Targeting the polyamine biosynthetic enzymes: a promising approach to therapy of African sleeping sickness, Chagas’ disease, and leishmaniasis

Summary. Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor α-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.     

Characterization of transgenic mice with overexpression of spermidine synthase

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.     

Trypanosoma brucei S-Adenosylmethionine Decarboxylase N Terminus Is Essential for Allosteric Activation by the Regulatory Subunit Prozyme

Human African trypanosomiasis is caused by a single-celled protozoan parasite, Trypanosoma brucei. Polyamine biosynthesis is a clinically validated target for the treatment of human African trypanosomiasis. Metabolic differences between the parasite and the human polyamine pathway are thought to contribute to species selectivity of pathway inhibitors. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the production of the polyamine spermidine. We previously showed that trypanosomatid AdoMetDC differs from other eukaryotic enzymes in that it is regulated by heterodimer formation with a catalytically dead paralog, designated prozyme, which binds with high affinity to the enzyme and increases its activity by up to 10³-fold. Herein, we examine the role of specific residues involved in AdoMetDC activation by prozyme through deletion and site-directed mutagenesis. Results indicate that 12 key amino acids at the N terminus of AdoMetDC are essential for prozyme-mediated activation with Leu-8, Leu-10, Met-11, and Met-13 identified as the key residues. These N-terminal residues are fully conserved in the trypanosomatids but are absent from other eukaryotic homologs lacking the prozyme mechanism, suggesting co-evolution of these residues with the prozyme mechanism. Heterodimer formation between AdoMetDC and prozyme was not impaired by mutation of Leu-8 and Leu-10 to Ala, suggesting that these residues are involved in a conformational change that is essential for activation. Our findings provide the first insight into the mechanisms that influence catalytic regulation of AdoMetDC and may have potential implications for the development of new inhibitors against this enzyme.     

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product

S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t _0.5∼10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K _0.5∼100 μM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine. Received: 4 August 1999 / Accepted: 14 February 2000     

Effect of polyamines and L-ornithine on the development of proembryogenic masses of Cryptomeria japonica

We examined the effects of polyamines, namely, putrescine, spermidine and spermine, and of amino acids, such as l-arginine and l-ornithine, as part of our efforts to identify factors that stimulate the development of proembryogenic masses (PEMs) of Cryptomeria japonica. We maintained two distinct types of PEM designated PEMs A, which consisted of normal embryogenic cells as single embryos with elongated suspensor cells, and PEMs B, which consisted of abnormal embryogenic cells with coalesced embryos on modified Campbell and Durzan medium (mCD) supplemented with individual polyamines at 0–100 μM or amino acids at 0–16.4 mM. All additives had a stimulatory/suppressive effect. Microscopy and image-processing techniques revealed that the regions of authentic embryos of PEMs that were treated with l-ornithine were remarkably enlarged and that the suspensor cells had elongated in the same direction. When all PEMs A were transferred to maturation medium (mCD that contained abscisic acid and maltose at various concentrations), only PEMs that had been treated with l-ornithine matured into somatic embryos and were able to germinate on hormone-free mCD. Our results indicate that l-ornithine is an important stimulator of the development of PEMs to the pre-filamentous stage in C. japonica.     

Identification of a suppressor gene for the arginine-auxotrophic <Emphasis Type="Italic">argJ</Emphasis> mutation in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We recently proposed a metabolic engineering strategy for l-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance l-ornithine production in a well-defined recombinant strain of Corynebacterium glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg⁻¹ protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted l-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during l-ornithine production in C. glutamicum.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Inhibitors of N α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency

A series of N ^α-acyl (alkyl)- and N ^α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N ^α-acetyl-l-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N ^α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC₅₀ values in the μM range toward ArgE, indicating that they are moderately strong inhibitors. N ^α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC₅₀ value of 85 μM while N ^α-trifluoroacetyl-l-ornithine (1f), N ^α-ethoxycarbonyl-l-ornithine (2b), and N ^α-acetyl-d-ornithine (1a) weakly inhibited ArgE activity providing IC₅₀ values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N ^α-acetyl-d-ornithine (1a) and N ^α-fluoro- (1f), N ^α-chloro- (1g), N ^α-dichloro- (1h), and N ^α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC₅₀ values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Molecular Dynamics Reveal Binding Mode of Glutathionylspermidine by Trypanothione Synthetase

The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two Mg²⁺ ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows N¹-glutathionylation of N⁸-glutathionylspermidine, classifying N⁸-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for N⁸-glutathionylspermidine was characterised as druggable.     

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.     

Studies on metabolic role of 5′-methylthioadenosine in Ochromonas malhamensis and other microorganisms

Several compounds containing a thiomethyl group were found to replace vitamin B₁₂ in a protozoan, Ochromonas malhamensis. The order of the effectiveness was as follows: 5-methylthioadenosine > S-adenosylmethionine > 5-methylthioribose > L-methionine. A similar order was obtained with respect to the permeability of these compounds into the protozoan cells, except for S-adenosylmethionine. 5-Methylthioadenosine and 5-methylthioribose as well as l-methionine markedly increased the intracellular content of l-methionine. The level of S-adenosylmethionine was also increased by them, but to a lesser degree. The thiomethyl group of the compounds was established to be incorporated into S-adenosylmethionine. The metabolic fate of the thiomethyl group of 5-methylthioadenosine cannot be distinguished from that of l-methionine. A high activity of 5-methylthioadenosine nucleosidase was detected in the cell-free extracts of the protozoan. These results strongly suggest that 5-methylthioadenosine would be metabolized to l-methionine via 5-methylthioribose and then the l-methionine would be converted to S-adenosylmethionine. Like l-methionine and vitamin B₁₂, 5-methylthioadenosine and 5-methylthioribose may play an important role in maintenance of the C-1 pool in Ochromonas malhamensis.Neither 5-methylthioadenosine nor 5-methylthioribose replaced vitamin B₁₂ in some vitamin B₁₂-requiring bacteria. This result is consistent with the fact that neither compounds was significantly taken up by these bacteria.Abbreviations MTA 5-methylthioadenosine - AdoMet S-adenosylmethionine - MTR 5-methylthioribose - TCA trichloroacetic acid Paper II in the series. The first paper of the series has been published (Sugimoto and Fukui, 1974)     

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)₂)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays K_m values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as K_i values for GSH and T(SH)₂ of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a K_m value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system''s biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)₂ inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.     

Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well‐studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S‐adenosylmethionine and a short‐chain polyamine (putrescine) to make a medium‐chain polyamine (spermidine) and 5′‐deoxy‐5′‐methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S‐adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose‐dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X‐ray crystallography at 2.0 Å resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K_d of 1.1 ± 0.3 μM in the absence of putrescine and 3.2 ± 0.1 μM in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.     

N5-(l-1-Carboxyethyl)-l-ornithine: NADP+ oxidoreductase inStreptococcus lactis: Distribution,constitutivity, and regulation

N⁵-(l-1-Carboxyethyl)-l-ornithine: NADP⁺ oxidoreductase [N⁵-(CE)ornithine synthase] catalyzes the NADPH-dependent reductive condensation between pyruvic acid and the terminal amino group ofl-ornithine andl-lysine to yield N⁵-(l-1-carboxyethyl)-l-ornithine and N⁶-(l-1-carboxyethyl)-l-lysine respectively. Polyclonal antibodies against N⁵-(CE)ornithine synthase purified fromStreptococcus lactis K1 have been used for the immunochemical (Western blot) detection and sizing of this enzyme in various lactic acid bacteria. The enzyme was confined to about one-half of the strains ofS. lactis examined. N⁵-(CE)ornithine synthase is constitutive, and in strains K1, 6F3, and (plasmid-free)H₁-4125 the native enzyme is a tetramer composed of identical subunits of M_r=38,000. However, in other strains, including 133 (ATCC 11454), C₁₀, and ML₈, the molecular weight of the native enzyme is approximately 130,000 and the corresponding subunit M_r=35,000. Analyses of the amino acid pool components maintained byS. lactis K1 during growth in medium containing [¹⁴C] labeled and unlabeled arginine have revealed that (i) exogenous arginine is the precursor of intracellular ornithine, citrulline, and N⁵-(CE)ornithine, and (ii) the rates of turnover of ornithine and citrulline were considerably faster than that of N⁵-(CE)ornithine. These data account for the biosynthesis and accumulation of N⁵-(CE)ornithine byS. lactis.     

Rational design of peptide-based inhibitors of trypanothione reductase as potential antitrypanosomal drugs

Summary The rational design of ligands for the substrate-binding site of a homology-modelled trypanothione reductase (TR) was performed. Peptides were designed to be selective for TR over human glutathione reductase (GR). The design process capitalized on the proposed differences between the activesites of TR and human GR, subsequently confirmed by the TR crystal structure. Enzyme kinetics confirmed that forT. cruzi TR benzoyl-Leu-Arg-Arg-ß-naphthylamide was an inhibitor (K_i 13.8µM) linearly competitive with the native substrate, trypanothione disulphide, and did not inhibit glutathione reductase.