Arginine Analogues Research Articles

Exogenous Nitric Oxide Confers Tolerance to Cr(VI) in Maize (Zea mays L.) Seedlings by Modulating Endogenous Oxido-Nitrosative Events

The use of exogenous compounds such as ‘gasotransmitter’ molecules is a well-established agronomic strategy to improve the crop tolerance to environmental stresses. In this current work, when Cr (200 µM) was combined with the nitric oxide (NO) generator sodium nitroprusside SNP, 500 µM) there was a suppression of metal-induced alterations in embryo growth. Exogenous NO produced by SNP reduced the accumulation of toxic hydrogen peroxide and methylglyoxal and stress-linked proline in Cr-treated seedlings. Chromium increased thiol and S-nitrosothiol levels but this was restored to control levels by SNP, in spite of a competing NO reaction leading to increase of S-nitrosoglutathione content. However, added complexity was indicated by addition of arginine analogue N (ω)-nitro-L-arginine methyl ester (L-NAME, 500 µM) in the germinating medium to suppress endogenous NO production. This suppressed endogenous NO production but superoxide dismutase (SOD) was suppressed not enhanced in Cr + L-NAME treatments. In addition, Cr + L-NAME significantly decreased the content of spermidine and spermine in epicotyls as compared to Cr treatment alone. Similarly, exposure to Cr + SNP decreased spermidine and spermine levels in both radicles and epicotyls. This is important as polyamines have been suggested as route for NO production. Thus, our observations suggest that exogenous NO mitigates Cr-induced damage and confers seedling tolerance to Cr through suppression of NADPH oxidase activity and increased GSNO contents. This may act to prevent an excess of methylglyoxal and hydrogen peroxide. However, the reduction in polyamine mediated cellular NO generation could also promote increased viability under Cr stress.

Partial Purification and Biochemical Characterization of Horse Gram Arginase

Plant arginase that catalyses the hydrolysis of arginine to ornithine and urea is known to play an important role in nitrogen metabolism. Recently, we reported a highly stable arginase from cilantro and its sensitivity to biotic and abiotic stress. During this investigation, we found horse gram also possessing a stable arginase among legumes. Hence, we partially purified arginase from horse gram seedlings by conventional chromatographic techniques with 869-fold purity, and a specific activity of 13752 nmoles of urea formed/mg of protein/min. The enzyme is relatively heat stable and requires Mn2+ for its activity and is sensitive to reducing agents and EDTA similar to cilantro arginase. The optimum pH and temperature for partially purified horse gram arginase was found to be 7.87 and 37° C - 60° C respectively. Arginine-derived polyamines and amino acids can regulate horse gram arginase in vitro. Partially purified arginase hydrolyses L-arginine and is incapable of hydrolysing other arginine analogues except L-homoarginine, a property that distinguishes horse gram arginase from cilantro arginase. The Km for partially purified arginase was found to be 5.47 ± 0.34 mM with respect to L-arginine. As plant arginases are not stable and their subunit organization differs from source to source, for further purification and biochemical characterization horse gram serves as an ideal and easily available source.

Interaction strength of osmolytes with the anion of a salt-bridge determines its stability.

In order to understand the role of osmolytes in regulating physicochemical behavior of proteins, we investigated the influence of protein destabilizing (urea and guanidinium chloride) and stabilizing osmolytes (TMAO, glycerol, and betaine) on a model salt-bridge (SB) formed between structural analogues of arginine and glutamate/aspartate sidechains in a solvent continuum using first-principles quantum chemical calculations based on DFT and MP2 methods. The binding strength of the osmolyte with the SB is found to be in the order of betaine > TMAO > Gdm+ > glycerol > urea. The osmolytes (TMAO and betaine) that preferentially bind to the SB cation have a marginal influence on SB stability. Also, pure π-π stacking interaction between Gdm+ and the SB cation plays an insignificant role in destabilizing the SB. In fact, the interaction strength of osmolytes with the SB anion mainly determines the stability of SB. For instance, a competition between Gdm+ and the SB cation to bind with the SB anion is responsible for instability and subsequent dissociation of the SB. The competition provided by other osmolytes is too weak to break the SB. Exploiting this information, we designed three structural derivatives of Gdm+, all having a stronger interaction with SB anion, and thereby show a stronger SB dissociation potential. Furthermore, we find an excellent linear anti-correlation between SB interaction energy and the energy of interaction between osmolyte and the SB anion, which suggests that by knowing only the strength of osmolyteacetate interaction, one can predict the influence of osmolytes on the salt-bridge instability. This information is useful in fine-tuning the SB dissociation power of Gdm+, which has a practical significance in obtaining the mechanistic insight into the influence of GdmCl on protein stability. Our results also provide a basis for understanding the chemistry of other ion-pairs formed between a cationic hydrogen donor and an anionic acceptor.

Dual role of ER stress in response to metabolic co-targeting and radiosensitivity in head and neck cancer cells

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.

Combinatory Treatment of Canavanine and Arginine Deprivation Efficiently Targets Human Glioblastoma Cells via Pleiotropic Mechanisms.

Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We directly observed canavanine incorporation into nascent proteins by using quantitative proteomics. Although canavanine in the absence of arginine readily incorporated into polypeptides, no motif preference for such incorporation was observed. Our findings provide a strong rationale for further developing the proposed modality based on canavanine and arginine deprivation as a potential antiglioblastoma metabolic therapy independent of the blood–brain barrier.

Differentiating COVID-19 Pneumonia From Acute Respiratory Distress Syndrome and High Altitude Pulmonary Edema: Therapeutic Implications.

Differentiating COVID-19 Pneumonia From Acute Respiratory Distress Syndrome and High Altitude Pulmonary Edema: Therapeutic Implications.

Indospicine combined with arginine deprivation triggers cancer cell death via caspase‐dependent apoptosis

Arginine-deprivation therapy is a rapidly developing metabolic anticancer approach. To overcome the resistance of some cancer cells to this monotherapy, rationally designed combination modalities are needed. In this report, we evaluated for the first time indospicine, an arginine analogue of Indigofera plant genus origin, as potential enhancer compound for the metabolic therapy that utilizes recombinant human arginase I. We demonstrate that indospicine at low micromolar concentrations is selectively toxic for human colorectal cancer cells only in the absence of arginine. In arginine-deprived cancer cells indospicine deregulates some prosurvival pathways (PI3K-Akt and MAPK) and activates mammalian target of rapamycin, exacerbates endoplasmic reticulum stress and triggers caspase-dependent apoptosis, which is reversed by the exposure to translation inhibitors. Simultaneously, indospicine is not degraded by recombinant human arginase I and does not inhibit this arginine-degrading enzyme at its effective dose. The obtained results emphasize the potential of arginine structural analogues as efficient components for combinatorial metabolic targeting of malignant cells.

Solid-Phase Synthesis of Substrate-Based Dipeptides and Heterocyclic Pseudo-dipeptides as Potential NO Synthase Inhibitors.

More than 160 arginine analogues modified on the C-terminus via either an amide bond or a heterocyclic moiety (1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,4-triazole) were prepared as potential inhibitors of NO synthases (NOS). A methodology involving formation of a thiocitrulline intermediate linked through its side-chain on a solid support followed by modification of its carboxylate group was developed. Finally, the side-chain thiourea group was either let unchanged, S-alkylated (Me, Et) or guanidinylated (Me, Et) to yield respectively after TFA treatment the corresponding thiocitrulline, S-Me/Et-isothiocitrulline and N-Me/Et-arginine substrate analogues. They all were tested against three recombinant NOS isoforms. Several compounds containing a S-Et- or a S-Me-Itc moiety and mainly belonging to both the dipeptide-like and 1,2,4-oxadiazole series were shown to inhibit nNOS and iNOS with IC50 in the 1-50 μM range. Spectral studies confirmed that these new compounds interacted at the heme active site. The more active compounds were found to inhibit intra-cellular iNOS expressed in RAW264.7 and INS-1 cells with similar efficiency than the reference compounds L-NIL and SEIT.

Effects of Arginine and Its Deprivation on Human Glioblastoma Physiology and Signaling.

The observations that numerous cancers are characterized by impairment in arginine synthesis and that deficit of exogenous arginine specifically affects their growth and viability are the ground for arginine deprivation-based anticancer treatment strategy. This review addresses molecular mechanisms of the human glioblastoma cell response to arginine deprivation. Our earlier studies have shown that arginine deprivation specifically impairs glioblastoma cell motility, adhesion and invasiveness. These changes were evoked by alterations in the actin cytoskeleton organization resulting from a decreased arginylation of β-actin isoform. Moreover, deficit of arginine induces prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response, not leading, however, to a massive apoptosis in glioblastoma cells. Our current research indicates that cell death could be augmented by other compounds such as modulators of ER stress, for example arginine analogue of plant origin, canavanine. Implication of these studies on the development of new anti-glioma metabolic therapeutic modalities are discussed.

290 Arginine Analogues as Biomarkers of Myocardial Ischaemia, Assessed with Cardiac Magnetic Resonance in Chronic Kidney Disease

290 Arginine Analogues as Biomarkers of Myocardial Ischaemia, Assessed with Cardiac Magnetic Resonance in Chronic Kidney Disease

Adaptation by Loss of Heterozygosity in Saccharomyces cerevisiae Clones Under Divergent Selection.

Loss of heterozygosity (LOH) is observed during vegetative growth and reproduction of diploid genotypes through mitotic crossovers, aneuploidy caused by nondisjunction, and gene conversion. We aimed to test the role that LOH plays during adaptation of two highly heterozygous Saccharomyces cerevisiae genotypes to multiple environments over a short time span in the laboratory. We hypothesized that adaptation would be observed through parallel LOH events across replicate populations. Using genome resequencing of 70 clones, we found that LOH was widespread with 5.2 LOH events per clone after ∼500 generations. The most common mode of LOH was gene conversion (51%) followed by crossing over consistent with either break-induced replication or double Holliday junction resolution. There was no evidence that LOH involved nondisjunction of whole chromosomes. We observed parallel LOH in both an environment-specific and environment-independent manner. LOH largely involved recombining existing variation between the parental genotypes, but also was observed after de novo, presumably beneficial, mutations occurred in the presence of canavanine, a toxic analog of arginine. One highly parallel LOH event involved the ENA salt efflux pump locus on chromosome IV, which showed repeated LOH to the allele from the European parent, an allele originally derived by introgression from S. paradoxus Using CRISPR-engineered LOH we showed that the fitness advantage provided by this single LOH event was 27%. Overall, we found extensive evidence that LOH could be adaptive and is likely to be a greater source of initial variation than de novo mutation for rapid evolution of diploid genotypes.

Synthesis and biological evaluation of [18F](2S,4S)4-(3-fluoropropyl) arginine as a tumor imaging agent

Designing novel 18F-labeled amino acid derivatives for targeted amino acid transporters is an attractive strategy for the development of therapeutic and diagnostic agents for cancer therapy. In this work, we have developed a novel 3-fluoropropyl analog of arginine, namely, (2S,4S)4-[18F]FPArg, [18F]1, to be used as a probe for studying arginine metabolism. Optically pure and labeled with 18F and 19F, (2S,4S)4-(3-fluoropropyl)arginine was synthesized and isolated in high radiochemical purity (>95%). Invitro uptake assays in human MCF-7 cells revealed that [18F]1 enters cells mainly via sodium-independent cationic amino acid transporters and was inhibited >62% by arginine. [18F]1 showed a high cellular uptake of 7.3 ± 0.24% and 6.07 ± 0.3% uptake/100 mg protein after incubation in MCF-7 and MDA-MB-231 cells for 120 min, respectively. Invivo biodistribution studies demonstrated that [18F]1 provided high tumor uptake and high tumor to muscle ratios (5:1 at the 30 and 60 min time points). Invivo PET imaging studies demonstrated tumor-specific uptake in nude mice bearing MCF-7 breast tumors with an excellent tumor-to-muscle ratio. These results suggest that [18F]1 is a promising tracer for clinical breast cancer imaging and may be used to diagnose and monitor diseases that are associated with arginine metabolism.

Canavanine-Induced Decrease in Nitric Oxide Synthesis Alters Activity of Antioxidant System but Does Not Impact S-Nitrosoglutathione Catabolism in Tomato Roots.

Canavanine (CAN) is a nonproteinogenic amino acid synthesized in legumes. In mammalians, as arginine analogue, it is an inhibitor of nitric oxide synthase (NOS) activity. The aim of this study was to investigate the impact of CAN-induced nitric oxide level limitation on the antioxidant system and S-nitrosoglutathione (GSNO) metabolism in roots of tomato seedlings. Treatment with CAN (10 or 50 µM) for 24–72 h led to restriction in root growth. Arginine-dependent NOS-like activity was almost completely inhibited, demonstrating direct effect of CAN action. CAN increased total antioxidant capacity and the level of sulphydryl groups. Catalase (CAT) and superoxide dismutase (SOD) activity decreased in CAN exposed roots. CAN supplementation resulted in the decrease of transcript levels of genes coding CAT (with the exception of CAT1). Genes coding SOD (except MnSOD and CuSOD) were upregulated by CAN short treatment; prolonged exposition to 50-µM CAN resulted in downregulation of FeSOD, CuSOD, and SODP-2. Activity of glutathione reductase dropped down after short-term (10-µM CAN) supplementation, while glutathione peroxidase activity was not affected. Transcript levels of glutathione reductase genes declined in response to CAN. Genes coding glutathione peroxidase were upregulated by 50-µM CAN, while 10-µM CAN downregulated GSHPx1. Inhibition of NOS-like activity by CAN resulted in lower GSNO accumulation in root tips. Activity of GSNO reductase was decreased by short-term supplementation with CAN. In contrast, GSNO reductase protein abundance was higher, while transcript levels were slightly altered in roots exposed to CAN. This is the first report on identification of differentially nitrated proteins in response to supplementation with nonproteinogenic amino acid. Among nitrated proteins differentially modified by CAN, seed storage proteins (after short-term CAN treatment) and components of the cellular redox system (after prolonged CAN supplementation) were identified. The findings demonstrate that due to inhibition of NOS-like activity, CAN leads to modification in antioxidant system. Limitation in GSNO level is due to lower nitric oxide formation, while GSNO catabolism is less affected. We demonstrated that monodehydroascorbate reductase, activity of which is inhibited in roots of CAN-treated plants, is the protein preferentially modified by tyrosine nitration.

Nitric oxide synthases (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [11] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.

New horizons in arginine metabolism, ageing and chronic disease states.

The elucidation of the metabolic pathways of the amino acid arginine and their role in health and disease have been an intensive focus of basic and clinical research for over a century. The recent advent of robust analytical techniques for biomarker assessment in large population cohorts has allowed the investigation of the pathophysiological role of specific arginine metabolites in key chronic disease states in old age, particularly those characterised by a reduced synthesis of endothelial nitric oxide, with consequent vascular disease and atherosclerosis. Two arginine metabolites have been increasingly studied in regard to their potential role in risk stratification and in the identification of novel therapeutic targets: the methylated arginine asymmetric dimethylarginine (ADMA) and the arginine analogue homoarginine. Higher circulating concentrations of ADMA, a potent inhibitor of nitric oxide synthesis, have been shown to predict adverse cardiovascular outcomes. By contrast, there is emerging evidence that homoarginine might exert cardioprotective effects. This review highlights recent advances in the biological and clinical role of ADMA and homoarginine in cardiovascular disease and other emerging fields, particularly chronic obstructive pulmonary disease, dementia, and depression. It also discusses opportunities for future research directions with the ultimate goal of translating knowledge of arginine metabolism, and its role in health and disease, into the clinical care of older adults.

The Goldilocks effect of respiration on canavanine tolerance in Saccharomyces cerevisiae.

When glucose is available, Saccharomyces cerevisiae prefers fermentation to respiration. In fact, it can live without respiration at all. Here, we study the role of respiration in stress tolerance in yeast. We found that colony growth of respiratory-deficient yeast (petite) is greatly inhibited by canavanine, the toxic analog of arginine that causes proteotoxic stress. We found lower amounts of the amino acids involved in arginine biosynthesis in petites compared with WT. This finding may be explained by the fact that petite cells exposed to canavanine show reduction in the efficiency of targeting of proteins required for arginine biosynthesis. The retrograde (RTG) pathway signals mitochondrial stress. It positively controls production of arginine precursors. We show that canavanine abrogates RTG signaling especially in petite cells, and mutants in the RTG pathway are extremely sensitive to canavanine. We suggest that petite cells are naturally ineffective in production of some amino acids; combination of this fact with the effect of canavanine on the RTG pathway is the simplest explanation why petite cells are inhibited by canavanine. Surprisingly, we found that canavanine greatly inhibits colony formation when WT cells are forced to respire. Our research proposes a novel connection between respiration and proteotoxic stress.

Levels of chemerin and asymmetric dimethylarginine (ADMA) in obese type 2 diabetic patients.

Diabetes Mellitus (D.M) has reached epidemic proportion and has become one of the most challenging health problems of the 21st century. Moreover, it is the 4th leading cause of death by disease globally, where every 10 seconds a person dies from diabetes-related causes. Obesity, insulin resistance, dyslipidemia and hypertension are associated with (T2DM) termed together as a metabolic syndrome. Mobilization of free fatty acids and secretion of certain inflammatory adipokines from adipose tissue promote insulin resistance in obese diabetics by interfere with insulin sensitivity, glucose and lipid metabolism. Chemerin, a specific adipokine, has an essential role in glucose and lipid metabolism. It plays a role in pathogenesis of obesity and T2DM also has important roles in energy metabolism, adipogenesis, and inflammation. Asymmetric dimethyl arginine (ADMA) consider a natural analogue of the essential amino acid arginine and a metabolic by-product obtained from processes of protein turnover of all human cells cytoplasm.

Catecholamine Vasopressor Support Sparing Strategies in Vasodilatory Shock.

Shock syndromes are associated with unacceptably high rates of mortality in critically ill patients despite advances in therapeutic options. Vasodilatory shock is the most common type encountered in the intensive care unit. It is manifested by cardiovascular failure, peripheral vasodilatation, and arterial hypotension leading to tissue hypoperfusion and organ failure. Hemodynamic support is typically initiated with fluid resuscitation strategies and administration of adrenergic vasopressor agents in nonresponsive patients to restore arterial pressure with subsequent adequate organ reperfusion. Unfortunately, high catecholamine dosing requirements may be necessary to achieve targeted hemodynamic goals that may increase the risk of vasopressor-induced adverse events. The purpose of this article is to review the clinical efficacy and safety data and potential role in therapy for catecholamine-sparing agents in vasodilatory shock. Adjunctive therapeutic options to reduce vasoactive support requirements without compromising arterial pressure include arginine vasopressin and analogs, corticosteroids, midodrine, methylene blue, and angiotensin II. Although concomitant vasopressin and corticosteroids have a more defined role in evidence-based guidelines for managing shock, clinicians may consider other potential catecholamine-sparing agents.

Seeking mTORC1 Inhibitors Through Molecular Dynamics Simulation of Arginine Analogs Inhibiting CASTOR1.

Hyperactivity of the mechanistic target of rapamycin complex 1 (mTORC1) is implicated in a variety of diseases such as cancer and diabetes. Treatment may benefit from effective mTORC1 inhibition, which can be achieved by preventing arginine from disrupting the cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1)-GTPase-activating proteins toward RAGS subcomplex 2 (GATOR2) complex through binding with CASTOR1. An attractive idea is to determine analogues of arginine that are as competent as arginine in binding with CASTOR1, but without disrupting the CASTOR1-GATOR2 interaction. Molecular dynamics simulations were performed for binding of arginine analogues with CASTOR1 and binding free energy, hydrogen bond formation, and root mean squared deviation and root mean square fluctuation kinetics were then calculated. The binding free energy calculations revealed that Nα-acetyl-arginine, citrulline, and norarginine have sufficient binding affinity with CASTOR1 to compete with arginine. The hydrogen bond analysis revealed that norarginine, Nα-acetyl-arginine and D-arginine have proficient H-bonds that can facilitate their entering the narrow binding pocket. Norarginine and Nα-acetyl-arginine are the top drug candidates for mTORC1 inhibition, with Nα-acetyl-arginine being the best choice.

Pharmacological activation of dimethylarginine dimethylaminohydrolase (DDAH) activity by inorganic nitrate and DDAH inhibition by NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline: results and overview.

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of nitric oxide (NO) synthase. SDMA is excreted in the urine without major metabolization. About 10% of daily produced ADMA are excreted unchanged in the urine. The major elimination route of ADMA (about 90%) involves its hydrolysis to dimethylamine (DMA) and L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and excretion of DMA in the urine. High circulating and low excretory concentrations of ADMA are considered risk factors. Experimentally, DDAH activity can be inhibited by SH-specific agents such as inorganic and organic mercury compounds, and by S-nitrosothiols which block the SH group of a particular cysteine moiety of DDAH that is essential for its hydrolytic activity. Alternatively, DDAH activity can be inhibited by organic compounds that compete with the substrate ADMA for DDAH. Arginine analogs that contain substituents on guanidine nitrogen atom(s) (NG) represent a class of DDAH inhibitors. In the present study, we investigated the effects of physiological and natural amino acid derivatives of L-arginine and L-citrulline as well as of nitrate and nitrite, the major circulating and excretory metabolites of NO and NO donating drugs. Here, we report for the first time that the physiological NG-hydroxy-L-arginine, an isolable intermediate in NO synthesis, inhibits recombinant DDAH-1 activity (IC50 ≈ 100µM). Two plant L-citrulline derivatives, i.e., Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline (connatin), were found to inhibit almost completely hepatic DDAH activity in vitro in rat homogenate at a concentration of 100µM each. At pharmacological concentrations (i.e., 1mM), inorganic nitrate, but not inorganic nitrite, was found to increase rat liver DDAH activity. In urine of 18 patients with Becker's muscular dystrophy, nitrate was found to correlate closely with DMA (Spearman, r = 0.73, p = 0.002). In summary, NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline are novel inhibitors of DDAH activity. This article provides an overview of amino acid-based DDAH inhibitors and discusses potential underlying inhibition mechanisms.