Response of the septic vasculature to prolonged vasopressor therapy with N(omega)-monomethyl-L-arginine and epinephrine in canines.

OBJECTIVE: To investigate the effect of blocking nitric oxide production on cardiovascular function and survival in canine septic shock treated with or without a conventional vasopressor. DESIGN: Randomized, controlled trial. SETTING: An animal research laboratory at the National Institutes of Health. SUBJECTS: Sixty purpose-bred beagles. INTERVENTIONS: Fibrin clots containing Escherichia coli were surgically placed into the peritoneal cavity. N(omega)-monomethyl-L-arginine (L-NMMA) 10 mg/kg followed by 0.5, 1.0, or 4.0 mg/kg/hr), epinephrine (1 microg/kg/min), both, or neither were infused for 24 hrs beginning 6 hrs after the onset of infection. All animals received fluid and antibiotic therapy. MEASUREMENTS AND MAIN RESULTS: Serum nitric oxide metabolites, nitrite and nitrate, increased with infection (p = .024) and decreased with L-NMMA (p = .004, all doses combined). Myocardial nitric oxide synthase activity was ranked as follows: nonsurvivors > survivors > noninfected controls (p < .01). Other tissues examined showed the same pattern. L-NMMA produced sustained increases in systemic vascular resistance index and mean arterial pressure 9 and 24 hrs after the onset of infection (p < or = .04). Left ventricular ejection fraction was depressed by septic shock (p = .01) and further decreased by L-NMMA (p = .02). However, control and L-NMMA cardiac index values were similar (p > .4), perhaps because L-NMMA increased pulmonary artery occlusion pressure (p = .02). From 9 to 24 hrs, epinephrine, in the absence or presence of L-NMMA, blunted recovery of cardiac index (p < .02) and had a diminishing vasopressor effect (p = .05). Neither L-NMMA nor epinephrine, individually or combined, significantly altered survival rates at the doses investigated (p > or = .69). CONCLUSIONS: The tested doses showed that nitric oxide production was inhibited by L-NMMA in canine septic shock, but mortality and myocardial depression were unaffected. These results suggest that if L-NMMA has a beneficial effect on survival rates in septic shock, it is small.

Guanine, pyrazolo[3,4-d]pyrimidine, and triazolo[4,5-d]pyrimidine (8-azaguanine) phosphonate acyclic derivatives as inhibitors of purine nucleoside phosphorylase.

Phosphonate acyclic derivates of guanines, pyrazolo[3,4-d]pyrimidines, and triazolo[4,5-d]-pyrimidines (8-azaguanines) are inhibitors of the enzyme purine nucleoside phosphorylase (PNPase) with Ki' values ranging from 0.05 to 1.6 microM. These compounds are enzymatically stable congeners of the potent PNPase inhibitor acyclovir diphosphate (53).

[[(Guaninylalkyl)phosphinico]methyl]phosphonic acids. Multisubstrate analogue inhibitors of human erythrocyte purine nucleoside phosphorylase.

A series of [[(guaninylalkyl)phosphinico]methyl]phosphonic acids, 2, was synthesized and tested as inhibitors of human erythrocyte purine nucleoside phosphorylase (PNPase). The target (phosphinicomethyl)phosphonic acids 2 were synthesized in six or seven steps from alkenylphosphonates 4. The latter were converted to the intermediate alkylmesylates 9 in a series of steps that included (1) conversion of the diethyl phosphonates 4 to the (phosphinoylmethyl)-phosphonates 7 and (2) conversion of the terminal double bond of [(alkenylphosphinoyl)methyl]-phosphonates 7 to the alkylmesylates 9. The pure 9-isomers 2 were obtained by alkylation of 2-amino-6-(2-methoxyethoxy)-9H-purine with alkylmesylates 9 followed by hydrolysis of the protecting groups with concentrated hydrochloric acid and ion exchange chromatography to give 2 as hydrated ammonium salts. The most potent inhibitor of human erythrocyte PNPase, [[[5-(2-amino-1,6-dihydro-6-oxo-9H-purin-9- yl)pentyl]phosphinico]methyl]phosphonic acid (2b), was a multisubstrate analogue inhibitor with a Ki' of 3.1 nM. Optimum PNPase inhibitory activity required the presence of zinc ions in the assay medium. These potent inhibitors of PNPase exhibited only weak activity against human leukemic T-cells in vitro.

Studies of inhibitor binding to Escherichia coli purine nucleoside phosphorylase using the transferred nuclear Overhauser effect and rotating-frame nuclear Overhauser enhancement.

NMR studies of the adenosine analog tubercidin have been carried out in the presence of Escherichia coli purine nucleoside phosphorylase (PNP) in order to characterize the conformation of the enzyme-complexed nucleoside. Although analysis of transferred NOE data at various enzyme/inhibitor ratios indicated a predominantly syn nucleoside conformation in the enzyme-complexed state, the results, particularly the 8(1') and 8(3') NOE interactions, were not quantitatively consistent with any single bound conformation. Dissociation rate constants for the tubercidin-PNP complex were determined based on analysis of chemical shift and line width data as a function of enzyme/inhibitor ratio, Carr-Purcell-Meiboom-Gill measurements of the transverse relaxation rate as a function of pulse rate, and T1 rho experiments as a function of the spin-lock field strength. Dissociation rate constants of 2100 s-1 at 20 degrees C and 1400 s-1 at 10 degrees C were determined using the latter two methods. These rates are sufficiently high to justify the validity of the transferred NOE method for an enzyme as large as PNP. The possible significance of spin diffusion was investigated by the use of the deuterated analog [2'-2H]tubercidin, for which many of the intraligand spin diffusion pathways are eliminated, and by performing a series of transferred ROE experiments. A comparison of data obtained using transferred NOE and ROE measurements provides a basis for separating direct and indirect relaxation pathways. Both approaches indicated that the relatively significant 8(3') NOE interaction was not dominated by spin diffusion. Furthermore, analysis of chemical shift and transverse relaxation data for the tubercidin H-2 resonance gave inconsistent results for the chemical shift of the bound species and was inconsistent with the assumption of a single, bound conformation. These results were interpreted in terms of a 2:1 ratio of a syn, 3'-exo:anti, 3'-endo geometry for bound tubercidin. Ligand competition experiments using 9-deazainosine show that all of the tubercidin TRNOE effects are reversed by addition of the second nucleoside, suggesting that the TRNOE data for tubercidin arise due to interactions at the active sites of PNP rather than as a consequence of nonspecific binding to the enzyme.

Purification and characterization of the constitutive nitric oxide synthase from human placenta.

Human endothelial nitric oxide synthase (NOS) mRNA was detected in human placenta. In contrast, mRNAs for human neuronal NOS or for human inducible NOS were not detected in placenta. Subsequently, NOS was purified over 3800-fold from placental extract to greater than 80% homogeneity. A single band with an apparent molecular weight of 135 kDa was identified by [125I] calmodulin binding to proteins in a sodium dodecyl sulfate-polyacrylamide gel, which is consistent with the predicted size of the endothelial NOS. Furthermore, the sequence of eight internal peptides derived from this 135-kDa protein was identical to the published sequence of human endothelial NOS. As has been shown for all constitutive NOS isozymes, the purified NOS was absolutely dependent on calcium and calmodulin. NOS was also purified from human umbilical vein endothelial cells and, on the basis of similar kinetic parameters and dependence upon calcium and calmodulin, appeared to be the same as the purified placental NOS. Together, these data indicate that the placental NOS is the constitutive NOS isozyme from endothelial tissue.

Influence of stereochemistry on antiviral activities and resistance profiles of dideoxycytidine nucleosides.

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.

9-[(Phosphonoalkyl)benzyl]guanines. Multisubstrate analogue inhibitors of human erythrocyte purine nucleoside phosphorylase.

A series of 9-[(phosphonoalkyl)benzyl]guanines was synthesized and tested for inhibition of human erythrocyte purine nucleoside phosphorylase (PNPase). Inhibitors of PNPase should be T-cell selective, immunosuppressive agents with potential clinical utility in the treatment of a wide variety of disorders in which T-lymphocytes are pathogenic. An initial set of six analogues of the weak PNPase inhibitor 9-benzylguanine (2) contained a phosphonic acid group linked to the ortho, meta, or para position of the aryl moiety via two- or three-atom spacers. These compounds allowed us to probe for a favorable interaction with the phosphate-binding domain. Several additional meta phosphonoalkyl substituents were examined in an effort to optimize the spacer. The two most potent compounds, [[3-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl] benzyl]oxy]-methylphosphonic acid (3f) and [[3-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-methyl)methyl] benzyl]-thio]methylphosphonic acid (3j), were inhibitors of PNPase with Ki's of 5.8 and 1.1 nM, respectively. These inhibitors displayed competitive kinetics with respect to inosine and inorganic phosphate, which showed that these compounds possess binding determinants for both the purine- and phosphate-binding domains of the enzyme, characteristics that are consistent with 3f and 3j being multisubstrate analogue inhibitors of PNPase. The potency of 9-benzylguanine (2) was enhanced more than 6000-fold by linking a phosphonic acid residue with a (methylthio)methyl spacer to the meta position of 2 to give 3j, which illustrates the potent enzyme inhibitory properties available to multisubstrate analogue inhibitors.

Purine 2'-deoxy-2'-fluororibosides as antiinfluenza virus agents.

Twenty purine 2'-deoxy-2'-fluororibosides were synthesized by enzymic pentosyl transfer from 2'-deoxy-2'-fluorouridine. Each nucleoside analogue was assayed for cytotoxicity in uninfected Madin-Darby canine kidney cells and for their ability to suppress influenza A virus infections in these cells. The most potent antivirial activity was observed with analogues having an amino group in the 2-position of the purine moiety. All 2-unsubstituted analogues were less potent than their 2-amino counterparts. Furthermore, 2-methyl,2-methoxy, or 2-fluoro substitution obliterated antivirial activity. The most cytotoxic member of the series was the 2-fluoro-6-amino analogue (IC50 = 120 microM). 2'-Deoxy-2'-fluoroguanosine and those congeners readily converted to it by adenosine deaminase showed the most potent antivirial activity (IC50 = 15-23 microM). Little cytotoxicity was observed with this subgroup of analogues which renders them worthy of further investigation as potential antiinfluenza agents.

Nucleotide analogue inhibitors of purine nucleoside phosphorylase.

The diphosphate of the antiherpetic agent acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] has been shown to inhibit purine nucleoside phosphorylase with unique potency (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069). A major factor contributing to the superior inhibition by this diphosphate over the corresponding mono- and triphosphates is revealed here. Homologues of acyclovir mono- and diphosphate that extend the ethoxy moiety by one to four methylene groups were synthesized. These homologues were evaluated for their ability to inhibit human purine nucleoside phosphorylase. Within the diphosphate series, the Ki values increased progressively with increasing chain length. With the monophosphates, the Ki values reached a minimum with the homologue containing a pentoxy moiety. A plot of chain length versus Ki values for both mono- and diphosphates showed that both series had similar optimal distances between the aminal carbon and the terminal oxygen anion. Monophosphates with optimal positioning were somewhat less potent than diphosphates with similar positioning. Nevertheless, it was clear that a major factor in determining potency of inhibition was the distance of the terminal phosphate from the guanine moiety.

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase.

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.

Effects of acyclovir and its metabolites on hypoxanthine-guanine phosphoribosyltransferase.

Acyclovir [9-(2-hydroxyethoxymethyl)guanine], a clinically useful anti-herpesvirus agent, was a weak inhibitor (Ki = 190 microM) of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from human erythrocytes. Nevertheless, this acyclic nucleoside analog was a more effective inhibitor than were its natural counterparts, guanosine (Ki = 1400 microM) and deoxyguanosine (Ki = 570 microM). The two oxidized metabolites of acyclovir, 9-carboxymethoxymethylguanine (Ki = 720 microM) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (Ki greater than 2000 microM), were less inhibitory than was the parent drug. None of the phosphorylated metabolites of acyclovir was as potent an inhibitor of HGPRTase as was GMP (Ki = 4 microM). However, the Ki value for acyclovir monophosphate was similar to that of dGMP (12 microM). The Ki values for acyclovir diphosphate (8.3 microM) and triphosphate (30 microM) were less than those for dGDP (110 microM) and dGTP (140 microM). The levels of these phosphate esters of acyclovir in cultured monkey kidney (Vero) and human embryo fibroblast (WI38) cells exposed to therapeutic levels of the drug were well below the observed Ki values. However, in herpesvirus-infected WI38 cells the levels of the phosphate esters of acyclovir were high enough potentially to inhibit the enzyme. Although inhibition of this enzyme by the phosphorylated metabolites of acyclovir may occur in these infected cells, concentrations of the drug very much higher than the EC50 concentration were required to achieve inhibitory levels. It is, therefore, unlikely that this inhibition contributes significantly to the antiviral activity.

Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases.

Substrate-stabilization of uridine phosphorylase (uridine:orthophosphate ribosyltransferase, EC 2.4.2.3), thymidine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from Escherichia coli was investigated by heat-inactivation experiments. Nucleoside substrates stabilized uridine phosphorylase and purine-nucleoside phosphorylase, but not thymidine phosphorylase. Aglycone substrates stabilized only uridine phosphorylase. Phosphate or pentose-1-phosphate ester substrates stabilized all three enzymes. The appropriate pentose-1-phosphate ester was a more effective stabilizer than was phosphate with all three enzymes. In previous reports dealing with the kinetic analysis of these phosphorylases, sequential mechanisms were proposed. Each enzyme appeared to have different sequence of substrate addition. The substrate-stabilization patterns reported here are consistent with the proposed mechanisms.

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases.

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.

Deoxycytidine kinase from calf thymus. Substrate and inhibitor specificity.

Kinetic constants were determined for 34 nucleoside substrates of deoxycytidine kinase (EC 2.7.1.74) from calf thymus. Substrate efficiency was assessed by the ratio of Vmax to Km. Inhibition constants were determined for 61 nonsubstrate nucleosides or nucleoside analogues. The enzyme was relatively specific for the pentose moiety of nucleoside substrates. beta-D-2'-Deoxyribonucleosides were more efficient substrates than the corresponding beta-D-arabinonucleosides. Unexpectedly, the L isomer of the beta-arabinonucleoside of cytosine was a more efficient substrate than was the D isomer. beta-Cytidine and beta-5-azacytidine were the only beta-D-ribonucleosides studied that had detectable substrate activity. alpha-Cytidine was an inhibitor but not a substrate. Nucleosides containing a variety of sugar moieties other than those mentioned above did not have detectable substrate activity. The enzyme was relatively nonspecific for the base moiety of nucleoside substrates. 2'-Deoxyribonucleosides of a variety of pyrimidines, purines, and other heterocycles were substrates. Cytosine was the most preferred pyrimidine moiety. 5-Substitution, except with fluorine, decreased substrate efficiency with nucleosides of cytosine or uracil. 2-Fluoradenine was the most preferred purine moiety. The effects of various purine ring substituents were interdependent. Nucleosides containing bulky, hydrophobic substituents on either the base or the pentose moiety had no substrate activity but were relatively potent competitive inhibitors. This suggested the presence of a hydrophobic region on the surface of the enzyme near the active site.