title: Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.
abstract: Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Degang
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.|Center for Computational Biology and Bioinformatics, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.|Department of BioHealth Informatics, Indiana University School of Informatics and Computing , Indianapolis, Indiana 46202, United States.
authors.forename: David
authors.initials: D
authors.lastname: Xu
authors.affiliation: Fujian Institute of Research on the Structure of Matter, Chinese Academy of Science , Gulou District, Fuzhou, Fujian 3500002, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: William Eric
authors.initials: WE
authors.lastname: Knabe
authors.affiliation: Fujian Institute of Research on the Structure of Matter, Chinese Academy of Science , Gulou District, Fuzhou, Fujian 3500002, China.
authors.forename: Cai
authors.initials: C
authors.lastname: Yuan
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Donghui
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Fujian Institute of Research on the Structure of Matter, Chinese Academy of Science , Gulou District, Fuzhou, Fujian 3500002, China.
authors.forename: Mingdong
authors.identifier: 0000-0002-1377-6786
authors.initials: M
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.|Center for Computational Biology and Bioinformatics, Indiana University School of Medicine , Indianapolis, Indiana 46202, United States.
authors.forename: Samy O
authors.identifier: 0000-0002-3948-7499
authors.initials: SO
authors.lastname: Meroueh
mesh_terms: D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011485:Protein Binding / Q000187:drug effects; D011720:Pyrazoles / Q000138:chemical synthesis* / Q000494:pharmacology; D011758:Pyrroles / Q000138:chemical synthesis* / Q000494:pharmacology; D055293:Receptors, Urokinase Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000138:chemical synthesis* / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011720:Pyrazoles; D011758:Pyrroles; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D014568:Urokinase-Type Plasminogen Activator