Small-molecule arginase inhibitors (original) (raw)
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Inhibition of human arginase I by substrate and product analogues
Archives of Biochemistry and Biophysics, 2010
Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K d = 3.6 μM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K d = 517 nM (surface plasmon resonance) or K d ≈ 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 Å and 1.55 Å resolution structures, respectively, which are significantly improved in comparison with previously determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K d = 13 μM) is reported at 1.90 Å resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor α-carboxylate and α-amino groups as key specificity determinants of amino acid recognition in the arginase active site. Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of Larginine to generate L-ornithine and urea. This reaction is the key step of the urea cycle in the liver that allows for the excretion of nitrogenous waste resulting from protein catabolism; healthy adults excrete approximately 10 kg urea per year [1-4]. In extrahepatic tissues arginase serves to regulate L-arginine concentrations for other metabolic pathways. For example, arginase activity can decrease L-arginine concentrations utilized by nitric oxide synthase to generate NO; arginase inhibitors can increase L-arginine concentrations and thereby enhance NO biosynthesis and NO-dependent physiological processes such as smooth muscle relaxation [5]. Accordingly, arginase is a pharmaceutical target for the treatment of diseases associated with aberrant smooth muscle physiology such as erectile dysfunction [6,7] and asthma [8,9]. Among substrate analogue inhibitors of arginase, boronic acid and N-hydroxyguanidinium derivatives exhibit the highest affinity (selected inhibitors are shown in Table 1) [7,10-19]. The crystal structures of rat arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) [6], dehydro-ABH [17], and S-(2-boronoethyl)-L-cysteine (BEC) [7], human arginase I complexed with ABH and BEC [18], and human arginase II complexed with BEC [20] reveal that the planar boronic acid moiety of the inhibitor undergoes nucleophilic attack by the metalbridging hydroxide ion to yield a tetrahedral boronate anion that mimics the tetrahedral
The arginases catalyze the divalent cation dependent hydrolysis of L-arginine to produce L-ornithine and urea. Although traditionally considered in terms of its role as the final enzyme of the urea cycle, the enzyme is found in a variety of nonhepatic tissues. These findings suggest that the enzyme may have other functions in addition to its role in nitrogen metabolism. High-resolution crystal structures have been determined for recombinant rat liver (type I) arginase and for recombinant human kidney (type II) arginase, their variants, and complexes with products and inhibitors. Each identical subunit of the trimeric enzyme contains an active site that lies at the bottom of a 15 Å deep cleft. The 2 essential Mn(II) ions are located at the bottom of this cleft, separated by ϳ3.3 Å and bridged by oxygens derived from 2 aspartic acid residues and a solvent-derived hydroxide. This metal bridging hydroxide is proposed to be the nucleophile that attacks the guanidinium carbon of substrate arginine. On the basis of this proposed mechanism, boronic acid inhibitors of the enzyme have been synthesized and characterized kinetically and structurally. These inhibitors display slow-onset inhibition at the pH optimum of the enzyme, and are found as tetrahedral species at the active site, as determined by X-ray diffraction. The potent inhibition of arginases I and II by these compounds has not only delineated key enzyme-substrate interactions, but has also led to a greater understanding of the role of arginase in nonhepatic tissues. J.
Competitive metabolism of L -arginine: arginase as a therapeutic target in asthma
Journal of Biomedical Research, 2011
Exhaled breath nitric oxide (NO) is an accepted asthma biomarker. Lung concentrations of NO and its amino acid precursor, L-arginine, are regulated by the relative expressions of the NO synthase (NOS) and arginase isoforms. Increased expression of arginase I and NOS2 occurs in murine models of allergic asthma and in biopsies of asthmatic airways. Although clinical trials involving the inhibition of NO-producing enzymes have shown mixed results, small molecule arginase inhibitors have shown potential as a therapeutic intervention in animal and cell culture models. Their transition to clinical trials is hampered by concerns regarding their safety and potential toxicity. In this review, we discuss the paradigm of arginase and NOS competition for their substrate L-arginine in the asthmatic airway. We address the functional role of L-arginine in inflammation and the potential role of arginase inhibitors as therapeutics.
Pegylated arginase I: a potential therapeutic approach in TALL
Blood, 2010
Adult patients with acute lymphoblastic T cell leukemia (T-ALL) have a very poor prognosis and few effective therapeutic options. Therefore, novel therapies that increase the efficacy of the treatments and that prolong T-ALL patient survival are needed. Malignant T cells require high concentrations of nutrients to sustain their increased rate of proliferation. In this study, we determined whether L-Arginine depletion by the pegylated form of the L-Arginine-metabolizing en-zyme arginase I (peg-Arg I) impairs the proliferation of malignant T cells. Our results show that peg-Arg I depleted L-Arginine levels in vitro and in vivo. In addition, treatment of malignant T-cell lines with peg-Arg I significantly impaired their proliferation, which correlated with a decreased progression into the cell cycle, followed by the induction of apoptosis. Furthermore, peg-Arg I impaired the expression of cyclin D3, a fundamental protein in T-ALL proliferation, through a global arrest in protein synthesis. Injection of peg-Arg I plus chemotherapy agent Cytarabine prolonged survival in mice bearing T-ALL tumors. This antitumoral effect correlated with an inhibition of T-ALL proliferation in vivo, a decreased expression of cyclin D3, and T-ALL apoptosis. The results suggest the potential benefit of L-Arginine depletion by peg-Arg I in the treatment of T-cell malignancies. (Blood. 2010;115(25):5214-5221)
Arginase pathway in human endothelial cells in pathophysiological conditions
Journal of Molecular and Cellular Cardiology, 2004
Objective. -Arginase is a nitric oxide synthase-alternative pathway for L-arginine breakdown leading to biosynthesis of urea and L-ornithine. Arginase pathway is inducible by inflammatory molecules-such as cytokines and bacterial endotoxin-in macrophages and smooth muscle cells. The presence of an arginase pathway in human endothelial cells and its possible modulation by inflammation are unknown.
SLAS Discovery, 2018
Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-...
Human Recombinant Arginase Enzyme Reduces Plasma Arginine in Mouse Models of Arginase Deficiency
Human molecular genetics, 2015
Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures, and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arg...
PLOS Pathogens, 2010
Arginine is a crucial amino acid that serves to modulate the cellular immune response during infection. Arginine is also a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The generation of nitric oxide from arginine is responsible for efficient immune response and cytotoxicity of host cells to kill the invading pathogens. On the other hand, the conversion of arginine to ornithine and urea via the arginase pathway can support the growth of bacterial and parasitic pathogens. The competition between iNOS and arginase for arginine can thus contribute to the outcome of several parasitic and bacterial infections. There are two isoforms of vertebrate arginase, both of which catalyze the conversion of arginine to ornithine and urea, but they differ with regard to tissue distribution and subcellular localization. In the case of infection with Mycobacterium, Leishmania, Trypanosoma, Helicobacter, Schistosoma, and Salmonella spp., arginase isoforms have been shown to modulate the pathology of infection by various means. Despite the existence of a considerable body of evidence about mammalian arginine metabolism and its role in immunology, the critical choice to divert the host arginine pool by pathogenic organisms as a survival strategy is still a mystery in infection biology.
Expression, purification, assay, and crystal structure of perdeuterated human arginase I
Archives of Biochemistry and Biophysics, 2007
Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to yield Lornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2-amino-6-boronohexanoic acid (ABH) at 1.90 Å resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeutaration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study. Keywords protein crystallography; enzyme-inhibitor complex; isotopic labeling Arginase catalyzes the hydrolysis of L-arginine to yield L-ornithine and urea [1]. In humans there are two isozymes, arginase I and arginase II, that share ~60% sequence identity. Ornithine is the biosynthetic precursor of polyamines that facilitate cellular proliferation and tumor growth [2]. Additionally, arginase I is implicated in tumoral immune evasion, and arginase inhibitors therefore have significant chemotherapeutic potential [2].
Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design
2011
Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of R, R-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional R-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.