Jon P. Miller

Analogs of cyclic AMP and cyclic GMP: general methods of synthesis and the relationship of structure to enzymic activity.

Abstract The syntheses of a large variety of analogs of adenosine cyclic 3′, 5′-phosphate and guanosine cyclic 3′, 5′-phosphate are reviewed. The ability of these compounds to stimulate cyclic nucleotide-dependent protein kinases and to be hydrolyzed by cyclic nucleotide phosphodiesterases is discussed with an emphasis on the structure-activity relationships.

THE RELATIONSHIP BETWEEN THE METABOLISM OF RIBAVIRIN AND ITS PROPOSED MECHANISM OF ACTION

The synthetic nucleoside ribavirins has been shown to have broad spectrum antiviral activity both in vitro and in vivo. and recent clinical studies indicate that it is also efficacious in man.I-l2 Streeter et al.I3 have shown that the 5’-phosphate of ribavirin is a potent inhibitor of IMP-dehydrogenase, and have suggested that its antiviral effect may be mediated in part by the inhibition of this crucial biosynthetic enzyme. Streeter et aI.,l3 have also shown that ribavirin-5’-phosphate is found in the liver of rats after oral dosing, and that the enzyme most probably responsible for the phosphorylation of ribavirin is deoxyadenosine kinase. Further investigations, which are reported here, were directed towards determining if ribavirin-5’-phosphate could be further phosphorylated to the corresponding diand tri-phosphates, and towards finding out if any additional metabolites of ribavirin are produced in the whole animal. The primary purpose for studying the metabolism of ribavirin is to gain further understanding of the mechanism of action of the drug. Metabolites of ribavirin other than the 5’-phosphate may be acting at sites in the cell other than IMP-dehydrogenase. Knowledge of what metabolites are formed may give some insight into what those other sites may be.

The invitro inhibition of purine nucleotide biosynthesis by 2-β-D-ribofuranosylthiazole-4-carboxamide

Abstract A series of C-glycosylthiazoles were tested as inhibitors of purine nucleotide biosynthesis in in vitro cultures of Ehrlich ascites tumor cells. The thiazole C-nucleoside, 2-β-D-ribofuranosylthiazole-4-carboxamide, demonstrated the only significant activity of the series as a specific inhibitor of guanine nucleotide biosynthesis. At concentrations of 10–1000 μM the compound inhibits the activities of the enzymes IMP dehydrogenase and GDP kinase by 50–60% and 30–60%, respectively. The antiviral agent ribavirin demonstrated a similar pattern of enzyme inhibition at the same range of concentrations. The possible relevance of this inhibition to the recently discovered antitumor properties of 2-β-D-ribofuranosylthiazole-4-carboxamide is discussed.

Activation of cyclic AMP-dependent protein kinases I and II by cyclic 3′,5′-phosphates of 9-β-D-ribofuranosylpurine and 1-β-D-ribofuranosylbenzimidazole

Abstract Analogs of cyclic AMP lacking the 6-amino group—9-β-D-ribofuranosylpurine cyclic 3′,5′-phosphate (I)—or the 1- and 3-nitrogens as well as the 6-amino group—1-β-D-ribofuranosylbenzimidazole cyclic 3′,5′-phosphate (II)—were effective activators of both type I (cAKI) and type II (cAKII) isozymes of cAMP-dependent protein kinase. An analog with a pyrimidine ring fused to the benzimidazole ring system of II—3-β-D-ribofuranosyl-8-aminoimidazo[4,5-g]-quinazoline cyclic 3′,5′-phosphate (III)—was equipotent to I or II as an activator of cAKII but only 1 10 as potent as I or II as an activator of cAKII. The results show that neither cAKI nor cAKII requires the 6-amino group and that they may have different sensitivities to the effects of alterations in the electron distribution in the pyrimidine ring.

Activity of tubercidin-, toyocomycin-, and sangivamycin-3',5'-cyclic phosphates and related compounds with some enzymes of adenosine-3',5'-cyclic phosphate metabolism.

Abstract The effect of modifying adenosine 3′,5′-cyclic phosphate in the 7-position was studied by investigating tubercidin-, toyocomycin-, and sangivamycin-3′,5′-cyclic phosphates as activators of adenosine 3′,5′-cyclic phosphate-dependent protein kinases and as substrates for and inhibitors of adenosine 3′,5′-cyclic phosphate phosphodiesterases. The same decreasing order of activity was seen with the cyclic phosphates as kinase activators or phosphodiesterase substrates: tubercidin- > toyocomycin- > sangivamycin-3′,5′-cyclic phosphate. The I50 values of the heterocyclic base, nucleoside, nucleoside 5′-phosphate, and nucleoside 3′,5′-cyclic phosphate of tubercidin, toyocomycin, and sangivamycin were determined for the inhibition of adenosine 3′,5′-cyclic phosphate hydrolysis. The heterocyclic bases and the nucleoside 3′,5′-cyclic phosphates were very good inhibitors while the nucleosides and nucleoside 5′-phosphates were in general quite poor inhibitors.

Effect of modification of the 1-, 2-, and 6-positions of 9-β-d-ribofuranosylpurine cyclic 3′,5′-phosphate on the cyclic nucleotide specificity of adenosine cyclic 3′,5′-phosphate-and guanosine cyclic 3′,5′-phosphate-dependent protein kinases☆

Abstract A group of analogs of adenosine cyclic 3′,5′-phosphate (cAMP) and guanosine cyclic 3′,5′-phosphate (cGMP) with modifications in the 1-, 2- (or N 2 ), and 6 (or N 6 )-positions of the purine ring were compared as activators of a cAMP-dependent protein kinase [PK(cAMP)] from bovine brain and of cGMP-dependent protein kinase [PK(cGMP)] from lobster tail muscle. The results suggest that the 6-amino group of cAMP is not required for the activation of PK(cAMP) by cAMP and that the 6-oxygen and 2-amino moieties of cGMP are required for the activation of PK(cAMP) by cAMP aIn the case of PK(cGMP) activation by cGMP, the 6-oxygen apparently accepts a proton from the enzyme and the 2-amino group apparently donates a proton to the enzyme.

Mapping cyclic AMP binding sites on type I and type II cyclic AMP-dependent protein kinases using 2-substituted derivatives of cyclic AMP

Abstract Twenty-one derivatives of cyclic AMP with substituents or modification in the 2-position were examined for their ability to activate rabbit skeletal muscle type I cyclic AMP-dependent protein kinase (PK I) and bovine heart type II cyclic AMP- dependent protein kinase (PK II). PK I had stricter steric requirements than did PK II for the binding locale on the protein kinases adjacent to the 2 position of cyclic AMP. Derivatives with substituents that caused electron withdrawal from the purine ring were better than cyclic AMP as activators of PK I, but were less active than cyclic AMP as activators of PK II.

7-ribosyl-3-deazaguanine—Mechanism of antibacterial action

Abstract The mechanism by which 7-ribosyl-3-deazaguanine [7 R 3 DG , 6- amino -3-β- d - ribofuranosylimidazo [4,5-c] pyridin -4(5H)- one ] exerts its antibacterial effect was examined. Escherichia coli was found to contain an enzyme that exhibited the properties of a nucleoside phosphorylase and that converted 7R3DG to 3-deazaguanine (3DG, 6-aminoimidazo[4,5- c ]pyridin-4(5 H )-one], but no mammalian system that was examined (Erilch ascites, rat liver and human liver) was able to convert 7R3DG to 3DG. The 3DG arising from the phosphorolysis of 7R3DG was converted to 3-deaza-GMP [3DGMP, 6-amino-l-β- D -riboluranosylimidazo [4,5- c ]pyridin-4(5 H )-one-5′-phosphate] by the guanine phosphoribosyltransferase present in E. coli . A strain of E. coli , resistant to 7R3DG, was found to lack this enzyme and, therefore, was unable to convert 3DG to 3DGMP.

Activation of type I and type II cyclic AMP-dependent protein kinases by 2,8-disubstituted derivatives of cyclic AMP

Derivatives of cyclic AMP with substituents in both the 2-position (methyl or butyl) and the 8-position (bromo, benzylthio, p-chlorophenylthio or azido) and their singly modified parent compounds were examined for their abilities to activate type I isozymes of cyclic AMP-dependent protein kinases from rabbit and porcine muscle and type II isozymes of cyclic AMP-dependent protein kinases from bovine brain and heart. The specificity of 2-n-butyl-cyclic AMP for type II was substantially reduced or eliminated by the addition of 8-substituents. The lack of specificity of 2-methyl-cyclic AMP for either type I or II was not changed by the addition of 8-substituents.