Phosphorylation and anti-leishmanial activity of formycin B

The inosine analog formycin B (1–10 μM) inhibited the $\text{in vitro}$ growth of $\text{Leishmania}$ promastigotes and amastigotes. When administered to Syrian hamsters infected with $\text{Leishmania}\text{donovani}$, formycin B (10 mg qd × 5) decreased by greater than 90% the number of liver amastigotes, with a concomitant reduction in hepatosplenomegaly. Both extracts and intact cells of $\text{Leishmania}$, unlike mammalian cells, effectively phosphorylated formycin B. The resulting formycin B monophosphate inhibited dose dependently the conversion of IMP to adenylosuccinate in parasite extracts. This effect may be related to the potent anti-leishmanial activity of formycin B.     

Complex formation between polyadenylic acid and formycin B

Polyadenylic acid forms a 2:1 complex with the C-nucleoside formyein B at both pH 7.0, 0.15 m-Na⁺ and pH 6.0, 0.15 M-Na⁺. The formation of this complex has been followed by equilibrium dialysis, and by optical rotatory dispersion measurements in the range 333 to 450 nm. At pH 7, melting curves for thermal dissociation of the complex (followed by the optical rotation at 345 nm) show a strongly co-operative helix-coil transition. From the variation of T_m with the free formyein B concentration at this temperature, the partial molar enthalpy of formation of the complex, at the mid-point of the transition, has been calculated as -12.8 kcal./mol of formyein B. Viscometry and optical rotatory dispersion measurements indicate that the structure of the complex at pH 6 is the same as at pH 7, and that it may be formed in preference to the double-helical acid form of poly (A). The structure and properties of the complex are discussed.     

Interaction of formycin A-5'-triphosphate with pyruvate carboxylase

Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a competitive inhibitor of chicken liver pyruvate carboxylase with respect to ATP. The chicken liver enzyme is unable to utilise FTP as a substrate at a measureable rate, but FTP is a poor substrate for the sheep liver enzyme. When FTP binds to the enzyme, its fluorescence is enhanced and in this way the formation of enzyme-FTP complexes can be monitored. Using this property of FTP, the effect of Mg2+ and acetyl-CoA on the binding of nucleoside triphosphates to the chicken liver enzyme was examined. Mg2+ was found to enhance the binding of FTP whilst acetyl-CoA reduced the fluorescence intensity of a mixture of Mg2+, enzyme and FTP. Most probably, this was caused by a conformational change in the enzyme which changed the environment of the fluorophore.     

A molecular orbital study of the conformation of formycin

Semiempirical quantum mechanical calculations, using the iterative extended Huckel theory, are carried out for the evaluation of conformational energies, dipole moment and net atomic charges as a function of the rotation about the glycosidic bond. Torsion about the C(4′)-C(5′) bond has also been considered. The energy diagrams for either the gg or gt rotamers of formycin predict that neither the first or the second energy minimum fall in the classical anti or syn regions. The predicted energy difference between the two most preferred conformations is rather large (3 kcal/mole). In contrast adenosine is predicted to favor the anti conformation by less than 1 kcal/mole. Barriers to internal counter-clockwise rotation about the glycosidic bond are higher for adenosine.     

Biochemical genetic analysis of formycin B action in Leishmania donovani

Formycin B is cytotoxic toward Leishmania and is a potential chemotherapeutic agent for leishmaniasis. In order to determine the mechanism of action of formycin B, we have isolated and characterized clonal populations of formycin B-resistant Leishmania donovani. These formycin B-resistant clones are also cross-resistant to formycin A and allopurinol riboside-mediated growth inhibition. Incubation of the formycin B-resistant cells with [3H]formycin B indicates that, unlike wild type cells, the resistant populations cannot accumulate phosphorylated metabolites of exogenous [3H]formycin B. This is due to a defective transport system for formycin B in the resistant cells. However, wild type and mutant cells incorporate [3H]formycin A equally efficiently into [3H]formycin A-containing nucleotides and into RNA. These data suggest that formycin B cytotoxicity in Leishmania is not mediated by its incorporation as the adenosine analog into RNA. A plausible alternative hypothesis is proposed for the mechanism of action of the pyrazolo (4,3-d)pyrimidine C-nucleosides based upon depletion of an essential intracellular metabolite.     

Interaction energy studies of pyrazolopyrimidine antibiotics during transcription: formycin B

The interaction energies of various type of binding pattern of pyrazolopyrimidine antibiotics formycin B (laurusin) with nucleic acid fragments (bases and base pairs) have been computed and compared with the corresponding binding energy for normal bases. These results are discussed in the light of the incorporation model to explain the biological function of formycin B. These findings are in agreement with the experimentally observed evidence.     

High-performance liquid chromatographic assay of formycin A in plasma after solid-phase extraction

A new, simple and accurate high-performance liquid chromatography (HPLC) method for the determination of formycin A in plasma is presented. The samples were chromatographed on a LiChrosorb RP-18 column after purification using a Bakerbond SPE column. The mobile phase was methanol–0.067 M phosphate buffer, pH 4.20 (1:4, v/v) containing 0.005 M sodium hexanesulfonate. Azathioprine was applied as an internal standard. UV detection was carried out at 293 nm. The method was tested for linearity (over the range 0.1–9.0 μg/ml). The recovery was 91.89% (mean). The described method has been successfully applied to the quantitative determination of formycin A in plasma and should be useful for clinical and bioavailability investigations.     

Involvement of adenosine kinase in the phosphorylation of formycin B in CHO cells

In Chinese hamster ovary cells, [3H]formycin B is metabolized into formycin B-5'-monophosphate, formycin A-5'-monophosphate and higher phosphorylated derivatives of formycin A which are incorporated into RNA. Mutants of CHO cells independently selected for resistance to various adenosine analogs viz. toyocamycin, tubercidin, 6-methylmercaptopurine riboside, which contain no detectable activity of adenosine kinase (AK) in cell extracts, all exhibited between 2- to 3-fold increased resistance to formycin B. Formycin B-resistant mutants of CHO cells are also affected in AK, as indicated by the absence of AK activity in cell extracts. Both types of AK- mutants showed reduced uptake and phosphorylation of [3H]formycin B in comparison to the parental (AK+) cells. In addition, toxicity of formycin B towards CHO cells was reduced in presence of adenosine in a concentration dependent manner. These observations strongly indicate that in CHO cells, formycin B is phosphorylated via AK and that like other nucleoside analogs its phosphorylation may be essential for the drugs cellular toxicity.     

RNA synthesis in starfish embryos: developmental consequences of its inhibition by formycin

Embryos of the starfish Asterina pectinifera were examined with regard to their ability to undergo the early events of embryonic development in the presence of formycin, an analogue of adenosine and a reported inhibitor of RNA synthesis. It was shown that in normal embryos the pool of ribonucleoside 5'-triphosphates increased during the period of blastula formation. The increase of the UTP pool was blocked nearly completely by 25 micrograms/ml formycin, and that of the CTP pool was inhibited partially by the same concentration of the drug. On the other hand, the pools of ATP and GTP were the same for both control and formycin-treated embryos. The development of embryos cultured in the presence of 25 micrograms/ml formycin stopped at the early blastula stage. Addition of 100 micrograms/ml each of uridine and cytidine to cultures of embryos that had been placed in 25 micrograms/ml formycin at the onset of blastulation allowed gastrulation to occur, suggesting that the developmental arrest produced by formycin is due primarily to the inhibition of pyrimidine nucleotide biosynthesis.     

The effect of formycin B on mRNA translation and uptake of purine precursors in Leishmania mexicana

Formycin B is a structural analog of inosine that is a potent inhibitor of Leishmania multiplication. Formycin B is reportedly converted to formycin A nucleotides and incorporated into RNA by the organisms, and it is unclear whether the active form of the drug is the nucleoside itself or its several metabolites. We confirmed that formycin A nucleotides are formed by formycin B-exposed L. mexicana promastigotes, and determined that the intraparasite concentration of Formycin B and its metabolites was 6 times the extracellular formycin B concentration. Formycin B did not significantly inhibit purine nucleoside transport by intact promastigotes or purine base phosphoribosylation by parasite lysates. Thus, the nucleoside does not appear to inhibit these initial steps of purine nucleoside metabolism. Since RNA and protein synthesis in formycin B-treated intact promastigotes was found to be inhibited within 30 minutes, the effect of formycin A metabolites on leishmanial protein synthesis was investigated in in vitro protein synthesis experiments. Messenger RNA from formycin B-treated promastigotes was translated only 40% as efficiently as control promastigote mRNA by rabbit reticulocyte lysates. In addition, when formycin A-5'-triphosphate was preincubated with the rabbit reticulocyte lysates, translation of control mRNA was 86% inhibited. Formycin B toxicity to Leishmania promastigotes appears to be at least partially due to inhibition of protein synthesis by formycin A nucleotides and formycin A containing mRNA.     

Polynucleotides. XXVI. Complex formation of polynucleotides derived from formycin and laurusin with cyclonucleoside oligonucleotides

Poly(formycin phosphate) and poly(laurusin phosphate) were synthesized by polymerizing formycin and laurusin 5′-diphosphate by means of E. coli polynucleotide phosphorylase. The complex formation of these polynucleotides with cyclonucleoside polynucleotides were investigated. While poly(formycin phosphate) did not form the complex with an octanucleotide of 6,2′-anhydro-6-oxy-1-β-D-arabinofuranosyluracil, poly(laurusin phosphate) did form a 1: 1 complex with octanucleotide of 8,2′-anhydro-8-mercapto-9-β-D-arabinofuranosyladenine in the presence of 0.15M Na ion at neutrality and 3^o. CD spectrum of this complex showed a couple of a trough at 286 nm and a peak at 262 nm. This fact suggests that the complex has a left-handed helical conformation, which is opposite to the natural double helical polynucleotides. The cause of this phenomenon was discussed in connection with the complex of cyclonucleoside oligonucleotides.