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Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction
journal contribution
posted on 2017-02-10, 00:00 authored by Degang Liu, David Xu, Min Liu, William Eric Knabe, Cai Yuan, Donghui Zhou, Mingdong Huang, Samy O. MerouehProtein–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.
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pyrazole 3uPAR binding40 derivatives12 bindsmall-molecule inhibitors12 exhibitpyrrolinone 12small-molecule engagementurokinase receptorCompounds 3small-molecule inhibitorbinding partnerFree energy calculationsurokinase-type plasminogen activator uPA12 showcrystal structureSmall Molecules Engage Hot Spotshot-spot residuespyrrolinone 1peptide probeArg -53salt-bridge interactionsdynamics simulationsCooperative Binding
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