Self-assembly of a gonadotropin-releasing hormone antagonist-Teverelix

Peptide and protein aggregation (self-assembly) has been an important topic for many years, since it is implicated in a range of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s Diseases. At the same time, it also represents a significant challenge for the Pharmaceutical Industry, as many peptides and protein-based drugs have a propensity to self assemble into amorphous or highly structured aggregates. Such processes can occur during the manufacture, purification, processing or even storage of therapeutic peptides, as they are exposed to different conditions (e.g., agitation and pH changes) which can enhance the intrinsic aggregation propensity. Aggregation is associated with a number of adverse effects, including loss of biological activity and gain of cytotoxicity and/or immunogenicity. Although most aggregation processes are deleterious, there are a few examples where self-assembly can be useful, for example, when the aggregates themselves can be used directly in drug delivery, as slow-release depots thus enhancing drug half-life in vivo. In this work, the self-assembly of teverelix, a synthetic peptide which has been shown to be an effective treatment for prostate cancer, was studied. It was known that at high concentrations teverelix (in the form of a TFA salt) forms a microcrystalline suspension, which is compatible with subcutaneous injection, however, under other conditions it was known to form fibrillar structures. The mechanism of formation of either state, and the factors affecting the stability and rate of formation of these states was largely unknown. Since the behaviour of Tv at low and high concentrations is significantly different, the studies performed within this Thesis can be divided into two parts. First, low peptide concentrations (< 10 mg/mL), were studied and fibrils found to be the main aggregated species. The amyloid identity of Tv fibrils was shown by X-ray fibre diffraction and some structural information on the Tv fibrils was extracted from the diffraction pattern. In parallel, the morphology and dimensions of the Tv fibrils were studied by transmission electron microscopy. The kinetics of fibril formation were investigated and factors including Tv concentration, pH, ionic strength and TFA concentration were demonstrated to have a considerable effect. Oligomeric species formed in freshly prepared solutions of Tv was studied by size-exclusion chromatography and Tv molecules found to be largely dimeric with some slightly larger oligomers also populated. By combining all the information obtained, a mechanism of Tv fibril formation is proposed. At high Tv concentrations (~75 mg/mL), microcrystals were found to the main species. The morphology and dimensions of the microcrystals was studied by transmission and scanning electron microscopy. All microcrystals had the rectangular-shaped morphology with the width varying from 500 nm to 2.5 m, length from 500 nm to 5 m. In addition, the stability of Tv microcrystals was studied using dilution experiments and the subsequent rapid formation of fibrils investigated. Considerable time and effort were put into determining the structure of Tv in its crystalline state. Unfortunately, all the crystal screens and seeding experiments under a wide variety of conditions did not produce crystals of sufficient size/stability to solve the structure. One incomplete diffraction dataset was obtained but there was insufficient data for structure determination. Lastly, preliminary studies were performed on Tv-Ac samples at both low and high concentrations (1 mg/mL & 75 mg/mL). At low concentration (pH 5.9-8.0), the behaviour of Tv-Ac is similar to Tv-TFA, and fibrils were the main aggregated species observed. A similar decrease in pH was observed after fibril formation by Tv-Ac and was attributed potentially to the deprotonation of the lysine side chain. At 75 mg/mL (pH 5.1), the behaviour of Tv-Ac was significantly different from Tv-TFA The cloudy microcrystalline suspension observed for Tv TFA was not seen for Tv-Ac even when the pH value was adjusted to 2.0 similar to that of 75 mg/mL Tv-TFA samples (pH was 1.74). Therefore, the TFA counterions play an important role in the formation of microcrystalline state of Tv.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Li, Xinyang

Advisor dc:contributor.advisor

• Jackson, Sophie

• Amyloid fibril
• Kinetics of fibril growth
• Peptide crystals
• Theraputic peptide

Licence

• https://www.repository.cam.ac.uk/bitstreams/fc5cda73-2c17-48b1-8c2f-e76873e2d6a5/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.124915

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/395385