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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Orthosteric inhibitors bind enzyme active sites, competing directly with substrates.
  • Substrate dependence is generally considered unlikely for orthosteric inhibitors due to their direct competition mechanism.

Purpose of the Study:

  • To investigate the unexpected substrate-dependent potency of CSN5i-3, an orthosteric inhibitor of the COP9 signalosome (CSN).
  • To elucidate the molecular mechanism underlying this substrate dependence and establish a new class of enzyme inhibitors.

Main Methods:

  • Enzyme inhibition assays to confirm CSN5i-3's activity against CSN.
  • Cryo-electron microscopy (Cryo-EM) to determine the structure of the enzyme-substrate-inhibitor complex.
  • Biochemical assays to analyze protein-protein interactions and binding affinities.

Main Results:

  • CSN5i-3 inhibits the COP9 signalosome (CSN) by binding to the active site of CSN5, competing with the substrate.
  • Despite low affinity for free CSN, CSN5i-3 achieves nanomolar potency by acting as a molecular glue, stabilizing the interaction between CSN5 and NEDD8.
  • Cryo-EM structures revealed that CSN5i-3 bridges the substrate and enzyme, enhancing binding affinity through a cooperative tri-molecular assembly.

Conclusions:

  • A molecular glue mechanism confers substrate-dependent potency to the orthosteric inhibitor CSN5i-3.
  • This mechanism highlights that molecular glues can function effectively with modest individual protein binding affinities.
  • The study establishes "orthosteric molecular glue inhibitors" as a novel class of substrate-dependent enzyme antagonists.