In situ kinetic characterization of methylthioadenosine transport by the adenosine transporter (P2) of the African Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense.

African trypanosomes are parasitic flagellates that live in the connective tissues of the host. Trypanosomes must obtain from their host adenine/adenosine and other nucleosides that can be salvaged through enzymatic cleavage. Methylthioadenosine (MTA) is a byproduct of polyamine metabolism, formed from the donation of an aminopropyl moiety by decarboxylated S-adenosylmethionine (dcAdoMet) to form spermidine. MTA is then cleaved phosphorolytically by MTA phosphorylase to methylthioribose-1-phosphate (MTR-1-P) and adenine. The uptake of MTA was compared with that of adenosine in two strains: Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense. The K(m) values for MTA and adenosine (with 5 mM inosine) transport by T. b. brucei were 1.4 and 0.175 mM, and the V(max) values were 70 and 7.8 micromol/L/min, respectively. The K(m) values for T. b. rhodesiense MTA and adenosine (with 5 mM inosine) transport were 1.2 and 0.11 mM, and the V(max) values were 52.6 and 2.9 micromol/L/min, respectively. Since MTA was not competitive with either AdoMet (100 microM), inosine (100 microM), or the methionine precursor ketomethylthiobutyrate (100 microM), it appears that MTA enters through the P(2) (adenosine/adenine) transport site. From this study and our previous work, we determined that these organisms transport adenylated intermediates of methionine metabolism found in sera for purine salvage and as an ancillary source of methionine. The significant ability of African trypanosomes to transport MTA and related intermediates is an important consideration in the design and development of selective chemotherapeutic agents.

The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site.

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.

Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.

A key step in the predicted mechanism of enzymatic transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the existence of this dihydrocytosine intermediate. Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonucleotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhibitors of DNA methylation than oligodeoxyribonucleotides identical in sequence containing 5-fluorocytosine. Crystallographic analysis of a ternary complex involving M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer oligodeoxyribonucleotide containing DZCyt at the target position showed that the analog is flipped out of the DNA helix in the same manner as cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a covalent bond was detected between the sulfur atom of the catalytic site nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results indicate that DZCyt can occupy the active site of M.HhaI as a transition state mimic and, because of the high degree of affinity of its interaction with the enzyme, it can act as a potent inhibitor of methylation.

Metabolic effects of a methylthioadenosine phosphorylase substrate analog on African trypanosomes.

The effects of 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5'-deoxy-5'-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and >800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.

Effects of intermediates of methionine metabolism and nucleoside analogs on S-adenosylmethionine transport by Trypanosoma brucei brucei and a drug-resistant Trypanosoma brucei rhodesiense.

The effects of purine nucleoside analogs, polyamines, and established trypanocidal agents on the uptake of [8-14C]adenosine and S-[methyl-3H]adenosylmethionine (AdoMet) by bloodform trypanosomes of drug-susceptible Trypanosoma brucei brucei and a drug-resistant Trypanosoma brucei rhodesiense clinical isolate were compared. AdoMet uptake was not antagonized by omithine or methionine (500 microM), adenosine (100 microM), or other purine nucleosides, including methylthioadenosine (MTA) at 500 microM. Hydroxyethylthioadenosine (HETA), a trypanocidal analog of methylthioadenosine, and sinefungin, an analog of AdoMet, were competitive with AdoMet transport in both isolates. Dipyridamole, an antagonist of the adenosine P2 transporter, also competed with AdoMet transport in both isolates. The trypanocidal diamidines pentamidine, Berenil, CGP 40215, and the decarboxylated S-adenosylmethionine (dAdoMet) analog MDL 73811 (5'-¿[(Z)-4-amino-2-butenyl]¿methyl-amino¿-5'-deoxyadenosine) competed with P2 adenosine transport but did not inhibit AdoMet transport at 100 microM. Methylglyoxalbis(guanylhydrazone) (MGBG), an analog of dAdoMet, was a strong competitive inhibitor of adenosine transport at 100 microM, but did not inhibit AdoMet transport. The polyamines putrescine, spermine, and spermidine (1 mM) were examined for competition with adenosine and AdoMet transport. Putrescine significantly inhibited P2 adenosine transport in both strains (in the presence of saturating inosine), but AdoMet transport was not affected by these polyamines. P2 adenosine transport in both strains was highly inhibited by melarsen oxide and melamine, its key organic component, whereas AdoMet uptake was not affected by these agents. These findings further characterize distinguishing features of the unique AdoMet transporter in African trypanosomes, and indicate that the P2 adenosine transporter remains functional in melarsen- and diamidine-resistant clinical isolates.

In vivo efficacies of 5'-methylthioadenosine analogs as trypanocides.

5'-Deoxy-5'-(methylthio)adenosine (MTA), a key by-product of polyamine biosynthesis, is cleaved by MTA phosphorylase and is salvaged as adenine and, through conversion of the ribose moiety, methionine. An analog of MTA, 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), is a substrate for trypanosome MTA phosphorylase and is active in vitro and in vivo against Trypanosoma brucei brucei, an agent of bovine trypanosomiasis. In this study, HETA and three O-acylated HETA derivatives were examined for their activities against model infections of T. b. brucei and Trypanosoma brucei rhodesiense, the agent of East African sleeping sickness. HETA was curative (>60%) for infections caused by 5 of 11 clinical isolates of T. b. rhodesiense when it was given to mice at 200 mg/kg of body weight for 7 days as a continuous infusion in osmotic pumps. HETA at 150 to 200 mg/kg also extended the life spans of the mice infected with four additional isolates two- to fivefold. Di- and tri-O-acetylated derivatives of HETA also proved curative for the infections, while a tri-O-propionyl derivative, although also curative, was not as effective. This study indicates that substrate analogs of MTA should be given important consideration for development as novel chemotherapies against African trypanosomiasis.

Antitrypanosomal activity of purine nucleosides can be enhanced by their conversion to O-acetylated derivatives.

Fifteen purine nucleosides and their O-acetylated ester derivatives were examined for in vitro antitrypanosomal activity against the LAB 110 EATRO isolate of Trypanosoma brucei brucei and two clinical isolates of Trypanosoma brucei rhodesiense. Initial comparisons of activity were made for the LAB 110 EATRO isolate. Three nucleoside analogs exhibited no significant activity (50% inhibitory concentrations [IC50s] of > 100 microM), whether they were O acetylated or unacetylated; three nucleosides showed almost equal activity (IC50s of < 5 microM) for the parent compound and the O-acetylated derivative; nine nucleosides showed significantly improved activity (> or = 3-fold) upon O acetylation; of these nine analogs, six displayed activity at least 10-fold greater than that of their parent nucleosides. The most significant results were those for four apparently inactive compounds which, upon O acetylation, displayed IC50s of < or = 25 microM. When the series of compounds was tested against T. brucei rhodesiense isolates (KETRI 243 and KETRI 269), their antitrypanosomal effects were comparable to those observed for the EATRO 110 strain. Thus, our studies of purine nucleosides have determined that O acetylation consistently improved their in vitro antitrypanosomal activity. This observed phenomenon was independent of their cellular enzyme targets (i.e., S-adenosylmethionine, polyamine, or purine salvage pathways). On the basis of our results, the routine preparation of O-acetylated purine nucleosides for in vitro screening of antitrypanosomal activity is recommended, since O acetylation transformed several inactive nucleosides into compounds with significant activity, presumably by improving uptake characteristics. O-acetylated purine nucleosides may offer in vivo therapeutic advantages compared with their parent nucleosides, and this possibility should be considered in future evaluations of this structural class of trypanocides.

Methionine recycling pathways and antimalarial drug design.

5'-Deoxy-5'-(methylthio)adenosine (MTA) is an S-adenosylmethionine metabolite that is generated as a by-product of polyamine biosynthesis. In mammalian cells, MTA undergoes a phosphorolytic cleavage catalyzed by MTA phosphorylase to produce adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTRP). Adenine is utilized in purine salvage pathways, and MTRP is subsequently recycled to methionine. Whereas some microorganisms metabolize MTA to MTRP via MTA phosphorylase, others metabolize MTA to MTRP in two steps via initial cleavage by MTA nucleosidase to adenine and 5-deoxy-5-(methylthio)ribose (MTR) followed by conversion of MTR to MTRP by MTR kinase. In order to assess the extent to which these pathways may be operative in Plasmodium falciparum, we have examined a series of 5'-alkyl-substituted analogs of MTA and the related MTR analogs and compared their abilities to inhibit in vitro growth of this malarial parasite. The MTR analogs 5-deoxy-5-(ethylthio)ribose and 5-deoxy-5-(hydroxyethylthio)ribose were inactive at concentrations up to 1 mM, and 5-deoxy-5-(monofluoroethylthio)ribose was weakly active (50% inhibitory concentration = 700 microM). In comparison, the MTA analogs, 5'-deoxy-5'-(ethylthio)adenosine,5'-deoxy-5'-(hydroxyethylthio)ade nosine (HETA), and 5'-deoxy-5'-(monofluoroethylthio)adenosine, had 50% inhibitory concentrations of 80, 46, and 61 microM, respectively. Extracts of P. falciparum were found to have substantial MTA phosphorylase activity. Coadministration of MTA with HETA partially protected the parasites against the growth-inhibitory effects of HETA. Results of this study indicate that P. falciparum has an active MTA phosphorylase that can be targeted by analogs of MTA.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Differential kinetic properties of L-2-amino-4-methylthio-cis-but-3-enoic acid, a methionine analog inhibitor of S-adenosylmethionine synthetase.

The L-methionine analog, L-2-amino-4-methylthio-cis-but-3-enoic acid (L-cisAMTB), was examined as a potential inhibitor of the enzyme, S-adenosylmethionine (AdoMet) synthetase. The rational design of L-cisAMTB was based on previously observed potent enzyme inhibitory activity for its closely related structural analog, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB). The kinetic behavior of L-cisAMTB was studied using AdoMet synthetase isozymes I and II fractionated from L1210 murine leukemia cells. L-cisAMTB, which was a competitive inhibitor with respect to L-methionine, gave apparent Ki values of 21 and 5.7 microM for isozymes I and II, respectively. These values indicate that L-cisAMTB was slightly less inhibitory than L-cisAMB. L-cisAMTB was also a substrate for the AdoMet synthetase reaction, with respective Km values of 555 and 33 microM for isozymes I and II. In the absence of added inhibitors, the activity of isozyme II, but not isozyme I, was stimulated 2.5-fold by the presence of 10% DMSO. This preferential stimulation of isozyme II and the highly significant difference in Km values of L-cisAMTB for isozymes I and II point to possible physical differences in these tumor isozymes that were not apparent in earlier studies.

S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei.

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10-250 microM the Km for methionine was 20 microM, and this changed to 200 microM for the range 0.5-5.0 mM. In the range 10-250 microM the Km for ATP was 53 microM, and this changed to 1.75 mM for the range 0.5-5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 microM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-alpha-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration approximately 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.

Targeting 5'-deoxy-5'-(methylthio)adenosine phosphorylase by 5'-haloalkyl analogues of 5'-deoxy-5'-(methylthio)adenosine.

A series of 5'-haloalkyl-modified analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), a nucleoside byproduct of polyamine biosynthesis, has been synthesized: 5'-deoxy-5'-[(2-monofluoroethyl)thio]adenosine (10), 5'-deoxy-5'-[(2-chloroethyl)thio]adenosine (4), 5'-deoxy-5'-[(2-bromoethyl)thio] adenosine (5), and 5'-deoxy-5'-[(3-monofluoropropyl)thio]adenosine (13). On the basis of their abilities to serve as substrates of MTA phosphorylase prepared from mouse liver, several of these analogues were characterized for their growth inhibitory effects in MTA phosphorylase-containing (murine L5178Y and human MOLT-4) and MTA phosphorylase-deficient (murine L1210 and human CCRF-CEM) leukemia cell lines. The MTA phosphorylase-containing tumor cell lines, especially of human origin, were found to be more sensitive to treatment by these analogues. Of the analogue series, 10 was the most potent inhibitor of growth in each of the cell lines tested. The analogues, especially compound 10, displayed a reduced capacity to alter polyamine pools relative to MTA, mechanistically indicating a decreased potential for interactions at sites other than MTA phosphorylase. The results indicate that of the analogues tested, compound 10 displayed the best inhibitor/substrate interaction with MTA phosphorylase, which, in turn, correlated with more potent growth inhibition in tumor cell lines containing MTA phosphorylase. Overall, this supports the concept that MTA phosphorylase plays a role in the activation of such analogues.

5'-Alkyl-substituted analogs of 5'-methylthioadenosine as trypanocides.

5'-Deoxy-5'-(methylthio)adenosine (MTA) is a by-product of polyamine metabolism and is phosphoryolytically cleaved to adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTR-1-P) by MTA phosphorylase. In eukaryotes, adenine is subsequently salvaged and converted to nucleotides, while MTR-1-P is converted to methionine. We examined 5'-deoxy-5'-substituted analogs of MTA for trypanocidal activity in vitro and in vivo. 5'-Deoxy-5'-(hydroxyethyl)thioadenosine (HETA) and its 5'-bromo,5'-chloro and 5'-fluoro derivatives were cleaved by extracts of the African trypanosome Trypanosoma brucei brucei (Km for MTA, 11.5 microM; Km for HETA, 13.2 microM) in a phosphate-dependent reaction. HETA and the three halo analogs were 50% inhibitory to growth at 0.5 to 5.0 microM in vitro. Inhibition of growth was reversible by exogenous methionine and 2-keto-4-methylthiobutyric acid, an intermediate in methionine synthesis from MTR-1-P. HETA was selected for further study in vivo. When administered by miniosmotic pump (25 to 150 mg/kg/day for 7 days) to mice infected with T. brucei brucei, HETA effected 70 to 90% cure rates. Results of this study indicate that these analogs of MTA are converted to trypanocidal MTR-1-P analogs and that this approach deserves further consideration in the development of novel chemotherapy of trypanosomiasis.

Combined modulation of S-adenosylmethionine biosynthesis and S-adenosylhomocysteine metabolism enhances inhibition of nucleic acid methylation and L1210 cell growth.

Biochemical modulation of methylation processes can be accomplished by agents which either reduce pools of S-adenosylmethionine (AdoMet), the principal methyl donor, or alternatively, which raise levels of S-adenosylhomocysteine (AdoHcy), a potent product inhibitor of methyltransferase reactions. Both strategies have apparent limitations arising from their direct interference with only one determinant of the intracellular AdoHcy/AdoMet ratio, a parameter proposed to be indicative of methylation inhibition. The biological consequences of maximally altering this ratio have been examined by the combined use of an inhibitor of AdoMet synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), with inhibitors of AdoHcy hydrolase, 9-(trans-2',trans-3'-di-hydroxycyclopent- 4'-enyl)adenine (DHCA) and neplanocin A. At concentrations which inhibited growth of L1210 cells by 50% at 48 h, L-cisAMB alone rapidly depleted AdoMet pools, while neplanocin A or DHCA alone led to an accumulation of AdoHcy. When L-cisAMB was combined with either neplanocin A or DHCA, AdoHcy increased and, concomitantly, AdoMet pools decreased. The resultant AdoHcy/AdoMet ratios for up to 48 h ranged from 2.2 to 3.6-a value 4-fold greater than those achieved with neplanocin A or DHCA alone. This elevation in the AdoHcy/AdoMet ratio was accompanied by marked and sustained interference with DNA and RNA methylation and with a near-total inhibition of cell growth for a period of 24 to 96 h. Thus, the combined treatment with these two types of mechanistically different methylation inhibitors resulted in significantly enhanced interference with nucleic acid methylation and cell growth, both of which correlated directly with unprecedented increases in the AdoHcy/AdoMet ratio. This approach may have therapeutic implications in antiviral and/or antitumor strategies targeting methylation.

Synthesis and antiproliferative effects of novel 5'-fluorinated analogues of 5'-deoxy-5'-(methylthio)adenosine.

5'-Deoxy-5'-[(monofluoromethyl)thio]adenosine (9) and 5'-deoxy-5'-fluoro-5'-(methylthio)adenosine (10), two novel analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), have been synthesized and evaluated for their substrate and inhibitory activities toward MTA phosphorylase and for their biological effects in L1210 (MTA phosphorylase deficient) and L5178Y (MTA phosphorylase containing) murine leukemia cell lines. Compound 9 was a potent competitive inhibitor of MTA phosphorylase with a Ki value of 3.3 microM and was also a substrate, with activity approximately 53% that of MTA. Compound 10 was significantly less inhibitory toward the phosphorylase with a Ki value of 141 microM; its lack of substrate activity was attributed to rapid nonenzymatic degradation. The 50% growth inhibitory concentrations (48 h) of 9 were 300 and 200 microM in L1210 and L5178Y cells, respectively; for 10, these respective values were 2 and 0.7 microM. The initial characterization of 9 in these systems reveals that it differs from MTA by not acting as a product regulator of the polyamine biosynthetic pathway.

Modulation of polyamine-biosynthetic activity by S-adenosylmethionine depletion.

The methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), was used to study the early effects of AdoMet depletion on polyamine biosynthesis. In the presence of decreased methionine (30 microM) in the medium, treatment of cultured L1210 cells with 1 mM-L-cisAMB resulted in a near-total (95%) depletion of cellular AdoMet pools by 4 h. This was accompanied by a 3-fold increase in ornithine decarboxylase (ODC) activity, a 2.5-fold increase in AdoMet decarboxylase (AdoMetDC) activity and a 20% decrease in spermidine and spermine pools. The increase in enzyme activities seemed to be partially due to prolongation of enzyme activity half-life, since that of ODC was extended from 30 to 50 min and that of AdoMetDC from 65 to 310 min. By temporal sequence characterization (0-4 h), the onset of elevations of enzyme activity (0.5-1 h) seemed to be causally related to an earlier (0-0.5 h) decline in AdoMet pools, as opposed to the 20% decrease in spermidine and spermine pools, which occurred much later (2-4 h); the latter are known to regulate decarboxylase activities negatively. Drug-induced elevations in ODC and, to a lesser extent, AdoMetDC activities were reversed by later treatment with exogenous AdoMet. However, because the latter also increased spermine pools (which could not be prevented with various enzyme inhibitors), the reversal of elevations in enzyme activities could not be directly linked to AdoMet. Although not definitive, the data raise the interesting possibility that, in addition to being negatively regulated by polyamines, ODC and AdoMetDC activities may also be subject to negative control by cellular AdoMet (or an AdoMet metabolite). The net effect of either or both of these influences would be to conserve polyamine-biosynthetic activity in the face of declining AdoMet supplies.

Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis.

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.

Interference with polyamine biosynthesis and/or function by analogs of polyamines or methionine as a potential anticancer chemotherapeutic strategy.

The obvious goal in cancer chemotherapy is selectivity. Highly cytotoxic agents abound but their usefulness as anticancer agents extends only so far as their specificity for tumor cells and tissues. In this context, we have reviewed those aspects of polyamine and AdoMet metabolism and function which might contribute to their potential as target sites for chemotherapeutic intervention. Although largely untested to date and far from unequivocal, these various considerations seem to provide sufficient rationale for continued evaluation of the therapeutic potential of these sites. Polyamine analogs and methionine analogs designed to modulate polyamine biosynthesis directly or through AdoMet formation have been discussed as strategies to effect this goal and previous studies with similar analogs have been reviewed. Progress achieved thus far with analogs derived from our own laboratories provides novel insights into polyamine and AdoMet metabolism and/or function as well as new leads towards the design of more effective agents and drug combinations. More detailed reading of the biochemistry of polyamines in eukaryotes and prokaryotes is available in several very excellent current reviews (6-9, 77).

Comparison of the biological effects of four irreversible inhibitors of ornithine decarboxylase in two murine lymphocytic leukemia cell lines.

The effects of the enzyme-activated irreversible inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine, alpha-(fluoromethyl)dehydroornithine, alpha-(fluoromethyl)dehydroornithine methyl ester, and (2R,5R)-6-heptyne-2,5-diamine (RR-MAP), on cell growth and parameters related to polyamine biosynthesis were compared in L5178Y and L1210 cells under identical culture conditions. The two lines are murine lymphocytic leukemia cells which differ in their ability to metabolize 5'-methylthioadenosine, the by-product of polyamine biosynthesis: L5178Y cells contain a specific 5'-methylthioadenosine phosphorylase; L1210 cells do not. In L1210 cells, the 50% inhibitory concentrations (lC50S) of the various analogues were 3.0 mM for alpha-difluoromethylornithine, 0.2 mM for alpha-(fluoromethyl)dehydroornithine, 0.1 mM for alpha-(fluoromethyl)dehydroornithine methyl ester, and 0.01 mM for RR-MAP. L5178Y cells were somewhat more sensitive to the inhibitors with lC50 values of 0.5 mM for alpha-difluoromethylornithine, 0.06 mM for alpha-(fluoromethyl)dehydroornithine, 0.03 mM for alpha-(fluoromethyl)dehydroornithine methyl ester, and 0.002 mM for RR-MAP. In all cases, growth inhibition was fully prevented by exogenous putrescine. The effects of the inhibitors on parameters related to polyamine metabolism were compared at drug concentrations approximating the average of lC50 values for the two cell lines. Under these treatment conditions, polyamine pools were similarly affected by the various inhibitors. Typically, putrescine and spermidine were depleted, but effects on spermine pools differed according to the cell line, increasing slightly in L1210 cells and decreasing by about 50% in L5178Y cells. Spermine pools in L1210 cells could be reduced by RR-MAP at concentrations higher than the lC50 (i.e., 0.1 mM). Clonogenicity in soft agar was decreased about 50% by putrescine and spermidine depletion and was not further affected by spermine depletion. The inhibitors elevated S-adenosylmethionine decarboxylase activity in both cell lines with a 2-fold greater increase in L5178Y cells than in L1210 cells. Finally, the inhibitors decreased S-adenosylmethionine pools in L1210 cells by about 50% but had little effect on these pools in L5178Y cells with the exception of RR-MAP, which decreased S-adenosylmethionine pools by about 40%. Whether the different polyamine responses of the two cell lines are related to their ability to metabolize 5'-methylthioadenosine is uncertain. It is apparent, however, that the presence or absence of methylthioadenosine phosphorylase does not substantially modulate the antiproliferative activity of ornithine decarboxylase inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth inhibition by methionine analog inhibitors of S-adenosylmethionine biosynthesis in the absence of polyamine depletion.

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cis-AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC50 levels, the analogs significantly reduced AdoMet pools by approximately 50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L-cis-AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.