Computational therapeutic repurposing of tavaborole targeting arginase-1 for venous leg ulcer.

Venous leg ulcers (VLUs) pose a growing healthcare challenge due to aging, obesity, and sedentary lifestyles. Despite various treatments available, addressing the complex nature of VLUs remains difficult. In this context, this study investigates repurposing boronated drugs to inhibit arginase 1 activity for VLU treatment. The molecular docking study conducted by Schrodinger GLIDE targeted the binuclear manganese cluster of arginase 1 enzyme (2PHO). Further, the ligand-protein complex was subjected to molecular dynamic studies at 500 ns in Gromacs-2019.4. Trajectory analysis was performed using the GROMACS simulation package of protein RMSD, RMSF, RG, SASA, and H-Bond. The docking study revealed intriguing results where the tavaborole showed a better docking score (-3.957 Kcal/mol) compared to the substrate L-arginine (-3.379 Kcal/mol) and standard L-norvaline (-3.141 Kcal/mol). Tavaborole interaction with aspartic acid ultimately suggests that the drug molecule binds to the catalytic site of arginase 1, potentially influencing the enzyme's function. The dynamics study revealed the compounds' stability and compactness of the protein throughout the simulation. The RMSD, RMSF, SASA, RG, inter and intra H-bond, PCA, FEL, and MMBSA studies affirmed the ligand-protein and protein complex flexibility, compactness, binding energy, van der waals energy, and solvation dynamics. These results revealed the stability and the interaction of the ligand with the catalytic site of arginase 1 enzyme, triggering the study towards the VLU treatment.

Py-CoMFA, docking, and molecular dynamics simulations of Leishmania (L.) amazonensis arginase inhibitors.

Leishmaniasis is a disease caused by a protozoan of the genus Leishmania, affecting millions of people, mainly in tropical countries, due to poor social conditions and low economic development. First-line chemotherapeutic agents involve highly toxic pentavalent antimonials, while treatment failure is mainly due to the emergence of drug-resistant strains. Leishmania arginase (ARG) enzyme is vital in pathogenicity and contributes to a higher infection rate, thus representing a potential drug target. This study helps in designing ARG inhibitors for the treatment of leishmaniasis. Py-CoMFA (3D-QSAR) models were constructed using 34 inhibitors from different chemical classes against ARG from L. (L.) amazonensis (LaARG). The 3D-QSAR predictions showed an excellent correlation between experimental and calculated pIC50 values. The molecular docking study identified the favorable hydrophobicity contribution of phenyl and cyclohexyl groups as substituents in the enzyme allosteric site. Molecular dynamics simulations of selected protein-ligand complexes were conducted to understand derivatives' interaction modes and affinity in both active and allosteric sites. Two cinnamide compounds, 7g and 7k, were identified, with similar structures to the reference 4h allosteric site inhibitor. These compounds can guide the development of more effective arginase inhibitors as potential antileishmanial drugs.

Novel orally bioavailable piperidine derivatives as extracellular arginase inhibitors developed by a ring expansion.

Arginase is a multifaced enzyme that plays an important role in health and disease being regarded as a therapeutic target for the treatment of various pathological states such as malignancies, asthma, and cardiovascular disease. The discovery of boronic acid-based arginase inhibitors in 1997 revolutionized attempts of medicinal chemistry focused on development of drugs targeting arginase. Unfortunately, these very polar compounds had limitations such as analysis and purification without chromophores, synthetically challenging space, and poor oral bioavailability. Herein, we present a novel class of boronic acid-based arginase inhibitors which are piperidine derivatives exhibiting a different pharmacological profile compared to our drug candidate in cancer immunotherapy -OATD-02 - dual ARG1/2 inhibitor with high intracellular activity. Compounds from this new series show low intracellular activity, hence they can inhibit mainly extracellular arginase, providing different therapeutic space compared to a dual intracellular ARG1/2 inhibitor. The disclosed series showed good inhibitory potential towards arginase enzyme in vitro (IC50 up to 160 nM), favorable pharmacokinetics in animal models, and encouraging preliminary in vitro and in vivo tolerability. Compounds from the new series have moderate-to-high oral bioavailability (up to 66 %) and moderate clearance in vivo. Herein we describe the development and optimization of the synthesis of the new class of boronic acid-based arginase inhibitors via a ring expansion approach starting from the inexpensive chirality source (d-hydroxyproline). This upgraded methodology facilitated a gram-scale delivery of the final compound and eliminated the need for costly and time-consuming chiral resolution.

Effect of the Macromolecular Crowding Agents on the Structure and Function of Human Arginase-I, a Therapeutically Important Enzyme.

Macromolecular crowding has been known to influence the structure and function of many enzymes through excluded volume effects and/or soft interactions. Here, we employed two synthetic macromolecular crowders, Dextrans and poly(ethylene glycol)s (PEGs) with varying molecular masses, to examine how they affected the structure and function of a therapeutically important enzyme, human arginase-I that catalyzes the conversion of l-arginine to l-ornithine and urea. Except at greater concentrations of Dextran 200, Dextrans were observed to slightly reduce the enzymatic activity, indicating that they exert their influence mainly through the excluded volume effects. Similar outcomes were seen with PEGs, with the exception of PEG 1000, where the activity decreased with increasing PEG concentrations, showing the maximum effect at a 20 g/L concentration. This finding suggests that the enzyme function is reduced by the soft interactions of this macromolecule with the enzyme, supported by the binding measurement. Secondary and local tertiary structures and thermodynamic stability were also affected, suggesting that PEG 1000 has an impact on the protein's structure. Furthermore, molecular dynamics simulation studies suggest that the catalytic pocket is disturbed, presumably by the unwinding of neighboring helix 9. As a result, the positioning of nearby Glu277 is altered, which prevents His141 and Glu277 from making contact. This hampers the proton transfer from the catalytic His141 to the intermediate species to form ornithine, a crucial step for the substrate hydrolysis reaction by this arginase. Overall, the knowledge gained from this study might be helpful for understanding how different enzymes work in a crowded/cellular environment.

Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1.

We report an enzyme cascade with horseradish peroxidase-based readout for screening human arginase-1 (hArg1) activity. We combined the four enzymes hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase in a reaction cascade that generated colorimetric or fluorescent signals in response to hArg1 activity and used this cascade to assay wild-type and variant hArg1 sequences as soluble enzymes and displayed on the surface of Escherichia coli. We screened a curated 13-member hArg1 library covering mutations that modified the electrostatic environment surrounding catalytic residues D128 and H141, and identified the R21E variant with a 13% enhanced catalytic turnover rate compared to wild type. Our scalable one-pot single-step arginase assay with continuous kinetic readout is amenable to high-throughput screening and directed evolution of arginase libraries and testing drug candidates for arginase inhibition.

Illuminating the structure-function landscape of an evolutionary nonconserved motif in the arginases of Helicobacter gastric pathogens.

The bimetallic enzyme arginase catalyses the conversion of L-arginine to L-ornithine and urea. In Helicobacter pylori (a known human gastric pathogen), this enzyme is an important virulence factor. In spite of the conservation of the catalytic and the metal-binding residues, the H. pylori homolog possesses a 13-residue motif (-153 ESEEKAWQKLCSL165 -) present in the middle of the protein sequence, whose role was recently elucidated. Despite several reviews available on arginases, no report has thoroughly illustrated the underlying basis for the importance of the above motif of the H. pylori enzyme in structure and function. In this review, we systematically describe a mechanistic basis for its importance in structure and function based on the known data. This motif of the H. pylori enzyme is present exclusively in the arginases of other Helicobacter gastric pathogens, where the critical residues are conserved, implying that the nonconserved stretch has been selected during the evolution of the enzyme in these gastric pathogens in a specific manner to perform its role in the structure and function. The combined information can be useful for understanding the function of arginases in other Helicobacter gastric pathogens. Additionally, this knowledge can be utilised to screen and design new small molecule inhibitors, specific to the arginases of these pathogens.

Discovery of non-boronic acid Arginase 1 inhibitors through virtual screening and biophysical methods.

Inhibiting Arginase 1 (ARG1), a metalloenzyme that hydrolyzes l-arginine in the urea cycle, has been demonstrated as a promising therapeutic avenue in immuno-oncology through the restoration of suppressed immune response in several types of cancers. Most of the currently reported small molecule inhibitors are boronic acid based. Herein, we report the discovery of non-boronic acid ARG1 inhibitors through virtual screening. Biophysical and biochemical methods were used to experimentally profile the hits while X-ray crystallography confirmed a class of trisubstituted pyrrolidine derivatives as optimizable alternatives for the development of novel classes of immuno-oncology agents targeting this enzyme.

L-arginase from Streptomyces diastaticus MAM5 as a potential therapeutic agent in breast cancer: Purification, characterization, G1 phase arrest and autophagy induction.

Targeting cancer metabolic processes has increased interest over the last century. Cancer cells have an enhanced proliferation rate that requires high quantities of amino acids, including arginine. Therefore, arginine deprivation by L-arginase impairs tumor growth resulting in cell death. In the present study, L-arginine amidinohydrolase (L-arginase) from Streptomyces diastaticus was purified successfully by heat treatment, ethanol precipitation, and Sephadex G75-120 column. The molecular mass of the purified enzyme was 39 kDa. It showed maximum activity at pH 9.0 and a temperature of 50 °C. Moreover, the enzyme stability was observed at temperatures up to 50 °C and a pH range of 7.5 to 9.0. Then, the potential cytotoxicity of L-arginase was examined. L-arginase has an IC50 value of 595 µg/ml for MCF-7 (breast adenocarcinoma cells), 915 µg/ml for HepG2 (hepatocellular carcinoma cells), and 1200 µg/ml for SW620 cells (colorectal carcinoma cells) at 72 h post-treatment. Noteworthy, MCF-7 showed the lowest IC50 value of arginase treatment, therefore was further investigated for the underlying cytotoxic mechanisms using flow cytometric analysis of cell-cycle distribution, apoptosis, and autophagy. Moreover, SI values indicating a high selective cytotoxicity of arginase toward MCF-7 cells. L-arginase induced significant cell cycle arrest at the G1 phase, and no apparent apoptosis was detected. Interestingly, arginine deprivation by arginase leads to a prominent activation of autophagy in the apoptosis defected MCF-7 cells. Moreover, treatment with arginase significantly attenuated MCF-7 cell migration compared with control medium-treated cells. Collectively, L-arginase might potentially be involved in treating breast cancer.

Sequence Divergence in the Arginase Domain of Ornithine Decarboxylase/Arginase in Fusobacteriacea Leads to Loss of Function in Oral Associated Species.

A number of species within the Fusobacteriaceae family of Gram-negative bacteria uniquely encode for an ornithine decarboxylase/arginase (ODA) that ostensibly channels l-ornithine generated by hydrolysis of l-arginine to putrescine formation. However, two aspartate residues required for coordination to a catalytically obligatory manganese cluster of arginases are substituted for a serine and an asparagine. Curiously, these natural substitutions occur only in a clade of Fusobacterium species that inhabit the oral cavity. Herein, we expressed and isolated full-length ODA from the opportunistic oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components. The crystal structure of the arginase domain reveals that it adopts the classical α/ß arginase-fold, but metal ions are absent in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated arginase domain. However, engineering of the complete metal coordination environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency for l-arginine was low (60-100 M-1 s-1). Full-length ODA and the isolated ODC component were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but kcat/KM of l-ornithine was ∼20-fold higher compared to l-arginine. We discuss environmental conditions that may have led to the natural selection of an inactive arginase in the oral associated species of Fusobacterium.

Catalytic mechanism of DcsB: Arginase framework used for hydrolyzing its inhibitor.

DcsB, an enzyme produced from the d-cycloserine biosynthetic gene cluster, displays moderate similarity to arginase in the sequence and three-dimensional structure. Arginase is a ubiquitous enzyme hydrolyzing l-arginine to generate l-ornithine and urea, whereas DcsB hydrolyzes Nω -hydroxy-l-arginine (l-NOHA), an arginase inhibitor, to generate l-ornithine and hydroxyurea. We determined the crystal structure of DcsB associated with l-ornithine and that with the tetrahedral derivative of 2(S)-amino-6-boronohexanoic acid, whose boron atom forms a covalent bond with an oxygen atom bridging two manganese ions at the active center. The substrate-binding pocket of DcsB is narrower than that of arginase, suggesting that DcsB is unsuitable for the binding of l-NOHA in an inhibitory manner. The transition state-like structure demonstrated that Asp210 and Glu241 have a role to trap a positively charged ion near the dimanganese cluster. Kinetic analysis using the mutated DcsB showed that the enzyme employs different catalytic mechanisms under the neutral and alkaline pH conditions. Glu241 in DcsB is likely involved in the recognition of the hydroxyguanidino group of l-NOHA, whereas Asp210, in cooperation with Glu241, seems to contribute to the reactivity toward the protonated l-NOHA, which is a preferable species under the neutral pH conditions. After entering of the protonated l-NOHA to the substrate-binding pocket of DcsB, a hydronium ion may be trapped at the positive ion-binding site. Then, the ion serves as a specific acid catalyst to facilitate the collapse of the tetrahedral intermediate of l-NOHA.

A synthetic peptide as an allosteric inhibitor of human arginase I and II.

Arginine metabolism mediated by arginases plays a critical role in cell and tissue function. The arginine hydrolysis is deeply involved in the urea cycle, which helps the kidney excrete ammonia from blood. Upregulation of arginases affects microenvironment stability due to the presence of excess urea in blood. To regulate the arginase activities properly, a synthetic peptide based on the structure of human arginase I was designed and assessed. Preliminary data shows it inhibits human arginase I and II with an IC50 of 2.4 ± 0.3 and 1.8 ± 0.1 mmol, respectively. Our kinetic analysis indicates the inhibition is not competitive with substrate - suggesting an allosteric mechanism. This result provides a step towards specific inhibitors design.

An evolutionary non-conserved motif in Helicobacter pylori arginase mediates positioning of the loop containing the catalytic residue for catalysis.

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.

Changes in arginase isoforms in a murine model of neonatal brain hypoxia-ischemia.

BACKGROUND: Arginases (ARG isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair; however, their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown. METHODS: C57BL/6 mice subjected to the Vannucci procedure on postnatal day (P9) were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically. RESULTS: ARG isoform expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 days. ARG activity during neurodevelopment remained unchanged, but increased at 1 day with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons. CONCLUSIONS: ARG isoform expression increases with age from P9 to P17, but is suppressed by injury specifically in the hippocampus and not in the cortex. Both levels and activity of ARG isoforms increase with H, while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG isoforms in the brain differ in spatiotemporal localization, expression, and activity during neurodevelopment and after injury. IMPACT: Arginase isoforms change during neurodevelopment and after neonatal brain HI. This is the first study investigating the key enzymes of inflammation and tissue repair called arginases following murine neonatal brain HI. The highly region- and cell-specific expression suggests the possibility of specific functions of arginases. ARG-1 in microglia at the injury site may regulate neuroinflammation, while ARG-2 in neurons of developmental structures may impact neurodevelopment. While further studies are needed to describe the exact role of ARGs after neonatal brain HI, our study adds valuable data on anatomical localization and expression of ARGs in brain during development and after stroke.

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.

Posttranslationally Acting Arginases Provide a Ribosomal Route to Non-proteinogenic Ornithine Residues in Diverse Peptide Sequences.

Ornithine is a component of many bioactive nonribosomal peptides but is challenging to incorporate into ribosomal products. We recently identified OspR, a cyanobacterial arginase-like enzyme that installs ornithines in the antiviral ribosomally synthesised and posttranslationally modified peptide (RiPP) landornamide A. Here we report that OspR belongs to a larger family of peptide arginases from diverse organisms and RiPP types. In E. coli, seven selected enzymes converted arginine into ornithine with little preference for the leader type. A broad range of peptide sequences was modified, including polyarginine repeats. We also generated analogues of ornithine-containing nonribosomal peptides using RiPP technology. Five pseudo-nonribosomal products with ornithines at the correct positions were obtained, including a brevicidine analogue containing ornithine and a d-amino acid installed by the peptide epimerase OspD. These results suggest new opportunities for peptide bioengineering.

Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection.

Macrophages are professional phagocytes known to play a vital role in controlling Mycobacterium tuberculosis (Mtb) infection and disease progression. Here we compare Mtb growth in mouse alveolar (AMs), peritoneal (PMs), and liver (Kupffer cells; KCs) macrophages and in bone marrow-derived monocytes (BDMs). KCs restrict Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections through enhanced autophagy. A metabolomics comparison of Mtb-infected macrophages indicates that ornithine and imidazole are two top-scoring metabolites in Mtb-infected KCs and that acetylcholine is the top-scoring in Mtb-infected AMs. Ornithine, imidazole and atropine (acetylcholine inhibitor) inhibit Mtb growth in AMs. Ornithine enhances AMPK mediated autophagy whereas imidazole directly kills Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or the two together restricts Mtb growth. Our study demonstrates that the metabolic differences between Mtb-infected AMs and KCs lead to differences in the restriction of Mtb growth.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

Cytotoxicity of [HuArgI (co)-PEG5000]-induced arginine deprivation to ovarian Cancer cells is autophagy dependent.

In this study, we assess arginine auxotrophy in ovarian cancer cells and attempt to target them using arginine deprivation induced by a pegylated recombinant human Arginase I cobalt [HuArgI (Co)-PEG5000]. Ovarian cancer cells were sensitive to [HuArgI (Co)-PEG5000]-induced arginine deprivation with IC50 values in the low pM range. Addition of excess L-citrulline rescued only one of three cell lines tested, indicating that the majority of cell lines are completely auxotrophic for arginine. The expression pattern of argininosuccinate synthetase (ASS1) confirmed the degree of auxotrophy of ovarian cancer cell lines with completely auxotrophic cells not expressing ASS1 and partially auxotrophic cells expressing the enzyme. Ovarian cancer cell lines were negative for annexinV staining while showing loss of membrane integrity and absence of caspase activation, indicating caspase-independent, non-apoptotic cell death. [HuArgI (Co)-PEG5000]-induced arginine deprivation led to extensive and prolonged activation of autophagy, which proved to be deleterious to cell survival since its inhibition led to a significant decrease in cytotoxicity. This indicates that the activation of autophagy following arginine-deprivation, rather than being protective, mediates cell cytotoxicity leading to death by autophagy.

Functional analysis of the Mn2+ requirement in the catalysis of ureohydrolases arginase and agmatinase - a historical perspective.

Ureohydrolases form a conserved family of enzymes with a strict requirement for divalent metal ions for catalytic activity. They catalyze the hydrolysis of the guanidino group and produce urea. In their active sites six highly conserved amino acid residues form a binding pocket for two catalytically essential metal ions that are needed to activate a water molecule to initiate the hydrolysis of the guanidino group in a nucleophilic attack. Focus in this review is on two members of the ureohydrolase family, the Mn2+-dependent arginase and agmatinase, which play important roles in functions related to replication and cell survival. We will focus in particular on Mn2+ binding interactions, and on how this metal ion contributes to the reaction catalyzed by these enzymes. We also include the agmatinase-like protein (ALP) because it is functionally closely related to agmatinase, also requires at least one Mn2+ ion for catalytic activity, but may possess an active site that differs significantly from all other known ureohydrolases.