Insight into the Evolution of uPAR Function and Optimization of uPAR-targeting Probes

Julie Maja Leth

Abstract

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that plays a central role in surface-associated plasminogen activation. It focalizes plasminogen activation at the cell-surfaces via high-affinity binding to the urokinase-type plasminogen activator
(uPA) and this promotes pericellular proteolysis and regulates extravascular fibrinolysis. In addition, uPAR facilitates cell adhesion via a low-affinity interaction with the extracellular matrix protein vitronectin (VN). The protein uPAR consists of three homologous Ly-6/uPAR (LU) domains. These domains contain five conserved disulfide bonds that give rise to a characteristic three-finger fold. The first LU domain in uPAR (DI) differs from the latter two domains by i) being highly flexible and important for the multi-domain assembly of the uPA-binding cavity, and ii) by lacking one of the LU consensus disulfide bonds, which is normally essential for correct folding of other single LU-domain
proteins. In study I, we investigated the structural and functional impact of reintroducing the LU consensus disulfide bond in uPAR DI. Using a variety of structural characterization methods, including hydrogendeuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS), and size exclusion chromatography (SEC), we showed that introducing the disulfide bond in uPAR DI impairs the flexible multi-domain assembly in uPAR. Furthermore, surface plasmon resonance (SPR) analyses revealed that the disulfide bond in uPAR DI reduces the binding affinity of uPAR to uPA by 40-fold.
This reduction was mainly caused by a faster dissociation rate constant showing that the introduced disulfide bond in uPAR DI decreases the complex stability. Based on these studies, we propose that an evolutionary deletion of this particular disulfide bond may have facilitated the multi-domain assembly of a high-affinity uPA-binding cavity.
The expression level of uPAR is high in many chronic inflammatory diseases including cancer. High levels of uPAR expression in most human solid cancers – combined with a generally low or absent
expression in healthy tissue – makes uPAR an attractive molecular target for anti-cancer therapy and molecular imaging within clinical oncology for visualization of diseased areas.
In study II, we investigated a novel small molecule inhibitor of the uPA•uPAR interaction. A structurebased virtual screening approach identified the small molecule inhibitor and predicted it to bind within the uPA-binding cavity. In study II, we probed the binding site- and mode of this compound on uPAR
by applying various single-site alanine mutants of uPAR in an SPR competition assay. We aimed to confirm the involvement of the uPA-binding cavity in uPAR and, in addition, our studies assisted in identifying the most likely binding mode. We determined the potency of this compound to be very low (IC50~60 μM); however, we discovered that the potency increased 10-fold against a uPAR mutant that mimicked the closed uPAR conformation of the uPA-bound state. These results were consistent with the design approach using the uPA-bound uPAR structure as a search template; however, it shows that this choice represents a sub-optimal design for targeting a flexible protein as uPAR. We
suggest developing small molecules that also target other conformations of uPAR.
In study III and IV, we investigated different strategies to optimize a uPAR-targeting small peptide as an imaging probe by either modifying the uPAR-targeting core sequence (study III) or changing the imaging reporter (study IV). In study III, we demonstrated that we could increase the helical propensity of the small peptide by cyclization as probed by NMR and HDX-MS studies. We showed with SPR that this peptide displayed similar affinity to uPAR as the original peptide; however, the thermo-stability of the peptide-uPAR complex increased with ~5°C – as assessed by nano-differential scanning fluorimetry (nano-DSF). In addition, we showed that amidation of the peptide C-terminus improved
the affinity of the original peptide while it did not affect the cyclized peptide. From testing these modifications in vivo using 64Cu-DOTA-conjugated peptides for positron emission tomography (PET)- imaging, we discovered that peptide cyclization may improve tumor-uptake compared to the original peptide. Study IV tested the small peptide as an optical imaging probe for fluorescence-guided surgery (FGS). Results from this study show that altering the imaging agent can improve features including affinity to uPAR as well as provide higher imaging contrast.
Collectively, the results in this thesis provide i) new insight into the important role of uPAR DI and the evolution of uPAR function and ii) different strategies to further optimize probes targeting uPAR – both as small molecule inhibitors and as peptide probes for molecular imaging of uPAR expression.
OriginalsprogEngelsk
Antal sider139
StatusUdgivet - jan. 2023

Citationsformater