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| Targets |
KPT-6566 specifically targets the peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1). PIN1 is a phosphorylation-specific enzyme that catalyzes the cis-trans isomerization of peptide bonds preceding proline, thereby modulating the conformation and function of numerous signaling proteins. KPT-6566 acts as a covalent inhibitor, forming an irreversible bond with the catalytic site of PIN1, specifically the Cys113 residue. This binding is so effective that it leads to the selective degradation of the PIN1 protein. The compound exhibits potent inhibitory activity against the PIN1 PPIase domain, with an IC₅₀ of 640 nM and a Ki of 625.2 nM.
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| ln Vitro |
KPT-6566 (1–5 μM; 0–8 days) suppresses the proliferation of WT fibroblasts [1]. In a PIN1-dependent way, KPT-6566 (0-10 μM; 48 hours) reduces the viability of cancer cells and normal breast epithelial cells [1]. Cyclin D1, PIN1 concentrations, and hyperphosphorylated pRB levels are all impacted by KPT-6566 (0-10 μM; 48 hours) [1]. The mut-p53, NOTCH1, and NRF2 pathways are inhibited by KPT-6566 (2.5–5 μM; 48 hours) [1]. In a PIN1-dependent way, KPT-6566 (0-5 μM; 48 hours) causes damage to DNA [1].
In vitro, KPT-6566 demonstrates potent and selective inhibition of PIN1's enzymatic activity. The compound's potency is quantified by its IC₅₀ of 640 nM and Ki of 625.2 nM against the PIN1 PPIase domain. Mechanistically, its covalent binding to PIN1's catalytic site leads to the release of a quinone-mimicking byproduct that generates reactive oxygen species (ROS) and induces DNA damage. This activity is highly selective for cancer cells, which overexpress PIN1. KPT-6566 (1-5 μM; 0-8 days) inhibits wild-type fibroblast proliferation, reduces the viability of cancer cells and normal breast epithelial cells in a PIN1-dependent manner (0-10 μM; 48 hours), and impacts cyclin D1 and hyperphosphorylated pRB levels. |
| ln Vivo |
Mice are not harmful to KPT-6566 (5 mg/kg; intraperitoneally administered once daily for 26 days) [1].
In vivo, KPT-6566 has demonstrated significant anti-cancer activity by effectively impairing PIN1-dependent cancer phenotypes. Specifically, treatment with KPT-6566 has been shown to inhibit the growth of lung metastases in preclinical models. The compound's ability to generate reactive oxygen species and DNA damage selectively in cancer cells translates into a potent anti-tumor effect in living organisms. Furthermore, KPT-6566 induces apoptotic cell death and suppresses the tumorigenicity of testicular germ cell tumors. |
| Enzyme Assay |
Non-cell-based assays for KPT-6566 typically involve measuring its ability to inhibit PIN1's enzymatic activity. The standard assay is the chymotrypsin-coupled PPIase assay, which measures the cis-trans isomerization activity of PIN1 on a synthetic peptide substrate. In this assay, recombinant human PIN1 protein is incubated with a substrate peptide (Suc-Ala-Glu-Pro-Phe-p-nitroanilide) in the presence of varying concentrations of KPT-6566. The reaction is initiated by adding the enzyme and α-chymotrypsin, which cleaves the trans isomer of the substrate to release a chromogenic product measured at 390 nm. The rate of the reaction is proportional to PIN1 activity, and the IC₅₀ is calculated by plotting inhibition against the logarithm of the inhibitor concentration. Mass spectrometry analyses have confirmed that KPT-6566 covalently modifies PIN1 via conjugate addition.
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| Cell Assay |
Cell proliferation assay[1]
Cell Types: WT and Pin1 KO mouse embryo-derived immortalized fibroblasts Tested Concentrations: 1 and 5 μM Incubation Duration: 0-8 days Experimental Results: Dose-dependent inhibition of proliferation of WT fibroblasts, whereas Pin1 KO had no effect on fibroblasts. Cell viability assay[1] Cell Types: MCF10A, HMEC, HeLa, LNCaP, SKOV-3, PANC-1, PC-3, MDA-MB-468 and MDA-MB-231 Cell Tested Concentrations: 0-10 μM Incubation Duration: 48 hour Experimental Results: Inhibited the viability of normal breast epithelial cells and cancer cells even at low concentrations, and increased the concentration of PIN1 in MDA-MB-468, SKOV-3, PC-3, LNCaP and PANC-1. Western Blot Analysis[1] Cell Types: Immortalized fibroblasts derived from WT and Pin1 KO mouse embryos and PIN1 KO MDA-MB-231 cells Tested Concentrations: 0-10 μM Incubation Duration: 48 hrs (hours) Experimental Results: Hyperphosphorylated pRB and Cyclin D1 levels, dose diminished - and promoted PIN1 degradation over time. Western Blot Analysis[1] Cell Types: MDA-MB-231, MCF10A, MDA-MB-468, and Cellular assays for KPT-6566 are performed using various cancer cell lines to assess its anti-proliferative and cytotoxic effects. In cell viability assays, such as the MTT assay, cancer cells are treated with KPT-6566 at concentrations ranging from 0 to 10 μM for 48 hours. The compound reduces cell viability in a PIN1-dependent manner. Colony formation assays are used to assess long-term anti-proliferative effects, with KPT-6566 showing an IC₅₀ of 1.2 μM for inhibiting colony formation in MDA-MB-231 cells. The compound also impairs secondary mammosphere formation, a marker of cancer stem cell activity. Western blot analysis is used to confirm target engagement and downstream effects, such as decreased cyclin D1 and hyperphosphorylated pRB levels. |
| Animal Protocol |
Animal/Disease Models: 6weeks old female mice, injected with 1 million MDA-MB-231Luc6 cells [1]
Doses: 5 mg/kg Route of Administration: intraperitoneal (ip) injection; 5 mg/kg, one time/day; for 26 days Experimental Results:Passed Postmortem morphological analysis revealed no evidence of local or systemic and organ toxicity. In vivo animal models for KPT-6566 are used to evaluate its anti-tumor efficacy. In a key study, KPT-6566 treatment was shown to impair the growth of lung metastasis in vivo. Furthermore, the compound has demonstrated efficacy in testicular germ cell tumor models, where it induced apoptotic cell death and suppressed tumorigenicity. The compound is typically administered at doses such as 5 mg/kg in these models. These studies confirm the in vivo relevance of PIN1 inhibition as a therapeutic strategy and validate KPT-6566 as a potent anti-cancer agent. |
| ADME/Pharmacokinetics |
KPT-6566 has a molecular weight of 443.54 g/mol and a molecular formula of C₂₂H₂₁NO₅S₂. Its CAS number is 881487-77-0. The formal name is 2-[[4-[[[4-(1,1-dimethylethyl)phenyl]sulfonyl]imino]-1,4-dihydro-1-oxo-2-naphthalenyl]thio]-acetic acid. It is supplied as a solid with a purity of ≥98% and is soluble in DMSO. The compound has UV absorbance maxima at 224, 260, and 324 nm. Storage conditions are typically -20°C, protected from light and under nitrogen. In solvent, it is stable at -80°C for 6 months and at -20°C for 1 month.
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| Toxicity/Toxicokinetics |
Detailed toxicological data for KPT-6566 are not extensively reported in the available literature. As a research compound, its safety profile would need to be established through standard preclinical toxicity assessments. The compound is intended for research use only and is not for human consumption. Standard laboratory safety precautions should be followed when handling the compound. The compound's mechanism, which involves the generation of reactive oxygen species and DNA damage, suggests potential genotoxicity concerns that would require careful evaluation.
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| References | |
| Additional Infomation |
KPT-6566 is a potent and selective covalent inhibitor of the peptidyl-prolyl cis-trans isomerase PIN1. It was identified through a mechanism-based screening approach and is described in a seminal publication in Nature Communications. The compound's defining feature is its ability to covalently bind to the catalytic site of PIN1, leading to its inhibition and subsequent degradation. This interaction releases a quinone-mimicking byproduct that generates ROS and DNA damage, selectively inducing cell death in cancer cells. KPT-6566 is widely used in cancer research to study the role of PIN1 in tumorigenesis, cancer stem cell expansion, and metastasis. It serves as a critical tool compound for validating PIN1 as a therapeutic target.
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| Molecular Formula |
C22H21NO5S2
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| Molecular Weight |
443.535843610764
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| Exact Mass |
443.086
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| CAS # |
881487-77-0
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| PubChem CID |
6022998
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| Appearance |
White to yellow solid powder
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| LogP |
4.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
840
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(CSC1C(=O)C2C(=CC=CC=2)C(=NS(C2C=CC(C(C)(C)C)=CC=2)(=O)=O)C=1)O
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| InChi Key |
BXWWOKYIKNEEHJ-PTGBLXJZSA-N
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| InChi Code |
InChI=1S/C22H21NO5S2/c1-22(2,3)14-8-10-15(11-9-14)30(27,28)23-18-12-19(29-13-20(24)25)21(26)17-7-5-4-6-16(17)18/h4-12H,13H2,1-3H3,(H,24,25)/b23-18+
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| Chemical Name |
2-[(4E)-4-(4-tert-butylphenyl)sulfonylimino-1-oxonaphthalen-2-yl]sulfanylacetic acid
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| Synonyms |
KPT6566; KPT 6566; KPT-6566
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~19.23 mg/mL (~43.36 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1.92 mg/mL (4.33 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 19.2 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. Solubility in Formulation 2: ≥ 1 mg/mL (2.25 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 10.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2546 mL | 11.2729 mL | 22.5459 mL | |
| 5 mM | 0.4509 mL | 2.2546 mL | 4.5092 mL | |
| 10 mM | 0.2255 mL | 1.1273 mL | 2.2546 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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