Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand

Abstract

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA.

Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor

“…39 While the small molecule does not directly bind to uPA, it is believed that the blocking of the uPAR•uPA interaction at the cell surface will likely result in less activation of uPA. Chloromethylketone (CMK) showed irreversible inhibition of uPA activity with nearly 50 and 60% inhibition at 10 and 50 µM of inhibitor, respectively (Fig.…”

Structure-Driven Design of Radionuclide Tracers for Non-Invasive Imaging of uPAR and Targeted Radiotherapy. The Tale of a Synthetic Peptide Antagonist

“…The importance of such considerations and the consequences of ignoring them are illustrated by the following case-story: In an attempt to develop small molecules as antagonists of the uPA•uPAR interaction, large chemical libraries were originally screened using a peptide surrogate of uPA as a reporter. Unfortunately, this peptide was unconsciously 125 I-labeled at a position corresponding to the hotspot residue Tyr 24 39, and the claimed potency of the selected lead compounds could accordingly not be confirmed in subsequent cell-binding assays by an independent group 40. Along the same lines, some precaution should always be exercised when modifying ATF extensively with reporter groups using traditional N-hydroxysuccinimide-based chemistry, as Lys 23 in the β-hairpin of GFD is particularly prone to this modification leading to loss of uPAR binding 41.…”