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 Meroueh

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1768-1784
Number of pages17
JournalBiochemistry
Volume56
Issue number12
DOIs
StatePublished - Mar 28 2017

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Molecules
Proteins
Urokinase-Type Plasminogen Activator
Cations
Cell signaling
Derivatives
Molecular Dynamics Simulation
Free energy
Molecular dynamics
Salts
Crystal structure
Peptides
Computer simulation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Small Molecules Engage Hot Spots through Cooperative Binding to Inhibit a Tight Protein-Protein Interaction. / Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy.

In: Biochemistry, Vol. 56, No. 12, 28.03.2017, p. 1768-1784.

Research output: Contribution to journalArticle

Liu, Degang ; Xu, David ; Liu, Min ; Knabe, William Eric ; Yuan, Cai ; Zhou, Donghui ; Huang, Mingdong ; Meroueh, Samy. / Small Molecules Engage Hot Spots through Cooperative Binding to Inhibit a Tight Protein-Protein Interaction. In: Biochemistry. 2017 ; Vol. 56, No. 12. pp. 1768-1784.
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