Angiotensin-converting enzyme 2 (ACE2). DX600 is a selective ACE2-specific inhibitor with a KD of 1.3 nM (affinity constant) and does not cross-react with ACE. It binds to the active site of ACE2, blocking its enzymatic activity. ACE2 is a membrane-bound carboxypeptidase that converts angiotensin II (Ang II) to angiotensin-(1-7) (Ang-(1-7)), which has vasodilatory, anti-inflammatory, antifibrotic, and cardioprotective effects. By inhibiting ACE2, DX600 reduces Ang-(1-7) production, leading to an increase in Ang II levels and exacerbation of Ang II-mediated pathological effects such as vasoconstriction, inflammation, oxidative stress, and fibrosis. In addition to its enzymatic role, ACE2 serves as the cellular receptor for SARS-CoV-2, the virus that causes COVID-19, making DX600 a valuable tool for studying viral entry and pathogenesis. DX600 has a Ki of 2.8 nM as an ACE2-specific peptide inhibitor.

DX600 (1 μM) has a pIC50 of 8.0 and reduces rhACE2 activity by 47%[4]. 42% of ACE2 activity in human mononuclear cells (MCNs) is inhibited by DX600 (10 μM) [4]. When LPS and osthole are present, DX600 (100 nM, 4 hours) inhibits the growth of NR 8383 cells and raises TNF-a and IL-6 levels in the supernatant [5].

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DX600 is a selective ACE2-specific inhibitor with a KD of 1.3 nM and does not cross-react with ACE. In vitro, DX600 (1 microM) has a pIC50 of 8.0 and reduces recombinant human ACE2 (rhACE2) activity by 47%. In human mononuclear cells (MCNs), DX600 (10 microM) inhibits 42% of ACE2 activity. In NR8383 cells (rat alveolar macrophages), DX600 (100 nM, 4 hours) inhibits cell growth and raises TNF-alpha and IL-6 levels in the supernatant when LPS and osthole are present. These results confirm that DX600 effectively inhibits ACE2 enzymatic activity in cell-free and cell-based systems, leading to increased inflammation and oxidative stress. The compound is highly specific for ACE2 and does not inhibit ACE, making it a valuable tool for dissecting the distinct roles of ACE and ACE2 in the renin-angiotensin system. The binding affinity (KD = 1.3 nM) indicates high potency for ACE2 inhibition.

In diabetic rats treated with streptozotocin, DX600 (5 μg/kg/day, intraperitoneally, everyday for 4 weeks) TFA exacerbates the cardiovascular dysfunction caused by diabetes[2]. In a rat thrombosis model, DX600 (0.1 µmol/L/kg, intravenous administration) TFA enhanced thrombus weight by 30%[5].

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In diabetic rats treated with streptozotocin (STZ), DX600 (5 microg/kg/day, intraperitoneally, every day for 4 weeks) exacerbates the cardiovascular dysfunction caused by diabetes. The compound increases cardiac and renal NADPH oxidase (NOX) activity, leading to increased oxidative stress. DX600 (0.1 micromol/L/kg, intravenous administration) enhances thrombus weight by 30% in a rat thrombosis model, indicating that ACE2 activity is protective against thrombosis. In STZ-treated diabetic rats, DX600 treatment results in increased cardiac and renal NOX activity and exacerbation of diabetes-induced cardiovascular dysfunction. These in vivo effects confirm that ACE2 plays a protective role in diabetes-induced cardiovascular and renal injury and that inhibition of ACE2 by DX600 worsens disease outcomes. The compound is used in animal models to study the pathophysiology of ACE2 in various diseases, including hypertension, heart failure, diabetic nephropathy, and thrombosis. DX600 is often administered intraperitoneally or intravenously in these studies. Doses used in animal studies: 5 microg/kg/day IP for 4 weeks (in diabetic rats) or 0.1 micromol/L/kg IV (in thrombosis model).

ACE2 enzymatic activity assay using a fluorogenic substrate: Vary concentrations of DX600 (0.1-10,000 nM) are pre-incubated with recombinant human ACE2 (rhACE2) enzyme in assay buffer (e.g., 75 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 microM ZnCl2, 0.01% Brij-35) for 10-30 minutes at 37degC. The fluorogenic substrate Mca-Ala-Pro-Lys(Dnp)-OH (10-50 microM) is added to start the reaction. The reaction is carried out for 1-4 hours at 37degC in the dark. Fluorescence is measured at excitation/emission 320/405 nm (Mca excitation/emission). The cleavage of the substrate by ACE2 results in an increase in fluorescence due to relief of FRET quenching. The rate of fluorescence increase (deltaF/min) is calculated. IC₅0 values are calculated from concentration-response curves, and Ki values are determined from Lineweaver-Burk plots or by fitting to competitive inhibition equations. DX600 has a KD of 1.3 nM and a Ki of 2.8 nM in this assay. The assay buffer should contain Zn2+ as a cofactor for ACE2. A standard curve using known amounts of the cleavage product can be used to quantify absolute ACE2 activity. This protocol has been used in published studies to characterize DX600 as an ACE2 inhibitor. Alternatively, a radiometric assay using 3H-labeled angiotensin II as a substrate can be used to measure ACE2 activity, with the product Ang-(1-7) separated by HPLC and quantified by liquid scintillation counting. The fluorogenic assay is simpler and suitable for high-throughput screening. The assay should be performed in black 96-well or 384-well plates to minimize background fluorescence. Control wells without enzyme are used to measure background fluorescence. DX600 specificity for ACE2 over ACE is confirmed by performing the same assay using ACE enzyme with a different substrate (e.g., Abz-FRK(Dnp)-P). No inhibition of ACE should be observed at DX600 concentrations up to 10,000 nM. This confirms that DX600 does not cross-react with ACE.

For cell-based ACE2 activity assays, cells expressing ACE2 (e.g., Calu-3 lung epithelial cells, Caco-2 intestinal epithelial cells, Huh-7 hepatoma cells, or primary human mononuclear cells (MCNs)) are seeded in culture plates. Cells are treated with DX600 (0.1-10,000 nM) for 1-4 hours. ACE2 activity in cell lysates or membrane fractions is measured using the same fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH) as in the cell-free assay. Alternatively, ACE2 activity can be assessed by measuring the conversion of angiotensin II to angiotensin-(1-7) by LC-MS/MS. Cell surface expression of ACE2 is measured by flow cytometry or Western blotting using anti-ACE2 antibodies. For studies on SARS-CoV-2 entry, cells expressing human ACE2 are treated with DX600 (1-1000 nM) for 1 hour prior to infection with pseudotyped SARS-CoV-2 or live virus. Viral entry is measured by detecting viral proteins (e.g., spike protein), viral RNA (by qPCR), or reporter gene expression (e.g., luciferase) in the cells after infection. DX600 inhibits ACE2 activity and can block viral entry. The compound is also used to study the role of ACE2 in cell growth, inflammation, and oxidative stress. Inflammatory markers (TNF-alpha, IL-6) in cell culture supernatants are measured by ELISA. ROS levels are measured by DCFH-DA probe. Cell viability is assessed by MTT or CCK-8 assay. All experiments should be performed in triplicate with appropriate controls (vehicle-treated cells, ACE2 knockdown controls, or ACE2-specific antibody blocking). This cell-based assay is important for validating the functional inhibition of ACE2 in a cellular context and for studying downstream signaling and pathophysiological effects of ACE2 inhibition. DX600 has been used in Calu-3, Caco-2, and Huh-7 cells to evaluate cell surface expression of ACE2 by flow cytometry and Western blot.

Animal/Disease Models: STZ-treated diabetes rats[2]
Doses: 5 μg/kg/day
Route of Administration: ip, daily for 4 weeks
Experimental Results: Increased cardiac and renal NOX activity.

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Animal models: For the diabetes-induced cardiovascular dysfunction model, male Sprague-Dawley rats are injected with streptozotocin (STZ, 55-65 mg/kg, intraperitoneally) to induce diabetes. Diabetic rats are confirmed by blood glucose levels (>250 mg/dL). After 2-4 weeks of diabetes induction, animals are treated with DX600 (5 microg/kg/day, intraperitoneally) or vehicle (saline) for 4 weeks. At the end of the study, cardiac function is assessed by echocardiography (e.g., ejection fraction, fractional shortening, E/A ratio). Blood pressure is measured by tail-cuff plethysmography. Blood samples are collected for measurement of inflammatory markers (TNF-alpha, IL-6, IL-1beta by ELISA), oxidative stress markers (MDA, SOD, GSH), and plasma renin activity and angiotensin levels (Ang II, Ang-(1-7) by ELISA or LC-MS/MS). Heart and kidney tissues are collected for histopathology (H&E, Masson's trichrome for fibrosis), immunohistochemistry (ACE2 expression, NADPH oxidase subunits), and protein expression (Western blotting for ACE2, NOX1, NOX2, NOX4). NADPH oxidase activity in cardiac and renal tissues is measured by lucigenin-enhanced chemiluminescence. DX600 treatment should exacerbate cardiovascular and renal dysfunction compared to vehicle-treated diabetic controls, confirming the protective role of ACE2. For the thrombosis model, male rats are anesthetized and the carotid artery is isolated. DX600 (0.1 micromol/L/kg) or vehicle is administered intravenously. Thrombosis is induced by applying filter paper saturated with 10% FeCl3 to the carotid artery. Time to occlusion (thrombosis) is measured using a flow probe. Thrombus weight is measured. DX600 enhances thrombus weight by 30% compared to vehicle, indicating that ACE2 activity is protective against thrombosis. Animal studies should be conducted in accordance with institutional animal care and use guidelines. Doses, routes, and treatment durations should be optimized based on the specific model. DX600 is often administered intraperitoneally or intravenously due to its peptide nature, which may limit oral bioavailability. The compound is typically dissolved in sterile saline or PBS for injection. For intraperitoneal administration, a volume of 100-200 microL per animal is typical. For intravenous administration, a smaller volume (e.g., 50-100 microL) is used. The stability of DX600 in solution should be tested, and freshly prepared solutions are recommended. Store stock solutions in aliquots at -20degC or -80degC and avoid repeated freeze-thaw cycles.

DX600 has a molecular weight of 3074.32 and molecular formula C141H1₈₅N3₅O40S2 (for the peptide; the TFA salt may have a different molecular weight). The compound is a 29-amino acid peptide with an amide group at the C-terminus. Solubility: soluble in water (100 mg/mL) and in DMSO. For in vitro studies, stock solutions are prepared in water or DMSO at concentrations of 10-100 mM. For in vivo studies, dissolve in sterile saline or PBS. Avoid using organic solvents for in vivo injection due to potential toxicity. Storage: Lyophilized powder at -20degC (stable for 1-2 years). Reconstituted solutions should be stored at -20degC or -80degC in small aliquots to avoid repeated freeze-thaw cycles. The solution is stable for several months when stored frozen. Avoid storage at 4degC for extended periods due to possible degradation. Pharmacokinetic properties: As a 29-amino acid peptide, DX600 is expected to be rapidly degraded by proteases in the bloodstream and tissues, leading to a short half-life (likely minutes to hours). The compound is usually administered intravenously or intraperitoneally to achieve systemic exposure. Oral bioavailability is likely negligible due to degradation in the gastrointestinal tract. Detailed PK parameters (t½, Cmax, AUC, clearance) have not been extensively reported in the literature, but in vivo studies have demonstrated efficacy with intraperitoneal administration (5 microg/kg/day) and intravenous administration (0.1 micromol/L/kg), suggesting that sufficient exposure is achieved with these routes. The compound is stable in the specified storage conditions. Always check the certificate of analysis (COA) for purity (typically >95-98% by HPLC) and storage recommendations. The peptide may aggregate if not stored properly. Reconstituted solutions should be centrifuged before use to remove any aggregates. The compound is for research use only, not for human or veterinary use. DX600 is available from multiple research chemical suppliers. Always follow the manufacturer‘s instructions for handling and storage. Dispose of waste according to local regulations. The compound is typically supplied as a lyophilized powder. Upon receipt, store at -20degC or -80degC in a desiccator. Let the vial warm to room temperature before opening to prevent moisture condensation. Dissolve the powder in an appropriate solvent (water, PBS, or saline) to achieve the desired concentration. Avoid vortexing to prevent denaturation. For long-term storage, aliquot and store at -80degC. Use within 1-3 months after reconstitution. The compound is stable at room temperature for a few days during shipping. Perform a stability test if long-term storage in solution is required. The compound is sensitive to light and should be protected from light. The peptide may contain a disulfide bond between two cysteine residues; reducing agents should be avoided as they may disrupt the disulfide bond and inactivate the compound. The compound is a potent ACE2 inhibitor and should be handled with appropriate care. Avoid contact with skin and eyes. Use appropriate personal protective equipment (lab coat, gloves, safety glasses). Work in a well-ventilated area. Wash hands thoroughly after handling. In case of accidental exposure, flush with water and seek medical advice if necessary.

The compound should not be used in humans. It is intended for research purposes only. Long-term toxicity studies have not been performed. However, since DX600 exacerbates diabetes-induced cardiovascular and renal dysfunction, it should be used with caution in animal models, especially those with pre-existing cardiovascular or renal disease. Monitor animals closely for signs of toxicity. In the diabetes model, DX600 treatment was well-tolerated at 5 microg/kg/day IP for 4 weeks, with no apparent signs of acute toxicity. In the thrombosis model, a single IV dose of 0.1 micromol/L/kg was well-tolerated. Nevertheless, comprehensive safety and toxicological studies (e.g., acute toxicity, chronic toxicity, genotoxicity) have not been performed. The compound is not for clinical use. Researchers should be aware that inhibition of ACE2, which is a protective component of the RAS, could lead to deleterious effects in certain disease contexts, which is indeed the intended purpose of using DX600 as a tool to study the consequences of ACE2 loss-of-function. Therefore, the compound itself may cause adverse effects in experimental animals, reflecting the biology of ACE2 rather than off-target toxicity. Baseline health and disease status of animals should be carefully considered when using DX600. The compound is not intended for use in healthy animals without proper justification and oversight. Researchers should design experiments with appropriate endpoints to monitor animal welfare.

[1]. Development of an enzymatic assay for the detection of neutralizing antibodies against therapeutic angiotensin-converting enzyme 2 (ACE2).J Immunol Methods. 2013 Mar 29;389(1-2):52-60.

[2]. Characterization of Angiotensin-(1-7) effects on the cardiovascular system in an experimental model of type-1 diabetes. Pharmacol Res. 2012 Sep;66(3):269-75.

[3]. Extrinsic stabilization of antiviral ACE2-Fc fusion proteins targeting SARS-CoV-2. Commun Biol. 2023 Apr 8;6(1):386.

[4]. Angiotensin converting enzyme versus angiotensin converting enzyme-2 selectivity of MLN-4760 and DX600 in human and murine bone marrow-derived cells. Eur J Pharmacol. 2016 Mar 5;774:25-33.

[5]. ACE2 activation promotes antithrombotic activity. Mol Med. 2010 May-Jun;16(5-6):210-5.

DX600 is a highly specific and potent ACE2 inhibitor that serves as a valuable tool for studying the physiological and pathophysiological roles of ACE2 in the renin-angiotensin system (RAS), cardiovascular and renal diseases, diabetes, thrombosis, and infectious diseases such as COVID-19 (as ACE2 is the cellular receptor for SARS-CoV-2). The compound has been used in numerous research studies to demonstrate the protective effects of ACE2. By inhibiting ACE2, DX600 increases angiotensin II levels and decreases angiotensin-(1-7) levels, shifting the RAS balance toward the pro-inflammatory, pro-fibrotic, vasoconstrictive arm. This makes DX600 useful for studying the consequences of ACE2 deficiency or inhibition. The peptide inhibitor is highly selective, does not cross-react with ACE, allowing researchers to isolate the specific effects of ACE2 inhibition. DX600 is available as a research-grade peptide (typically as the TFA salt) from multiple commercial suppliers. It is not a marketed drug and is not approved for human use. The compound is for research use only. It is often used in combination with ACE inhibitors to study the distinct roles of ACE and ACE2. The sequence of DX600 is Ac-Gly-Asp-Tyr-Ser-His-Cys-Ser-Pro-Leu-Arg-Tyr-Tyr-Pro-Trp-Trp-Lys-Cys-Thr-Tyr-Pro-Asp-Pro-Glu-Gly-Gly-Gly-amide. The peptide contains a disulfide bridge between the two cysteine residues (positions 6 and 17 from the N-terminus). The amide group at the C-terminus increases stability against carboxypeptidase degradation. The molecular weight is approximately 3074 Da. The TFA salt is often supplied for research use. The compound is stable when stored as a lyophilized powder at -20degC or -80degC. Reconstituted solutions should be stored frozen in small aliquots. Avoid repeated freeze-thaw cycles. The compound is sensitive to light; store in a dark container or protect from light. Use appropriate personal protective equipment when handling. DX600 should not be used in humans. Additional research is ongoing to explore the therapeutic potential of targeting ACE2 in diseases such as hypertension, heart failure, diabetic nephropathy, and COVID-19. DX600 is a critical tool for these investigations.

C141H185N35O40S2

3074.32

3187.292

478188-26-0

DX600 TFA

146158982

White to off-white solid powder

41

50

63

225

7360

0

C([C@H]1C(N[C@H](C(N[C@H](C(N[C@@H](CSSC[C@@H](C(N[C@H](C(N2CCC[C@@]2([H])C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N2CCC[C@@]2([H])C(N1)=O)=O)CC1C=CC(=CC=1)O)=O)CC1C=CC(=CC=1)O)=O)CCCNC(N)=N)=O)CC(C)C)=O)=O)CO)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)CNC(=O)C)CC1C=CC(=CC=1)O)CC1NC=NC=1)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC1C=CC(=CC=1)O)C(N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)NCC(=O)NCC(=O)NCC(=O)N)=O)=O)=O)CCCCN)=O)CC1=CNC2=CC=CC=C12)=O)C1=CNC2=CC=CC=C12

OXNRLFLIPDHJEQ-UHFFFAOYSA-N

InChI=1S/C141H185N35O40S2.C2HF3O2/c1-72(2)50-93-122(198)157-91(21-11-45-147-141(144)145)120(196)159-94(51-75-26-34-82(181)35-27-75)123(199)165-100(53-77-30-38-84(183)39-31-77)137(213)173-46-13-23-108(173)134(210)164-97(56-80-61-150-89-19-8-6-17-87(80)89)126(202)161-96(55-79-60-149-88-18-7-5-16-86(79)88)125(201)156-90(20-9-10-44-142)121(197)170-106(131(207)172-118(73(3)179)136(212)167-101(54-78-32-40-85(184)41-33-78)138(214)174-47-14-25-110(174)135(211)166-102(59-117(193)194)139(215)175-48-12-22-107(175)132(208)158-92(42-43-115(189)190)119(195)153-65-113(187)152-64-112(186)151-63-111(143)185)70-218-217-69-105(130(206)169-104(68-178)140(216)176-49-15-24-109(176)133(209)163-93)171-127(203)98(57-81-62-146-71-154-81)162-129(205)103(67-177)168-124(200)95(52-76-28-36-83(182)37-29-76)160-128(204)99(58-116(191)192)155-114(188)66-148-74(4)180;3-2(4,5)1(6)7/h5-8,16-19,26-41,60-62,71-73,90-110,118,149-150,177-179,181-184H,9-15,20-25,42-59,63-70,142H2,1-4H3,(H2,143,185)(H,146,154)(H,148,180)(H,151,186)(H,152,187)(H,153,195)(H,155,188)(H,156,201)(H,157,198)(H,158,208)(H,159,196)(H,160,204)(H,161,202)(H,162,205)(H,163,209)(H,164,210)(H,165,199)(H,166,211)(H,167,212)(H,168,200)(H,169,206)(H,170,197)(H,171,203)(H,172,207)(H,189,190)(H,191,192)(H,193,194)(H4,144,145,147);(H,6,7)

4-[[1-[2-[[1-[2-[[2-[[24-[[2-[[2-[[2-[[2-[(2-acetamidoacetyl)amino]-3-carboxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-32-(4-aminobutyl)-9-(3-carbamimidamidopropyl)-21-(hydroxymethyl)-3,6-bis[(4-hydroxyphenyl)methyl]-35,38-bis(1H-indol-3-ylmethyl)-12-(2-methylpropyl)-2,5,8,11,14,20,23,31,34,37,40-undecaoxo-26,27-dithia-1,4,7,10,13,19,22,30,33,36,39-undecazatricyclo[39.3.0.015,19]tetratetracontane-29-carbonyl]amino]-3-hydroxybutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-carboxypropanoyl]pyrrolidine-2-carbonyl]amino]-5-[[2-[[2-[(2-amino-2-oxoethyl)amino]-2-oxoethyl]amino]-2-oxoethyl]amino]-5-oxopentanoic acid;2,2,2-trifluoroacetic acid

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)

H2O: 50 mg/mL (16.26 mM)

Solubility in Formulation 1: 50 mg/mL (16.26 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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