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Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase.

Abstract
Protein prenylation is one of the most common post-translational modifications affecting hundreds of eukaryotic proteins. Rab geranylgeranyl transferase prenylates exclusively the GTPases of Rab family, and inhibition of this enzyme induces apoptosis in cancer cells, making it an attractive anticancer target. To efficiently test for possible inhibitors of this enzyme, a robust high-throughput assay is required. Here, we present protocols for the synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate NBD-FPP. We utilized this fluorescent probe to design a high-throughput fluorometric assay of Rab prenylation. This continuous fluorometric assay offers the advantage of being sensitive, cost-effective and amendable to miniaturization. The protocol includes the synthesis of the fluorescent substrate, setup of the assay, assay procedure and data analysis. The procedure for the Rab geranylgeranyl transferase (RabGGTase) plate assay depends on the number of compounds in the screen but generally can be performed within a day.
AuthorsYao-Wen Wu, Kirill Alexandrov, Luc Brunsveld
JournalNature protocols (Nat Protoc) Vol. 2 Issue 11 Pg. 2704-11 ( 2007) ISSN: 1750-2799 [Electronic] England
PMID18007605 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • 3,7,11-trimethyl-12-(7-nitrobenzo(1,2,5)oxadiazo-4-ylamino)dodeca-2,6,10-trien-1-yl pyrophosphate
  • Diphosphates
  • Fluorescent Dyes
  • Polyisoprenyl Phosphates
  • Alkyl and Aryl Transferases
  • Rab geranylgeranyltransferase
  • 4-Chloro-7-nitrobenzofurazan
  • geranylgeranyl pyrophosphate
Topics
  • 4-Chloro-7-nitrobenzofurazan (analogs & derivatives, chemical synthesis, chemistry)
  • Alkyl and Aryl Transferases (analysis, antagonists & inhibitors)
  • Diphosphates (chemical synthesis, chemistry)
  • Fluorescent Dyes (chemical synthesis, chemistry)
  • Fluorometry (methods)
  • Polyisoprenyl Phosphates (chemistry)

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