title: A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.
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.
authors: Degang
authors: D
authors: Liu
authors: Donghui
authors: D
authors: Zhou
authors: §Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis (IUPUI), Indianapolis, Indiana 46202, United States.
authors: Bo
authors: B
authors: Wang
authors: William Eric
authors: WE
authors: Knabe
authors: §Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis (IUPUI), Indianapolis, Indiana 46202, United States.
authors: Samy O
authors: SO
authors: Meroueh
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding; D011758:Pyrroles / Q000138:chemical synthesis / Q000494:pharmacology*; D020239:Receptor Cross-Talk / Q000187:drug effects; D055293:Receptors, Urokinase Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000138:chemical synthesis / Q000494:pharmacology*; D013816:Thermodynamics; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019096:Vitronectin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D039302:Integrin alphaVbeta3; D016023:Integrins; D010455:Peptides; D011758:Pyrroles; C107953:RAC1 protein, human; D055293:Receptors, Urokinase Plasminogen Activator; D015842:Serine Proteinase Inhibitors; D054852:Small Molecule Libraries; D019096:Vitronectin; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D014568:Urokinase-Type Plasminogen Activator; D020830:rac1 GTP-Binding Protein