous cyclization of peptides enabled by nonstandard residue incorporation at the N-terminus, such as chloroacetyl-Tyr. Adapted from Saha, Suga, and Brik (2023) (Saha et al., 2023).

Figure 2.. Macrocycle identity and affinity measurements.

a, Sequence and structure of macrocycles validated by SPR. Assessment of HTTQ23 and HTTQ23-HAP40 binding by, b, SPR and, c, FP of exemplary macrocycle HL2 and summary tables for remaining macrocycles. Detailed affinity measurements for all macrocycles by SPR and FP available in Supplementary Figure 2. Both SPR and FP results indicate that HHL1 and HHD3 require the presence of HAP40 to interact with HTT. By contrast, HL5 interacts with apo HTT more tightly than HTT-HAP40 suggesting a potential binding site proximal to the interface between HTT and HAP40. Both HL2 and HD4 bound apo or HTT-HAP40, suggested a binding pocket distal to the HTT-HAP40 interface. Notably, significant selectivity for polyQ length was not observed since affinities fell into a similar range, suggesting the N-terminal polyQ tract did not play a major role in binding.

Figure 3.. Mapping macrocycle binding interfaces.

a, Determination of macrocycle affinity for HTT structure-rationalized subdomains by FP. Each plot represents a technical triplicate of each macrocycle tested against HTT’s CTD, NTD, and CTD-HAP40. The summary table reports the mean of a biological duplicate (n=2) ± σ. N.B. indicates no binding could be accurately estimated or detected at the concentrations tested. b, Differential Hydrogen-Deuterium Exchange Mass Spectrometry (ΔHDX-MS) of HTTQ23-HAP40 + HHD3, HHL1, HD4, or HL2, and HTTQ23 + HL5. Cumulative differences (0.5, 5, 30 mins) in fractional uptake exceeding cumulative error are shown on the HTT-HAP40 A3F model as increases (red), decreases (blue), statistically insignificant (white/green), and no coverage (grey).

Figure 4.. Cryo-EM resolved binding interactions of HL2, HD4, HHL1, and HHD3.

a, Full complex of HTT-HAP40 and macrocycles HHL1, HD4 and HL2, showing binding interface and binding interactions as red dashed lines. b, HTT-HAP40 complex bound to HHL1, HD4 and HL2 showing interface and binding interactions.

Figure 5.. Macrocycles selectively bind endogenous HTT in MP assays.

a. Schematic representation of the macrocycle precipitation (MP) assay workflow. HEK293T cells were lysed, and macrocycles were added to the lysate to bind to endogenous HTT in complex with other binding partners. Streptavidin beads were then introduced to capture macrocycle-bound complexes. After incubation, the beads were washed to remove unbound proteins, and HTT complexes were eluted for downstream analysis. b. Volcano plot of MP results showing log2 fold change (FC) and −log10 P values derived from normalized protein spectral counts

Figure 6.. HTT and HAP40 form a constitutive complex across different polyQ lengths in NPCs.

Volcano plot of HL2(PEG11) macrocycle pulldown experiments for Q30/Q19, Q45/Q19, and Q81/Q27 HTT-expressing NPCs. Results are shown in log₂ fold change (FC) and −log10 p-values derived from normalized protein spectral counts.