Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide.
Leflunomide is currently in phase-III clinical trials for the treatment of rheumatoid arthritis. In this study, we have focused our efforts on the study of the mechanism of action of the active metabolite of leflunomide, A77 1726, in cells and tissue of human origin. The human high-affinity binding protein for radiolabelled A77 1726 was purified from solubilized U937 membranes by following the binding activity through the purification process and was characterized as the mitochondrial enzyme dihydro-orotate dehydrogenase (DHO-DH). The human and murine enzyme displayed identical pI and molecular mass values on SDS/PAGE (43 kDa), which contrasts notably with previous reports suggesting a molecular mass of 50 kDa for the human enzyme. DHO-DH activity was inhibited by A77 1726 and its analogue HR325 with similar potency in U937 and human spleen membrane preparations. HR325 was found to be anti-proliferative for phytohaemagglutinin-stimulated human peripheral blood mononuclear cells, at the same concentrations that caused accumulation of DHO and depletion of uridine. Supplementation of the cultures with exogenous uridine led to partial abrogation of the anti-proliferative effect. This is in line with our recent demonstration that the anti-proliferative effect in vitro of A77 1726 on lipopolysaccharide-stimulated mouse spleen cells was mediated by DHO-DH inhibition [Williamson, Yea, Robson, Curnock, Gadher, Hambleton, Woodward, Bruneau, Hambleton, Moss et al., (1995) J. Biol. Chem. 270, 22467-22472]. Thus, DHO-DH inhibition by A77 1726 and its analogues is responsible for the anti-proliferative effects in vitro of the compounds on human cells and is likely to be responsible for some of its effects in vivo.
Bruneau
J M
JM
Immunology Domain, Hoechst Roussel, 93235 Romainville, France.
Yea
C M
CM
Spinella-Jaegle
S
S
Fudali
C
C
Woodward
K
K
Robson
P A
PA
Sautès
C
C
Westwood
R
R
Kuo
E A
EA
Williamson
R A
RA
Ruuth
E
E
D000814:Aniline Compounds / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003437:Crotonates; D000090722:Dihydroorotate Dehydrogenase; D006801:Humans; D006885:Hydroxybutyrates / Q000378:metabolism / Q000494:pharmacology*; D008223:Lymphoma / Q000201:enzymology; D009570:Nitriles; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013154:Spleen / Q000201:enzymology; D014052:Toluidines; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism / Q000494:pharmacology
D000814:Aniline Compounds; D003437:Crotonates; D000090722:Dihydroorotate Dehydrogenase; C107042:HR 325; D006885:Hydroxybutyrates; D009570:Nitriles; D014052:Toluidines; C527525:teriflunomide; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D014529:Uridine