Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase.
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.
Wu
Yao-Wen
YW
Department of Physical Biochemistry, Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Alexandrov
Kirill
K
Brunsveld
Luc
L
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D019883:Alkyl and Aryl Transferases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011756:Diphosphates / Q000138:chemical synthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry
C526599:3,7,11-trimethyl-12-(7-nitrobenzo(1,2,5)oxadiazo-4-ylamino)dodeca-2,6,10-trien-1-yl pyrophosphate; D011756:Diphosphates; D005456:Fluorescent Dyes; D011106:Polyisoprenyl Phosphates; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; D009327:4-Chloro-7-nitrobenzofurazan; C002963:geranylgeranyl pyrophosphate