Dendritic Self-assembled Structures from Therapeutic Charged Pentapeptides

[Image: see text] CRE(N)KA [Cys-Arg-(NMe)Glu-Lys-Ala, where (NMe)Glu refers to N-methyl-Glu], an anti-cancer pentapeptide that induces prostate tumor necrosis and significant reduction in tumor growth, was engineered to increase the resistance to endogenous proteases of its parent peptide, CREKA (Cys-Arg-Glu-Lys-Ala). Considering their high tendency to aggregate, the self-assembly of CRE(N)KA and CREKA into well-defined and ordered structures has been examined as a function of peptide concentration and pH. Spectroscopic studies and atomistic molecular dynamics simulations reveal significant differences between the secondary structures of CREKA and CRE(N)KA. Thus, the restrictions imposed by the (NMe)Glu residue reduce the conformational variability of CRE(N)KA with respect to CREKA, which significantly affects the formation of well-defined and ordered self-assembly morphologies. Aggregates with poorly defined morphology are obtained from solutions with low and moderate CREKA concentrations at pH 4, whereas well-defined dendritic microstructures with fractal geometry are obtained from CRE(N)KA solutions with similar peptide concentrations at pH 4 and 7. The formation of dendritic structures is proposed to follow a two-step mechanism: (1) pseudo-spherical particles are pre-nucleated through a diffusion-limited aggregation process, pre-defining the dendritic geometry, and (2) such pre-nucleated structures coalesce by incorporating conformationally restrained CRE(N)KA molecules from the solution to their surfaces, forming a continuous dendritic structure. Instead, no regular assembly is obtained from solutions with high peptide concentrations, as their dynamics is dominated by strong repulsive peptide–peptide electrostatic interactions, and from solutions at pH 10, in which the total peptide charge is zero. Overall, results demonstrate that dendritic structures are only obtained when the molecular charge of CRE(N)KA, which is controlled through the pH, favors kinetics over thermodynamics during the self-assembly process.