A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction

dc.contributor.authorLiu, Degang
dc.contributor.authorZhou, Donghui
dc.contributor.authorWang, Bo
dc.contributor.authorKnabe, William Eric
dc.contributor.authorMeroueh, Samy O.
dc.contributor.departmentDepartment of Biochemistry & Molecular Biology, IU School of Medicineen_US
dc.date.accessioned2016-10-05T17:04:07Z
dc.date.available2016-10-05T17:04:07Z
dc.date.issued2015-06-19
dc.description.abstractThe 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.en_US
dc.eprint.versionAuthor's manuscripten_US
dc.identifier.citationLiu, D., Zhou, D., Wang, B., Knabe, W. E., & Meroueh, S. O. (2015). A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction. ACS Chemical Biology, 10(6), 1521–1534. http://doi.org/10.1021/cb500832qen_US
dc.identifier.issn1554-8937en_US
dc.identifier.urihttps://hdl.handle.net/1805/11097
dc.language.isoen_USen_US
dc.publisherAmerican Chemical Societyen_US
dc.relation.isversionof10.1021/cb500832qen_US
dc.relation.journalACS chemical biologyen_US
dc.rightsPublisher Policyen_US
dc.sourcePMCen_US
dc.subjectPyrrolesen_US
dc.subjectpharmacologyen_US
dc.subjectReceptors, Urokinase Plasminogen Activatoren_US
dc.subjectantagonists & inhibitorsen_US
dc.subjectSerine Proteinase Inhibitorsen_US
dc.subjectSmall Molecule Librariesen_US
dc.subjectUrokinase-Type Plasminogen Activatoren_US
dc.subjectVitronectinen_US
dc.titleA new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interactionen_US
dc.typeArticleen_US
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