Key Findings
The study successfully designed ICLIST-11, a mini-protein that exhibits high affinity for Pituitary adenylate cyclase-activating peptide 38 (PACAP-38) with a $K_D$ of 4.51 nM. In cell-based functional assays, ICLIST-11 showed concentration-dependent neutralizing activity against PACAP-38-induced PAC1R activation with an $IC_{50}$ of 10.6 nM, significantly outperforming the initial design, ICLIST-2.
Technical Details
The development process utilized a computational pipeline involving Bindcraft and ProteinMPNN:
- Initial Design: Bindcraft was used to identify ICLIST-2, a 63-residue peptide with a $K_D$ of 26.5 nM.
- Optimization: Alanine scanning and structural analysis of ICLIST-2 identified residues for optimization. Using ProteinMPNN, researchers implemented nine amino acid substitutions to create ICLIST-11.
- Stability and Affinity: ICLIST-11 achieved a $T_m$ value 14 °C higher than its predecessor. Molecular dynamics (MD) simulations suggest that the substitution of three residues with hydrophobic residues (E30F, M31L, and R54W) contributed to this increased stability. Interaction analysis further suggests that three specific mutations were critical for enhancing binding affinity.
Background & Context
PACAP-38 is a vasoactive peptide identified as a key mediator of migraine and a target for next-generation treatments. While antibodies against PACAP-38 have shown promising results as ligand trap medicines in clinical trials, this research explores the potential of medium-molecular-weight drugs as an alternative therapeutic modality.
Significance & Outlook
The transition from ICLIST-2 to ICLIST-11 demonstrates the efficacy of combining de novo design tools with structural optimization to improve both the affinity and thermal stability of mini-proteins. These results establish a computational framework for the discovery of protein drugs targeting PACAP-38.
Source: https://www.biorxiv.org/content/10.64898/2026.10.30.763049v1
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