Differential effects of inhibitors of purine metabolism on two trichomonad species

Tritrichomonas foetus and Trichomonas vaginalis are both incapable of de novo purine nucleotide synthesis. Previous studies indicated that T. foetus relies mainly on the salvage of hypoxanthine and subsequent conversion of IMP to AMP and GMP, whereas T. vaginalis depends on direct conversions of exogenous adenosine to AMP and guanosine to GMP without much interconversion between the two nucleotides. These two different types of purine salvage suggest the possibility of differential sensitivities between the two species of trichomonad flagellates toward different purine antimetabolites. Mycophenolic acid, hadacidin, 8-azaguanine, and formycin B inhibited the growth of T. foetus but had no effect on T. vaginalis. Mycophenolic acid acted by blocking conversion of IMP to GMP, hadacidin inhibited conversion of IMP to AMP, and 8-azaguanine was incorporated into the T. foetus nucleotide pool, likely via hypoxanthine phosphoribosyl transferase. Formycin B was converted to 5'-monophosphate in T. foetus and inhibited the conversion of IMP to AMP. Its precise mechanism of action on T. foetus remains, however, to be elucidated. Alanosine, whose ribonucleotide derivative is a potent inhibitor of adenylosuccinate synthetase, had no effect on the growth or hypoxanthine incorporation in T. foetus, which may be due to the lack of conversion of alanosine to the ribonucleotide because of the absence of de novo purine nucleotide synthesis in parasites. Four adenosine analogs, adenine arabinoside, tubercidin, sangivamycin, and toyocamycin, were found inhibitory to the growth of T. vaginalis but showed little effect on T. foetus growth. Further investigations suggested that these four compounds acted on T. vaginalis by blocking incorporation of adenosine into the adenine nucleotide pool.     

阿糖腺苷全合成工艺改进

报道了抗病毒药阿糖腺苷(Ara-A,1)的全合成法,总收率为32％(按噁唑烷2计算);其中关键步骤——N-烃化和还原反应进行了工艺改进:以甲醇钠代替氢化钠,以铝箔在原位生成铝汞齐,革除大量有机溶剂,简化操作,适合工业生产。     

Characterization of adenosine deaminase from the malarial parasite, Plasmodium lophurae, and its host cell,the duckling erythrocyte

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purified and characterized from the malarial parasite, Plasmodium lophurae, and its host cell, the duck (Anas domesticus) erythrocyte, using chromatofocusing (Pharmacia) and adenosine affinity columns. Gel filtration of the enzymes gave molecular weights of 33,800 (P. lophurae) and 36,500 (duck erythrocyte); both enzymes had broad pH optima (pH 6.8 to 8.0), similar stabilities when stored as crude lysates, and like K_m values with adenosine: 2.74 ± 0.88 × 10^?5 M (parasite) and 1.74 ± 0.27 × 10^?5 M (erythrocyte). The P. lophurae adenosine deaminase had a pI of 5.37 ± 0.09, and the duck erythrocyte enzyme had a pI of 4.72 ± 0.09, as determined by chromatofocusing. The parasite enzyme exhibited a specific activity in the crude lysate that was an average 60-fold higher than that of the erythrocyte enzyme. The pattern of elution from the adenosine affinity column, as well as kinetic studies with three adenosine analogs, revealed distinct differences in the binding characteristics of the two enzymes. The P. lophurae adenosine deaminase was weakly retarded by the affinity column, whereas the duck erythrocyte enzyme was strongly retarded. With 9-β-d-arabinosyladenine as substrate, the K_m values were similar (2.29 ± 0.98 × 10^?4 M for P. lophurae and 1.10 ± 0.21 × 10^?4 M for the duck erythrocyte). Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was a potent inhibitor of the duck erythrocyte enzyme with 100% inhibition at 1.3 μM, whereas the parasite adenosine deaminase was not inhibited at 422 μM even when incubated for 24 hr. Inhibitor studies with coformycin, a tight-binding inhibitor, resulted in K_i values of 7.14 × 10^?11 M for P. lophurae and 1.86 × 10^?10 M for the duck erythrocyte. The molar equivalencies, E_t, and catalytic numbers, k₃, were slightly different for both enzymes. The E_t values were 2.80 × 10^?10 M (P. lophurae) and 3.13 × 10^?10 M (duck erythrocyte); the k₃ values were 5.18 × 10³ min^?1 and 4.36 × 10³ min^?1 respectively.     

Pharmacokinetics of inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA mice

The pharrnacokinetics oferythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) inhibition of adenosine deaminase (ADA) was measured in vivo in CBA mice. The in vivo assay utilized injection of 10–100 nmoles [2-³H]adenosine and measurement of blood ³H₂O 20 min later. A single oral dose of EHNA (50 mg/kg) totally inhibited ADA for 4 hr and caused a large increase in conversion of [2-³H]adenosine to [2-³H]ATP. EHNA (3 mg/kg) decreased deamination by 50% for 2–6 hr, depending on the dose of adenosine used. Mice dosed with EHNA (100 mg/kg) once daily for 7 days showed the same ADA recovery rate as mice dosed only once. High single oral doses of EHNA had no effect on blood ATP and GTP pools.     

Protective effects of interferon, various anti-viral drugs and combinations of these on Herpes simplex virus infection in mice

Young adult mice were inoculated intraperitoneally with 30LD₅₀ of Herpes simplex virus type 1 (Miyama strain). They died within 14 days after the inoculation. Mice were equally protected by intraperitoneal injection of L cell interferon (L-IF) or mouse brain IF (MB-IF) given 1 day before, simultaneously and 1 day after the viral inoculation. Mice were also protected when 100μg of polyinosinic acid-polycytidylic acid (Poly I:C) was given intraperitoneally 1 day before the viral inoculation or on the same day, but not if given on the following day. When adenine arabinoside (ara-A) was administered every day from the 1st to the 4th day after the viral inoculation, the protective effect also occurred. However, lipopolysaccharide (LPS), amphotericin B methyl ester (AME) and gamma globulin (GG) were ineffective in reducing mortality. The protective effect could be also demonstrated by combining poly I:C and L-IF at doses which were individually ineffective. On the other hand, no synergism between L-IF and ara-A was observed.     

Metabolism of arabinosyladenine in herpes simplex virus-infected and uninfected cells: Correlation with inhibition of DNA synthesis and role in antiviral selectivity

The metabolism of 9-β-D-arabinofuranosyladenine (ara-A, vidarabine) and its effects on DNA synthesis were compared in uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells. In the absence of an inhibitor of adenosine deaminase, ara-A was deaminated to 9-β-D-arabinofuranosylhypoxanthine and phosphorylated to ara-A-5′-mono-, di- and triphosphates in both types of cells. When an inhibitor of adenosine deaminase (coformycin) was added to cell cultures, nucleotides were the only metabolites detected—primarily the 5′-triphosphate of ara-A (aATP). Detailed studies performed in the presence of coformycin established that the net rate and extent of aATP formation were the same in uninfected and HSV-1-infected cells. After a 12-hr exposure to 50 μM ara-A, intracellular concentrations of aATP were approximately 40 μM. Levels of aATP correlated directly with inhibition of total DNA synthesis. Approximately 0.7 μM aATP was required for 50% inhibition of total DNA synthesis in both uninfected and HSV-1-infected cells. Following removal of ara-A-containing culture medium, aATP levels in uninfected cells declined with a half-life of 3.2 hr. In marked contrast, the half-life in HSV-1-infected cells was 9.3 hr; this may explain why as little as a 3-hr exposure to ara-A resulted in a significant HSV-1 titer reduction. Taken together, the data show that when ara-A was removed from culture medium, levels of aATP persisted longer in HSV-1-infected cells thereby prolonging antiviral activity. This effect could be important in vivo where levels of ara-A oscillate with dosing schedule.     

Evaluation of the anti-herpesvirus drug combinations: virazole plus arabinofuranosylhypoxanthine and virazole plus arabinofuranosyladenine

Combinations of Virazole plus arabinofuranosylhypoxanthine (ara-Hx) and Virazole plus arabinofuranosyladenine (ara-A) were investigated in KB or BHK cells infected with types 1 or 2 herpes viruses. Combinations of Virazole and ara-Hx exhibited significant synergy as evaluated graphically (isobolograms) or by fractional inhibitory concentration (FIC) indices. Optimal ratios for the combination were 1:1 to 1:10 for Virazole to ara-Hx. At these ratios, FIC indices in the range of 0.5-0.2 were commonly observed. Combinations of Virazole and ara-A were antagonistic when observed in the presence of pentostatin, an adenosine deaminase inhibitor. In the absence of pentostatin, the minimum inhibitory concentration (MIC) of ara-A and degree of synergy with Virazole were variable.