Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction Degang Liu David Xu Min Liu William Eric Knabe Cai Yuan Donghui Zhou Mingdong Huang Samy O. Meroueh 10.1021/acs.biochem.6b01039.s001 https://acs.figshare.com/articles/journal_contribution/Small_Molecules_Engage_Hot_Spots_through_Cooperative_Binding_To_Inhibit_a_Tight_Protein_Protein_Interaction/4762213 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 <b>1</b>, 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 <b>3</b> and to pyrrolinone <b>12</b>. While both <b>3</b> and <b>12</b> bind to uPAR and compete with a fluorescently labeled peptide probe, only <b>12</b> and its derivatives inhibit the full uPAR·uPA interaction. Compounds <b>3</b> and <b>12</b> mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, <b>12</b> is involved in a π–cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that <b>3</b> and <b>12</b> exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or <b>12</b> show that Arg-53 interacts with uPA or with <b>12</b> 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. 2017-02-10 00:00:00 pyrazole 3 uPAR binding 40 derivatives 12 bind small-molecule inhibitors 12 exhibit pyrrolinone 12 small-molecule engagement urokinase receptor Compounds 3 small-molecule inhibitor binding partner Free energy calculations urokinase-type plasminogen activator uPA 12 show crystal structure Small Molecules Engage Hot Spots hot-spot residues pyrrolinone 1 peptide probe Arg -53 salt-bridge interactions dynamics simulations Cooperative Binding