Probing the metabolic stabilities and binding affinities of model peptide-based radiopharmaceuticals

Peptide-based radiopharmaceuticals offer a powerful tool for the diagnosis and treatment of various cancers, neurological disorders, and cardiovascular diseases. However, the use of peptides in this way can be challenging as unmodified peptides often show poor in vivo stability due to rapid degradation by endogenous proteolytic enzymes. A number of approaches have been developed to increase the metabolic stability of peptide-based radiopharmaceuticals. These include modifications of the C- and/or N-termini, introduction of D- or other unnatural amino acids, PEGylation and alkyl chain incorporation, cyclisation, and peptide bond substitution. Despite a wide diversity of available modifications, most studies seeking to improve the metabolic stability of a peptide-based radiopharmaceuticals have only investigated one or two of these modifications. Additionally, the methods used to assess metabolic stability have varied greatly between different studies, preventing their direct comparison. Due to this, there is little consensus on which of these modifications yields the greatest increase to metabolic stability. It is also unknown if the performance of these modifications in one peptide or peptidomimetic system is comparable to the performance of the same modifications on in a different peptide or peptidomimetic system.

The work presented in this thesis aimed to compare the impact of different linkages on the metabolic stability of a model peptide system relevant to radiopharmaceutical applications. EuK (glutamic acid-urea-lysine) was chosen as the peptide system of interest, with its conjugation (via the lysine free amine) to the 4-fluorobenzoyl (FB) moiety modified with an amide linkage, and sulfonamide, β-alanine, D-alanine, N-methyl-L-alanine, PEG4, and L-alanine linkages, which have all been reported to enhance metabolic stability when compared to the simple amide linkage. To minimise impact to the binding affinity of EuK the structure of EuK was otherwise not modified.

The EuK-FB conjugates with the selected linkages were synthesised in moderate yields and good purity using procedures adapted from the literature. The metabolic stability of these EuK-FB conjugates were assessed using rat serum diluted in phosphate buffered saline for 24 hours at 37 °C. All compounds were shown to be fully stable. A control known to degrade in rat serum did show degradation, confirming the rat serum was functional.

The binding affinities of the EuK-FB conjugates towards their biological target, prostate specific membrane antigen (PSMA), were assessed using a fluorescence-based PSMA inhibition assay. All compounds showed a dose dependant inhibition of PSMA in the nanomolar range with IC50 values between 28.6 and 122 nM. To further assess the impact of the different linkers on their ability to bind to PSMA, molecular docking studies were performed using the Molecular Operating Environment (MOE) software. These studies identified that all compounds except for the conjugate linked by PEG4 showed very good and similar binding scores. All of the docked compounds also showed the same orientation and similar poses within the PSMA binding cavity. The lipophilicity of the EuK-FB conjugates was also predicted in silico and all compounds showed a similar range of hydrophilic LogD values (-1.41 to -0.33). The similarity in the performance of the compounds in terms of metabolic stability, binding affinity, and lipophilicity suggests that these linkages are not significantly altering the properties of the target compounds. This may indicate that they could also be used interchangeably as linkers in a different peptide-based systems or for different applications, such as in medicinal chemistry and chemical biology for maintaining metabolic stability of drugs and chemical probes.