Aggregation Behavior of Structurally Similar Therapeutic Peptides Investigated by (1)H NMR and All-Atom Molecular Dynamics Simulations

[Image: see text] Understanding of peptide aggregation propensity is an important aspect in pharmaceutical development of peptide drugs. In this work, methodologies based on all-atom molecular dynamics (AA-MD) simulations and (1)H NMR (in neat H(2)O) were evaluated as tools for identification and investigation of peptide aggregation. A series of structurally similar, pharmaceutically relevant peptides with known differences in aggregation behavior (D-Phe(6)-GnRH, ozarelix, cetrorelix, and degarelix) were investigated. The (1)H NMR methodology was used to systematically investigate variations in aggregation with peptide concentration and time. Results show that (1)H NMR can be used to detect the presence of coexisting classes of aggregates and the inclusion or exclusion of counterions in peptide aggregates. Interestingly, results suggest that the acetate counterions are included in aggregates of ozarelix and cetrorelix but not in aggregates of degarelix. The peptides investigated in AA-MD simulations (D-Phe(6)-GnRH, ozarelix, and cetrorelix) showed the same rank order of aggregation propensity as in the NMR experiments. The AA-MD simulations also provided molecular-level insights into aggregation dynamics, aggregation pathways, and the influence of different structural elements on peptide aggregation propensity and intermolecular interactions within the aggregates. Taken together, the findings from this study illustrate that (1)H NMR and AA-MD simulations can be useful, complementary tools in early evaluation of aggregation propensity and formulation development for peptide drugs.