ADAM17:11.1 nM (IC50); ADAM10:748 nM (IC50); MMP2:717 nM (IC50); MMP13:930 nM (IC50); MMP14:2140 nM (IC50); MMP8:2200 nM (IC50); MMP9:5410 nM (IC50); MMP17:7100 nM (IC50); MMP3:9760 nM (IC50)
ADAM17 (IC50: 11.1 nM).
ADAM10 (IC50: 748 nM).
MMP1 (IC50: >100,000 nM).
MMP2 (IC50: 717 nM).
MMP3 (IC50: 9,760 nM).
MMP8 (IC50: 2,200 nM).
MMP9 (IC50: 5,410 nM).
MMP13 (IC50: 930 nM).
MMP14 (IC50: 2,140 nM).
MMP17 (IC50: 7,100 nM). [1]

KP-457 is a selective metalloproteinase 17 (ADAM17) inhibitor, with higher selectivity for ADAM17 than for other MMPs and ADAM10, and IC50s are 11.1 nM (ADAM17), 748 nM (ADAM10), 717 nM (MMP2), 9760 nM (MMP3), 2200 nM (MMP8), 5410 nM (MMP9), 930 nM (MMP13), 2140 nM (MMP14), and 7100 nM (MMP17), respectively. KP-457 blocks Zn2+ chelation of the catalytic domain of ADAM17. KP-457 (15 μM) retains the expression of GPIbα on iPSC-derived platelets[1].

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In a cell-free enzyme activity assay, KP-457 potently inhibited ADAM17-catalyzed cleavage of a TNF-α sequence-based substrate with an IC50 of 11.1 nM, which was about 10 times more potent than the pan-metalloproteinase inhibitor GM-6001 (IC50: 163 nM). KP-457 showed high selectivity for ADAM17 over other MMPs and ADAM10 (selectivity >50-fold compared to ADAM10 and most MMPs). [1]
In a cellular assay using washed human platelets, KP-457 dose-dependently inhibited carbonyl cyanide m-chlorophenylhydrazone (CCCP, 100 μM)-induced shedding of glycoprotein Iba (GPIba) with greater potency than GM-6001. At 15 μM, KP-457 completely blocked GPIba shedding, maintaining GPIba levels comparable to fresh platelets, while the p38 MAPK inhibitors SB203580 and BIRB796 provided only partial inhibition. [1]
During the in vitro generation of human induced pluripotent stem cell (iPSC)-derived platelets at 37°C, the addition of KP-457 (15 μM) to the culture medium effectively preserved GPIba expression on CD41a+ platelets without significantly affecting the yield of megakaryocytes (MKs) or total platelet numbers. In contrast, p38 MAPK inhibitors increased MK and total platelet yields but failed to preserve GPIba expression. The combination of KP-457 and the p38 MAPK inhibitor BIRB796 (10 μM) additively increased the yield of GPIba+ iPSC-derived platelets. [1]
KP-457 (15 μM) did not adversely affect the aggregation of human platelets induced by thrombin or collagen, whereas p38 MAPK inhibitors SB203580 and BIRB796 diminished collagen-induced platelet aggregation. [1]
iPSC-derived platelets generated in the presence of KP-457 exhibited improved von Willebrand factor (vWF)/GPIba-dependent aggregation in response to ristocetin in a flow cytometry-based assay, with performance comparable to fresh human platelets derived from peripheral blood. [1]
Transmission electron microscopy analysis suggested that imMKCL (immortalized megakaryocyte progenitor cell line)-derived platelets generated with KP-457 had improved structural properties. [1]

In a thrombus formation model using immunodeficient mice after platelet transfusion, induced pluripotent stem cells (iPSCs) platelets generated with KP-457 exerts good hemostatic function[1].

A fluorescence-based enzymatic activity assay was used to measure the inhibitory effects of KP-457 and GM-6001 on ADAM17. Recombinant human ADAM17 enzyme was incubated with a fluorogenic substrate peptide (Nma-LAQAVRSSK(Dnp)-NH2), which is based on the cleavage site of TNF-α. Inhibitors at various concentrations were pre-incubated with the enzyme. The enzymatic reaction was carried out under defined conditions, and the fluorescence signal resulting from substrate cleavage was measured. The half-maximal inhibitory concentration (IC50) was calculated from the dose-response curves. [1]
Additionally, the inhibitory activity of KP-457 against ADAM17-mediated cleavage of a GPIba-based peptide substrate was measured using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The assay utilized a synthetic GPIba-based substrate peptide (KKTIPELDQPPKLRGVLGGHLESSRNDPFLHPDF), a C-terminal-based standard peptide (VLGGHLESSRNDPFLHPDF), and an internal standard peptide (VTTGKGQDHSPFWGF). The reaction mixture containing ADAM17 and the substrate was incubated with or without the inhibitor. The formation of the C-terminal cleavage product was quantified by LC/MS/MS to determine the inhibitory potency (IC50). [1]

Briefly, 3 × 104 hematopoietic progenitor cells (HPCs) derived from iPS-sacs on C3H10T1/2 feeder cells in the presence of 20 ng/mL vascular endothelial growth factor are transferred on day 14 of culture onto C3H10T1/2 feeder cells in differentiation medium supplemented with 50 ng/mL stem cell factor, 100 ng/mL thrombopoietin, 25 U/mL heparin sodium, and KP-457 at 24°C or 37°C. The medium is refreshed every 3 days, and nonadherent cells are collected and analyzed on days 22-24[1].

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To assess the inhibition of GPIba shedding in a cellular context, washed human platelets were prepared and suspended in Tyrode-HEPES buffer. Platelets were treated with 100 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce GPIba shedding, in the presence or absence of various concentrations of KP-457 or other inhibitors. The mixture was incubated overnight at 37°C. After incubation, the platelets were stained with fluorescently labeled antibodies against CD41a and GPIba (CD42b) and analyzed by flow cytometry to determine the percentage of CD41a+ platelets that retained GPIba expression. [1]
For generating iPSC-derived platelets, hematopoietic progenitor cells (HPCs) derived from iPSCs were cultured on feeder cells in differentiation medium supplemented with stem cell factor, thrombopoietin, and heparin. KP-457 (15 μM) or other inhibitors were added to the culture medium during both the megakaryocyte differentiation and platelet production phases at 37°C. The medium was refreshed periodically. Non-adherent cells were collected on days 22-24 and analyzed by flow cytometry using antibodies against CD41a, GPIX (CD42a), and GPIba (CD42b) to quantify MKs, total platelets, and the GPIba+ platelet population. [1]
A flow cytometry-based platelet aggregation assay was performed to evaluate vWF/GPIba-dependent function. Aliquots of iPSC-derived platelets or fresh human platelets were stained with different fluorescent markers (anti-CD9-APC or anti-CD9-V450). Equal numbers of platelets stained with each marker were mixed and suspended in Tyrode-HEPES solution containing a thrombin inhibitor, human plasma, and calcium chloride. The mixed platelet suspension was stimulated with ristocetin (2 mg/ml) to induce vWF/GPIba-mediated aggregation for 10 minutes at 37°C while swirling. The reaction was stopped by fixation, and the samples were analyzed by flow cytometry. Platelet aggregation was quantified by calculating the percentage of double-colored events (platelets positive for both fluorescent markers) relative to the total platelet population. [1]

The in vivo hemostatic function of iPSC-derived platelets generated with KP-457 was evaluated in a thrombus formation model using immunodeficient NOD/SCID/IL-2Rg-null (NOG) mice. The mice were first irradiated to induce thrombocytopenia. Equal numbers (1 x 10^7 cells) of tetramethylrhodamine-labeled CD41a+ iPSC-derived platelets generated with or without KP-457 (15 μM during culture) were transfused into the mice intravenously. To induce thrombus formation, mesenteric capillaries were exposed and injured by laser irradiation in the presence of hematoporphyrin (a photosensitizer that generates reactive oxygen species upon laser exposure). The mice were also administered FITC-dextran (to visualize blood vessels) and Hoechst33342 (a nuclear stain). The dynamics of platelet attachment and thrombus formation at the injury site in mesenteric capillaries were visualized and recorded in real-time using a confocal laser scanning microscope. The number of transfused iPSC platelets participating in thrombus formation per unit length of blood vessel was quantified from the images. [1]

Good laboratory practice (GLP) studies using KP-457 showed that in dogs, KP-457 did not exhibit either genotoxicity or systemic toxicity at intravenous doses up to 3 mg/kg once daily for 4 weeks (maximum test dose). [1]

[1]. Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets. Stem Cells Transl Med. 2017 Mar;6(3):720-730.

KP-457 is a novel selective ADAM17 inhibitor with a trans-hydroxyxamic acid structure designed to inhibit the shedding of platelet glycoprotein Iba (GPIba). Its chemical name is N-(2-((4-(but-2-yn-1-oxy)phenyl)sulfonyl)-1-(4-(methylsulfonamide methyl)phenyl)ethyl)-N-hydroxyformamide. [1] Studies have shown that selective inhibition of ADAM17 using KP-457 (rather than inhibition of upstream regulators such as p38 MAPK) is an effective and safe strategy to maintain GPIba expression during in vitro generation of human iPSC-derived platelets at 37°C, which is crucial for efficient platelet production. Preservation of GPIba improves vWF-dependent platelet aggregation in vitro and enhances its hemostatic function in vivo. [1]
KP-457 is considered an ideal process aid for the clinical-scale production of functional iPSC-derived platelet concentrates for transfusion due to its selectivity, potency, and established safety in animal toxicity studies. [1]

C21H24N2O7S2

480.554463386536

480.102

1365803-52-6

56598918

White to off-white solid powder

1.2

2

8

10

32

865

0

CS(NCC1=CC=C(C(N(C=O)O)CS(C2=CC=C(OCC#CC)C=C2)(=O)=O)C=C1)(=O)=O

VIWDSTUVCAZZDF-UHFFFAOYSA-N

InChI=1S/C21H24N2O7S2/c1-3-4-13-30-19-9-11-20(12-10-19)32(28,29)15-21(23(25)16-24)18-7-5-17(6-8-18)14-22-31(2,26)27/h5-12,16,21-22,25H,13-15H2,1-2H3

N-[2-(4-but-2-ynoxyphenyl)sulfonyl-1-[4-(methanesulfonamidomethyl)phenyl]ethyl]-N-hydroxyformamide

KP457 KP 457KP-457

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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