Joan A. Harrington

5-ethynyluracil (776C85): Inactivation of dihydropyrimidine dehydrogenase in vivo

5-Ethynyluracil (776C85), a potent, mechanism-based, irreversible inactivator (Porter et al., J Biol Chem 267:5236-5242, 1992) of purified dihydropyrimidine dehydrogenase (DPD, uracil reductase, EC 1.3.1.2), readily inactivated DPD in vivo. DPD was assayed in tissue extracts by measuring the release of 14CO2 from [2-14C]uracil with an improved method. Specific activities from 0.1 to > 1000 U/mg protein were reproducibly measured. After rats were orally dosed with 20 micrograms/kg 5-ethynyluracil, liver, intestinal mucosa, lung, and spleen DPD were inactivated by 83-94%. The dose required to inactivate rat liver, rat brain, and mouse liver DPD by 50% was 1.8, 11, and 8.9 micrograms/kg, respectively. Rat liver DPD was inactivated completely within 25 min after an oral dose of 500 micrograms/kg 5-ethynyluracil. New DPD was synthesized with a half-time of 63 hr. We also developed an assay based on stoichiometric inactivation of DPD by 5-ethynyluracil to measure 5-ethynyluracil in plasma samples. Samples containing 5-ethynyluracil were incubated with rat liver extract for 24 hr at 12 degrees and then assayed for DPD. DPD activity decreased linearly with the concentration of 5-ethynyluracil (between 0 and 20 nM 5-ethynyluracil). The assay could detect 5-ethynyluracil at concentrations as low as 6 nM in human plasma and was not affected by high concentrations of uracil.

Herpes and human ribonucleotide reductases: Inhibition by 2-acetylpyridine 5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone (348U87)

The mode of inactivation of herpes simplex virus type 1 and human ribonucleotide reductases by 2-acetylpyridine 5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone++ + (348U87) was determined and compared to that described previously [Porter et al. Biochem Pharmacol 39: 639-646, 1990] for 2-acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U). 348U87 inactivated herpes ribonucleotide reductase faster than did A1110U. Moreover, iron-complexed 348U87 was a considerably more potent inactivator than iron-complexed A1110U. It appeared to efficiently form an initial complex with the viral enzyme prior to rapid enzyme inactivation. The combination of 348U87 and iron-complexed 348U87 inactivated with a rate constant that was slightly greater than the sum of their individual rate constants of inactivation. The corresponding combination of A1110U species inactivated with a rate constant that was much greater than the sum of the individual rate constants of inactivation. Herpes ribonucleotide reductase that had been inactivated by either species of 348U87 was reactivated by diluting the enzyme and inactivators into assay medium containing excess iron. 348U87 was also an effective inactivator of herpes simplex virus type 2 and varicella zoster virus ribonucleotide reductases. The iron-complexed forms of 348U87 and A1110U exhibited very different modes of inactivation of human ribonucleotide reductase. Iron-complexed 348U87 was a tight-binding inactivator, whereas iron-complexed A1110U was only a weak, non-inactivating, inhibitor. Furthermore, the inactivation by iron-complexed 348U87 was not stimulated by either 348U87 or A1110U, whereas the weak inhibition by iron-complexed A1110U was converted to rapid inactivation by A1110U. Excess iron prevented the inactivation by iron-complexed 348U87. Uncomplexed 348U87 was similar to uncomplexed A1110U in that it was not an inhibitor of the human enzyme.

5-Ethynyl-2(1H)-pyrimidinone: Aldehyde oxidase-activation to 5-ethynyluracil, a mechanism-based inactivator of dihydropyrimidine dehydrogenase

5-Ethynyluracil is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) in vitro (Porter et al., J Biol Chem 267: 5236-5242, 1992) and in vivo (Spector et al., Biochem Pharmacol, 46: 2243-2248, 1993. 5-Ethynyl-2(1H)-pyrimidinone was rapidly oxidized to 5-ethynyluracil by aldehyde oxidase. The substrate efficiency (kcat/Km) was 60-fold greater than that for N-methylnicotinamide. In contrast, xanthine oxidase oxidized 5-ethynyl-2(1H)-pyrimidinone to 5-ethynyluracil with a substrate efficiency that was only 0.02% that of xanthine. Because 5-ethynyl-2(1H)-pyrimidinone did not itself inactivate purified DPD in vitro and aldehyde oxidase is predominately found in liver, we hypothesized that 5-ethynyl-2(1H)-pyrimidinone could be a liver-specific inactivator of DPD. We found that 5-ethynyl-2(1H)-pyrimidinone administered orally to rats at 2 micrograms/kg inactivated DPD in all tissues studied. Although 5-ethynyl-2(1H)-pyrimidinone produced slightly less inactivation than 5-ethynyluracil, the two compounds showed fairly similar patterns of inactivation of DPD in these tissues. At doses of 20 micrograms/kg, however, 5-ethynyl-2-pyrimidinone and 5-ethynyluracil produced equivalent inactivation of DPD. Thus, 5-ethynyl-2(1H)-pyrimidinone appeared to be an efficient, but not highly liver-selective prodrug of 5-ethynyluracil.

Enzymatic elimination of fluoride from α-fluoro-β-alanine

Abstract Rat liver homogenates catalyzed the elimination of fluoride from ( R , S )- α -fluoro- β -alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial l -alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with l -alanine and glyoxylate as substrates, copurified with the α-fluoro-β-alanine-defluorinating enzyme. The NH 2 -terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of l -alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of k cat = 17 sec −1 , K L -Ala = 3.2 mM, and K glyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of ( R )- α -fluoro- β -alanine and ( S )- α -fluoro- β -alanine were k cat = 6.2 and 2.6 min −1 , respectively, and K m = 2.7 and 0.88 mM, respectively. l -Alanine potently inhibited the defluorination reaction with an apparent K i of 0.024 mM. ( R , S )- α -fluoro- β -alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridox-amino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with ( R , S )- α -fluoro- β -alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of ( R , S )- α -fluoro- β -alanine but was inactivated completely during dehalogenation of β-chloro- l -alanine.

(R)-5-Fluoro-5,6-dihydrouracil : kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase

The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.

Herpes simplex virus type 1 ribonucleotide reductase: Selective and synergistic inactivation by a1110u and its iron complex

2-Acetylpyridine-5-[(dimethylamino)thiocarbonyl]thiocarbonohydr azone (A1110U) inactivated herpes simplex virus Type 1 ribonucleotide reductase (EC 1.17.4.1) by a first-order process (kinact) which had a maximum value (Mkinact) of 8 hr-1 and a Kd that was less than 1 microM. The stable complex between iron and A1110U, (A1110U)2Fe+i, inactivated this enzyme with a Mkinact of 7 hr-1 and a Kd of 7 microM. Free A1110U and its iron-complex synergized as inactivators of the enzyme. For example, the kinact for the combination of 2 microM A1110U and 1 microM (A1110U)2Fe+i as independent inactivators was calculated to be about 9 hr-1, while the observed value was 32 hr-1. The bimolecular rate constant for inactivation of the viral enzyme by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was 2.5 10(7) M-1 hr-1 at 30 degrees. Human ribonucleotide reductase was less sensitive to the inhibitory effects of A1110U and its iron-complex. This enzyme was neither inhibited nor inactivated by A1110U and was weakly inhibited by (A1110U)2Fe+i. Furthermore, inactivation required the combination of A1110U and (A1110U)2Fe+i. The bimolecular rate constant for inactivation of human ribonucleotide reductase by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was considerably smaller (3.8 10(6) M-1 hr-1 at 37 degrees) than the analogous constant for the viral enzyme. Several iron-chelating reagents with unrelated structures substituted for free A1110U in its various roles with both enzymes. However, the iron complexes of these alternative chelators did not substitute for (A1110U)2Fe+i. The rates of inactivation of both enzymes were independent of the oxidation state of iron in (A1110U)2Fe+i and of CDP concentration. The inactivated enzymes were reactivated rapidly by FeSO4, but were not reactivated by CoCl2, CuSO4, or NiCl2. MnCl2 inhibited reactivation of the viral enzyme by FeSO4.

Rapid sampling of multiple enzyme reactions

A simple method of initiating and sampling six simultaneous reactions was devised. A commercially available vial rack was fitted with a Plexiglas overlaying sheet to stabilize the vials for the addition and sampling procedures. Glass vials were routinely used because of their thermal conductivity advantages. Samples were added and removed, and the reactions were mixed with a multichannel pipet using every other channel. The data showing six simultaneous progress curves for the rapid inactivation of herpes simplex virus ribonucleotide reductase were presented and analyzed. In addition, the time course of 12 reactions catalyzed by varicella zoster virus thymidine kinase were assayed at one min intervals generating 96 data points within 8.5 min. A second experiment generated data points every 30 s for six simultaneous replicate thymidine kinase reactions. The ease of use and high reproducibility of the method are demonstrated by these data.