Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a stable and potent inhibitor of firefly luciferase

Bioorg Med Chem Lett. 2005 Sep 1;15(17):3860-4. doi: 10.1016/j.bmcl.2005.05.115.

Abstract

In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5'-O-[(N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent (Ki = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Monophosphate / analogs & derivatives*
  • Animals
  • Drug Stability
  • Firefly Luciferin / analogs & derivatives*
  • Kinetics
  • Luciferases, Firefly / antagonists & inhibitors*
  • Molecular Mimicry
  • Peptide Fragments / analysis
  • Peptide Hydrolases / metabolism
  • Protein Conformation
  • Structure-Activity Relationship
  • Sulfonic Acids / chemical synthesis*
  • Sulfonic Acids / pharmacology

Substances

  • Peptide Fragments
  • Sulfonic Acids
  • Adenosine Monophosphate
  • Firefly Luciferin
  • sulfamic acid
  • Luciferases, Firefly
  • Peptide Hydrolases