Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.
Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.
Coxon
F P
FP
Department of Medicine and Therapeutics, Medical Microbiology, and Pathology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. f.p.coxon@abdn.ac.uk
Helfrich
M H
MH
Larijani
B
B
Muzylak
M
M
Dunford
J E
JE
Marshall
D
D
McKinnon
A D
AD
Nesbitt
S A
SA
Horton
M A
MA
Seabra
M C
MC
Ebetino
F H
FH
Rogers
M J
MJ
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors*; D000818:Animals; D002460:Cell Line; D004164:Diphosphonates / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008854:Microscopy, Electron; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D017368:Protein Prenylation; D011725:Pyridines / Q000494:pharmacology*; D011817:Rabbits
C410028:2-(3-pyridinyl)-1-hydroxyethylidene-1,1-phosphonocarboxylic acid; D004164:Diphosphonates; D004791:Enzyme Inhibitors; D011725:Pyridines; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase