Abstract
Teverelix is a short non-natural peptide, which is a gonadotropin releasing hormone antagonist and used as a treatment for prostate cancer. Teverelix is formulated as a trifluoroacetic acid salt, which, at the high concentrations used for parenteral injection, forms a microcrystalline suspension. At low concentrations and immediately after injection, teverelix self-assembles into a fibrillar species thought to be important for the slow-release kinetics and long-acting action of this peptide in vivo. In this paper, we confirmed the amyloid-like identity of teverelix fibrils using X-ray fiber diffraction and transmission electron microscopy. The inter-β-sheet packing distance was found to be larger than that of typical amyloid fibrils and this was attributed to the large non-natural side chains within the peptide. Using data from numerous biophysical experiments, a model of the structure of teverelix within the fibril is proposed. The kinetics of fibril formation were investigated using standard ThT assays, and teverelix found to fibrillate rapidly over a wide range of conditions. The fibrillation rate was shown to depend critically upon pH, peptide, and trifluoroacetic acid concentration. Fibrillation was accompanied by a drop in pH, which we attribute to the fact that the pyridinium side chain must be deprotonated before self-assembly. Based on our results, we propose a nucleation-polymerization mechanism in which dimers of teverelix rapidly self-assemble into amyloid-like fibrils with little change in the secondary structure but burial of some of the aromatic acid side chains. Interestingly, the fibrils can, under certain conditions, align to create a highly ordered array. To the best of our knowledge, this is the first paper studying teverelix in detail from a biophysical perspective, and it is directly relevant to the aggregation of the peptide observed in vivo.