HCH6-1 is a competitive antagonist of Formyl Peptide Receptor 1 (FPR1).
IC50 for inhibition of superoxide anion generation in fMLF (FPR1 agonist)-activated human neutrophils: 0.32 ± 0.03 µM [1]
IC50 for inhibition of elastase release in fMLF-activated human neutrophils: 0.57 ± 0.07 µM [1]

HCH6-1, with an IC50 of 0.32 μM, markedly reduced the production of superoxide anion in fMLF (FPR1 agonist)-activated neutrophils in a cell-impermeable cytochrome c reduction assay. HCH6-1, with an IC50 of 4.98±0.27 μM and 17.68±2.77 μM, respectively, has a slight inhibitory effect on neutrophils activated by WKYMVm (a FPR1/FPR2 dual agonist) and MMK1 (a FPR2 agonist) [1]. HCH6-1 has no cytotoxic effect on human neutrophils because it does not cause LDH release, not even at 30 μM. In cell-free systems, HCH6-1 has no effect on the levels of DPPH radicals, xanthine/xanthine oxidase superoxide anion, or either of these [1]. At an IC50 of 0.57 μM, HCH6-1 dramatically reduces the release of elastase from fMLF-activated neutrophils. Nevertheless, HCH6-1 inhibits elastase release at higher concentrations in WKYMVm or MMK1-triggered neutrophils (IC50 of 5.22±0.69 μM and 10.00±0.65 μM, respectively) [1].

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HCH6-1 selectively inhibited extracellular superoxide anion generation induced by the FPR1 agonist fMLF in human neutrophils (IC50 0.32 µM). It showed much weaker inhibition against superoxide generation induced by the dual FPR1/FPR2 agonist WKYMVm (IC50 4.98 µM) and the FPR2 agonist MMK1 (IC50 17.68 µM). [1]
HCH6-1 significantly inhibited intracellular superoxide anion generation (measured by hydroethidine oxidation and flow cytometry) in fMLF-activated human neutrophils but not in MMK1-activated cells. [1]
HCH6-1 selectively inhibited elastase release from human neutrophils activated by fMLF (IC50 0.57 µM), with weaker effects against WKYMVm- (IC50 5.22 µM) and MMK1- (IC50 10.00 µM) induced release. [1]
HCH6-1 did not inhibit superoxide anion generation induced by direct G-protein activator (NaF) or protein kinase C activator (PMA). It also failed to significantly inhibit elastase release induced by NaF, leukotriene B4 (BLT1 agonist), or interleukin-8. [1]
HCH6-1 dose-dependently diminished fMLF-induced upregulation of adhesion molecule CD11b expression and inhibited fMLF-induced neutrophil migration in a chemotaxis assay. It did not affect MMK1-induced responses. [1]
HCH6-1 (up to 30 µM) did not cause lactate dehydrogenase (LDH) release from human neutrophils, indicating no cytotoxicity at tested concentrations. [1]
In cell-free systems, HCH6-1 did not scavenge superoxide anion (tested in xanthine/xanthine oxidase system with WST-1) or DPPH radicals, ruling out direct antioxidant effects. [1]
HCH6-1 competitively inhibited the binding of a fluorescent fMLF analog (FNLFNYK) to FPR1 on human neutrophils, differentiated THP-1 cells (neutrophil-like), and hFPR1-transfected HEK293 cells, as shown by flow cytometry. NMR analysis indicated no direct structural interaction between HCH6-1 and fMLF. [1]
HCH6-1 dose-dependently and competitively attenuated the transient increase in intracellular calcium concentration ([Ca2+]i) induced by fMLF in human neutrophils, but did not affect calcium mobilization induced by MMK1, LTB4, or IL-8. [1]
HCH6-1 competitively attenuated fMLF-induced phosphorylation of ERK, p38 MAPK, JNK, and Akt proteins in human neutrophils, as shown by immunoblotting. [1]
HCH6-1 did not increase intracellular cAMP levels in human neutrophils. The PKA inhibitor H89 did not reverse the inhibitory effects of HCH6-1 on fMLF-induced responses, indicating that the cAMP/PKA pathway is not involved in its mechanism. [1]

Cavity inflammation was not induced by HCH6-1 alone (intraperitoneal injection; 50 mg/kg; 1 hour before or 30 minutes after LPS nebulization). In the presence of LPS, pretreatment with HCH6-1 decreased inflammatory cell infiltration and lung structural deformation. In LPS-induced ALI mice, HCH6-1 post-treatment demonstrated an inhibitory effect on neutrophil accumulation and lung damage [1].

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In a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model, intraperitoneal pretreatment with HCH6-1 (50 mg/kg, 1 hour before LPS) significantly ameliorated lung injury. It reduced LPS-induced lung congestion, inflammatory cell infiltration, inter-alveolar septal thickening, and interstitial edema observed in histopathological sections (H&E staining). [1]
Pretreatment with HCH6-1 significantly reduced LPS-induced elevation of myeloperoxidase (MPO) activity and total protein concentration in lung tissues, indicating reduced neutrophil infiltration and pulmonary edema. [1]
Immunofluorescence staining for Ly6G (a neutrophil marker) showed that HCH6-1 pretreatment reduced the number of Ly6G-positive cells infiltrating the lungs after LPS challenge. [1]
Post-treatment with HCH6-1 (50 mg/kg, intraperitoneal injection 30 minutes after LPS) also showed protective effects, reducing neutrophil accumulation (lower MPO activity) and lung damage in the ALI model. [1]

Superoxide Anion Generation Assay: Extracellular superoxide anion production by activated human neutrophils was measured by superoxide dismutase-inhibitable ferricytochrome c reduction. Briefly, neutrophils were preincubated with ferricytochrome c and Ca2+, then treated with test compounds for 5 minutes before activation by various stimuli (fMLF, WKYMVm, MMK1, NaF, PMA) in the presence or absence of cytochalasin B priming. The change in absorbance at 550 nm was continuously monitored. [1]
Intracellular Superoxide Anion Detection: Neutrophils were labeled with hydroethidine (HE) for 15 minutes, incubated with test drugs for 5 minutes, and then stimulated. The fluorescence intensity of oxidized ethidium was measured by flow cytometry to assess intracellular superoxide production. [1]
Elastase Release Assay: Degranulation was measured by elastase release using methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as the substrate. Neutrophils were mixed with substrate and CaCl2, treated with test compounds for 5 minutes, and then activated by various stimuli. The release of elastase was monitored spectrophotometrically at 405 nm. [1]
Chemotaxis Assay: Neutrophil migration was assessed using a 24-well microchemotaxis chamber with a 3 µm pore membrane. Neutrophils pretreated with test drugs were placed in the top chamber, and chemoattractants were placed in the bottom chamber. The number of migrated cells was counted after incubation. [1]
Receptor Binding Assay: Binding of HCH6-1 to FPR1 was assessed by competitive inhibition of a fluorescent fMLF analog (FNLFNYK). Cells (human neutrophils, differentiated THP-1 cells, or hFPR1-transfected HEK293 cells) were incubated with test drugs for 5 minutes at 4°C and then labeled with FNLFNYK for 30 minutes. Binding was quantified by flow cytometry. [1]
Intracellular Calcium Measurement: Neutrophils were loaded with Fluo-3/AM for 30 minutes. After treatment with test drugs, stimulants were added, and the fluorescence emission at 520 nm (excitation 488 nm) was recorded in real-time using a spectrofluorometer to calculate the [Ca2+]i. [1]
Immunoblotting: Neutrophils preincubated with test drugs were stimulated with fMLF for 30 seconds, then lysed. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated and total forms of ERK, p38, JNK, and Akt. Bands were visualized by enhanced chemiluminescence. [1]

Animal/Disease Models: C57BL/6 mice (20-25 g, 7-8 weeks old) [1]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; 50 mg/kg; 1 hour before LPS spray or after LPS spray 30 min
Experimental Results: Improved ALI in LPS-induced mice. HCH6-1-mediated reduction in neutrophil recruitment is a protective mechanism in ALI mice.

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LPS-induced Acute Lung Injury (ALI) Model: Male C57BL/6 mice (7-8 weeks old, 20-25 g) were anesthetized with isoflurane. ALI was induced by intra-tracheal spray of 50 µg LPS (E. coli 0111:B4) dissolved in 40 µl normal saline. Control mice received saline spray. [1]
Drug Administration: HCH6-1 was dissolved in DMSO and administered via intraperitoneal injection at a dose of 50 mg/kg body weight. For pretreatment, the drug was given 1 hour before LPS challenge. For post-treatment, the drug was given 30 minutes after LPS challenge. Control groups received vehicle (DMSO). [1]
Sample Collection: Mice were euthanized 6 hours after LPS application. Lungs were removed. The left lobe was fixed for histology, and the right lobes were used for biochemical assays (MPO activity, total protein). [1]

In vitro studies showed that HCH6-1 at concentrations up to 30 µM did not induce significant release of lactate dehydrogenase (LDH) from human neutrophils, indicating that HCH6-1 was not acutely cytotoxic at the tested concentrations. [1] In vivo studies showed that intraperitoneal injection of HCH6-1 (50 mg/kg) alone into mice did not induce airway inflammation or significant lung injury, similar to the sham-operated control group. No other specific toxicity data (e.g., LD50, organ toxicity) were reported. [1]

[1]. Dipeptide HCH6-1 inhibits neutrophil activation and protects against acute lung injury by blocking FPR1. Free Radic Biol Med. 2017 May;106:254-269.

HCH6-1 (N-(N-benzoyl-L-tryptophanyl)-D-phenylalanine methyl ester) is a synthetic dipeptide derived from hesperidin acetate. [1] It is a selective competitive antagonist of formyl peptide receptor 1 (FPR1), a G protein-coupled receptor involved in neutrophil activation during inflammation. [1] Its mechanism of action involves competitive binding to FPR1, thereby inhibiting downstream signaling pathways, including calcium mobilization and phosphorylation of MAPK (ERK, p38, JNK) and Akt, which are essential for neutrophil functions such as superoxide production, degranulation, and chemotaxis. [1] This study suggests that HCH6-1 has therapeutic potential for treating neutrophil inflammatory diseases, particularly acute lung injury (ALI), by inhibiting FPR1-mediated neutrophil recruitment and activation. [1]

C28H27N3O4

469.531687021255

469.2

1435265-06-7

71697968

White to off-white solid powder

1.259±0.06 g/cm3(Predicted)

790.8±60.0℃at 760 mmHg

3.8

3

4

10

35

717

2

COC(=O)[C@@H](CC1=CC=CC=C1)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)C4=CC=CC=C4

MPFZAIDTRUPTSU-LOSJGSFVSA-N

InChI=1S/C28H27N3O4/c1-35-28(34)25(16-19-10-4-2-5-11-19)31-27(33)24(30-26(32)20-12-6-3-7-13-20)17-21-18-29-23-15-9-8-14-22(21)23/h2-15,18,24-25,29H,16-17H2,1H3,(H,30,32)(H,31,33)/t24-,25+/m0/s1

methyl (2R)-2-[[(2S)-2-benzamido-3-(1H-indol-3-yl)propanoyl]amino]-3-phenylpropanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~250 mg/mL (~532.45 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.43 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 20.8 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: ≥ 2.08 mg/mL (4.43 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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