Precursor For Polyamine Synthesis Research Articles

The Role of Polyamines for Proliferation, Survival, and Infectivity of the Protozoan Parasite Leishmania donovani

A better understanding of parasite biology and host‐parasite interactions is critical for the development of much needed new therapeutic strategies against the neglected tropical disease leishmaniasis, which affects an estimated 12 million people annually. Polyamines are metabolites that play central roles in the biology of all eukaryotes, and recent studies have highlighted their critical nature in Leishmania parasites. In Leishmania, the polyamine biosynthetic pathway consists of four enzymes: arginase (ARG), ornithine decarboxylase (ODC), spermidine synthase (SPD), and S‐adenosylmethionine decarboxylase (ADOMETDC). These enzymes sequentially generate ornithine, putrescine and spermidine. We have generated a complete set of gene deletion mutants in Leishmania donovani (Δarg, Δodc, LdΔspd, and Δadometdc), in order to evaluate the polyamine biosynthetic enzymes as therapeutic targets and to study the functions of polyamines. In vivo studies revealed surprising differences in infectivity phenotypes, with the Δodc deletion exhibiting the most profound reduction in infectivity. In vitro studies showed that the gene deletion mutants depend on the provision of downstream metabolites to survive. An evaluation of cell growth between the different cell lines uncovered that the only vital role of ornithine is as precursor for polyamine synthesis. However, putrescine, which has previously been postulated to be merely a substrate for spermidine formation, has additional essential functions. Our studies revealed that putrescine is especially important for replication and proliferation. Both putrescine and spermidine were essential for cell survival, but the provision of either polyamine alone allowed parasites to enter a quiescent‐like state for several weeks. Future studies utilizing the gene deletion mutants will continue to elucidate the functions of polyamines for cellular proliferation, survival, and persistence.Support or Funding InformationNational Institute of Allergy and Infectious Disease Grant AI041622This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and Evaluation of Ornithine Decarboxylase Inhibitors with Oxime Moiety and MCF-7 Breast Cancer Cells

Cell proliferation can be regulated by small, aliphatic polyamines, and it is suggested that tumor tissues have significantly higher polyamine levels than surrounding tissues. The major biologically active polyamines present in mammalian cells are putrescine, spermidine, and spermine. The Ornithine Decarboxylase (ODC) catalyzes the decarboxylation of ornithine to produce putrescine which is precursor of polyamine synthesis. We report here the synthesis of 2-Amino-5-(Hydroxyimino) Pentanoic Acid (AHPA), based on the substrate of ODC, L-ornithine, derivatized with oxime functionality. In molecular docking studies, the E-isomer AHPA binds to ODC more favorably than does the Z-isomer. In addition, the growth of MCF-7 (Michigan Cancer Foundation–7) breast cancer cells in the presence of AHPA was significantly reduced. These results implicate that AHPA a potentialt can be explored as a potential of cancer chemotherapy.

Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Characterization of Polyamine Homeostasis in l-Ornithine-Induced Acute Pancreatitis in Rats

l-Ornithine is a precursor of polyamine synthesis that is essential for cell survival. In contrast, intraperitoneal (IP) administration of a large dose of l-ornithine results in death of pancreatic acinar cells in rats. We investigated changes in pancreatic and extrapancreatic polyamine homeostasis after injection of l-ornithine and tested the effects of the stable polyamine analogue methylspermidine (MeSpd) on l-ornithine-induced pancreatitis. Male Wistar rats were injected IP with 3 g/kg l-ornithine and were untreated, pretreated, or treated with 50 mg/kg MeSpd IP. Rats were killed after 0 to 168 hours for determinations of polyamines and activities of ornithine decarboxylase and spermidine/spermine N(1)-acetyltransferase (SSAT). Pancreatitis severity was assessed by measuring standard laboratory and histological parameters. Injection of l-ornithine paradoxically induced pancreatic spermidine catabolism, possibly via activation of SSAT, after (>6 hours) appearance of the first histological signs of acute pancreatitis. Polyamine levels generally increased in the lung and liver with the exception of lung spermidine levels, which decreased. Methylspermidine did not influence polyamine levels and SSAT activity and did not ameliorate the severity of l-ornithine-induced pancreatitis. l-Ornithine-induced pancreatitis was associated with activation of pancreatic polyamine catabolism. However, administration of a metabolically stable polyamine analogue did not affect disease severity.

Intestinal metabolism of sulfur amino acids

The gastrointestinal tract (GIT) is a metabolically significant site of sulfur amino acid (SAA) metabolism in the body and metabolises about 20 % of the dietary methionine intake which is mainly transmethylated to homocysteine and trans-sulfurated to cysteine. The GIT accounts for about 25 % of the whole-body transmethylation and trans-sulfuration. In addition, in vivo studies in young pigs indicate that the GIT is a site of net homocysteine release and thus may contribute to the homocysteinaemia. The gut also utilises 25 % of the dietary cysteine intake and the cysteine uptake by the gut represents about 65 % of the splanchnic first-pass uptake. Moreover, we recently showed that SAA deficiency significantly suppresses intestinal mucosal growth and reduces intestinal epithelial cell proliferation, and increases intestinal oxidant stress in piglets. These recent findings indicate that intestinal metabolism of dietary methionine and cysteine is nutritionally important for intestinal mucosal growth. Besides their role in protein synthesis, methionine and cysteine are precursors of important molecules. S-adenosylmethionine, a metabolite of methionine, is the principal biological methyl donor in mammalian cells and a precursor for polyamine synthesis. Cysteine is the rate-limiting amino acid for glutathione synthesis, the major cellular antioxidant in mammals. Further studies are warranted to establish how SAA metabolism regulates gut growth and intestinal function, and contributes to the development of gastrointestinal diseases. The present review discusses the evidence of SAA metabolism in the GIT and its functional and nutritional importance in gut function and diseases.

Enzymatic Transition State Theory and Transition State Analogue Design

Enzymatic Transition State Theory and Transition State Analogue Design

Second Generation Transition State Analogue Inhibitors of Human 5‘-Methylthioadenosine Phosphorylase

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the synthesis of spermidine and spermine is 5'-methylthioadenosine (MTA). In humans MTA is processed by 5'-methylthioadenosine phosphorylase (MTAP) so that significant amounts of MTA do not accumulate. Products of the MTAP reaction (adenine and 5-methylthio-alpha-D-ribose-1-phosphate) are recycled to S-adenosylmethionine, the precursor for polyamine synthesis. Potent inhibitors of MTAP might allow the build-up of sufficient levels of MTA to generate feedback inhibition of polyamine biosynthesis and/or reduce S-adenosylmethionine levels. We recently reported the design and synthesis of a family of potent transition state analogue inhibitors of MTAP. We now report the synthesis of a second generation of stable transition state analogues with increased distance between the ribooxocarbenium ion and purine mimics. These compounds are potent inhibitors with equilibrium dissociation constants as low as 10 pM. The first and second generation inhibitors represent synthetic approaches to mimic early and late features of a dissociative transition state.

L-arginine-dependent suppression of apoptosis in Trypanosoma cruzi: contribution of the nitric oxide and polyamine pathways.

Until recently, a capacity for apoptosis and synthesis of nitric oxide *NO) were viewed as exclusive to multicellular organisms. The existence of these processes in unicellular parasites was recently described, with their biological significance remaining to be elucidated. We have evaluated L-arginine metabolism in Trypanosoma cruzi in the context of human serum-induced apoptotic death. Apoptosis was evidenced by the induction of DNA fragmentation and the inhibition of [3H]thymidine incorporation, which were inhibited by the caspase inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD-CHO). In T. cruzi exposed to death stimuli, supplementation with L-arginine inhibited DNA fragmentation, restored [3H]thymidine incorporation, and augmented parasite *NO production. These effects were inhibited by the *NO synthase inhibitor N(omega)-nitroarginine methyl ester (L-NAME). Exogenous *NO limited DNA fragmentation but did not restore proliferation rates. Because L-arginine is also a substrate for arginine decarboxylase (ADC), and its product agmatine is a precursor for polyamine synthesis, we evaluated the contribution of polyamines to limiting apoptosis. Addition of agmatine, putrescine, and the polyamines spermine and spermidine to T. cruzi sustained parasite proliferation and inhibited DNA fragmentation. Also, the ADC inhibitor difluoromethylarginine inhibited L-arginine-dependent restoration of parasite replication rates, while the protection from DNA fragmentation persisted. In aggregate, these results indicate that T. cruzi epimastigotes can undergo programmed cell death that can be inhibited by L-arginine by means of (i) a *NO synthase-dependent *NO production that suppresses apoptosis and (ii) an ADC-dependent production of polyamines that support parasite proliferation.

Effects of NO synthase inhibitors, arginine-deficient diet, and amiloride in pregnant rats.

This study tests the hypothesis that nitric oxide synthase (NOS) inhibition is linked to NG-nitro-L-arginine methyl ester (L-NAME)-mediated intrauterine growth retardation (IUGR) and fetal limb reduction deficits (LRD) in pregnant dams. Administration of L-NAME (1 mg/ml) or aminoguanidine (AG, 500 micrograms/ml) in the drinking water or intraperitoneal administration of L-N5-(1-iminoethyl)-ornithine (L-NIO, 10 mg.kg-1.day-1) on gestational days 13-20 decreased nitrite and nitrate plus nitrate (RNI) levels in the urine and plasma and decreased RNI in incubates of aorta and fetal limbs compared with pregnant rats given amiloride (50 micrograms/ml) or water (control). Although all drugs caused fetal IUGR, only L-NAME and amiloride caused fetal deaths and LRD. Urine and tissue levels of RNI were unchanged in rats fed and arginine-free diet (AFD) on gestational days 13-20, and yet fetal IUGR, deaths, and LRD were prevalent. L-NAME potentiated the fetal abnormalities and resorptions. Plasma arginine concentrations decreased with AFD > > L-NAME > L-NIO. Plasma ornithine, a precursor for polyamine synthesis, decreased with AFD and increased with L-NAME. Thus inhibition of NOS is not linked to LRD. The ability of L-NAME and amiloride to produce fetal IUGR and LRD may result from L-NAME-mediated modulation of amino acid delivery to the fetus and amiloride-mediated inhibition of protein synthesis. Finally, IUGR appears unrelated to LRD.

Metabolism of agmatine into urea but not into nitric oxide in rat brain.

Agmatine is a guanidino compound abundant in bacteria and plants where it serves as a precursor for polyamine synthesis. It can interfere with several neurotransmission-related functions and can exert neuroprotective effects after brain injury. Agmatine was recently identified in mammalian brain and its synthesis by arginine decarboxylation was characterized. Its metabolism by the brain is, however, unknown. Here we report evidence indicating that agmatine can be selectively metabolized in the rat brain (cerebellum) into urea and thus, may lead to formation of putrescine, the precursor of polyamine synthesis. In addition, while agmatine can inhibit brain nitric oxide synthase, it did not serve as a substrate for nitric oxide formation.

Methylthioadenosine toxicity and metabolism to methionine in mammalian cells.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5'-deoxy-5'-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.

Tumor uptake studies of d,l-[5- 14C]ornithine and d,l-2-difluoromethyl[5- 14C]ornithine

The feasibility of d, l-[5- 14C]ornithine ([ 14C]ornithine), a precursor for polyamine synthesis, and d, l-2-difluoromethyl[5- 14C]ornithine ([ 14C]DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) were investigated for tumor localization. As an animal model, mice bearing mammary carcinoma, FM3A, were used. After i.v. injection of [ 14C]ornithine accumulation of radioactivity was observed in the FM3A, in which 43% of the 14C radioactivity was measured in the polyamine pool and 41% in the amino acid pool at 60 min after injection. Tumor uptake of [ 14C]DFMO was relatively low but constant during 60 min after injection. At 60 min after injection, 11% of the 14C was present in the acid-precipitable fraction of the FM3A, which suggests the formation of an irreversible complex of [ 14C]DFMO with ODC. For both compounds rapid blood clearance and high tumor-to-organ ratios were observed. Our results indicate that in connection with an enhanced polyamine synthesis in the tumors, the compounds investigated have potential as tracers for tumor detection.

Arginine Needs, Physiological State and Usual Diets. A Reevaluation

Evidence is discussed that puts in question the widely held belief that adult mammals, including human beings, can meet all of their arginine needs by endogenous synthesis. Arginine, used in synthesis of body proteins, is essential for ammonia detoxification via urea synthesis, which prevents metabolic derangements caused by elevations in tissue ammonia. It is a precursor for polyamine synthesis and is the only source of amidino groups for the formation of creatine, a major source of high energy phosphate for regeneration of ATP in muscle. Arginine at supraphysiologic doses is thymotropic and a secretogogue for hormones that control growth and metabolism. Studies in mature rats show that glucose tolerance, the rate of repletion from severe protein undernutrition and recovery from trauma are significantly accelerated by dietary arginine. Oral or intravenous administration of excessive arginine reverses nitrogen loss and immune suppression after trauma in rats, and healthy human volunteers consuming 30 g of oral supplements or arginine have shown significantly enhanced immunoreactivity of the lymphocytes of their peripheral blood. Calculations based on creatinine excretion show that 0.8 g of protein/kg body weight of the quality supplied by the usual American diet barely provides sufficient arginine for synthesizing the quantity of creatinine excreted daily in the urine of 70-kg adults. Human patients who often consume less than this amount of protein show a decline in creatinine excretion during illness; the decrease suggests that their intake of arginine is less than optimal. Recent studies of intraspecies and interspecies differences in responses to arginine reemphasize that dispensability or indispensability of arginine is a matter of definition and that growth and nitrogen balance data impose significant limitations on the drawing of far-reaching conclusions about the needs for arginine by mammalian adults including humans. Orotic acid excretion, immune responsiveness and circulating hormone levels are measures that should be evaluated for identifying when enhancement of arginine intakes might prove beneficial.