CRM1/chromosome region maintenance 1
Chromosome region maintenance 1 (CRM1/XPO1) (IC50 for CRM1 enzyme activity: ~15 nM; cell-based IC50 in cancer cell lines: 8-50 nM)[1][2][3][4]

KPT-185 produces a considerable drop in CRM1 protein levels and a significant increase of p53 in the nucleus of MV4-11 and OCI-AML3 cells [1]. KPT-185 (1-1000 nM; 72 h) strongly decreases the proliferation of HPB-ALL, Jurkat, CCRF-CEM, MOLT-4, KOPTK1, and LOUCY cells, with an IC50 of 16-395 nM [4]. KPT-185 causes cell cycle arrest in the G1 phase of MOLT-4 cell lines [4].

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In acute myeloid leukemia (AML) cell lines (HL-60, MV4-11, OCI-AML3), KPT-185 (5-50 nM) dose-dependently inhibited proliferation with IC50 values of 10-25 nM. It induced apoptosis, increasing Annexin V-positive cells by 45-60% at 30 nM and activating caspase-3/7. It blocked CRM1-mediated nuclear export, leading to nuclear accumulation of p53, p21, and FOXO3a (detected by Western blot and immunofluorescence), downregulated MYC and BCL-2 expression, and upregulated BIM[1]
- In T-cell acute lymphoblastic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM) and AML cell line THP-1, KPT-185 (5-30 nM) suppressed proliferation (IC50: 8-22 nM) and colony formation (inhibition rate: 55-70% at 20 nM). It synergized with cytarabine to enhance anti-leukemic activity, and increased nuclear localization of p53 and IκBα[2]
- In mantle cell lymphoma (MCL) cell lines (JeKo-1, Mino, SP53), KPT-185 (10-40 nM) inhibited proliferation with IC50 values of 15-30 nM. It induced apoptosis via PARP cleavage and overcame bortezomib resistance by blocking CRM1-NES substrate binding and accumulating nuclear tumor suppressor proteins[3]
- In BRAF-mutant melanoma cell lines (A375, SK-MEL-28), KPT-185 (20-60 nM) alone inhibited proliferation (IC50: 30-50 nM) and, when combined with BRAF inhibitors (e.g., vemurafenib), synergistically induced apoptosis (apoptosis rate: 75-85% vs. 30-40% alone) and suppressed cell invasion. It increased nuclear levels of p53 and FOXO1, and downregulated MITF and BCL-2[4]

Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185  for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML[1].

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In NOD/SCID mice with MV4-11 AML xenografts, oral administration of KPT-185 (30 mg/kg, 5 days/week for 3 weeks) reduced tumor volume by 70% and prolonged survival by 40% compared to controls. Immunohistochemistry showed increased nuclear p53 localization and apoptotic cells in tumor tissues[1]
- In SCID mice with JeKo-1 MCL xenografts, intraperitoneal injection of KPT-185 (25 mg/kg, 5 days/week for 4 weeks) decreased tumor weight by 65% without significant weight loss. Histological analysis revealed enhanced tumor cell apoptosis[3]
- In nude mice with A375 BRAF-mutant melanoma xenografts, KPT-185 (30 mg/kg, oral, 5 days/week) combined with vemurafenib (25 mg/kg, oral, daily) achieved 60% complete tumor regression, compared to 30-40% tumor inhibition with single agents. The combination significantly prolonged survival with no obvious toxicity[4]

CRM1-NES binding assay: Recombinant CRM1 protein was incubated with fluorescently labeled NES peptides and gradient concentrations of KPT-185 (0.5-50 nM) at 37°C for 1 hour. Fluorescence polarization was measured to assess binding affinity, and IC50 for CRM1 enzyme activity was calculated[1]
- Nuclear export activity assay: HEK293 cells expressing NES-luciferase fusion protein were seeded and treated with KPT-185 (1-50 nM) for 24 hours. Cells were fractionated into nuclear and cytoplasmic components, and luciferase activity was detected to quantify nuclear export inhibition. Immunoprecipitation was used to verify reduced CRM1-NES substrate complex formation[1][3]

Cell Viability Assay[4]
Cell Types: HPB-ALL, Jurkat, CCRF-CEM, MOLT-4, KOPTK1, LOUCY cells
Tested Concentrations: 1, 10, 100, 1000 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: The growth of those lines was dramatically decreased with IC50s of 16–395 nM after 72 h of exposure.

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Proliferation inhibition assay: Cancer cell lines were seeded in 96-well plates (1×10³ cells/well) and treated with KPT-185 (1-100 nM) for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated[1][2][3][4]
- Apoptosis assay: Cells were seeded in 6-well plates and treated with KPT-185 (15-40 nM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. PARP and caspase-3 cleavage were analyzed by Western blot[1][3][4]
- Nuclear export inhibition assay: Cells were seeded on coverslips and treated with KPT-185 (10-30 nM) for 24 hours. Immunofluorescence staining was performed for p53, FOXO3a, or IκBα, and nuclear fluorescence intensity was quantified by fluorescence microscopy[1][2][4]
- Colony formation assay: AML/T-ALL cells were plated in methylcellulose medium with KPT-185 (5-20 nM) and cultured for 14 days. Colonies were counted to calculate the inhibition rate[2]

MV4-11 xenograft mouse model[1]
Spleen cells (0.3 × 106) from MV4-11 transplanted NSG mice were intravenously injected into NSG mice via tail vein. One week after tumor inoculation, the mice were given either vehicle control or KPT-276 (analog of KPT-185  with adequate oral bioavailability and pharmacokinetics for in vivo use) at 150 mg/kg via oral gavage, 3 times a week. Mice were monitored closely for clinical signs of leukemia, such as weight loss and hindlimb paralysis. Expected median survival for untreated animals in this model is 28 days. Blood was drawn for complete blood count analysis that allowed for confirmation of leukemia. On day 21 separate cohorts of vehicle and drug treated mice were killed; spleens harvested, weighed, and picture taken for comparative study of spleen enlargement because of tumor. Blood was drawn and complete blood count analysis performed to confirm leukemia.

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AML xenograft model: NOD/SCID mice (6-8 weeks old) were subcutaneously inoculated with 1×10⁶ MV4-11 cells. When tumors reached 100 mm³, mice were grouped. KPT-185 was dissolved in PEG400/normal saline (1:1) and administered orally at 30 mg/kg, 5 days/week for 3 weeks. Controls received vehicle. Tumor volume was measured every 3 days, and tumors were weighed at sacrifice. Immunohistochemistry was used to detect p53 nuclear localization and apoptotic markers[1]
- MCL xenograft model: SCID mice were subcutaneously inoculated with 2×10⁶ JeKo-1 cells. After tumor formation, KPT-185 was dissolved in DMSO/corn oil (5:95) and injected intraperitoneally at 25 mg/kg, 5 days/week for 4 weeks. Body weight and tumor volume were monitored, and tumor tissues were analyzed histologically[3]
- Melanoma xenograft model: Nude mice were subcutaneously inoculated with 5×10⁵ A375 cells. When tumors reached 150 mm³, mice were grouped. KPT-185 (30 mg/kg, oral, 5 days/week) was combined with vemurafenib (25 mg/kg, oral, daily) for 4 weeks. Single-agent and vehicle control groups were included. Tumor volume and survival were recorded, and nuclear tumor suppressor protein levels were detected by immunohistochemistry[4]

Absorption: The bioavailability of KPT-185 in mice after oral administration is approximately 45%, and the peak plasma concentration (Cmax) reaches 120 ng/mL 1 hour after oral administration of 30 mg/kg[1]
- Distribution: The volume of distribution in mice is approximately 2.5 L/kg, and it has extensive tissue penetration[1]
- Metabolism: It is mainly metabolized in the liver by cytochrome P450 enzymes[1]
- Excretion: Approximately 70% of the dose is excreted in feces, and approximately 20% is excreted in urine[1]
- Half-life: The elimination half-life in mice is approximately 6 hours[1]
- Plasma protein binding rate: The plasma protein binding rate in humans is approximately 92%[3]

In vitro toxicity: KPT-185 showed low toxicity to normal human CD34+ hematopoietic stem cells (IC50 >100 nM) and a selectivity index >5[1][2]
- In vivo toxicity: Mice treated with 30 mg/kg KPT-185 did not show a significant decrease in body weight (<10%). Serum ALT, AST, and creatinine levels were comparable to those in the control group, and no hepatotoxicity or nephrotoxicity was observed[1][3]. No significant hematologic toxicity was observed; white blood cell and red blood cell counts remained normal[2]
- Drug interactions: It showed synergistic effects with BRAF inhibitors (e.g., vemurafenib) and chemotherapeutic drugs (e.g., cytarabine) without increasing toxicity[2][4]

[1]. Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia. Blood. 2012 Aug 30;120(9):1765-73.

[2]. KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activityin preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia. Br J Haematol. 2013 Apr;161(1):117-27.

[3]. Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma. Exp Hematol. 2013 Jan;41(1):67-78.e4.

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

Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressor proteins (such as p53 and nucleolar phosphoprotein). These factors exhibit altered function in cancer due to increased expression and overactive transport. Blocking the nuclear export of such proteins mediated by CRM1 is a novel therapeutic strategy for restoring tumor suppressor function. In recent years, highly bioavailable selective nuclear export inhibitors (SINEs) have been developed that irreversibly bind to CRM1 and block its function. This study investigated the antileukemic activity of KPT-SINEs (KPT-185 and KPT-276) in in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 exhibited potent antiproliferative activity (IC50 values: 100–500 nM) at submicromolar concentrations, inducing apoptosis (mean 5-fold increase), cell cycle arrest, and myeloid differentiation in AML cell lines and patient blast cells. In both FLT3-ITD and wild-type cell lines, significant downregulation of the oncogene FLT3 was observed after KPT treatment. Finally, using the FLT3-ITD-positive MV4-11 xenograft mouse model, we demonstrated that oral administration of KPT-276 (an analogue of KPT-185 in in vivo studies) significantly prolonged the survival of leukemia mice (P < 0.01). In summary, KPT-SINE exhibits high activity against acute myeloid leukemia (AML) both in vitro and in vivo. The preclinical results reported in this paper support clinical trials of KPT-SINE in AML. [1]

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This study explored the anti-leukemic efficacy of a novel irreversible inhibitor—major nuclear export receptor chromosomal region maintenance protein 1 (CRM1, also known as XPO1). We found that these novel CRM1 antagonists, namely SINE (selective nuclear export inhibitors), induced rapid apoptosis in 14 T-cell acute lymphoblastic leukemia (T-ALL) cell lines representing different molecular subtypes at low nanomolar concentrations. To evaluate its in vivo anti-leukemic cell activity, we intravenously transplanted human T-ALL MOLT-4 cells carrying NOTCH1 and NRAS activating mutations, as well as loss-of-function CDKN2A, PTEN, and TP53 tumor suppressor genes and high expression of the oncogenic transcription factor TAL1, into immunodeficient mice. Importantly, we also examined the in vivo anti-leukemic efficacy of the clinical SINE compound KPT-330 against T-ALL and acute myeloid leukemia (AML) cells. These studies demonstrated that KPT-330 exhibits significant in vivo activity against T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells with minimal toxicity to normal mouse hematopoietic cells. In summary, our results indicate that SINE CRM1 antagonists are promising first-in-class drugs with novel mechanisms of action and broad therapeutic indices, suggesting their potential for targeted therapy in T-ALL and AML. [2] Overexpression of nuclear export protein 1 (more commonly known as chromosomal region maintenance protein 1, CRM1) is associated with the progression and mortality of malignant tumors. Therefore, activation of nuclear export may play an important role in the etiology of some human tumors and could serve as a novel therapeutic target for these cancers. Mantle cell lymphoma (MCL) is a highly aggressive B-cell non-Hodgkin lymphoma that remains incurable. This study aimed to explore the functional significance of CRM1 in MCL by evaluating the therapeutic effects of CRM1 inhibitors on mantle cell lymphoma (MCL) in vitro and in vivo. The results showed that CRM1 is highly expressed in MCL cells and participates in regulating cell growth and survival mechanisms through the key nuclear factor-κB (NF-κB) survival pathway, and this process is independent of p53 status. Two novel selective nuclear export inhibitors (SINEs), KPT-185 and KPT-276, significantly inhibited cell growth and induced apoptosis in MCL cells by inhibiting CRM1. KPT-185 also induces the accumulation of CRM1 in the nucleus, which in turn leads to the degradation of CRM1 by the proteasome. Oral administration of KPT-276 significantly inhibited tumor growth in a severe combined immunodeficiency (SCID) model of MCL mice without serious toxicity. Our data suggest that SINE CRM1 antagonists may be a potential new therapy for patients with mantle cell lymphoma (MCL), especially those with relapsed/refractory disease. [3]

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Due to the resistance to BRAF inhibitor therapy, it is crucial to develop BRAF inhibitor-based combination therapies 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, we will alter the function of these proteins, thereby reducing melanoma viability and enhancing the antitumor effects of BRAF inhibitors. To validate our hypotheses, we used the selective nuclear export inhibitor (SINE) analogues KPT-185, KPT-251, KPT-276, and KPT-330 to induce CRM1 inhibition. 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 inhibitors reduced melanoma cell proliferation, and this effect was independent of BRAF mutation status; furthermore, CRM1 inhibitors 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 inhibited 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 tumors. Mechanistic studies showed that CRM1 inhibitors are associated with p53 stabilization and the regulation of retinoblastoma protein (pRb) and survivin. Furthermore, we found that BRAF inhibitors can block phosphorylation of extracellular signal-regulated kinase (ERK) associated with CRM1 inhibitors, which may contribute to the synergistic effect of the combination therapy. In summary, CRM1 inhibitors inhibit the survival of both BRAF-mutant and wild-type melanomas. The combination of CRM1 and BRAF inhibitors produces a synergistic effect and induces regression in BRAF-mutant melanomas. [4]

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KPT-185 is a selective nuclear export inhibitor (SINE) that specifically targets CRM1 (XPO1)[1][2][3][4]
- Its core mechanism is to bind to the nuclear export signal (NES) binding pocket of CRM1, blocking the nuclear export of tumor suppressor proteins (p53, p21, FOXO family), which accumulate in the cell nucleus, activate the apoptosis pathway and inhibit tumor proliferation[1][3]
- It has shown activity against hematologic malignancies (acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), mantle cell lymphoma (MCL)) and solid tumors (BRAF mutant melanoma) in preclinical studies[1][2][3][4]
- It can overcome drug resistance (e.g., bortezomib resistance in MCL) and synergize with chemotherapeutic drugs or targeted drugs to enhance efficacy. Antitumor efficacy [2][3][4] - KPT-185 exhibits good selectivity for cancer cells relative to normal cells and is well tolerated in vivo, supporting its potential as an antitumor therapeutic drug [1][2][3]

C16H16F3N3O3

355.31

355.114

C, 54.09; H, 4.54; F, 16.04; N, 11.83; O, 13.51

1333151-73-7

53495165

White to off-white solid powder

1.3±0.1 g/cm3

458.8±55.0 °C at 760 mmHg

231.3±31.5 °C

0.0±1.1 mmHg at 25°C

1.526

4.24

0

8

6

25

485

0

CC(C)OC(=O)/C=C\N1C=NC(=N1)C2=CC(=CC(=C2)OC)C(F)(F)F

NLNGWFLRRRYNIL-PLNGDYQASA-N

InChI=1S/C16H16F3N3O3/c1-10(2)25-14(23)4-5-22-9-20-15(21-22)11-6-12(16(17,18)19)8-13(7-11)24-3/h4-10H,1-3H3/b5-4-

propan-2-yl (Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2-enoate

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)

Solubility in Formulation 1: 2.5 mg/mL (7.04 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.04 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.04 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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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 (Please use freshly prepared in vivo formulations for optimal results.)