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

12 Citations (Scopus)

Abstract

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
Volume10
Issue number6
DOIs
StatePublished - Jun 19 2015

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Vitronectin
Molecules
Integrins
Surface Plasmon Resonance
Urokinase-Type Plasminogen Activator
Surface plasmon resonance
Inhibitory Concentration 50
Extracellular Matrix
Proteins
Cells
Focal Adhesion Protein-Tyrosine Kinases
Phosphorylation
HEK293 Cells
GTP Phosphohydrolases
Cell adhesion
Cell Surface Receptors
Entropy
Serine Proteases
Molecular Dynamics Simulation
Static Electricity

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

A New Class of Orthosteric uPAR·uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR·Vitronectin Interaction. / Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O.

In: ACS chemical biology, Vol. 10, No. 6, 19.06.2015, p. 1521-1534.

Research output: Contribution to journalArticle

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abstract = "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 Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 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 IC50 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 VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB·uPAR interaction was less favorable in the VTNSMB·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.",
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AU - Knabe, William Eric

AU - Meroueh, Samy O.

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