A New Class of Orthosteric uPAR·uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR·Vitronectin Interaction

Degang Liu, Donghui Zhou, Bo Wang, William Eric Knabe, Samy O. Meroueh

Research output: Contribution to journalArticle

14 Scopus citations


The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant K<inf>i</inf> of 0.7 μM and inhibited the tight uPAR·uPA<inf>ATF</inf> interaction with an IC<inf>50</inf> of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC<inf>50</inf> values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN<inf>SMB</inf>·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTN<inf>SMB</inf> interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTN<inf>SMB</inf>·uPAR interaction was less favorable in the VTN<inf>SMB</inf>·uPAR·7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.

Original languageEnglish (US)
Pages (from-to)1521-1534
Number of pages14
JournalACS chemical biology
Issue number6
StatePublished - Jun 19 2015

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

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