Recombinant Human Arginase Research Articles

Recombinant human arginase I elicited immunosuppression in activated macrophages through inhibiting autophagy

Arginase I has been documented to impair T cell function and attenuate cellular immunity, however, there is little evidence to reveal the effect of arginase I on macrophage function. Recently, recombinant human arginase I (rhArg) has been developed for cancer therapy and is in clinical trial for hepatocellular carcinoma, whereas the potential immunosuppression induced by rhArg limited its therapeutic efficacy. To improve the clinical outcome of rhArg, addressing the immune suppression appears to be particularly important. In this study, we found that rhArg attenuated macrophage functions, including inhibiting macrophage cell proliferation, nitric oxide (NO) and reactive oxygen species (ROS) production, cytokine secretion, MHC-II surface expression, and phagocytosis, thereby inducing immunosuppression in lipopolysaccharides (LPS)/interferon-γ (IFN-γ)-activated macrophages. Notably, we observed that rhArg downregulated autophagy in activated macrophages. Moreover, application of trehalose (an autophagy inducer) significantly restored the impaired immune function in activated macrophages, suggesting the essential role of autophagy in rhArg-induced immunosuppression. To further illustrate the effect of autophagy in immunosuppression, we then observed the effect of 3-MA (an autophagy inhibitor) on the immune function of macrophages. As expected, inhibiting autophagy by 3-MA attenuated immune functions in activated macrophages. Collectively, this study elucidated that rhArg induced immunosuppression in activated macrophages via inhibiting autophagy, providing potential strategy to ameliorate the immune suppression which is of great significance to cancer therapy and facilitating the development of rhArg as a potential therapy for malignant carcinomas.

Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

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.

Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer.

Small cell lung cancer (SCLC) accounts for approximately 13% of all lung cancer cases. Small cell lung cancer is characterized by frequent relapse, and current treatments lack tumor specificity. Arginine is a non‐essential amino acid for human normal cells but critical to some tumor cells that cannot synthesize arginine. Therefore, arginine deprivation has become a potential therapeutic option for selected tumors. BCT‐100 is a pegylated arginase that has documented anticancer activity in arginine auxotrophic tumors, such as melanoma, hepatocellular carcinoma, and acute myeloid leukemia. One of the resistance mechanisms to arginase treatment is overexpression of argininosuccinate synthetase (ASS1) and ornithine transcarbamylase (OTC), two important enzymes in the urea cycle. We selected 9 SCLC and 1 non‐small cell lung carcinoma cell lines to determine the growth inhibition effects of BCT‐100 and established that cell lines with low expression of ASS1 and OTC are relatively sensitive to BCT‐100 treatment. Knocking down OTC in a H841 cell line could potentiate its sensitivity to BCT‐100 treatment. Arginine concentration was sharply decreased, accompanied by apoptosis through oxidative stress as well as G1 cell cycle arrest. In addition, BCT‐100 showed an anticancer effect on H446 and H510A xenograft models by lowering arginine levels and inducing apoptosis.

PO-065 Arginine deprivation through human recombinant arginase I (Co)-PEG5000 [HuArgI (Co)-PEG5000] activates autophagy and leads to autophagy-mediated cell death in glioblastoma cells

IntroductionArginine auxotrophy, secondary to a lack of argininosuccinate synthetase-1 (ASS-1) expression, is a hallmark of several tumour types allowing for their selective targeting using arginine deprivation. HuArgI (Co)-PEG5000, a recombinant PEGylated human arginase I in which the two active site Mn++ ions have been replaced by Co++ ions, degrades extracellular arginine leading to arginine deprivation and selective cytotoxicity to GBM cells. In this study, we investigate the mechanisms of arginine deprivation-induced cell death, particularly the contribution of autophagy, in glioblastoma (GBM) cells.Material and methodsFour GBM cell lines were used to determine extent and dynamics of autophagy activation using Cyto-ID staining on fluorescent microscopy and LC3-I to II conversion using western blot. The contribution of autophagy to arginine deprivation-induced cell death in GBM cells was investigated through inhibition of autophagy, at different time points, using Chloroquine (CQ).Results and discussions[HuArgI (Co)-PEG5000]-induced arginine deprivation led to a clear and time-dependent increase in the autophagic flux, as indicated by the increase in the number of autophagosomes on Cyto-ID and in the processing of LC3-I to LC3-II, in all cell lines, starting at 12 hours and increasing with time to peak at 96 and 120 hours post treatment. Blocking the autophagic flux using CQ, a downstream inhibitor of autophagy, led to a further accumulation of autophagosomes at all time points. Inhibiting autophagy, using CQ, had a binary, time-dependent effect on the cytotoxicity of [HuArgI (Co)-PEG5000]-induced arginine deprivation in GBM cells. At early time points (12, 24 and 48 hour), inhibition of autophagy increased cell sensitivity to arginine deprivation underlying the protective role of autophagy while at late time points (72, 96 and 120 hour), inhibition of autophagy significantly decreased or completely prevented cell death indicating that autophagy plays a deleterious role in GBM cells exposed to arginine deprivation for a prolonged period of time.ConclusionThis study demonstrates that arginine deprivation leads to significant and sustained activation of the autophagic flux in GBM cells. Our data also indicates that the observed over-activation of autophagy is protective to GBM cells at early time points but becomes deleterious at later time points leading to autophagy-induced cell death (death by autophagy), following prolonged arginine deprivation.

PO-248 The role of ER stress in arginine deprivation-induced radiosensitization and cell death of HNSCC cells

IntroductionPancreatic ductal adenocarcinoma (pdac) is one of the most lethal human cancers. about 20–30% of pdac are described to be auxotrophic for arginine and thus might be particularly sensitive to arginine deprivation therapy (adt). although radiotherapy for treatment of pdca is controversially discussed, the abnormal metabolism of pdac in combination with radiation is an attractive approach for targeted therapy. previously, we could show that adt is efficient in radiosensitizing auxotrophic pdacs to photons. recent clinical studies imply a benefit of proton radiotherapy for pdac patients. we therefore systematically compared the sensitivity of pdac cells to photon and proton irradiation in combination with adt as well as gemcitabine treatment.Material and methodsHuman and murine auxotrophic pdacs cells mimicking a patient cohort were studied. adt was achieved with 2 u/ml of recombinant human arginase +/- citrulline, a physiological precursor of arginine. single doses ≤ 30 gy of 200 kv x-rays or 150 mev double-scattered protons were applied. treatment response was assessed by 2-d clonogenic survival and/or 3-d spheroid control probability (scp) assays.results and discussionsadt led to a sensitization of pdac cells to x-rays in 2-d and 3-d culture. proton irradiation significantly reduced the scp and spheroid control dose 50% (scd50) relative to x-rays in the 3-d assay as reflected by a relative biological effectiveness (rbe) of 2.0. combining protons with adt further increased the rbe to 2.4. gemcitabine treatment did not improve radiotherapy outcome. however, the triple combination of adt, gemcitabine and proton irradiation had a highly synergistic effect (rbe = 3.0). extended mechanistic studies are ongoing.ConclusionFor pdac, adt in combination with gemcitabine and proton irradiation seems to be a very promising treatment strategy and should be further explored for translation into clinical trials.

PO-247 Beneficial therapeutic ménage à trois: enzymotherapeutic arginine-deprivation, gemcitabine and proton irradiation for pancreatic cancer treatment - a systematic in vitro study

IntroductionPancreatic ductal adenocarcinoma (pdac) is one of the most lethal human cancers. about 20–30% of pdac are described to be auxotrophic for arginine and thus might be particularly sensitive to arginine deprivation therapy (adt). although radiotherapy for treatment of pdca is controversially discussed, the abnormal metabolism of pdac in combination with radiation is an attractive approach for targeted therapy. previously, we could show that adt is efficient in radiosensitizing auxotrophic pdacs to photons. recent clinical studies imply a benefit of proton radiotherapy for pdac patients. we therefore systematically compared the sensitivity of pdac cells to photon and proton irradiation in combination with adt as well as gemcitabine treatment.Material and methodsHuman and murine auxotrophic pdacs cells mimicking a patient cohort were studied. adt was achieved with 2 u/ml of recombinant human arginase +/- citrulline, a physiological precursor of arginine. single doses ≤ 30 gy of 200 kv x-rays or 150 mev double-scattered protons were applied. treatment response was assessed by 2-d clonogenic survival and/or 3-d spheroid control probability (scp) assays.results and discussionsadt led to a sensitization of pdac cells to x-rays in 2-d and 3-d culture. proton irradiation significantly reduced the scp and spheroid control dose 50% (scd50) relative to x-rays in the 3-d assay as reflected by a relative biological effectiveness (rbe) of 2.0. combining protons with adt further increased the rbe to 2.4. gemcitabine treatment did not improve radiotherapy outcome. however, the triple combination of adt, gemcitabine and proton irradiation had a highly synergistic effect (rbe = 3.0). extended mechanistic studies are ongoing.ConclusionFor pdac, adt in combination with gemcitabine and proton irradiation seems to be a very promising treatment strategy and should be further explored for translation into clinical trials.

Refining Radiation for the Next Century.

![][1] Wilhelm Roentgen's report more than a century ago describing X-ray imaging fostered in the application of ionizing radiation (IR) for cancer therapy. Today, about half of all the patients with cancer are treated with radiotherapy and it remains one of our most effective

Arginine Deprivation Therapy: Putative Strategy to Eradicate Glioblastoma Cells by Radiosensitization.

Tumor cells-even if nonauxotrophic-are often highly sensitive to arginine deficiency. We hypothesized that arginine deprivation therapy (ADT) if combined with irradiation could be a new treatment strategy for glioblastoma (GBM) patients because systemic ADT is independent of local penetration and diffusion limitations. A proof-of-principle in vitro study was performed with ADT being mimicked by application of recombinant human arginase or arginine-free diets. ADT inhibited two-dimensional (2-D) growth and cell-cycle progression, and reduced growth recovery after completion of treatment in four different GBM cell line models. Cells were less susceptible to ADT alone in the presence of citrulline and in a three-dimensional (3-D) environment. Migration and 3-D invasion were not unfavorably affected. However, ADT caused a significant radiosensitization that was more pronounced in a GBM cell model with p53 loss of function as compared with its p53-wildtype counterpart. The synergistic effect was independent of basic and induced argininosuccinate synthase or argininosuccinate lyase protein expression and not abrogated by the presence of citrulline. The radiosensitizing potential was maintained or even more distinguishable in a 3-D environment as verified in p53-knockdown and p53-wildtype U87-MG cells via a 60-day spheroid control probability assay. Although the underlying mechanism is still ambiguous, the observation of ADT-induced radiosensitization is of great clinical interest, in particular for patients with GBM showing high radioresistance and/or p53 loss of function. Mol Cancer Ther; 17(2); 393-406. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

The arginine metabolome in acute lymphoblastic leukemia can be targeted by the pegylated-recombinant arginase I BCT-100.

Arginine is a semi‐essential amino acid that plays a key role in cell survival and proliferation in normal and malignant cells. BCT‐100, a pegylated (PEG) recombinant human arginase, can deplete arginine and starve malignant cells of the amino acid. Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, yet for patients with high risk or relapsed disease prognosis remains poor. We show that BCT‐100 is cytotoxic to ALL blasts from patients in vitro by necrosis, and is synergistic in combination with dexamethasone. Against ALL xenografts, BCT‐100 leads to a reduction in ALL engraftment and a prolongation of survival. ALL blasts express the arginine transporter CAT‐1, yet the majority of blasts are arginine auxotrophic due to deficiency in either argininosuccinate synthase (ASS) or ornithine transcarbamylase (OTC). Although endogenous upregulation or retroviral transduced increases in ASS or OTC may promote ALL survival under moderately low arginine conditions, expression of these enzymes cannot prevent BCT‐100 cytotoxicity at arginine depleting doses. RNA‐sequencing of ALL blasts and supporting stromal cells treated with BCT‐100 identifies a number of candidate pathways which are altered in the presence of arginine depletion. Therefore, BCT‐100 provides a new clinically relevant therapeutic approach to target arginine metabolism in ALL.

Abstract 1241: A preclinical study of the combined treatment of arginase and canavanine in pancreatic cancer

Abstract Background: Arginine deprivation is a novel approach for cancer treatment as some cancers were found to be arginine auxotrophic. PEG-BCT-100, a pegylated form of recombinant human arginase, was used to investigate the effect of arginine deprivation on pancreatic cell lines. On the other hand, Canavanine (CAV), a natural toxic analog of arginine isolated from leguminous plant, has also demonstrated its ability in inhibiting cancer cell proliferation. It alters the protein structure and causes critical metabolic defects in the cells by replacing arginine into newly synthesized proteins during translation. In this study, we have also tested the efficacy of combined arginase and CAV treatment on pancreatic cell lines. Methods: Pancreatic cell lines (MIA PaCa-2, CFPAC-1) and normal fibroblast cell line (WI-38), were either incubated in normal or arginine free condition. The corresponding IC50 of CAV was determined by cell viability assay. To evaluate the responsiveness to the combined treatment, cells were monitored by live cell imaging under the conditions of PEG-BCT-100 treatment alone (0.3IU/ml) or in combination with CAV (10μM or 50μM). The apoptotic effect of the combined treatment was examined by Western blotting and Annexin V assay. Results: Either arginase or CAV treatment could inhibit pancreatic cancer cell growth. The cells treated with CAV in arginine free condition demonstrated an inhibition of cell proliferation and its corresponding IC50 was significantly lowered from millimolar to micromolar concentration when arginine was withdrawn. Strong synergism of CAV and PEG-BCT-100 was observed after 48-hour treatment with enhanced apoptosis in pancreatic cell lines, but not in normal fibroblast cells. Both early and late apoptosis were observed in co-treated pancreatic cancer cells as indicated by Annexin V assay and the detection of polyADP ribosyl polymerase (PARP) in Western blot. Conclusion: Arginine deprivation by PEG-BCT-100 combined with CAV treatment showed a strong synergetic effect on inducing cell death in pancreatic cell lines, suggesting that the supplement of CAV could facilitate the treatment outcome of arginine deprivation by arginase. This combined treatment may serve as a treatment strategy for pancreatic cancer. Citation Format: Tsz Tung Kwong, Chi Hang Wong, Herbert Ho Fung Loong, Stephen Lam Chan. A preclinical study of the combined treatment of arginase and canavanine in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1241. doi:10.1158/1538-7445.AM2017-1241

P-142 - A phase 1 study of recombinant human arginase 1 (PEG-BCT- 100) in combination with capecitabine and oxaliplatin in advanced hepatocellular carcinoma patients

P-142 - A phase 1 study of recombinant human arginase 1 (PEG-BCT- 100) in combination with capecitabine and oxaliplatin in advanced hepatocellular carcinoma patients

A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor

Recombinant human arginase (rhArg), an enzyme capable of depleting arginine, has been shown to be an effective therapeutic approach for various cancers. Non-small-cell lung cancer (NSCLC), a histological subtype of pulmonary carcinoma, has a high rate of morbidity and mortality in the world. Thus, the need for novel and more effective treatment is urgent. In this study, it is the first time to report that rhArg could induce significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells. Subsequently, our research revealed that rhArg dramatically stimulated autophagic response in NSCLC cells, which was proved by the formation and accumulation of autophagosomes and the conversion of microtubule-associated protein light chain 3 (LC3) from LC3-I to LC3-II. Furthermore, blocking autophagy by chloroquine or LY294002 remarkably enhanced rhArg-induced cytotoxicity and caspase-dependent apoptosis, suggesting that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells. Further experiments showed that two signaling pathways including the Akt/mTOR and extracellular signal-regulated kinase pathway, and mitochondrial-derived reactive oxygen species (ROS) production were involved in rhArg-induced autophagy and apoptosis. Meanwhile, N-acetyl-L-cysteine, a common antioxidant, was employed to scavenge ROS, and we detected that it could significantly block rhArg-induced autophagy and cytotoxicity, indicating that ROS played a vital role in arginine degradation therapy. Besides, xenograft experiment showed that combination with autophagy inhibitor potentiated the anti-tumor efficacy of rhArg in vivo. Therefore, these results provided a novel prospect and viewpoint that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells, and treatment with rhArg alone or combined with autophagy inhibitor could be a novel and promising therapeutic approach for NSCLC in vivo and in vitro.

Abstract B045: Arginase inhibitor CB-1158 elicits immune-mediated antitumor responses as a single agent and in combination with other immunotherapies

Abstract Myeloid derived suppressor cells (MDSCs) and polymorphonuclear cells (PMNs) are mediators of tumor immune evasion, however there are no approved clinical agents that directly antagonize the immunosuppressive activity of these cells. One of the mechanisms by which MDSCs and PMNs suppress anti-tumor immunity is expression of the enzyme arginase, which reduces L-arginine levels rendering T-cells and natural killer (NK)-cells unable to proliferate and mount an effective anti-tumor response. As a rationale to pursue arginase inhibition as a potential therapeutic strategy, we investigated the prevalence of arginase in tumors and peripheral blood from patients with various types of cancer. Our results confirm reports that multiple tumor types have arginase-expressing PMN infiltrates and that cancer patients have higher levels of plasma arginase and lower levels of plasma arginine compared to healthy volunteers. We also confirmed that in vitro-activated primary human T-cells and NK-cells require arginine to proliferate, secrete pro-inflammatory cytokines, and express the intracellular signaling molecule CD3ζ and cell surface activation marker PD-1. We developed CB-1158 to be a potent, selective, and orally-bioavailable small molecule inhibitor of arginase. CB-1158 potently inhibits recombinant human arginase 1 (IC50 = 98 nM), recombinant human arginase 2 (IC50 = 249 nM), and endogenous arginase from human neutrophils (IC50 = 160 nM). In a co-culture system, neutrophils strongly suppressed T-cell proliferation. The addition of CB-1158 blocked arginase activity, maintained arginine levels in the media, and allowed T-cells to proliferate in the presence of suppressive myeloid cells, suggesting that arginase is a major mechanism of myeloid cell suppression of lymphocyte proliferation and that CB-1158 can reverse the suppression. To extend these findings to MDSCs, we isolated peripheral monocytic MDSCs (M-MDSCs) or granulocytic MDSCs (G-MDSCs) from cancer patients and found that G-MDSCs expressed higher levels of arginase, depleted more arginine from cell-culture media, and were more suppressive to T-cell proliferation compared to M-MDSCs. Incubation of G-MDSCs with CB-1158 resulted in media arginine levels that could support T-cell proliferation. CB-1158 has high oral bioavailability in mice and rats. Twice-daily oral dosing of CB-1158 produced dose-dependent pharmacodynamic increases in plasma and tumor arginine levels and resulted in single-agent anti-tumor efficacy in the murine syngeneic tumor models Lewis Lung carcinoma (LLC1), Madison-109 lung carcinoma, and B16F10 melanoma. Antibody-mediated depletion of either CD8+ T-cells or NK-cells partially abrogated the anti-tumor effect of CB-1158 in the LLC1 and B16F10 models indicating that the mechanism of action of CB-1158 is immune cell-mediated through the actions of both T-cells and NK cells. Corroborating the immune-mediated anti-tumor efficacy in CB-1158-treated LLC1 tumors, following dosing with CB-1158 we observed increases in tumor infiltrating CD8+ T-cells; increased levels of tumor Th1 T-cell cytokines; increased expression of T-cell and NK-cell activation markers; and increased interferon-inducible genes. Based on the novel mechanism of action of CB-1158, there is potential for enhanced therapeutic benefit by combining CB-1158 with other immunotherapies. We observed improved anti-tumor activity by combining CB-1158 with either epacadostat or anti-PD-L1 in the B16F10 model, with low dose ionizing radiation in the Madison-109 model, and with gemcitabine in the LLC1 model. These results support the development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology agent targeting the immunosuppressive effects of tumor-infiltrating myeloid cells. Citation Format: Susanne M. Steggerda, Mark Bennett, Jason Chen, Ethan Emberley, Matthew Gross, Tony Huang, Weiqun Li, Andy MacKinnon, Amani Makkouk, Gisele Marguier, Silinda Neou, Alison Pan, Tracy Wang, Melissa Works, Jing Zhang, Winter Zhang, Francesco Parlati. Arginase inhibitor CB-1158 elicits immune-mediated antitumor responses as a single agent and in combination with other immunotherapies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B045.

Cinnamide Derivatives as Mammalian Arginase Inhibitors: Synthesis, Biological Evaluation and Molecular Docking.

Arginases are enzymes that are involved in many human diseases and have been targeted for new treatments. Here a series of cinnamides was designed, synthesized and evaluated in vitro and in silico for their inhibitory activity against mammalian arginase. Using a microassay on purified liver bovine arginase (b-ARG I), (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide, also named caffeic acid phenylamide (CAPA), was shown to be slightly more active than our natural reference inhibitor, chlorogenic acid (IC50 = 6.9 ± 1.3 and 10.6 ± 1.6 µM, respectively) but it remained less active that the synthetic reference inhibitor Nω-hydroxy-nor-l-arginine nor-NOHA (IC50 = 1.7 ± 0.2 µM). Enzyme kinetic studies showed that CAPA was a competitive inhibitor of arginase with Ki = 5.5 ± 1 µM. Whereas the activity of nor-NOHA was retained (IC50 = 5.7 ± 0.6 µM) using a human recombinant arginase I (h-ARG I), CAPA showed poorer activity (IC50 = 60.3 ± 7.8 µM). However, our study revealed that the cinnamoyl moiety and catechol function were important for inhibitory activity. Docking results on h-ARG I demonstrated that the caffeoyl moiety could penetrate into the active-site pocket of the enzyme, and the catechol function might interact with the cofactor Mn2+ and several crucial amino acid residues involved in the hydrolysis mechanism of arginase. The results of this study suggest that 3,4-dihydroxycinnamides are worth being considered as potential mammalian arginase inhibitors, and could be useful for further research on the development of new arginase inhibitors.

Biotech in Hong Kong: How Biologist-Entrepreneurs Pursued “Hong Kong's Bioscience Dream”

This article aims to put Hong Kong on the map of East Asia's biotech studies by identifying the major themes of biotech innovation specific to the city-state's colonial past and postcolonial present and future. I provide evidence of a concerted effort led by biologist-entrepreneurs to promote the biotech industry in postcolonial Hong Kong. Through assessing scientific literature, media representations, business strategies, and programmatic visions of key scientist-entrepreneurs in Hong Kong's biotech enterprise, I highlight how factors such as local history and geopolitical considerations shape the emerging “Hong Kong's bioscience dream.” Using the recent discovery of a new recombinant anticancer drug, pegylated human recombinant arginase (BCT-100), as a case in point, I explore how biotech research and applications are marketed and interpreted in relation to Hong Kong's unique historical, political, and cultural context, thus making it distinct from other Asian Tigers. My analysis of the featured recombinant drug reveals a set of uniquely Hong Kong–centered cultural meanings attributed to biopharmaceutical research and benefits. This case reflects how genetic engineering and biotech research are configured and imagined within the context of the postcolonial Hong Kong Special Administrative Region.

Low expressions of ASS1 and OTC in glioblastoma suggest the potential clinical use of recombinant human arginase (rhArg)

Low expressions of ASS1 and OTC in glioblastoma suggest the potential clinical use of recombinant human arginase (rhArg)

Development and evaluation of a host-targeted antiviral that abrogates herpes simplex virus replication through modulation of arginine-associated metabolic pathways

Since their inception five decades ago, most antivirals have been engineered to disrupt a single viral protein or process that is essential for viral replication. This approach has limited the overall therapeutic effectiveness and applicability of current antivirals due to restricted viral specificity, a propensity for development of drug resistance, and an inability to control deleterious host-mediated inflammation. As obligate intracellular parasites, viruses are reliant on host metabolism and macromolecular synthesis pathways. Of these biosynthetic processes, many viruses, including Herpes simplex viruses (HSV), are absolutely dependent on the bioavailability of arginine, a non-essential amino acid that is critical for many physiological and pathophysiological processes associated with either facilitating viral replication or progression of disease. To assess if targeting host arginine-associated metabolic pathways would inhibit HSV replication, a pegylated recombinant human Arginase I (peg-ArgI) was generated and its in vitro anti-herpetic activity was evaluated. Cells continuously treated with peg-ArgI for over 48 h exhibited no signs of cytotoxicity or loss of cell viability. The antiviral activity of peg-ArgI displayed a classical dose-response curve with IC50’s in the sub-nanomolar range. peg-ArgI potently inhibited HSV-1 and HSV-2 viral replication, infectious virus production, cell-to-cell spread/transmission and virus-mediated cytopathic effects. Not unexpectedly given its host-targeted mechanism of action, peg-ArgI showed similar effectiveness at controlling replication of single and multidrug resistant HSV-1 mutants. These findings illustrate that targeting host arginine-associated metabolic pathways is an effective means of controlling viral replicative processes. Further exploration into the breadth of viruses inhibited by peg-ArgI, as well as the ability of peg-ArgI to suppress arginine-associated virus-mediated pathophysiological disease processes is warranted.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Molecular basis and current strategies of therapeutic arginine depletion for cancer.

Renewed interest in the use of therapeutic enzymes combined with an improved knowledge of cancer cell metabolism, has led to the translation of several arginine depletion strategies into early phase clinical trials. Arginine auxotrophic tumors are reliant on extracellular arginine, due to the downregulation of arginosuccinate synthetase or ornithine transcarbamylase-key enzymes for intracellular arginine recycling. Engineered arginine catabolic enzymes such as recombinant human arginase (rh-Arg1-PEG) and arginine deiminase (ADI-PEG) have demonstrated cytotoxicity against arginine auxotrophic tumors. In this review, we discuss the molecular events triggered by extracellular arginine depletion that contribute to tumor cell death.