Kallikrein-related peptidases (KLKs) are a family of 15 secreted serine proteases, which form a network – the KLK activome – with an important role in proteolysis and signalling. KLK activity is decoupled from abundance by several mechanisms of activation and inhibition, and active KLK molecules promote tumour progression and drug resistance through multiple biochemical pathways. To date no technology exists for specific assessment of KLK activities in a complex biological setting, presenting a challenge to realise the potential of KLKs as drug targets or novel biomarkers.
In this study, we have developed a technology platform that enables the identification of potent and highly selective activity-based probes (ABPs) for KLK proteases. Our approach consists of grafting optimal substrate sequences, identified from peptide combinatorial libraries composed of unnatural amino acids, into a peptidyl diphenyl phosphonate scaffold.
We employed our optimal ABPs to quantify KLK2, 3 and 14 activity in prostate cancer (PCa) cells and patient tissue, and demonstrated that active KLK molecules are present in the prostate cancer/bone niche. In addition, we used a specialised PCa/osteoblast co-culture model to demonstrate that dysregulation of the KLK activome facilitates resistance to anti-androgen therapy and drives metastasis of tumour cells to bone.
Finally, by employing a novel anthraquinone-conjugated mixed alkyl aryl phosphonate moiety, we transformed our ABPs into quenched activity-based probe (qABP) derivatives, which become fluorescent only after covalent modification of a KLK, and thus present uniquely excellent signal-to-background applicable to imaging.
Given the promising results obtained in this thesis, additional studies are warranted to assess the utility of targeting the proteolytic activity of KLK2, 3 and 14 for treatment of metastatic PCa and for demarcating tumour margins during prostatectomy surgery. In addition, we propose that our technology platform can be used to develop selective chemical tools for any KLK protease.