Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS.
The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC-MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A, a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects on other enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis.
Academic Medical Center, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Amsterdam, The Netherlands.
Linda
L
Henneman
Arno G
AG
van Cruchten
Willem
W
Kulik
Hans R
HR
Waterham
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D050071:Bone Density Conservation Agents / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002853:Chromatography, Liquid; D004164:Diphosphonates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors; D051231:Farnesyltranstransferase / Q000037:antagonists & inhibitors; D005347:Fibroblasts / Q000378:metabolism; D056945:Hep G2 Cells / Q000378:metabolism; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008214:Lymphocytes / Q000378:metabolism; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D000077268:Pamidronate; D054876:Prenylation; D015398:Signal Transduction / Q000187:drug effects*; D053719:Tandem Mass Spectrometry; D013729:Terpenes / Q000378:metabolism*; D014233:Tricarboxylic Acids / Q000494:pharmacology; D000077211:Zoledronic Acid
D050071:Bone Density Conservation Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004164:Diphosphonates; D004791:Enzyme Inhibitors; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007093:Imidazoles; D013729:Terpenes; D014233:Tricarboxylic Acids; C075117:squalestatin 1; C045174:6-fluoromevalonolactone; D000077211:Zoledronic Acid; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D013186:Farnesyl-Diphosphate Farnesyltransferase; D051231:Farnesyltranstransferase; D000077268:Pamidronate; D008798:Mevalonic Acid