Arginase
nor-NOHA monoacetate targets arginase enzymes, specifically arginase-1 (Arg1) and arginase-2 (Arg2). Arginase is a key enzyme in the urea cycle that converts L-arginine into L-ornithine and urea. By competitively inhibiting arginase, this compound prevents the breakdown of L-arginine, thereby increasing its availability for nitric oxide synthase (NOS) to produce nitric oxide (NO), a critical vasodilator and signaling molecule. This mechanism is particularly relevant in the tumor microenvironment, where arginase activity suppresses T-cell receptor expression and anti-tumor immunity.

nor-NOHA (0.1-1 mM, 72 h) induces apoptosis of K562 cells in a dose-dependent manner under hypoxia (1.5% O2)[1]. nor-NOHA (1 mM, 72 h) can attenuate hypoxia-mediated imatinib resistance in K562 or KCL22 cells[1].

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nor-NOHA monoacetate exhibits potent inhibitory activity in vitro, with a Ki of 0.5 µM against rat liver arginase and an IC50 of 2 µM. It displays approximately 10-fold selectivity for human type II arginase (Arg2) over type I (Arg1). In functional assays, it enhances the effect of acetylcholine on isolated aortic and mesenteric arterial rings, demonstrating its ability to potentiate NO-mediated vasodilation. It also inhibits the growth of lung carcinoma implants, confirming its anti-tumor potential.

nor-NOHA (100 mg/kg, intravenous injection, once) can significantly reduce infarct size in male Sprague-Dawley rats [2]. nor-NOHA (100 mg/kg IV once) increases plasma citrulline and nitrite levels and decreases plasma ornithine levels in male Sprague-Dawley rats [2]

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In vivo, nor-NOHA monoacetate has demonstrated significant biological activity. Its ability to inhibit arginase in animal models has been linked to enhanced immune responses and reduced tumor growth. The compound is used in research to study the role of arginase in conditions such as cancer, cardiovascular disease, and immune dysfunction. By increasing NO availability, it can improve vascular function and modulate inflammatory responses, highlighting its therapeutic potential in preclinical disease models.

Arginase activity assay[1]
Arginase activity was analysed as described with modifications. Cells were counted, and equal numbers of cells were lysed in 50μl of lysis buffer (PBS with 1mM EDTA, 0.1% Triton X−100 and protease Inhibitors) and centrifuged for 15 minutes at 14,000g at 4°C. The supernatants were mixed with 50μl of freshly prepared activation buffer (10mM MnCl2, 50mM Tris-HCl pH7.5) and 50μl of 0.5M arginine, and heated for 10 minutes at 56°C. Thereafter, 800μl of acidic solution (H2SO4 (96%)/H3PO4 (85%)/H2O, 1/3/7, v/v/v) and 25μl of 9% α–isonitrosopropiophenone (in ethanol) were added to the mixture and heated for 15 minutes at 100°C. The mixture was allowed to develop colour in the dark. Finally, 250μl was transferred to a 96-well plate for OD measurements at 550nm.

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Arginase inhibitory activity is assessed using in vitro enzyme assays. Recombinant or tissue-derived arginase is incubated with its substrate L-arginine and varying concentrations of nor-NOHA monoacetate. The production of urea is measured colorimetrically, and the inhibition constant (Ki) or half-maximal inhibitory concentration (IC50) is calculated from dose-response curves. The competitive nature of the inhibition can be confirmed by varying the substrate concentration.

Measurement of cellular respiration by Seahorse Analyzer[1]
0.1x106 K562 cells were plated per well in poly–L–lysine-coated XF–24 well cell culture microplates in XF Assay media supplemented with 4.5 g/L glucose and 1mM sodium pyruvate. The cells were spin–immobilized to the microplates at 200g for 1 minute. The cellular oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and photon production rate (PPR) were obtained using an XF24 Analyzer from Seahorse Bioscience. The measurements were performed according to the manufacturer’s instructions, using Oligomycin, Carbonyl cyanide–4–(trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone & antimycin A (R/A; all from Sigma−Aldrich) at the specified concentrations. Data was analysed using the Seahorse XF software.

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The cellular activity of nor-NOHA monoacetate is evaluated in cell culture models, such as macrophages or cancer cell lines. Cells are treated with the compound, and its effect on arginase activity is measured by quantifying urea production in cell lysates. The impact on L-arginine metabolism and downstream signaling can be assessed by measuring NO production or by Western blot analysis of arginase expression. The compound's effect on cell proliferation and immune function is also assessed.

Sprague-Dawley rats were subjected to 30 min of coronary artery ligation, followed by 2 h of reperfusion. The animals were given either saline, or the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-NOHA) with or without the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) or the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) iv 15 min before ischaemia. The infarct size was 79 +/- 4% of the area at risk in the control group. Nor-NOHA treatment reduced the infarct size to 39 +/- 7% (P < 0.001). Administration of cPTIO or l-NMMA completely abolished the protective effect of nor-NOHA. Expression of arginase I was significantly (P < 0.05) increased in ischaemic myocardium. Nor-NOHA treatment resulted in higher plasma levels of nitrite (P < 0.05) and a 10-fold increase in the citrulline/ornithine ratio (P < 0.001), indicating a shift in arginine utilization towards NOS.
Conclusion: Inhibition of arginase protects from myocardial infarction by a mechanism that is dependent on NOS activity and bioavailability of NO by shifting arginine utilization from arginase towards NOS. These findings suggest that targeting of arginase is a promising future therapeutic strategy for protection against myocardial IR injury.[2]

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In animal studies, nor-NOHA monoacetate is typically administered via intraperitoneal (i.p.) or intravenous (i.v.) injection. Dosing regimens vary depending on the model; for example, in tumor models, it may be given daily to assess its effect on tumor growth. Efficacy endpoints include tumor volume, immune cell infiltration, and markers of arginase activity and NO production in target tissues. Pharmacodynamic biomarkers are measured to confirm target engagement.

nor-NOHA monoacetate is a small molecule (MW 236.23) that is soluble in water and DMSO. It is recommended to be stored as a powder at -20°C. Detailed pharmacokinetic parameters, such as half-life and bioavailability, are not extensively documented in publicly available resources, but its use in animal models indicates it is suitable for in vivo research.

Toxicology data for nor-NOHA monoacetate is limited, as it is a research tool not intended for human therapeutic use. In preclinical studies at efficacious doses, it is generally well-tolerated, but its safety profile has not been established in formal toxicology studies. As with all research chemicals, standard laboratory safety precautions should be followed.

[1]. The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target. PLoS One. 2018 Oct 11;13(10):e0205254.

[2]. Arginase inhibition mediates cardioprotection during ischaemia-reperfusion. Cardiovasc Res. 2010 Jan 1;85(1):147-54.

ω-hydroxy-n-L-arginine is an L-α-amino acid. N-hydroxy-n-L-arginine (nor-NOHA) is being investigated in the clinical trial NCT02009527 (arginase inhibitors in ischemia-reperfusion injury). Cancer cells, including chronic myeloid leukemia (CML), rely on hypoxia to persist in the host and evade immunotherapy. Therefore, drug development targeting cancer-specific hypoxia responses has attracted considerable interest. However, a major challenge in leukemia research is identifying differentially expressed, targetable hypoxia responses between leukemia cells and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme in the urea cycle, is overexpressed in CML cells but not in normal progenitor cells. ARG2 is a target of hypoxia-inducible factors (HIF1-α and HIF2-α) and is essential for polyamine production required for cell growth. Therefore, we investigated whether the clinically validated arginase inhibitor Nω-hydroxy-norarginine (nor-NOHA) was effective against leukemia cells under hypoxic conditions. Notably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxic conditions, but was ineffective under normoxic conditions. Combination therapy with a BCR-ABL1 kinase inhibitor overcame hypoxia-mediated resistance. While nor-NOHA itself shows promise in targeting the hypoxic response in leukemia, we unexpectedly found that its antileukemic activity was independent of ARG2 inhibition. ARG2 gene knockout using CRISPR/Cas9 had no effect on the viability of leukemia cells or their sensitivity to nor-NOHA. The different effects of ARG2 knockout and nor-NOHA on cellular respiration further confirmed this difference. In conclusion, we found that nor-NOHA possesses significant, but non-targeted, antileukemic activity in hypoxic cells expressing ARG2. Since nor-NOHA has been used in clinical trials and is widely used in studies of endothelial dysfunction, immunosuppression, and metabolism, its various biological effects must be carefully evaluated before attributing its activity to ARG inhibition. [1]

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nor-NOHA monoacetate (CAS: 2250019-93-1) is a well-characterized and widely used tool for studying arginase biology. Its water solubility and potent inhibitory activity make it a convenient and effective compound for both in vitro and in vivo experiments. The compound has been instrumental in elucidating the role of arginase in immunosuppression and vascular dysfunction, and it continues to be a key reference for the development of more selective arginase inhibitors.

C7H16N4O5

236.225741386414

236.112

C, 35.59; H, 6.83; N, 23.72; O, 33.86

2250019-93-1

189302-40-7; 1140844-63-8 (acetate); 291758-32-2 (HCl); 2250019-93-1 (nor-NOHA monoacetate)

131648256

White to off-white solid powder

6

7

5

16

213

1

OC([C@H](CC/N=C(\N)/NO)N)=O.OC(C)=O

RYUGHGOGNIYFKU-DFWYDOINSA-N

InChI=1S/C5H12N4O3.C2H4O2/c6-3(4(10)11)1-2-8-5(7)9-12;1-2(3)4/h3,12H,1-2,6H2,(H,10,11)(H3,7,8,9);1H3,(H,3,4)/t3-;/m0./s1

acetic acid;(2S)-2-amino-4-[[amino-(hydroxyamino)methylidene]amino]butanoic acid

Nor NOHA monoacetate; 2250019-93-1; nor-NOHA (monoacetate); nor-NOHA monoacetate; N-OMega-hydroxy-L-norarginine acetate salt; AKOS032962868; HY-112885B; N-OMega-hydroxy-L-norarginineacetatesalt;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~423.32 mM)
H2O : ~50 mg/mL (~211.66 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (423.32 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)