Insight into the interaction of heme with human calcitonin (hCT) and its inhibitory effect on hCT aggregation

Human calcitonin (hCT) is a peptide hormone with therapeutic potential for bone-related disorders, yet its clinical application is hindered by a strong propensity to form amyloid fibrils. Strategies that effectively suppress hCT aggregation are therefore essential for enabling its reintroduction into pharmaceutical development. In our previous work, heme emerged as a potent inhibitor of hCT fibrillation via binding to the peptide. However, the molecular basis of this interaction and long-term inhibitory efficacy remain unclear. Herein, we systematically investigated heme-hCT binding and compared its anti-aggregation performance with a series of pharmaceutically relevant metalloporphyrins (FeTPPS, FeTBAP, FeTMPyP, MnTPPS, MnTBAP, MnTMPyP). Electrochemical and spectroscopic analyses revealed that heme binds with high affinity (K_b ≈ (3.67 ± 0.19) × 10⁶ M^-1) and 1:1 stoichiometry to the N-terminal region of hCT (residues 8-22), with His20 identified as the key binding site. Molecular calculations further illustrated that heme stabilizes hCT through multiple noncovalent interactions, including coordination with His20, π-π stacking with Phe22, and ionic interaction with Lys18. Long-term inhibition studies (up to 72 h) using thioflavin T fluorescence, circular dichroism, Nu-PAGE, and TEM demonstrated that heme persistently suppresses hCT fibrillation and maintains its monomeric conformation, outperforming all tested metalloporphyrins. A clear inhibitory hierarchy was observed: heme > > FeTPPS ≈ FeTBAP > MnTPPS ≈ MnTBAP > FeTMPyP > MnTMPyP, highlighting the importance of metal center, peripheral substituents, and electrostatic compatibility. These findings provide a mechanistic foundation for the design of heme-inspired molecular modulators aimed at improving the stability and pharmaceutical applicability of hCT, and other aggregation-prone peptide therapeutics.