Shigeyasu Ichihara

Design, synthesis and antifungal activity of a novel water soluble prodrug of antifungal triazole

A highly potent water soluble triazole antifungal prodrug, RO0098557 (1), has been identified from its parent, the novel antifungal agent RO0094815 (2). The prodrug includes a triazolium salt linked to an aminocarboxyl moiety, which undergoes enzymatic activation followed by spontaneous chemical degradation to release 2. Prodrug 1 showed high chemical stability and water solubility and exhibited strong antifungal activity against systemic candidiasis and aspergillosis as well as pulmonary aspergillosis in rats.

Involvement of leukocytes in the oxygenation and chlorination reaction of phenylbutazone

A center carbon atom of 1,3-diketone moiety of phenylbutazone was oxidized to give three metabolites--4-hydroxyphenylbutazone (metabolite I), 4-hydroperoxyphenylbutazone (metabolite II) and 4-chlorophenylbutazone (metabolite III)--by the action of enzymes present in leukocyte extract obtained from peritoneal exudate of rats. Both metabolites II and III were produced by peroxidases, while metabolite I was produced by enzymes other than the peroxidases.

Synthesis of novel water soluble benzylazolium prodrugs of lipophilic azole antifungals

Water soluble N-benzyltriazolium or N-benzylimidazolium salt type prodrugs of several highly lipophilic triazole or imidazole antifungals have been synthesized. They were designed to undergo an enzymatic activation followed by a self-cleavage to release a parent drug. The prodrugs such as 16 had enough chemical stability and water solubility for parenteral use and were rapidly and quantitatively converted to the active substance in human plasma.

Involvement of Cystathionase in the Formation of Alkane-Thiols from Corresponding Cysteine Conjugates

1. Rat liver cystathionase [EC.4.4.1.1] mediated a cleavage reaction of the C-S bond of S-alkyl-L-cysteine conjugates to give equimolar amounts of corresponding thiols, ammonia, and pyruvic acid. Neither S-aryl- nor S-aralkyl-L-cysteine conjugates were acceptable substrates. 2. The Km value for S-(tert-butyl)-L-cysteine was 0.3 mM at pH 8.5 in Tris-HCl buffer. 3. The S-alkyl-L-cysteine lyase activity of cystathionase was inhibited with carbonyl reagents and dithiothreitol. 4. The present finding that cystathionase has activity towards cysteine conjugates may provide some insights into the role of this enzyme in the metabolism of xenobiotics.

Cassette dosing approach and quantitative structure-pharmacokinetic relationship study of antifungal N-myristoyltransferase inhibitors.

Pharmacokinetic (PK) parameters of N-myristoyltransferase (Nmt) inhibitors were measured, and a multivariate quantitative structure-pharmacokinetic relationship (QSPKR) model for predicting rat elimination half-life (t(1/2)) values was constructed. One hundred seven benzofuran derivatives have been selected as the data set for QSPKR analysis. The correlation between the t(1/2) values and 30 physicochemical descriptors was examined by a stepwise multiple linear regression method. The statistical analysis gives a significant QSPKR model (r = 0.843) with the following three variables: partial negative surface area (PNSA), atomic-based octanol/water partition coefficient (AlogP), and the number of rotational bonds (Rotlbonds). The QSPKR model obtained is predictive and simple, and would give a direction for designing new Nmt inhibitors having good PK profiles.

Purification and properties of methyl sulfoxide reductases from rat kidney

Two kinds of enzymes (tentatively designated methyl sulfoxide reductases I and II) responsible for the reduction of the methyl sulfoxide group on various xenobiotics have been purified about 223- and 155-fold, respectively, from rat kidney cytosol. The molecular weight was determined to be 12,000 +/- 1000 for methyl sulfoxide reductase I and 24,000 +/- 1000 for methyl sulfoxide reductase II. Thioredoxin or dithiothreitol is essential in order for the reducing activity to occur. The respective Km values of p-bromophenylmethyl sulfoxide were 2.75 and 1.30 mM for methyl sulfoxide reductases I and II. Replacement of the methyl group on the sulfur atom with a longer alkyl group or phenyl group caused a markedly low or negligible substrate activity.

Purification and Characterization of Cysteine Conjugate Transaminases from Rat Liver

1. Soluble cysteine-conjugate alpha-ketoglutarate transaminase (CAT-I) was purified about 670-fold from rat liver cytosol using s-(p-bromophenyl)-L-cysteine as amino acid substrate. The enzyme preparation of the final step of purification showed a single band in polyacrylamide gel electrophoresis. CAT-I accounted for 64% of the transaminase activity in cytosol. 2. The mol. wt of the enzyme was about 64,000 as determined by gel filtration. Respective Km values for s-(p-bromophenyl)-L-cysteine and alpha-ketoglutaric acid were 1.0 and 1.3 mM in Tris-acetate buffer (pH 7.0). Aminooxyacetic acid, hydroxylamine, and KCN inhibited the enzyme activity. 3. In addition to CAT-I, two isozymes (CAT-IIA and CAT-IIB) were partially purified from rat liver cytosol. In respect of mol. wt, substrate specificity towards cysteine conjugates, and several other properties, CAT-IIA and CAT-IIB were very similar to CAT-I. However, differences were observed for these enzymes in the rate of reverse reaction (formation reaction of cysteine conjugates and alpha-ketoglutaric acid) and substrate specificity towards L-aspartic acid and L-cysteinesulphinic acid.

The behavior of remaining enzyme activity in a suicidal enzyme system

We derived an equation which describes the plot of the remaining enzyme activity versus ratio of initial concentration of suicide substrate to that of enzyme to obtain a partition ratio from the time-course of remaining enzyme activity. The simulation data calculated from the representative kinetic model for a suicide substrate were used to verify this equation, which approximated steady state kinetics. Although the time-dependent loss of enzyme activity is usually characterized by pseudo-first-order kinetics, the present results show that pseudo-first-order kinetics are followed only when the ratio of initial concentration of suicide substrate to that of enzyme is greater than the partition ratio. Our results also show that the present method can be used to obtain the partition ratio of a suicide substrate from the time-course of the remaining enzyme activity when the suicide substrate is given an arbitrary concentration of one, where the ratio of initial concentration of suicide substrate to that of enzyme is less than the partition ratio. The theoretically verified equation was also checked against reported experimental data for a microsomal enzyme system.

Transamination of LTE4 by cysteine conjugate aminotransferase

Leukotriene E4 (LTE4) was transaminated to 5(S)-hydroxy-6(R)-S-(2-keto-3-thiopropionyl)-7,9-trans-11,14-cis- eicosatetraenoic acid (tentatively designated as LTG4) by cysteine conjugate aminotransferase I purified from rat liver supernatant in the presence of alpha-ketoglutaric acid or alpha-keto-gamma-methiolbutyric acid. The transamination activity was present in the kidney as well as in the liver, but not in the lung or leukocytes.