Effect of undermethylation on mRNA cytoplasmic appearance and half-life.

S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocysteine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. We investigated the effects of STH on HeLa cell mRNA metabolism. Dual labeling studies reveal that STH dramatically inhibits the methylation of HeLa mRNA in a dose-dependent manner. Analysis of the modified nucleosides and 5'-terminal cap structures in radiolabeled mRNA by high-pressure liquid chromatography indicated that internal N6-methylation of adenosine was reduced by 65% at 50 microM STH and by 83% at 500 microM STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structures lacking 2'-O-methylated nucleosides (m7GpppN, cap zero) were detected at the higher level of STH. To test the possibility that methylation affects mRNA stability, cytoplasmic mRNA half-life was measured in a pulse-chase experiment. The half-life of undermethylated mRNA, produced as a consequence of STH treatment, was unchanged compared with the control. To determine whether mRNA methylation is coupled to nuclear processing or transport, the time of cytoplasmic appearance of polyadenylated RNA in STH-treated HeLa cells was compared with untreated cells. STH caused a significant lag in the time of appearance of the polyadenylated RNA, suggesting that mRNA methylation may be required for efficient processing or transport.

Biochemical and growth inhibitory effects of the erythro and threo isomers of gamma-fluoromethotrexate, a methotrexate analogue defective in polyglutamylation.

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(gamma-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.

Stable multisubstrate adducts as enzyme inhibitors. Potent inhibition of ornithine decarboxylase by N-(5'-phosphopyridoxyl)-ornithine.

The synthesis of several N-(5'-phosphopyridoxyl)-amino acids is described. These compounds, analogs of the Schiff base intermediate involved in enzyme-catalyzed decarboxylation, are potent inhibitors of the cognate amino acid decarboxylases. Kinetic studies using partially purified rat liver ornithine decarboxylase, have shown that N-(5'-phosphopyridoxyl)-ornithine inhibits the enzyme in a non-competitive manner with respect to both ornithine and pyridoxal-5'-phosphate. These findings suggest that the inhibitor binds to the holoenzyme active site in place of the Schiff base intermediate.

Aldehyde and phosphinate analogs of glutathione and glutathionylspermidine: potent, selective binding inhibitors of the E. coli bifunctional glutathionylspermidine synthetase/amidase.

INTRODUCTION: The tripeptide glutathione is converted to glutathionylspermidine (Gsp) in Escherichia coli and in trypanosomatid parasites by an ATP-cleaving Gsp synthetase activity. In parasites, an additional glutathionylation forms bis-(glutathionyl)-spermidine, trypanothione, believed to be the major surveillance thiol involved in oxidant defense mechanisms in trypanosomatid parasites. In E. coli, the Gsp synthetase is part of a bifunctional enzyme opposed by the hydrolytic Gsp amidase. RESULTS: Gsp amidase and Gsp synthetase activities of the bifunctional E. coli enzyme can be separately targeted by potent, selective slow-binding inhibitors that induce time-dependent inhibition. The inhibitor gamma-Glu-Ala-Gly.CHO most probably captures Cys59 and accumulates as the tetrahedral adduct in the amidase active site. Inhibitory Gsp phosphinate analogs are phosphorylated by ATP to yield phosphinophosphate analogs in the synthetase active site. Binding of phosphinophosphate in the Gsp synthetase active site potentiates the inhibition affinity for the aldehyde at the Gsp amidase active site by two orders of magnitude. CONCLUSIONS: Time-dependent inhibition of the Gsp amidase activity by the aldehyde substrate analog supports previous work that suggests glutathionyl acyl-enzyme intermediate formation in the Gsp amidase reaction mechanism. Use of potent selective inhibitors against Gsp amidase (aldehyde) and Gsp synthetase (phosphinate) activities provides further evidence of interdomain communication in the bifunctional enzyme from E. coli.

Use of aminopropyltransferase inhibitors and of non-metabolizable analogs to study polyamine regulation and function.

The polyamines spermidine and spermine are essential for the growth of mammalian cells. This review describes the properties of the two aminopropyltransferases that are responsible for their biosynthesis, the synthesis and use of specific aminopropyltransferase inhibitors, and the use of analogs of the polyamines to investigate polyamine transport and function. Highly specific and potent multisubstrate adduct inhibitors of these enzymes have been synthesized while less potent inhibitors have been obtained by the synthesis of amines that bind at the active site. Studies with these inhibitors indicate that polyamines are needed for a normal rate of growth and that, although some of the functions of polyamines may be interchangeable, other functions may have a specific requirement for spermidine or spermine. Two groups of growth-promoting polyamine analogs can be distinguished: the many that are effective in short-term experiments compared to the few that can act over a prolonged period. The more stringent structural requirements for long-term growth are probably due to a need for spermidine, or a closely related analog, as a precursor of hypusine in the protein eIF-5A. Metabolically resistant polyamine analogs can be used as model substrates for studies of the polyamine transport system, which plays a critical role in maintaining normal cellular polyamine levels. The feedback regulation by high levels of polyamines that downregulates transport is essential to prevent the accumulation of polyamines at toxic levels. Such accumulation may be associated with apoptosis and, therefore, polyamine analogs are useful tools for investigating the mechanism(s) of polyamine-mediated toxicity.

Inhibition of bacterial aminopropyltransferases by S-adenosyl-1,8-diamino-3-thiooctane and by dicyclohexylamine.

Bacterial aminopropyltransferases from Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa were strongly inhibited by S-adenosyl-1,8-diamino-3-thiooctane (AdoDATO) and by dicyclohexylamine. The sensitivity to these drugs in vitro was comparable to that of mammalian spermidine synthase, but AdoDATO was much less potent in reducing spermidine content in the bacteria than in mammalian cells. Although AdoDATO was a stronger inhibitor than dicyclohexylamine in vitro, dicyclohexylamine was more active in reducing bacterial spermidine levels in vivo, suggesting that it is taken up better or is more stable in the cell and is the preferable compound for in vivo studies in microorganisms. The strong inhibition of spermidine synthases by AdoDATO which is a transition state analog supports the concept that these enzymes proceed by a single displacement reaction, rather than by a ping-pong mechanism.

Stereospecific synthesis of (R)- and (S)-S-adenosyl-1,8-diamino-3-thiooctane, a potent inhibitor of polyamine biosynthesis. Comparison of asymmetric induction vs enantiomeric synthesis.

Two diastereomers of the potent spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thiooctane have been prepared in high (greater than 96% de) stereochemical purity. Two synthetic routes were investigated, one based on asymmetric induction and the other involving an enantiomeric synthesis. The latter route gave the desired products in greater than 96% de, whereas the synthesis based on asymmetric induction resulted in only 80% de in the final product. Evaluation of the two diastereomers as inhibitors of spermidine synthase showed that the R diastereomer is a more potent inhibitor than the S diastereomer.

Analogues of S-adenosylhomocysteine as potential inhibitors of biological transmethylation. Synthesis of analogues with modifications at the 5'-thioether linkage.

The synthesis of S-adenosylhomocysteine analogues, in which the 5'-thioether linkage is replaced by an oxygen or nitrogen isostere, has been investigated. These compounds were disigned to be resistant to enzyme-catalyzed hydrolytic cleavage of the 5'-substituent. The amine analogue Id and two amide analogues 20 were prepared via alkylation or acylation of appropriately blocked adenosine derivatives. These new analogues were evaluated as inhibitors of catechol O-methyltransferase and tRNA methylases and found to have poor inhibitory activity.

Synthesis and evaluation of some stable multisubstrate adducts as specific inhibitors of spermidine synthase.

A new series of aminopropyltransferase inhibitors has been designed in which the nuclephilic aminopropyl acceptor is attached to the aminopropyl donor, S-adenosyl-1-(methylthio)-3-propylamine (decarboxylated S-adenosylmethionine), to form a "multisubstrate adduct". In the present case, S-adenosyl-1,8-diamino-3-thiooctane (2b) and the corresponding methysulfonium salt (3b) have been synthesized. Several compounds of this type were assayed as inhibitors of spermidine synthase, and both 2b and 3b were found to be potent inhibitors of the enzyme. The thioether 2b is the most potent inhibitor of spermidine synthase described to date and is almost totally devoid of inhibitory activity against the closely related aminopropyltransferase, spermine synthase. This type of compound should have use as a specific inhibitor of spermidine biosynthesis in vivo.

Synthesis and biochemical evaluation of adenosylspermidine, a nucleoside-polyamine adduct inhibitor of spermidine synthase.

The synthesis of a new class of multisubstrate adduct inhibitors of polyamine biosynthesis has been investigated. The first target compound, designed to inhibit spermidine synthase, was obtained and proved to be a very potent inhibitor of that enzyme. Two synthetic routes to effect the coupling of the polyamine spermidine to the nucleoside adenosine were studied. The first route involved a proposed Wittig or Julia olefination reaction to form the critical 5'-6' carbon-carbon bond between the nucleoside and polyamine moieties. This route failed due to a facile beta-elimination of a portion of the side chain from a carbanion intermediate during either coupling reaction. A second route involved a reductive amination approach and proved to be successful. The new inhibitor, given the trivial name adenosylspermidine, is the most potent inhibitor of spermidine synthase prepared to date.

Synthesis and biological evaluation of DL-4,4-difluoroglutamic acid and DL-gamma,gamma-difluoromethotrexate.

DL-4,4-Difluoroglutamic acid (DL-4,4-F2Glu) and its methotrexate analogue, DL-gamma,gamma-difluoromethotrexate (DL-gamma,gamma-F2MTX), were synthesized and evaluated as alternate substrates or inhibitors of folate-dependent enzymes. Synthesis of DL-4,4-F2Glu involved the nitroaldol reaction of ethyl nitroacetate with a difluorinated aldehyde ethyl hemiacetal as a key step. Attempted ligation of DL-4,4-F2Glu to methotrexate (MTX), catalyzed by human folylpoly-gamma-glutamate synthetase (FPGS), revealed that DL-4,4-F2Glu is a poor alternate substrate. DL-gamma,gamma-F2MTX was synthesized by a route proceeding through N-[4-(methylamino)benzoyl]-4,4-difluoroglutamic acid di-tert-butyl ester followed by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. DL-gamma,gamma-F2MTX was found to be neither a substrate nor an inhibitor of human FPGS. The fluorinated analogue of MTX, however, inhibits DHFR and cell growth with the same potency as MTX.

Synthesis and evaluation of some stable multisubstrate adducts as inhibitors of catechol O-methyltransferase.

A new series of methylase inhibitors has been designed in which the nucleophilic methyl acceptor is attached to the adenosine and/or homocysteine fragments of the methyl donor, S-adenosylmethionine, to form a "multisubstrate adduct". In the present case, catecholamine analogues attached through a phenethyl sulfide linkage to 5'-thioadenosine or homocysteine have been synthesized, together with the corresponding methylsulfonium salts. These compounds were assayed as inhibitors of catechol O-methyltransferase, and the adenosylsulfonium salts (4) were found to be inhibitors of the enzyme.

Synthesis and biological evaluation of N alpha-(4-amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithine.

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithi ne (AMPte-DL-4,4-F2Orn, 4) was synthesized and evaluated as an inhibitor of human folypoly-gamma-glutamate synthetase (FPGS), dihydrofolate reductase (DHFR), and cell growth. Synthesis of 4 involved the use of a protected form of DL-4,4-difluoroornithine 9 which was derived from DL-4,4-difluoroglutamic acid. Biological activities of 4 were compared directly to those of the corresponding nonfluorinated compound N alpha-(4-amino-4-deoxy-10-methylpteroyl)-L-ornithine (AMPte-L-Orn, 3). Although the fluorinated analogue is a potent inhibitor of DHFR, it is a poor inhibitor of FPGS. However, the compound is transported across the cell membrane and inhibits cell growth, presumably due to the inhibition of DHFR. The data obtained with the fluorinated analogue are in contrast to those of the corresponding nonfluorinated compound 3, which is a potent inhibitor of both FPGS and DHFR but shows very low cytotoxicity due to poor transport.

Polyamine biosynthesis in rat prostate. Substrate and inhibitor properties of 7-deaza analogues of decarboxylated S-adenosylmethionine and 5'-methylthioadenosine.

The 7-deaza analogue of 5'-methylthioadenosine, a nucleoside end product in polyamine biosynthesis, has been synthesized. This analogue has been shown to competitively inhibit the hydrolytic cleavage of the purine-ribose bond in methylthioadenosine with Ki congruent to Km. In addition, the 7-deaza analogue of decarboxylated S-adenosylmethionine, a cofactor in the biosynthesis of both spermidine and spermine, has been synthesized. This analogue has been shown to act as a substrate in the reaction catalyzed by spermidine synthase, in which severe substrate inhibition by both the normal nucleoside substrate and the 7-deaza analogue is observed. These results are discussed in terms of possible end product regulation of polyamine biosynthesis and the possible substitution of the nucleoside antibiotic, tubercidin, for adenosine in reactions involving S-adenosylmethionine and its metabolites.

alpha-Methyl polyamines: metabolically stable spermidine and spermine mimics capable of supporting growth in cells depleted of polyamines.

In order to assess the tolerance of the target enzyme spermine synthase for alpha-substituents on the aminopropyl moiety of the substrate spermidine, 1-methylspermidine (MeSpd, 2) was synthesized. It was determined that MeSpd is a poor substrate for spermine synthase and is not a substrate for spermidine N1-acetyltransferase, suggesting that alpha-methylated polyamines might be metabolically stable and therefore useful tools for studying polyamine effects in intact cells. On the basis of initial cellular results with 2, 1-methylspermine (MeSpm, 3) and 1,12-dimethylspermine (Me2Spm, 4) were also synthesized. When added to cells (L1210, SV-3T3, or HT29) depleted of both putrescine and spermidine by prior treatment with alpha-(difluoromethyl)ornithine (DFMO), these alpha-methylated polyamines were able to restore cell growth to that observed in the absence of DFMO. In accord with the enzyme data noted above, metabolic studies indicated a slow conversion of 2 to 3, but no metabolism of 4 in these cells. It was concluded from these results that the alpha-methylated polyamines are able to substitute for the natural polyamines spermidine and spermine in critical biochemical processes which involve polyamines for continued cell growth. In accord with the hypothesis, preliminary data indicate that MeSpd and Me2Spm are as effective as spermidine and spermine, respectively, in promoting the conversion of B-DNA to Z-DNA.

Design, synthesis, and biochemical evaluation of phosphonate and phosphonamidate analogs of glutathionylspermidine as inhibitors of glutathionylspermidine synthetase/amidase from Escherichia coli.

Three phosphapeptides designed to mimic two distinct tetrahedral intermediates formed during either the synthesis or hydrolysis of glutathionylspermidine (Gsp) were synthesized and evaluated as inhibitors of the bifunctional enzyme Gsp synthetase/amidase. While the polyamine-containing phosphapeptides were determined to be potent and selective inhibitors, they selectively inhibit the synthetase activity over the amidase domain. A phosphonate-containing tetrahedral mimic is a reversible mixed-type inhibitor of Gsp synthetase with an inhibition constant of 6 microM for the inhibitor binding to the free enzyme (Ki) and 14 microM for the inhibitor binding to the enzyme-substrate complex (Ki'). The corresponding phosphonamidate is a slow-binding inhibitor with a Ki of 24 microM and a Ki* (isomerization inhibition constant) of 0.88 microM. A non-polyamine-containing phosphonamidate exhibits no significant inhibition of the synthetase or amidase activity.

Synthesis and biological evaluation of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase.

As part of a continuing search for specific inhibitors of the enzymes involved in polyamine biosynthesis, we have designed and synthesized a multisubstrate adduct inhibitor, S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDATAD), in which critical portions of the nucleophilic aminopropyl acceptor are covalently linked to critical portions of the electrophilic aminopropyl donor to form a potent and specific inhibitor of spermine synthase. In addition, the corresponding desamino analogue which was designed to lack activity against spermine synthase on the basis of substrate structure-activity data has been synthesized as a control. Preliminary biological results demonstrate that AdoDATAD is a potent and specific inhibitor of mammalian spermine synthase in vitro, while being almost completely devoid of inhibitory activity toward the closely related aminopropyltransferase spermidine synthase. The desamino analogue, as predicted, showed no inhibitory activity against either enzyme. AdoDATAD represents an important addition to the arsenal of specific enzyme inhibitors available for blockade of the polyamine biosynthetic pathway at specific sites.

Synthesis of N-[N-(4-deoxy-4-amino-10-methylpteroyl)-4-fluoroglutamyl]- gamma-glutamate, an unusual substrate for folylpoly-gamma-glutamate synthetase and gamma-glutamyl hydrolase.

N-[N-(4-Deoxy-4-amino-10-methylpteroyl)-4-fluoroglutamyl]-ga mma-glutamate has been synthesized and its ability to serve as a substrate for folylpolyglutamate synthetase and gamma-glutamyl hydrolase has been investigated. It was anticipated that this compound would be a substrate for both of these enzymes. Although the title compound proved to be a good substrate for folylpolyglutamate synthetase, hydrolysis catalyzed by gamma-glutamyl hydrolase was unexpectedly slow. These results suggest the use of fluoroglutamate-containing peptides as hydrolase-resistant folates or antifols in a variety of chemotherapeutic regimens.

Synthesis and biological activity of folic acid and methotrexate analogues containing L-threo-(2S,4S)-4-fluoroglutamic acid and DL-3,3-difluoroglutamic acid.

The stereospecific syntheses of L-threo-gamma-fluoromethotrexate (1t) and L-threo-gamma-fluorofolic acid (3t) are reported. Compounds 1t and 3t have no substrate activity with folylpoly-gamma-glutamate synthetase isolated from CCRF-CEM human leukemia cells, and compound 1t inhibits human dihydrofolate reductase at similar levels as methotrexate. The synthesis of DL-3,3-difluoroglutamic acid (6) and its incorporation into DL-beta,beta-difluorofolic acid (4) are also reported. Compound 4 acts as a better substrate for human CCRF-CEM folylpoly-gamma-glutamate synthetase than folic acid (V/K = ca. 7-fold greater). Thus, replacement of the glutamate moiety of methotrexate and folic acid with 4-fluoroglutamic acid and 3,3-difluoroglutamic acid results in folates and antifolates with altered polyglutamylation activity.

Effect of N-(n-butyl)-1,3-diaminopropane on polyamine metabolism, cell growth and sensitivity to chloroethylating agents.

The effects of N-(n-butyl)-1,3-diaminopropane (BDAP) on cell growth and polyamine content were examined in L1210, SV-3T3 and HT-29 cells. In all cases, BDAP was a specific and highly effective inhibitor of spermine synthesis, and spermine levels were greatly suppressed in the presence of 50 microM BDAP. At the same time, there was a parallel increase in spermidine, which equalled or exceeded the fall in spermine so that total polyamine levels were not reduced. Cell growth was not affected in short-term experiments but culture of L1210 cells for 72-144 hr in the presence of BDAP did lead to an effect on growth that was reversed by the addition of spermine. These results suggest that, in the short term, a normal growth rate is maintained by spermidine but that a function or cellular component critically dependent on spermine becomes depleted at longer times. BDAP was a weak inducer of spermidine/spermine-N1-acetyltransferase and this enzyme may be responsible for excretion or degradation of the inhibitor. The reduction of spermine produced by BDAP led to a substantial increase in the activity of S-adenosylmethionine decarboxylase (AdoMetDC) showing that the repression of this enzyme by spermine is greater than the repression by spermidine. Although higher concentrations were required, BDAP was as effective an inhibitor of spermine synthesis as the mechanism-based inhibitor, S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDATAD), and produced similar decreases in spermine and increases in AdoMetDC. Prior treatment of HT-29 human colon carcinoma cells with BDAP increased the killing by chloroethylating agents but to a much smaller extent than the increase brought about by the DNA repair inhibitor, O6-benzylguanine. The effect of BDAP is likely to be due to an increased interaction of chloroethylating drugs with nuclear DNA in the absence of spermine since BDAP treatment sensitized cells even in the presence of O6-benzylguanine, which prevents repair of these lesions.