Document Detail


Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide.
MedLine Citation:
PMID:  9820804     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
J M Bruneau; C M Yea; S Spinella-Jaegle; C Fudali; K Woodward; P A Robson; C Sautès; R Westwood; E A Kuo; R A Williamson; E Ruuth
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The Biochemical journal     Volume:  336 ( Pt 2)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1998 Dec 
Date Detail:
Created Date:  1999-01-26     Completed Date:  1999-01-26     Revised Date:  2012-05-25    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  299-303     Citation Subset:  IM    
Affiliation:
Immunology Domain, Hoechst Roussel, 93235 Romainville, France.
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MeSH Terms
Descriptor/Qualifier:
Aniline Compounds / metabolism,  pharmacology*
Binding Sites
Binding, Competitive
Cell Division / drug effects
Cell Membrane / drug effects,  enzymology
Humans
Hydroxybutyrates / metabolism,  pharmacology*
Lymphoma / enzymology
Oxidoreductases / antagonists & inhibitors*,  isolation & purification*,  metabolism
Oxidoreductases Acting on CH-CH Group Donors*
Spleen / enzymology
Tumor Cells, Cultured
Uridine / metabolism,  pharmacology
Chemical
Reg. No./Substance:
0/Aniline Compounds; 0/HR 325; 0/Hydroxybutyrates; 108605-62-5/A 771726; 58-96-8/Uridine; EC 1.-/Oxidoreductases; EC 1.3.-/Oxidoreductases Acting on CH-CH Group Donors; EC 1.3.5.2/dihydroorotate dehydrogenase
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