8-aminoquinolines as anticoccidials - Part III.

Analogues of the antimalarial pentaquine, 1, in which the nature of the side-chain on the 8-amino position was varied, were prepared and evaluated for anticoccidial activity both in vitro and in vivo. Specifically, both the inter-nitrogen distance and the nature of the terminal amino group were investigated. Novel analogues of equal or improved efficacy in vitro and in vivo to pentaquine were discovered.

8-Aminoquinolines as anticoccidials--II.

During a chemistry program aimed at finding a novel analogue of pentaquine with improved in vivo activity, a number of hypotheses concerning the way this drug acts in the chicken were investigated. Consideration of the products of monoamine oxidase metabolism of pentaquine suggested that pentaquine aldehyde is the likely active metabolite. Although isolation of this unstable compound was not possible, oxime and cyclic acetal and ketal derivatives were obtained and shown to possess in vitro anticoccidial activity.

Enzymes of energy metabolism in the bradyzoites and tachyzoites of Toxoplasma gondii.

The bradyzoite and tachyzoite forms of Toxoplasma gondii, purified from infected animals, were analysed for their activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD(+)- and NADH-linked isocitrate dehydrogenases, and succinic dehydrogenase. Both developmental stages contained high activities of phosphofructokinase (specific for pyrophosphate rather than ATP), pyruvate kinase and lactate dehydrogenase, suggesting that energy metabolism in both forms may centre around a high glycolytic flux linked to lactate production. The markedly higher activity of the latter two enzymes in bradyzoites suggests that lactate production is particularly important in this developmental form. NAD(+)-specific isocitrate dehydrogenase was not detectable in either stage of the parasite (and proved useful as a measure of the purity of the bradyzoite preparation), whereas both parasite forms contained low activities of NADP(+)-linked isocitrate dehydrogenase. The results are consistent with the bradyzoites lacking a functional TCA cycle and respiratory chain and are suggestive of a lack of susceptibility of this developmental stage to atovaquone.

Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii.

Oocysts of Cryptosporidium parvum were shown to contain a pyrophosphate-dependent phosphofructokinase (PPi-PFK) similar to those previously described for Eimeria tenella and Toxoplasma gondii. PPi-PFK of C. parvum displayed simple hyperbolic kinetics with respect to its substrate fructose 6-phosphate and was not affected by fructose 2,6-bisphosphate, the major allosteric activator of most ATP-PFKs. Inorganic pyrophosphatase was not detectable in any of the three parasites. T. gondii tachyzoites and C. parvum cysts both contained a pyruvate kinase (PK) specific for ADP rather than PPi/AMP. The PK of T. gondii was similar to that of E. tenella in that it displayed strong positive cooperativity with respect to its substrate phosphoenolpyruvate and was heterotropically activated by glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. PK of C. parvum showed no evidence of allosteric properties. The results suggest that the three coccidia are similar in depending heavily on anaerobic energy production via glycolysis but that the mechanisms for regulating glycolysis are not common to all species.

Eimeria tenella contains a pyrophosphate-dependent phosphofructokinase and a pyruvate kinase with unusual allosteric regulators.

Sporozoites and unsporulated oocysts of Eimeria tenella were shown to contain a pyrophosphate-dependent phosphofructokinase (PPi-PFK) but apparently lack an ATP-specific activity. The PPi-PFK resembles those that occur in a number of other protists in being reversible and not subject to metabolic control. In contrast, the ADP-utilising pyruvate kinase, present in two developmental stages of the parasite, exhibited strong positive cooperativity with respect to its substrate, phosphoenolpyruvate, and was shown to be allosterically activated by glucose 6-phosphate, fructose 6-phosphate and AMP. It is suggested that the PPi-PFK represents an adaptation of the parasite towards life in an environment containing only low concentrations of oxygen and that the unusual allosteric regulation of pyruvate kinase evolved to compensate for glycolysis not being controlled at the PPi-PFK step.

The identification of a variant form of cystathionine beta-synthase in nematodes.

Characterization of the physical and catalytic properties of the enzyme responsible for nematode "activated L-serine sulfhydrase" activity (L-cysteine + R-SH-->cysteine thioether + H2S) has led to its identification as a novel, variant form (allelozyme) of cystathionine beta-synthase that is distinct from a mammalian-type synthase also present in nematodes. Additional work has demonstrated the ability of live Panagrellus redivivus to produce H2[35S] from exogenous L-[35S]cysteine and 2-mercaptoethanol, thus providing preliminary evidence for the in vivo operation of the activated L-serine sulfhydrase reaction in nematodes.

The effects of inhibitors of sulphur-containing amino acid metabolism on the growth of Trichomonas vaginalis in vitro.

A range of inhibitors of enzymes catalysing the metabolism of sulphur-containing amino acids were tested for efficacy against Trichomonas vaginalis in vitro. Sinefungin, tubercidin, ara A, bithionol, hexachlorophene, dichlorophene and 5-azacytidine were found to be effective antitrichomonal agents. Combinations of any two of these inhibitors were, in most cases, no more effective than one inhibitor used alone, but marked synergy was apparent with monothioglycerol and methionine. None of the inhibitors investigated was as potent as metronidazole, the drug of choice for the treatment of trichomoniasis.

Trichomonas species: homocysteine desulphurase and serine sulphydrase activities.

Homocysteine desulphurase (EC 4.4.1.2) and serine sulphydrase (EC 4.2.1.22) activities in various lines of Trichomonas vaginalis, both metronidazole resistant and sensitive, and other trichomonad species were assessed. T. vaginalis contained the highest homocysteine desulphurase and serine sulphydrase activities of all the species. Although the levels of the enzyme activity in T. vaginalis isolates differed, no correlation between the activities and sensitivity to metronidazole was apparent. T. vaginalis homocysteine desulphurase catalysed both the hydrolysis of homocysteine to hydrogen sulphide, ammonia, and 2-oxoacid, and an exchange reaction between homocysteine and 2-mercaptoethanol. Homocysteine desulphurase was detected as a single enzyme band on isoelectric focusing, whereas several isoenzymes of serine sulphydrase were found. There were large differences in serine sulphydrase isoenzyme patterns between T. vaginalis lines and between species. Several isoenzymes were amplified in cells grown with 10(-5) M DL-propargylglycine for 24 hr. T. vaginalis homocysteine desulphurase and serine sulphydrase activities were inhibited by bithionol, hexachlorophene, and dichlorophene. These compounds also inhibited growth in vitro of T. vaginalis at concentrations similar to those that inhibited the enzymes.

L-methionine catabolism in trichomonads.

Trichomonas vaginalis growing in complex medium produced volatile thiols at a rate of 0.7 nmol min-1 (mg protein)-1 and the parasite suspended in PBS with L-methionine excreted volatile thiols, including methanethiol, and alpha-keto acid. Cell-free extracts of the parasite also produced volatile thiols from L-methionine, at the rate of 5.4 nmol min-1 (mg protein)-1. Thiol production was not detectable with living cells or cell-free extracts of Tritrichomonas foetus, Trichomitus batrachorum or Pentatrichomonas hominis and homogenates of a range of trypanosomatids and mouse liver also failed to produce volatile thiols from L-methionine. Approximately equimolar concentrations of alpha-keto acid and volatile thiols were produced from L-methionine by cell-free extracts of Trichomonas vaginalis; the release of ammonia, however, was not detectable. The parasite enzyme catabolised a range of substrates and was inhibited by several compounds, including bithionol and DL-propargylglycine. Parasites grown in the presence of 10(-5) M DL-propargylglycine had no detectable L-methionine-catabolising enzyme activity. These findings indicate that T. vaginalis is significantly different from other trichomonads, a range of trypanosomatids and mouse liver in L-methionine catabolism, and that the parasite enzyme responsible for the breakdown of L-methionine in T. vaginalis appears to be similar in several ways to bacterial L-methionine-gamma-lyase (EC 4.4.1.11) and trichomonal homocysteine desulphurase (EC 4.4.1.2).

S-adenosylmethionine and transmethylation reactions in trichomonads.

S-Adenosylmethionine (SAM) levels in trichomonads, a range of trypanosomatids and mouse liver were measured using HPLC techniques. The concentrations were found to be similar in each with the exception of Herpetomonas muscarum ingenoplastis, which contained approximately ten-fold more. Living trichomonads were found to incorporate exogenous L-methionine into intracellular SAM and its methyl carbon was also detected in lipids and nucleic acids, presumably through its involvement in transmethylation reactions. Norleucine and cycloleucine inhibited L-methionine uptake and incorporation into living Trichomonas vaginalis. Both the rates of incorporation of exogenous L-methionine into intracellular SAM and its involvement in transmethylation reactions were greater for Trichomonas vaginalis than for Tritrichomonas foetus. The results suggest that Trichomonas vaginalis and other trichomonads contain enzymes equivalent to SAM synthetase (EC 2.5.1.6) and SAM-dependent methyltransferases (EC 2.1.1).

Comparative study of ferredoxin-linked and oxygen-metabolizing enzymes of trichomonads.

1. The activities of pyruvate:methyl viologen oxidoreductase (EC 1.2.7.1), hydrogenase (EC 1.18.99.1), NADH:methyl viologen oxidoreductase (EC 1.6.99.3), NADPH:methyl viologen oxidoreductase (EC 1.6.99.1), NADH oxidase (EC 1.6.99.3) and NADPH oxidase (EC 1.6.99.1) were determined for Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum. 2. The three trichomonad species were found to differ significantly, especially with respect to NADH oxidase and NADH:methyl viologen oxidoreductase activities. 3. The species differences in ferredoxin-linked and oxygen-metabolising enzymes may be related to the ways in which the trichomonads are adapted for growth in their respective hosts.

S-Adenosylhomocysteine hydrolase activity in Trichomonas vaginalis and other trichomonads.

S-Adenosylhomocysteine hydrolase has been detected in crude homogenates of Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum at activities of 14, 1.2 and 3.3 nmol min-1 mg-1 protein, respectively. The enzyme from T. vaginalis was found to be soluble with pH optimum of 8.0 and apparent Km values for adenosine and homocysteine of 100 and 155 microM, respectively. Ara A was shown to inhibit the T. vaginalis enzyme but only at relatively high concentration (I50 100 microM), whereas sinefungin and 2'-deoxyadenosine had only small inhibitory effects. EDTA (I50 6 mM) and various divalent cations also inhibited the enzyme from T. vaginalis.