Haruhiro Okuda

Suicidal inactivation of human dihydropyrimidine dehydrogenase by (E)-5-(2-bromovinyl)uracil derived from the antiviral, sorivudine.

An enzymatic study was performed to clarify the mechanism of 18 acute deaths in patients who had received the new oral antiviral drug, sorivudine (SRV), during anticancer chemotherapy with 5-fluorouracil (5-FU) prodrugs. Human dihydropyrimidine dehydrogenase (hDPD), playing a key role in the liver as the rate-limiting enzyme in catabolism of 5-FU, was expressed in E. coli, purified and incubated in the presence of NADPH with SRV or (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of SRV produced by human gut flora. hDPD was rapidly and irreversibly inactivated by BVU, but not by SRV. Radioactivity of [14C]BVU was incorporated into hDPD in the presence of NADPH in a manner reciprocal to the enzyme inactivation. In the absence of NADPH, hDPD was not inactivated by BVU, nor radiolabeled with [14C]BVU. Thus, as we demonstrated previously with studies using the rat, the acute deaths were strongly suggested to be attributable to markedly elevated tissue 5-FU levels which were responsible for irreversible inhibition of hDPD by covalent binding of a reduced form of BVU as a suicide inactivator.

Long-chained 1-mercapto-n-alkanes as potent inhibitors toward liver alcohol dehydrogenase

Long-chained 1-mercapto-n-alkanes showed potent inhibitory effects on horse liver alcohol dehydrogenase (HLADH). The inhibitory effect of the thiols was enhanced by increasing the number of the alkyl carbon atoms up to 10-12 and steeply lowered by further increase in the carbon number. The HLADH activity was almost completely inhibited in competitive manner by an equivalent concentration of 1-mercapto-n-decane or -n-dodecane to that of the subunit of the dimeric zinc enzyme; inhibition constant Ki was 0.55 nM for the former. The present study strongly suggests that the thiols interact simultaneously with at least two sites of HLADH; the primary one could be the zinc atom in the active site of the enzyme, interacting with the sulfhydryl groups, and the other a hydrophobic binding site for the their alkyl carbons.

Metabolic activation of carcinogens by conjugation reactions.

Conjugations, as well as oxidations by P-450 and fp3, are main activation mechanisms for chemical carcinogens, though the conjugation of xenobiotics has been know ordinarily to be a process to make them water-soluble for excretion. O-Sulfation of arylhydroxamic acids, hydroxylamines, and hydroxymethylarenes by sulfotransferases transform these carcinogens to highly reactive sulfate esters which act as good leaving groups with concomitant formation of carbonium and/or nitrenium ions which can react readily with DNA, RNA, and protein, and consequently exert carcinogenicity. Acetyl-CoA-dependent acetylation and N, O-transacylation also activate arylhydroxylamines and hydroxamic acids, respectively, by the same mechanism as proposed for the sulfation. Aminoacylation of arylhydroxylamines by aminoacyl-tRNA synthetases, which are not drug-metabolizing enzymes, is in the same category of activation of carcinogens by acylation. Despite of its importance in detoxifying a variety of electrophiles formed in vivo, GSH conjugation is recognized to activate a certain type of compounds, such as 1, 1-, 1, 2- di-and poly-haloalkanes, of which the 1, 2-dihaloalkanes form reactive episulfonium ions of GSH. A possible participation of reactive glucuronides and phosphoric acid esters of carcinogens are discussed in their carcinogenesis mechanisms.