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Purity: ≥98%
INCB053914 is a novel, potent, selective, and ATP-competitive small molecule pan-inhibitor of PIM (Proviral Integration site of Moloney murine leukemia virus) kinases with IC50 values of 0.24 nM, 30 nM and 0.12 nM for PIM1, PIM2 and PIM3 respectively in biochemical assays. In cell proliferation assays, INCB053914 is active as a single agent in the majority of cell lines derived from different hematological malignancies, including MM, AML, DLBCL, MCL and T-ALL, with IC50values ranging from 3 - 300 nM. INCB053914 synergizes with a variety of cytotoxic and targeted agents, reducing the viability of a panel of hematological tumor cell lines. In pharmacodynamic assays, INCB053914 inhibits phosphorylation of S6RP, P70S6K, 4E-BP-1 and BAD, known PIM kinase targets. INCB053914 may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.
| Targets |
All multiple myeloma (MM) cell lines examined are inhibited in their ability to proliferate by usansertib; typical GI50 values for AML, MM, DLBCL, MCL, and T-ALL cell lines range from 13.2 nM to 230.0 nM [1]. In MOLM-16 (AML) phosphorylated), Pfeiffer (DLBCL), and KMS-12-PE/BM (MM) cell lines, umansertib (0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 nM) inhibits downstream PIM kinase substrates (p70S6K/S6 and 4E-BP1) in a dose-dependent manner [1]. In MOLM-16 and KMS-12-BM cells, PIM kinase-mediated BAD phosphorylation is especially susceptible to Uzansertib inhibition (average IC50 of 4 nM and 27 nM, respectively) [1].
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| ln Vitro |
All multiple myeloma (MM) cell lines examined are inhibited in their ability to proliferate by usansertib; typical GI50 values for AML, MM, DLBCL, MCL, and T-ALL cell lines range from 13.2 nM to 230.0 nM [1]. In MOLM-16 (AML) phosphorylated), Pfeiffer (DLBCL), and KMS-12-PE/BM (MM) cell lines, umansertib (0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 nM) inhibits downstream PIM kinase substrates (p70S6K/S6 and 4E-BP1) in a dose-dependent manner [1]. In MOLM-16 and KMS-12-BM cells, PIM kinase-mediated BAD phosphorylation is especially susceptible to Uzansertib inhibition (average IC50 of 4 nM and 27 nM, respectively) [1].
INCB053914 inhibited proliferation in a panel of hematologic malignancy cell lines, including multiple myeloma (MM), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and T-cell acute lymphoblastic leukemia (T-ALL), with mean GI₅₀ values <100 nM in 50% of tested cell lines. Particularly sensitive lines included MOLM-16 (AML, GI₅₀ 3.3 nM), Kasumi-3 (AML, GI₅₀ 4.9 nM), Pfeiffer (DLBCL, GI₅₀ 19.5 nM), and all MM lines tested (GI₅₀ range 13.2–230 nM)[1] Treatment with INCB053914 dose-dependently inhibited phosphorylation of downstream PIM kinase substrates (p70S6K/S6 and 4E-BP1) in MOLM-16 (AML), Pfeiffer (DLBCL), and KMS-12-PE/BM (MM) cell lines[1] INCB053914 potently inhibited phosphorylation of the pro-apoptotic protein BAD in MOLM-16 (IC₅₀ = 4 nM) and KMS-12-BM (IC₅₀ = 27 nM) cells[1] INCB053914 increased PIM2 protein expression in KG-1a (AML), Pfeiffer (DLBCL), and KMS-12-PE (MM) cell lines[1] Ex vivo treatment of primary bone marrow blasts from AML patients with INCB053914 decreased phosphorylation of p70S6K and 4E-BP1 and increased PIM2 expression[1] Ex vivo treatment of peripheral blood mononuclear cells (PBMCs) from AML patients with INCB053914 resulted in a concentration-dependent increase in PIM2 expression[1] INCB053914 dose-dependently inhibited neoplastic erythroid colony formation in primary cell cultures from patients with JAK2 V617F-positive myeloproliferative neoplasms (polycythemia vera, essential thrombocythemia, primary myelofibrosis) at concentrations as low as 0.5 nM[1] |
| ln Vivo |
Uzansertib (25–100 mg/kg; oral; twice daily; for 15 days) dose-dependently suppresses tumor growth in mice expressing KMS-12-BM (MM) or MOLM-16 (AML)[1]. Four hours after injection, uzansertib showed dose-dependent reduction of BAD phosphorylation in comparison to vehicle (IC50=70 nM for MOLM-16 tumors and IC50=145 nM for KMS-12-BM tumors) [1].
In SCID mice bearing MOLM-16 (AML) xenografts, oral administration of INCB053914 (1-30 mg/kg, twice daily) for 15 days dose-dependently inhibited tumor growth, with maximal 96% inhibition at 30 mg/kg (6/8 mice showed partial regression)[1] In SCID mice bearing KMS-12-BM (MM) xenografts, oral administration of INCB053914 (25-100 mg/kg, twice daily) for 15 days dose-dependently inhibited tumor growth (43%-88% inhibition)[1] A single oral dose of INCB053914 (up to 100 mg/kg) dose-dependently inhibited intratumoral BAD phosphorylation in MOLM-16 (IC₅₀ = 70 nM) and KMS-12-BM (IC₅₀ = 145 nM) xenograft models at 4 hours post-dose[1] Combination of INCB053914 (30 mg/kg once daily) with the PI3Kδ inhibitor INCB050465 (10 mg/kg once daily) synergistically inhibited tumor growth in Pfeiffer (DLBCL) xenografts, resulting in 7 partial and 1 complete regression among 8 mice[1] Combination of INCB053914 (20 mg/kg twice daily) with cytarabine (20 mg/kg twice weekly, intraperitoneal) showed additive inhibition of tumor growth in KG-1 (AML) xenografts[1] Combination of INCB053914 (100 mg/kg twice daily) with the JAK1-selective inhibitor itacitinib (60 mg/kg twice daily) synergistically inhibited tumor growth in INA-6 (MM) xenografts[1] |
| Enzyme Assay |
The inhibitory activity of INCB053914 against PIM1 and PIM3 kinases was measured using an AlphaScreen assay. In this assay, the kinase reaction utilizes biotinylated peptide substrates. Upon phosphorylation, streptavidin-coated donor beads and anti-phospho-substrate antibody-coated acceptor beads bind, generating a luminescent signal in close proximity. Inhibition of kinase activity reduces this signal[1]
The inhibitory activity of INCB053914 against PIM2 kinase was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. This assay typically involves a fluorescently labeled kinase, a terbium-labeled anti-phospho-antibody, and a substrate. Phosphorylation brings the donor (terbium) and acceptor (fluorescent label) into proximity, enabling FRET. Inhibition reduces FRET signal[1] |
| Cell Assay |
The antiproliferative effects of INCB053914 were assessed in various hematologic tumor cell lines. Cells were seeded in culture plates and treated with a concentration range of INCB053914. After a defined incubation period (typically 3-5 days), cell viability/proliferation was measured using a metabolic dye reduction assay (e.g., AlamarBlue or CellTiter-Glo). The concentration causing 50% growth inhibition (GI₅₀) was calculated[1]
To assess inhibition of PIM kinase substrate phosphorylation, cells (e.g., MOLM-16, Pfeiffer, KMS-12-PE/BM) were incubated with a concentration range of INCB053914 for 2 hours. Cells were then lysed, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against phosphorylated forms of BAD (S112), p70S6K (T389), S6 (S235/236), 4E-BP1 (S65), and total PIM2. Actin was used as a loading control[1] Quantification of phosphorylated BAD (pBAD S112) was performed using an enzyme-linked immunosorbent assay (ELISA). Cells were treated with INCB053914 for 2.5 hours, lysed, and the pBAD protein concentration in lysates was determined using a commercial ELISA kit following the manufacturer's protocol[1] Erythroid colony formation assays were performed using primary peripheral blood cells from patients with JAK2 V617F-positive myeloproliferative neoplasms. Cells were cultured in methylcellulose-based medium containing cytokines, with or without INCB053914. After an incubation period (e.g., 14 days), erythroid colonies were counted[1] |
| Animal Protocol |
Animal/Disease Models: Female immunocompromised (severe combined immunodeficiency [SCID]) mice (5-9 weeks old) [1] carrying MOLM-16 (AML) or KMS-12-BM (MM)
Doses: 25, 50 , 75, 100 mg/kg. Doses: Oral; twice a day; for 15 days. Experimental Results: Inhibited tumor growth in mice in a dose-dependent manner. For efficacy and pharmacodynamic studies, female severe combined immunodeficiency (SCID) mice were inoculated subcutaneously in the flank with tumor cells (e.g., MOLM-16, KMS-12-BM) suspended in a mixture of Dulbecco's phosphate-buffered saline and Matrigel, or with tumor fragments (e.g., INA-6, Pfeiffer)[1] When tumors reached a specified volume range, mice were grouped and treatment commenced. INCB053914 was administered orally (by gavage), typically twice daily (BID), as a suspension in vehicle containing 5% dimethylacetamide and 0.5% methylcellulose[1] In some studies, INCB053914 was administered via continuous subcutaneous infusion using osmotic pumps[1] Tumor dimensions were measured regularly, and volume was calculated. For pharmacodynamic studies, mice were sacrificed at specified times post-dose, and tumors were collected for analysis of biomarker modulation (e.g., pBAD by ELISA or Western blot)[1] Blood samples were collected at various time points for pharmacokinetic analysis of INCB053914 plasma concentrations[1] For combination studies, INCB053914 was administered orally alongside other agents: PI3Kδ inhibitor INCB050465 (oral), cytarabine (intraperitoneal), or JAK inhibitor itacitinib (oral), following their respective dosing schedules[1] |
| ADME/Pharmacokinetics |
After oral administration to tumor-bearing mice, the plasma concentration of INCB053914 showed a dose-proportional relationship within the tested dose range [1]. When the plasma trough concentration of INCB053914 exceeded the in vitro pBAD inhibition IC₅₀, maximum antitumor activity was observed in the xenograft model [1].
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| Toxicity/Toxicokinetics |
In reported xenotransplantation studies, INCB053914 was well tolerated at all tested doses, with no unexpected deaths reported. Animal welfare was monitored during the study, and pre-defined, humane endpoints (e.g., weight loss >20%, tumor burden criteria) were established [1]
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| References | |
| Additional Infomation |
Uzansertib is being investigated in the clinical trial NCT02587598 (INCB053914, a study in patients with advanced malignant tumors). Uzansertib is an orally administered, small-molecule selective ATP-competitive pan-inhibitor that inhibits the proviral integration site (PIM) kinase of Moloney murine leukemia virus, exhibiting potential antitumor activity. After oral administration, Uzansertib binds to and inhibits the activity of three PIM subtypes: PIM1, PIM2, and PIM3. This prevents phosphorylation of its downstream targets and inhibits the proliferation of PIM-overexpressing cells. PIM kinases are constitutively active proto-oncogene serine/threonine kinases, upregulated in various cancers, and play a crucial role in tumor cell proliferation and survival.
INCB053914 is a novel ATP-competitive small molecule pan-PIM kinase (PIM1, PIM2, PIM3) inhibitor[1] PIM kinases are overexpressed in hematologic malignancies and integrate signals from multiple pathways (including JAK/STAT and PI3K/AKT/mTOR) and share downstream substrates such as BAD and 4E-BP1[1] Inhibition of one PIM isoenzyme can lead to upregulation of other isoenzymes; therefore, pan-PIM inhibitors are considered beneficial for therapeutic effects[1] INCB053914 showed high selectivity for PIM kinases in a wide range of kinase screenings (selectivity >475-fold for 56 kinases). It exhibits moderate activity against RSK2 (IC₅₀ = 7.1 µM) and comparable activity against PAS kinase to that against PIM2 [1]. Based on preclinical data, a phase I/II dose-escalation trial (NCT02587598) was initiated to evaluate the efficacy of INCB053914 as a monotherapy or in combination with INCB050465, azacitidine, cytarabine, or ruxolitinib in the treatment of advanced malignancies [1]. |
| Molecular Formula |
C26H26F3N5O3
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| Molecular Weight |
513.511556148529
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| Exact Mass |
513.198
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| CAS # |
1620012-39-6
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| Related CAS # |
Uzansertib phosphate;2088852-47-3
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| PubChem CID |
90279868
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
1.7
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
37
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| Complexity |
803
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| Defined Atom Stereocenter Count |
4
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| SMILES |
C[C@H]1CN(C[C@H]([C@@H]1O)N)C2=C3CC[C@H](C3=NC=C2NC(=O)C4=NC(=C(C=C4)F)C5=C(C=CC=C5F)F)O
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| InChi Key |
PTORCEYGCGXHDH-OVMXCRKPSA-N
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| InChi Code |
InChI=1S/C26H26F3N5O3/c1-12-10-34(11-17(30)25(12)36)24-13-5-8-20(35)22(13)31-9-19(24)33-26(37)18-7-6-16(29)23(32-18)21-14(27)3-2-4-15(21)28/h2-4,6-7,9,12,17,20,25,35-36H,5,8,10-11,30H2,1H3,(H,33,37)/t12-,17+,20+,25+/m0/s1
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9474 mL | 9.7369 mL | 19.4738 mL | |
| 5 mM | 0.3895 mL | 1.9474 mL | 3.8948 mL | |
| 10 mM | 0.1947 mL | 0.9737 mL | 1.9474 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.
Structure of INCB053914 and IC50values for the inhibition of PIM isozymes by INCB053914 in biochemical assays.PLoS One.2018 Jun 21;13(6):e0199108. |
INCB053914 inhibits cellular proliferation in hematologic tumor cell lines (A), inhibits phosphorylation of PIM substrates (B), including pBAD (C), and increases PIM2 expression (D) in hematologic tumor cell lines.PLoS One.2018 Jun 21;13(6):e0199108. |
INCB053914 inhibits phosphorylation of PIM substrates and increases PIM2 expression in primary bone marrow (BM) blasts (A), and increases PIM2 expression in PBMCs derived from whole blood samples from patients with AML (B).PLoS One.2018 Jun 21;13(6):e0199108. |
INCB053914 inhibits erythroid colony formation in patients with JAK2 V617F-positive MPNs.PLoS One.2018 Jun 21;13(6):e0199108. |
INCB053914 inhibits the phosphorylation of BAD in mice bearing MOLM-16 (AML) (A) or KMS-12 (MM) tumors (B), and inhibits growth of MOLM-16 (AML) (C) and KMS-12 (MM) (D) tumorsin vivo. Mean INCB053914 plasma concentrations were determined at 2, 4, 8, and 16 hours post oral administration in mice bearing MOLM-16 tumors (E) or KMS-12-BM tumors (F).PLoS One.2018 Jun 21;13(6):e0199108. |
Effects of the selective PI3Kδ inhibitor, INCB050465, on PIM isozyme expression in Pfeiffer DLBCL cells (A). Effects of INCB053914 alone, or in combination with INCB050465, on thein vitroproliferation of Pfeiffer DLBCL cells (B). Effects of INCB053914 alone or in combination with INCB050465 on tumor growth in a DLBCL xenograft model (C); with cytarabine on tumor growth in an AML xenograft model (D); with the JAK1-selective inhibitor, itacitinib, on BAD, STAT3 phosphorylation, and MYC levels (E), and on tumor growth in an INA-6 MM xenograft model (F).PLoS One.2018 Jun 21;13(6):e0199108. |
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