The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 A resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.     

2'-Deoxy-2'-azidocytidine, a new inhibitor of DNA replication in mammalian cells.

Cell growth is reversibly inhibited by the nucleoside analogue, 2'-deoxy-2'-azidocytidine and the inhibition is a result of interference with DNA replication. The 5'-diphosphate of the analogue was earlier shown to specifically inactivate the enzyme ribonucleotide reductase in vitro. However, measurements of the pools of deoxyribonucleoside triphosphates in cells incubated in azidocytidine showed only minor changes which appeared to result from and not to be the cause of the inhibition of DNA replication. The DNA synthesized in polyoma-infected cells after incubation in azidocytidine showed a sedimentation pattern quite different from that seen after inhibition of DNA synthesis with arabinosyl cytosine or hydroxyurea. Experiments with nuclei isolated from azidocytidine-inhibited, polyoma-infected cells indicated (a) that the number of replicating molecules is decreased during the inhibition and (b) that upon incubation of the nuclei there is a rapid synthesis of DNA occurring in a new class of DNA molecules which are at a very early state of replication. Neither the 5'-triphosphate of azidocytidine nor the nucleoside itself inhibit DNA synthesis in vitro in isolated nuclei from polyoma-infected cells and at present the nature of the DNA-synthesis-inhibiting compound acting in the cells after addition of azidocytidine is unknown. Taken together the results suggest that azidocytidine inhibits DNA synthesis at an early stage, possible by blocking the initiation of DNA synthesis at the origin or by interfering with the elongation of newly initiated DNA molecules.     

Acarbose is a competitive inhibitor of mammalian lysosomal acid alpha-D-glucosidases

Intraperitoneal injections (approximately 400 mg/kg of body weight) of acarbose, an inhibitor of acid (1----4)-alpha-D-glucosidase, perturb the metabolism of glycogen in the liver, resulting in excess storage of lysosomal glycogen. The metabolism of skeletal muscle glycogen was unaffected, suggesting that acarbose either does not enter the tissue or that the muscle alpha-D-glucosidase is not inhibited. The hydrolysis of maltose and glycogen by the acid alpha-D-glucosidases from rat liver, rat skeletal muscle, and human placenta was inhibited competitively by acarbose. Thus, the lack of effect of acarbose upon the metabolism of muscle glycogen is due to its inability to enter the tissue.     

Synthesis of N-chlorosulfonyl dicyclohexylamine as a potent inhibitor for spermidine synthase and its effects on human leukemia Molt4B cells

N-Chlorosulfonyl dicyclohexylamine (CSD) was synthesized as a potent inhibitor of spermidine synthase and analyzed for antiproliferative effects on leukemic cells. The compound specifically inhibited spermidine synthase in a competitive mode with the substrate putrescine (Ki, 1.8 X 10(-7) M). When human leukemia Molt4B cells were cultured in the presence of the inhibitor, the intracellular level of spermidine and the rate of cell proliferation were markedly depressed. In these polyamine depleted and growth retarded cells the synthesis of protein, but not of DNA or RNA, was found to be significantly diminished.     

Effect of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase, on polyamine metabolism in mammalian cells

The effects of the potent spermine synthase inhibitor S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDatad) on polyamine biosynthesis have been studied in transformed mouse fibroblasts (SV 3T3 cells) and in mouse leukemia cells (L1210). A dose-dependent decrease in intracellular spermine concentration was observed in both cell lines when grown in the presence of the inhibitor. A major difference in the effects seen in these two cell lines was the cytotoxicity observed in L1210 cells exposed to the inhibitor, which contrasted with little or no effects on growth of SV 3T3 cells treated similarly. Oxidative metabolism of the drug in L1210 cells was suggested by the fact that addition of aminoguanidine, an amine oxidase inhibitor, to the cell cultures ablated the cytotoxic effects of the inhibitor. Complete analysis of intracellular polyamines was carried out, together with analysis of S-adenosylmethionine, decarboxylated S-adenosylmethionine, and the inhibitor. These analyses revealed that, although the inhibitor had a dramatic effect on spermine biosynthesis in the cells studied, a compensatory increase in spermidine biosynthesis was observed. This resulted in no change in total polyamine concentrations in cells treated with inhibitors of either spermine synthase or spermidine synthase (Pegg et al., 1982) alone or in combination. In all cases, the concentration of the aminopropyl donor decarboxylated S-adenosylmethionine increased dramatically, thus allowing for the observed maintenance of total polyamine levels even in the presence of either one or both potent inhibitors of the aminopropyltransferases. Oxidative metabolism of the inhibitor complicates the interpretation of experiments carried out in the absence of amine oxidase inhibitors such as aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermidine synthase from bovine brain.

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.     

Mirroring perfection: the structure of methylglyoxal synthase complexed with the competitive inhibitor 2-phosphoglycolate

The crystal structure of the transition-state analogue 2-phosphoglycolate (2PG) bound to methylglyoxal synthase (MGS) is presented at a resolution of 2.0 A. This structure is very similar to the previously determined structure of MGS complexed to formate and phosphate. Since 2PG is a competitive inhibitor of both MGS and triosephosphate isomerase (TIM), the carboxylate groups of each bound 2PG from this structure and the structure of 2PG bound to TIM were used to align and compare the active sites despite differences in their protein folds. The distances between the functional groups of Asp 71, His 98, His 19, and the carboxylate oxygens of the 2PG molecule in MGS are similar to the corresponding distances between the functional groups of Glu 165, His 95, Lys 13, and the carboxylate oxygens of the 2PG molecule in TIM. However, these spatial relationships are enantiomorphic to each other. Consistent with the known stereochemical data, the catalytic base Asp 71 is positioned on the opposite face of the 2PG-carboxylate plane as Glu 165 of TIM. Both His 98 of MGS and His 95 of TIM are in the plane of the carboxylate of 2PG, suggesting that these two residues are homologous in function. While His 19 of MGS and Lys 13 of TIM appear on the opposite face of the 2PG carboxylate plane, their relative location to the 2PG molecule is quite different, suggesting that they probably have different functions. Most remarkably, unlike the coplanar structure found in the 2PG molecule bound to TIM, the torsion angle around the C1-C2 bond of 2PG bound to MGS brings the phosphoryl moiety out of the molecule's carboxylate plane, facilitating elimination. Further, the superimposition of this structure with the structure of MGS bound to formate and phosphate suggests a model for the enzyme bound to the first transition state.     

Aflatoxin B1, a selective inhibitor of DNA synthesis in mammalian cells.

Aflatoxin B1 in concentrations between 0.01 and 0.1 microgram/ml inhibits DNA synthesis in African green monkey cells in culture, but has little effect on RNA synthesis and no effect on protein synthesis. The drug even at concentrations up to 1.0 microgram/ml does not interfere with DNA repair promoted by ultraviolet irradiation nor does it induce DNA repair. The inhibition of DNA synthesis attains maximum values 3 h after addition of aflatoxin B1 and is irreversible upon removal of the drug. Profiles of pulselabeled DNA sedimented in alkaline sucrose gradients indicate that aflatoxin B1 blocks initiation of replication rather than elongation.     

Validation of spermidine synthase as a drug target in African trypanosomes

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.     

1-Aminooxy-3-aminopropane, a new and potent inhibitor of polyamine biosynthesis that inhibits ornithine decarboxylase, adenosylmethionine decarboxylase and spermidine synthase

1-Aminooxy-3-aminopropane was shown to be a potent competitive inhibitor (Ki = 3.2 nM) of homogenous mouse kidney ornithine decarboxylase, a potent irreversible inhibitor (Ki = 50 microM) of homogeneous liver adenosylmethionine decarboxylase and a potent competitive (Ki = 2.3 microM) of homogeneous bovine brain spermidine synthase. It did not inhibit homogeneous bovine brain spermine synthase and it did not serve as a substrate for spermidine synthase. The compound did not inhibit tyrosine aminotransferase, alanine aminotransferase or aspartate aminotransferase, which are pyridoxal phosphate-containing enzymes like ornithine decarboxylase. The inactivation of adenosylmethionine decarboxylase was partially prevented by pyruvate, which is the coenzyme of adenosylmethionine decarboxylase, and by the substrate, adenosylmethionine. 1-Aminooxy-3-aminopropane at 0.5 mM concentration inhibited the growth of HL-60 promyelocytic leukemia cells and this inhibition was prevented by spermidine but not by putrescine.     

Studies of inhibition of rat spermidine synthase and spermine synthase

1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5′-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70–98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5′-Methylthioadenosine, 5′-ethylthioadenosine and 5′-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10–15μm and 50% inhibition of spermidine synthase at 30–45μm. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20μm respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5′-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.     

Decyl-thio-trifluoropropanone, a competitive inhibitor of moth pheromone receptors

An earlier study (Pophof 1998) showed that the esterase inhibitor decyl-thio-trifluoropropanone inhibited the responses of two receptor neurons of the moth Antheraea polyphemus tuned to straight-chain pheromone components, an acetate and an aldehyde, respectively. Here we report that decyl-thio-trifluoropropanone also inhibited the responses of two pheromone receptor neurons of Bombyx mori to bombykol and bombykal. In contrast, decyl-thio-trifluoropropanone activated receptor neurons of the moth Imbrasia cytherea tuned to the pheromone component (Z)-5-decenyl 3-methyl-butanoate. However, decyl-thio-trifluoropropanone did not affect the responses of two receptor neurons of B. mori females specialized to the plant volatiles benzoic acid and linalool, respectively. These results indicate that decyl-thio-trifluoropropanone, besides inhibiting the sensillar esterase, interferes with proteins involved specifically in the excitation of pheromone receptor neurons. In binding studies with radiolabelled decyl-thio-trifluoropropanone, the inhibitor was bound by the pheromone-binding protein of A. polyphemus. However, the amount of decyl-thio-trifluoropropanone causing response inhibition was 300 times lower than the amount of pheromone-binding protein present in the sensilla. Since the amount of decyl-thio-trifluoropropanone adsorbed corresponded to about the maximum number of receptor molecules calculated per sensillum, we expect that decyl-thio-trifluoropropanone, probably in complex with pheromone-binding protein, competitively inhibits the pheromone receptor molecules. Accepted: 8 January 2000     

Putrescine derivatives as substrates of spermidine synthase

1. Derivatives of 1,4-butanediamine (putrescine) were studied in vitro and in vivo as potential substrates of spermidine synthase. 2. Substituents in the 1-position decreased the reaction rate by steric hindrance, and in the case of electron withdrawing groups there was an additional decrease due to the lowered basicity of the vicinal amino group. 3. Substituents in the 2-position are tolerated; under saturating conditions reaction rates are comparable to those of putrescine. 4. Compounds which were identified as substrates of spermidine synthase in vitro formed derivatives of spermidine and spermine in vivo. Exception: compounds, such as 1-methylputrescine formed in vivo only a spermidine derivative, because the second aminopropylation was sterically hindered by the substituent on the carbon atom next to the amino group. 5. Administration of 2-hydroxyputrescine to alpha-difluoromethylornithine-pretreated chick embryos produced spermidine and spermine analogues in amounts exceeding spermidine and spermine formation from putrescine under comparable conditions. 6. Since the concentration of 2-hydroxyputrescine in the embryo was higher than that of putrescine and all other putrescine analogues, it appears that uptake of the polyamine precursor from the yolk may be rate limiting. 7. Three days after administration of 5 mM alpha-difluoromethylornithine there is a near-to-complete arrest of embryonal growth. 8. A series of diamines supported growth under these conditions, even if they were not substrates of spermidine synthase. 9. Survival of chick embryos was, however, only supported if the diamines were capable of forming significant amounts of spermidine and spermine analogues.     

Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I

Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.     

Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol alpha) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol alpha. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as delta and epsilon; it also showed no effect even on the pol alpha activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 microm. Dehydroaltenusin-induced inhibition of mammalian pol alpha activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol alpha, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol alpha. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol alpha, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.     

Thioglycolate, competitive inhibitor of urocanase.

Urocanase was inhibited by thioglycolate, 2-mercaptoethanol, dithioerythritol, and 3-mercaptopropionate. Thioglycolate inhibited competitively at low concentrations (K_i, 0.1 mM) and protected the active site from modification by sulfite. The inhibited enzyme was reactivated by dialysis. A difference spectrum peak of 328 nm for the thioglycolate-urocanase complex compared to the 327 nm absorption maximum of the NAD-thioglycolate adduct. Several nucleophiles are known to inhibit urocanase. We conclude that thioglycolate, as a nucleophilic agent, inhibits by forming an adduct with the tightly bound NAD of urocanase. These results provide indirect evidence that NAD may be the locus of substrate binding in urocanase.