De novo design of mini-proteins as specific binders to PACAP-38, a key mediator of migraine
- 1Imperial College London
Abstract
Pituitary adenylate cyclase-activating peptide 38 (PACAP-38) is a vasoactive peptide that is attracting attention as a potential target for next-generation migraine treatments. Antibodies against PACAP-38 have recently shown promising results as a ligand trap medicine in clinical trials. In this study, we investigated the de novo design of miniproteins to evaluate the potential for developing a medium-molecular-weight drug that binds to PACAP-38. Using Bindcraft, we designed binders against PACAP-38 and identified ICLIST-2, a 63-residue peptide with affinity for PACAP-38 (KD = 26.5 nM). Furthermore, by alanine scanning and structural analysis of ICLIST-2, we determined residues that were suitable for further optimization using ProteinMPNN. This resulted in the design of ICLIST-11, which incorporates nine amino acid substitutions relative to ICLIST-2, resulting in a KD of 4.51 nM. Interaction analysis based on a structural model suggested that three mutations in ICLIST-11 may be important for enhancing affinity. Moreover, ICLIST-11 exhibits a Tm value that is 14 {degrees}C higher, and MD simulations suggest that the substitution of three residues with hydrophobic residues (E30F, M31L, and R54W) is one reason for this. In cell-based functional assays, ICLIST-11 demonstrated potent, concentration-dependent neutralizing activity against PACAP-38-induced PAC1R activation (IC50 = 10.6 nM), significantly outperforming the parental ICLIST-2. Taken together, these results provide a framework for protein drug discovery targeting PACAP-38.
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BibTeX
@misc{yamaguchi2026novo,
title = {{De novo design of mini-proteins as specific binders to PACAP-38, a key mediator of migraine}},
author = {H. Yamaguchi and S. Suzuki and Y. Fujiyoshi and M. Isalan},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.30.763049},
url = {https://www.biorxiv.org/content/10.64898/2026.10.30.763049v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - De novo design of mini-proteins as specific binders to PACAP-38, a key mediator of migraine
AU - H. Yamaguchi
AU - S. Suzuki
AU - Y. Fujiyoshi
AU - M. Isalan
PY - 2026
DA - 2026/10/30
PB - bioRxiv
DO - 10.64898/2026.10.30.763049
UR - https://www.biorxiv.org/content/10.64898/2026.10.30.763049v1
AB - Pituitary adenylate cyclase-activating peptide 38 (PACAP-38) is a vasoactive peptide that is attracting attention as a potential target for next-generation migraine treatments. Antibodies against PACAP-38 have recently shown promising results as a ligand trap medicine in clinical trials. In this study, we investigated the de novo design of miniproteins to evaluate the potential for developing a medium-molecular-weight drug that binds to PACAP-38. Using Bindcraft, we designed binders against PACAP-38 and identified ICLIST-2, a 63-residue peptide with affinity for PACAP-38 (KD = 26.5 nM). Furthermore, by alanine scanning and structural analysis of ICLIST-2, we determined residues that were suitable for further optimization using ProteinMPNN. This resulted in the design of ICLIST-11, which incorporates nine amino acid substitutions relative to ICLIST-2, resulting in a KD of 4.51 nM. Interaction analysis based on a structural model suggested that three mutations in ICLIST-11 may be important for enhancing affinity. Moreover, ICLIST-11 exhibits a Tm value that is 14 {degrees}C higher, and MD simulations suggest that the substitution of three residues with hydrophobic residues (E30F, M31L, and R54W) is one reason for this. In cell-based functional assays, ICLIST-11 demonstrated potent, concentration-dependent neutralizing activity against PACAP-38-induced PAC1R activation (IC50 = 10.6 nM), significantly outperforming the parental ICLIST-2. Taken together, these results provide a framework for protein drug discovery targeting PACAP-38.
ER -