CRM1/chromosome region maintenance 1
CRM1 (chromosome region maintenance 1 protein, also called exportin 1/Xpo1); binds covalently to Cys528 in the NES-binding groove [1]

On the central convex surface of the CRM1 ring, KPT-251 binds in the NES binding groove [1]. KPT-251 (for 72 hours) prevents the growth of melanoma cells [2]. The phosphorylation levels of p53, pRb, survivin, and ERK are regulated by KPT-251 (1 μM; 0-48 hours) [2]. Apoptosis and cell cycle arrest are induced by KPT-251 (0.1 and 1 μM; 0-72 hours) [2].

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Significant survival improvements are obtained by KPT-251 (75 mg/kg/day; i.e., three times per week for five weeks) which successfully suppresses the development of MV4-11 cells transplanted into NSG mice [1]. In a mouse melanoma xenograft model, KPT-251 (50 mg/kg; oral; every other day for 21 days) reduces the formation of tumors [2].

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KPT-251 (75 mg/kg) administered orally three times per week for 5 weeks to NOD-SCID-IL2Rγnull (NSG) mice engrafted with luciferase-expressing MV4-11 human AML cells led to striking suppression of leukemia growth as measured by bioluminescence imaging (BLI), with no increase in BLI signal during treatment, whereas vehicle-treated mice showed marked increase; KPT-251-treated mice exhibited significantly increased survival (Kaplan-Meier analysis, P<0.0001) with leukemia progression occurring only after cessation of treatment; histopathologic analysis of bone marrow and spleen after 35 days of treatment showed very low levels of leukemic cell infiltration and normal hematopoietic cell morphology and cellularity in KPT-251-treated mice, including developing cells within myeloid, erythroid and megakaryocytic lineages, while vehicle-treated mice showed extensive leukemia infiltration; spleens of KPT-251-treated mice showed prominent extramedullary hematopoiesis. [1]

The 2.2-Å crystal structure of KPT-251 bound to the ternary complex of Saccharomyces cerevisiae CRM1 (with T539C mutation to mimic human Cys528), S. cerevisiae RanBP1 and human RanGTP was determined. The complex was crystallized using reservoir solution containing 18% PEG3350, 200 mM ammonium nitrate, 100 mM Bis-Tris (pH 6.6) supplemented with 20% (v/v) glycerol for cryo-protection. X-ray diffraction data were collected at beamline 19ID, Advanced Photon Source. The structure was solved by molecular replacement using MolRep with the inhibitor-free CRM1-Ran-RanBP1 structure (PDB 3M11) as search model. Model building and refinement were performed with COOT and Refmac5, including TLS refinement. The refined structure shows that KPT-251 binds in the NES-binding groove of CRM1, with interactions almost entirely hydrophobic; the trifluoromethyl phenyl group penetrates much deeper into the groove than NES side chains. The structure is deposited under PDB ID 4GPT. [1]

Western Blot Analysis[2]
Cell Types: Melanoma BRAF WT (Mewo) and mutant cells (A375)
Tested Concentrations: 1 μM
Incubation Duration: 4, 8, 24 and 48 hrs (hours)
Experimental Results: Prevents cytoplasmic p53 degradation, decreases survivin levels, increases ERK phosphorylation pRb and p-pRb levels were diminished in BRAF WT and mutants.

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Cell cycle analysis[2]
Cell Types: Mewo and A375 Cell
Tested Concentrations: 1 μM
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: Shortening of S phase and G1 and/or G2 cell cycle arrest can be observed.

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Apoptosis analysis[2]
Cell Types: Mel-Juso, SK-MEL-28, SK-MEL-5 and A375 Cell
Tested Concentrations: 0.1 and 1 μM
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: caspase-3 and - 7 increased activity in the melanoma cell lines tested in a dose- and time-dependent manner.

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Animal/Disease Models: 7weeks old female NOD-SCID-IL2Rcγnull (NSG) mice, 2 × 106 MV4-11 cells expressing luciferase were introduced through tail vein injection [1]
Doses: 75 mg/kg/day
Route of Administration: gavage, 3 times weekly for 5 weeks
Experimental Results: demonstrated Dramatically increased survival, leukemia progression only after discontinuation of treatment, prevented leukemic cell infiltration into mouse bone marrow and spleen, and protected from normal hematopoiesis Cellular effects.

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Animal/Disease Models: Athymic nude mouse Nu/Nu, melanoma xenograft model [2]
Doses: 50 mg/kg
Route of Administration: Orally, once every other day for 21 days
Experimental Results: Inhibited tumor growth, increased cleaved caspase-3 and lower Ki67.

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Female 7-week-old NOD-SCID-IL2Rγnull (NSG) mice were injected via tail vein with 2×10^6 luciferase-expressing MV4-11 human AML cells. After leukemia establishment documented by bioluminescence imaging (BLI), mice were randomized into two groups of nine mice and treated by gavage either with vehicle control (Pluronic F-68/PVP-K29/32) or KPT-251 at 75 mg/kg/day three times per week for 5 weeks. Blood counts were obtained after 4.5 weeks of treatment. Survival was measured from initiation of therapy until moribund state and assessed by Kaplan-Meier analysis. Femur and spleen tissues were fixed in 10% formalin, sectioned, paraffin-embedded, stained with hematoxylin and eosin, and examined by microscopy. [1]

KPT-251 at 75 mg/kg three times weekly for 4.5 weeks showed minimal toxicity to normal hematopoietic cells: no significant differences in white blood cell counts (P<0.19), neutrophil counts (P<0.62), lymphocyte counts (P<0.75) or percent reticulocytes (P<0.09) between vehicle-treated and KPT-251-treated mice; platelet counts (P<0.0001) and hematocrit readings (P<0.0096) were significantly different but remained within normal ranges. Bone marrow histopathology after 35 days of treatment showed normal hematopoietic cell morphology and cellularity (myeloid, erythroid, megakaryocytic lineages) without evidence of toxicity. The dose-limiting toxicity of this class of CRM1 inhibitors in mice is weight loss, which was remediated by caloric supplementation at the dosages administered. [1]

[1]. Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells. Leukemia. 2013 Jan;27(1):66-74.

[2]. CRM1 and BRAF inhibition synergize and induce tumor regression in BRAF-mutant melanoma. Mol Cancer Ther. 2013 Jul;12(7):1171-9.

Drugs targeting chromosomal region maintenance protein 1 (CRM1), a major mediator of nuclear export, hold potential for treating leukemia. However, existing CRM1 inhibitors exhibit varying potencies and broad cytotoxicity. This article reports the structural analysis and antileukemic activity of a novel class of small-molecule CRM1 inhibitors. These compounds, named Selective Nuclear Export Inhibitors (SINEs), were developed using molecular modeling techniques to screen a small virtual library of compounds in search of inhibitors targeting the CRM1 nuclear export signal (NES) groove. The 2.2 Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies the portion of the CRM1 groove typically occupied by the NES, but its action extends deeper into the groove, thereby blocking CRM1-mediated protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis in 14 acute myeloid leukemia (AML) cell lines representing different molecular subtypes at nanomolar concentrations. When KPT-251 was administered orally to immunodeficient mice transplanted with human AML cells, the drug exhibited potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Therefore, KPT-SINE CRM1 antagonists represent a new class of drugs worthy of further testing in AML patients. [1]

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Resistance to BRAF inhibitor therapy makes the development of BRAF inhibitor-based combination therapies urgent to overcome primary resistance and prevent the emergence of acquired resistance mechanisms. The CRM1 receptor mediates the nuclear export of key proteins required for melanoma proliferation, survival, and resistance. We hypothesize that by inhibiting CRM1-mediated nuclear export, the function of these proteins can be altered, thereby reducing melanoma cell survival and enhancing the antitumor effects of BRAF inhibitors. To test this hypothesis, we used the selective nuclear export inhibitor (SINE) analogues KPT-185, KPT-251, KPT-276, and KPT-330 to inhibit CRM1. Simultaneously, we used the analogues PLX-4720 and PLX-4032 as BRAF inhibitors. We tested these compounds in xenograft tumor models and in vitro melanoma models. In vitro experiments showed that CRM1 inhibition reduced melanoma cell proliferation, and this effect was independent of BRAF mutation status; furthermore, CRM1 inhibition synergistically enhanced the inhibitory effect of BRAF inhibitors on BRAF-mutant melanoma by promoting cell cycle arrest and apoptosis. In melanoma xenograft models, CRM1 inhibitors reduced tumor growth, and this effect was independent of BRAF or NRAS status; when used in combination with BRAF inhibitors, CRM1 inhibitors induced complete regression of BRAF V600E-mutant tumors. Mechanistic studies showed that CRM1 inhibitors are associated with p53 stabilization and the regulation of retinoblastoma protein (pRb) and survivin. In addition, we found that BRAF inhibitors can eliminate extracellular signal-regulated kinase (ERK) phosphorylation associated with CRM1 inhibitors, which may contribute to the synergistic effect of this combination therapy. In summary, CRM1 inhibitors can reduce the survival rate of BRAF-mutant and wild-type melanomas. The combination of CRM1 inhibitors and BRAF inhibitors has a synergistic effect and can induce regression in BRAF-mutant melanomas. [2]

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KPT-251 is a second-generation selective inhibitor of nuclear export (SINE) that irreversibly blocks CRM1 by covalent modification of Cys528. It was developed through in silico molecular modeling screening against the NES groove of CRM1. The compound has an oxadiazole bioisostere replacing a labile ester to improve metabolic stability and bioavailability. KPT-251 binds in the NES-binding groove, and the inhibitor-bound groove is narrower and deeper than the NES-bound groove, indicating conformational plasticity. The trifluoromethyl phenyl group penetrates deeper into the groove than NES side chains, potentially outcompeting cargo proteins. KPT-330, closely related to KPT-251 but with superior pharmacokinetic properties, has entered phase 1 clinical trials in humans with advanced cancers. [1]

C14H7F6N5O

375.228702783585

375.055

C, 44.81; H, 1.88; F, 30.38; N, 18.66; O, 4.26

1388841-50-6

57519758

Off-white to yellow solid powder

3.993

0

11

3

26

491

0

C1=C(C=C(C=C1C(F)(F)F)C(F)(F)F)C2=NN(C=N2)/C=C\C3=NN=CO3

LDFXTRYMMZGKIC-UPHRSURJSA-N

InChI=1S/C14H7F6N5O/c15-13(16,17)9-3-8(4-10(5-9)14(18,19)20)12-21-6-25(24-12)2-1-11-23-22-7-26-11/h1-7H/b2-1-

(Z)-2-{2-[3-(3,5-Bis-trifluoromethyl-phenyl)-[1,2,4]triazol-1-yl]-vinyl}-[1,3,4]oxadiazole

KPT-251; KPT251; (Z)-2-(2-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)vinyl)-1,3,4-oxadiazole; KPT251; SCHEMBL11318201; KPT251?; LDFXTRYMMZGKIC-UPHRSURJSA-N; KPT 251

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.)