Synthesis and antiviral activity of formycin derivatives with anti-influenza virus activity.

In a screening using our unique natural product library, the C-nucleoside antibiotic formycin A, which exerts strong anti-influenza virus activity, was rediscovered. Aiming to develop a new type of anti-influenza virus drug, we synthesized new derivatives of formycin and evaluated its anti-influenza virus activity. Structural modifications were focused on the base moiety and sugar portion, respectively, and >40 novel formycin derivatives were synthesized. Modification of the C-7 position of the pyrazolopyrimidine ring strongly contributed to improve the activity. In particular, excellent anti-influenza virus activity was observed in the NHMe (10), SMe (12), and SeMe (15) derivatives, in which heteroatoms were introduced. In addition, in the modification of the sugar moiety, the presence of a hydroxyl group and its stereochemistry greatly affected both the expression and intensity of the activity. Furthermore, the evaluation results of the 7-SEt derivative (29) and the 2'-modified derivative (59) suggested that structural modifications may reduce cytotoxicity.

A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori.

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

Nucleolipids of the Nucleoside Antibiotics Formycins A and B: Synthesis and Biomedical Characterization Particularly Using Glioblastoma Cells.

Two lipophilic derivatives of formycin A (1) and formycin B (5) carrying an O-2',3'-(ethyl levulinate) ketal group have been prepared. These were base-alkylated at N(1) (for 1) and N(1) and N(6) (for 5) with both isopentenyl and all-trans-farnesyl residues. Upon the prenylation, side reactions were observed, resulting in the formation of nucleolipids with a novel tricyclic nucleobase (→4a, 4b). In the case of formycin B, O-2',3'-(ethyl levulinate) (6) farnesylation gave the double prenylated nucleolipid 7. All new compounds were characterized by 1 H-, 13 C-, UV/VIS and fluorescence spectroscopy, by ESI-MS spectrometry and/or by elemental analysis. Log P determinations between water and octanol as well as water and cyclohexane of a selection of compounds allowed qualitative conclusions concerning their potential blood-brain barrier passage efficiency. All compounds were investigated in vitro with respect to their cytotoxic activity toward rat malignant neuroectodermal BT4Ca as well as against a series of human glioblastoma cell lines (GOS 3, U-87 MG and GBM 2014/42). In order to differentiate between anticancer and side effects of the novel nucleolipids, we also studied their activity on PMA-differentiated human THP-1 macrophages. Here, we show that particularly the formycin A derivative 3b possesses promising antitumor properties in several cancer cell lines with profound cytotoxic effects partly on human glioblastoma cells, with a higher efficacy than the chemotherapeutic drug 5-fluorouridine.

Metabolism of formycin B by Leishmania amastigotes in vitro. Comparative metabolism in infected and uninfected human macrophages.

Formycin B is metabolized by cutaneous Leishmania amastigotes within cultured human macrophages to give formycin B 5'-monophosphate and formycin A 5'-mono-, di-, and triphosphates. Formycin A is also incorporated into RNA. The activity of formycin B against amastigotes was correlated with the levels of formycin A metabolites formed in the parasites. Uninfected macrophages also convert formycin B into the same products, but the levels are markedly lower than those seen in infected macrophages. The results suggest that a sufficient therapeutic index exists to warrant consideration of formycin B as an anti-leishmanial drug in humans.

Assessment of methylthioadenosine/S-adenosylhomocysteine nucleosidases of Borrelia burgdorferi as targets for novel antimicrobials using a novel high-throughput method.

BACKGROUND: Lyme disease is the most prevalent tick-borne disease in the USA with the highest number of cases (27 444 patients) reported by CDC in the year 2007, representing an unprecedented 37% increase from the previous year. The haematogenous spread of Borrelia burgdorferi to various tissues results in multisystemic disease affecting the heart, joints, skin, musculoskeletal and nervous system of the patients. OBJECTIVES: Although Lyme disease can be effectively treated with doxycycline, amoxicillin and cefuroxime axetil, discovery of novel drugs will benefit the patients intolerant to these drugs and potentially those suffering from chronic Lyme disease that is refractory to these agents and to macrolides. In this study, we have explored 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase as a drug target for B. burgdorferi, which uniquely possesses three genes expressing homologous enzymes with two of these proteins apparently exported. METHODS: The recombinant B. burgdorferi Bgp and Pfs proteins were first used for the kinetic analysis of enzymatic activity with both substrates and with four inhibitors. We then determined the antispirochaetal activity of these compounds using a novel technique. The method involved detection of the live-dead B. burgdorferi by fluorometric analysis after staining with a fluorescent nucleic acids stain mixture containing Hoechst 33342 and Sytox Green. RESULTS: Our results indicate that this method can be used for high-throughput screening of novel antimicrobials against bacteria. The inhibitors formycin A and 5'-p-nitrophenythioadenosine particularly affected B. burgdorferi adversely on prolonged treatment. CONCLUSIONS: On the basis of our analysis, we expect that structure-based modification of the inhibitors can be employed to develop highly effective novel antibiotics against Lyme spirochaetes.

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (Pi ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes Pi binding, while a three-site model is needed to characterize FA binding, irrespective of Pi presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with Pi and FA shows, however, that Pi binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that Pi moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. DATABASES: The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). ENZYMES: Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463.

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Functional characterization of nucleoside transporter gene replacements in Leishmania donovani.

Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine-pyrimidine nucleoside transporter and the LdNT2 inosine-guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity. The mutant alleles were verified by Southern blotting, and the effects of gene replacement on transport phenotype were evaluated by rapid sampling transport measurements and by drug resistance profiles. The Deltaldnt1, Deltaldnt2, and Deltaldnt1/Deltaldnt2 mutants were all capable of proliferation in defined culture medium supplemented with any of a spectrum of purine nucleobases or nucleosides, except that a Deltaldnt2 lesion conferred an inability to efficiently salvage exogenous xanthosine, a newly discovered ligand of LdNT2. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in J774 and bone marrow-derived murine macrophages. These genetic studies demonstrate: (1) that L. donovani promastigotes, axenic amastigotes, and tissue amastigotes are viable in the absence of nucleoside transport; (2) that nucleoside transporters are not essential for sustaining an infection in mammalian host cells; (3) that the phagolysosome of macrophages is likely to contain purines that are not LdNT1 or LdNT2 ligands, i.e., nucleobases. Furthermore, the Deltaldnt1, Deltaldnt2, and Deltaldnt1/Deltaldnt2 knockouts offer a unique genetically defined null background for the biochemical and genetic characterization of nucleoside transporter genes and cDNAs from phylogenetically diverse species and of genetically manipulated LdNT1 and LdNT2 constructs.

Inhibitors of purine nucleoside phosphorylase: effects of 9-deazapurine ribonucleosides and synthesis of 5'-deoxy-5'-iodo-9-deazainosine.

9-Deazapurine ribonucleosides constitute a new class of noncleavable purine nucleoside phosphorylase inhibitors that have at least 30-fold greater affinity for the enzyme than the corresponding C-nucleosides of the formycin B series. 9-Deazaguanosine, 9-deazainosine, and 5'-deoxy-5'-iodo-9-deazainosine competitively inhibited human erythrocytic purine nucleoside phosphorylase with Ki values of 29, 20, and 1.8 X 10(-7) M. The last compound is the most potent nucleoside inhibitor of the enzyme presently available and its synthesis is described. In contrast, 7,9-dideaza-7-thiainosine is a very weak inhibitor of the enzyme. When tested as an inhibitor of 2'-deoxyguanosine phosphorolysis in intact human erythrocytes and MOLT-3 human T-cell lymphoblastic leukemia cells, 5'-deoxy-5'-iodo-9-deazainosine was equipotent with 8-aminoguanosine (which is a precursor for 8-aminoguanine, Ki = 2 X 10(-7) M). Similarly, 5'-deoxy-5'-iodo-9-deazainosine and 8-aminoguanosine both potentiated the growth inhibition of human T-lymphocytic MOLT-3 cells by 2'-deoxyguanosine, reducing the 50% inhibitory concentration from approximately 2 X 10(-5) to approximately 2 X 10(-6) M.

Influence of formycin B on polyadenosine diphosphoribose synthesis in vitro and in vivo.

Formycin B inhibited growth of L5178Y mouse leukemia cells in concentrations of less than twice the concentration that inhibits cell proliferation at 50% by cytostasis; at higher concentrations (more than twice the 50% concentration mentioned), the cells were killed. In cells treated with the concentration that inhibits cell proliferation at 50%, the average cell volume did not change. The formycin B inhibitory effect on cell proliferation was reduced by coincubation with nicotinamide adenine diphosphate or adenosine. The biosyntheses of DNA,RNA, and protein in whole cells were sensitively inhibited by formycin B as checked by incorporation studies with radioactive precursors. In addition, the formation of polyadenosine diphosphoribose was reduced even with higher sensitivity; in particular the extent of adenosine diphosphate ribosylation of histone subfraction H1 was reduced. Formycin B has been shown to be an inhibitor for the polyadenosine diphosphoribose polymerase, isolated from oviduct nuclei of quails. Both the chromatin-bound and the soluble enzyme are inhibited competitively; the relative affinity (Ki/Km) of formycin B to the polyadenosine diphosphoribose polymerase is 1.5.

Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase.

Formycin B (FB), a moderate inhibitor (Ki approximately 100 microM) of mammalian purine nucleoside phosphorylase (PNP), and formycin A (FA), which is totally inactive vs. the mammalian enzyme, are both effective inhibitors of the bacterial (Escherichia coli) enzyme (Ki approximately 5 microM). Examination of a series of N-methyl analogues of FA and FB led to the finding that N(6)-methyl-FA, virtually inactive vs. the mammalian enzyme, is the most potent inhibitor of E. coli purine nucleoside phosphorylase (Ki approximately 0.3 uM) at neutral pH. Inhibition is competitive not only with respect to Ino, but also relative to 7-methyl-Guo and 7-methyl-Ado, as substrates. Both oxoformycins A and B are relatively poor inhibitors. For the most potent inhibitor, N(6)-methyl-FA, it was shown that the enzyme preferentially binds the neutral, and not the cationic, form. In accordance with this the neutral, but not the cationic form, of the structurally related N(1)-methyl-Ado was found to be an excellent substrate. Reported data on tautomerism of formycins were profited from, and extended, to infer which tautomeric species and ionic forms are the active inhibitors. A commercially available (Sigma) bacterial PNP, of unknown origin, was shown to differ from the E. coli enzyme by its inability to phosphorylase Ado; this enzyme was also resistant to FA and FB. These findings have been extended to provide a detailed comparison of the substrate/inhibitor properties of PNP from various microorganisms.

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer.

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes () affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Protein kinase activities in Leishmania aethiopica: control by growth, transformation and inhibitors.

Promastigotes of L. aethiopica express an ectokinase activity preferring histone V-S as substrate. A soluble kinase activity utilizing protamine and histone V-S, as well as a particulate fraction associated kinase activity preferring protamine are also expressed. The soluble histone kinase activity, but not the ectokinase, was expressed at a higher level in cells from late phases of growth, as compared to early log phase cultures. Transformation of L. aethiopica to an amastigote-like stage, resulted in almost complete loss of the kinase activities, with retained viability of the cells. Formycin-ATP only weakly inhibited the kinases while effectively inhibiting cell growth and thymidine incorporation. Staurosporin efficiently blocked the kinase activities and cell growth without affecting thymidine incorporation.

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.

Studies on the metabolic effects of methylthioformycin.

5'-Methylthioformycin, a structural analog of 5'-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5'-methylthioadenosine and is not metabolized by the cells. 5'-Methylthioformycin inhibits Friend erythroleukemia cells' growth: the effect is dose-dependent, fully reversible and not caused by cytotoxicity. Several enzymes related to methylthioadenosine metabolism are inhibited by methylthioformycin. Rat liver methylthioadenosine phosphorylase is competitively inhibited with a Ki value of 2 microM. Among the propylamine transferases tested only rat brain spermine synthase is significantly inhibited, while rat brain spermidine synthase is less sensitive. Rat liver S-adenosylhomocysteine hydrolase is irreversibly inactivated with 50% inhibition at 400 microM methylthioformycin. 5'-Methylthioformycin does not exert any significant effect on protein carboxyl-O-methyltransferase. Inferences about the mechanism of the antiproliferative effect of the drug have been drawn from the above results.

Dual effect of formycin A upon the hydrolysis of phosphoinositides in perifused pancreatic islets.

Formycin A (1.0 mM) caused a rapid, sustained and rapidly reversible inhibition of effluent radioactivity in rat pancreatic islets prelabelled with myo-[2-3H]inositol and perifused in the presence of 8.3 mM D-glucose. This coincided with a progressive decrease in islet ATP content and transient inhibition of insulin release. Thereafter, however, formycin A increased glucose-induced insulin release. Moreover, in islets that were preincubated with myo-[2-3H]inositol and then exposed during perifusion to a rise in D-glucose concentration from 2.8 to 16.7 mM, the release of insulin and 3H fractional outflow rate at both the low and high hexose concentrations were much higher when both the preincubation and perifusion were conducted in the presence, rather than absence, of formycin A. It is concluded that formycin A first inhibits and later enhances both the hydrolysis of phosphoinositides and release of insulin, these effects being possibly related to changes in the islet cell content of adenosine and/or formycin A triphosphates.

A QM-MD simulation approach to the analysis of FRET processes in (bio)molecular systems. A case study: complexes of E. coli purine nucleoside phosphorylase and its mutants with formycin A.

Predicting FRET pathways in proteins using computer simulation techniques is very important for reliable interpretation of experimental data. A novel and relatively simple methodology has been developed and applied to purine nucleoside phosphorylase (PNP) complexed with a fluorescent ligand - formycin A (FA). FRET occurs between an excited Tyr residue (D*) and FA (A). This study aims to interpret experimental data that, among others, suggests the absence of FRET for the PNPF159A mutant in complex with FA, based on novel theoretical methodology. MD simulations for the protein molecule containing D*, and complexed with A, are carried out. Interactions of D* with its molecular environment are accounted by including changes of the ESP charges in S1, compared to S0, and computed at the SCF-CI level. FRET probability W F depends on the inverse six-power of the D*-A distance, R da . The orientational factor 0 < k(2) < 4 between D* and A is computed and included in the analysis. Finally W F is time-averaged over the MD trajectories resulting in its mean value. The red-shift of the tyrosinate anion emission and thus lack of spectral overlap integral and thermal energy dissipation are the reasons for the FRET absence in the studied mutants at pH 7 and above. The presence of the tyrosinate anion results in a competitive energy dissipation channel and red-shifted emission, thus in consequence in the absence of FRET. These studies also indicate an important role of the phenyl ring of Phe159 for FRET in the wild-type PNP, which does not exist in the Ala159 mutant, and for the effective association of PNP with FA. In a more general context, our observations point out very interesting and biologically important properties of the tyrosine residue in its excited state, which may undergo spontaneous deprotonation in the biomolecular systems, resulting further in unexpected physical and/or biological phenomena. Until now, this observation has not been widely discussed in the literature.

Stimulation of proinsulin biosynthesis by purine-ribonucleosides and D-ribose.

Inosine and guanosine were potent stimuli of proinsulin biosynthesis ([3H]leucine incorporation) in isolated pancreatic islets of the rat. The effect was nearly abolished by formycin B, an inhibitor of purine nucleoside phosphorylase, but not by D-mannoheptulose. The corresponding bases had no effect on the rate of proinsulin biosynthesis. D-ribose enhance proinsulin biosynthesis at low concentrations )0.3-0.6mM) but concentrations above 5 mM were ineffective. The effect of all three compounds was highly specific for proinsulin biosynthesis, since incorporation of [3H]leucine into other islet proteins was not significantly stimulated. The data strongly indicate that metabolic signals regulate modulation of proinsulin biosynthesis in the beta cells.