IRE1α endoribonuclease
MKC-3946 causes mild growth inhibition in MM cell lines while having no harmful effects on healthy mononuclear cells. Crucially, it amplifies the cytotoxicity caused by 17-AAG or bortezomib considerably, even when external IL-6 or bone marrow stromal cells are present. XBP1s is induced by both 17-AAG and bortezomib, indicating ER stress, which is inhibited by MKC-3946. MKC-3946 increases the apoptosis brought on by these agents and is linked to an increase in CHOP. Treatment with MKC-3946 does not impede the function of IRE1α kinase or the binding of activated IRE1α to TRAF2 and subsequent JNK signaling[1].

MKC-3946 causes mild growth inhibition in MM cell lines while having no harmful effects on healthy mononuclear cells. Crucially, it amplifies the cytotoxicity caused by 17-AAG or bortezomib considerably, even when external IL-6 or bone marrow stromal cells are present. XBP1s is induced by both 17-AAG and bortezomib, indicating ER stress, which is inhibited by MKC-3946. MKC-3946 increases the apoptosis brought on by these agents and is linked to an increase in CHOP. Treatment with MKC-3946 does not impede the function of IRE1α kinase or the binding of activated IRE1α to TRAF2 and subsequent JNK signaling[1].

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MKC3946 is a novel small-molecule RNase inhibitor of IRE1. [1]
The compound was found to be very unstable in vitro; however, one of its two major hydrolyzed precursors, 2-hydroxy-1-naphthaldehyde (HNA), retained the IRE1α RNase inhibitory activity. [1]
Compared with MKC3946, HNA showed either the same or more potent ability to inhibit the activity of IRE1α to cleave XBP1 into the active spliced form (XBP1s) after tunicamycin (TM) induced activation in NB4 acute myeloid leukemia (AML) cells. [1]
HNA dose-dependently inhibited XBP1s expression induced by TM in AML cell lines and AML patient samples. [1]
HNA significantly decreased cellular viability of both AML cell lines (mean GI50=31 μM, n=8) and AML patient samples (mean GI50=35 μM, n=18) compared to untreated control samples. [1]
HNA caused a significant inhibition (mean GI50=6 μM, n=6) of clonogenic growth in soft agar of AML cells from patients. [1]
In contrast, HNA had very low toxicity against normal human marrow mononuclear cells (mean GI50=123 μM, n=4). [1]
HNA treatment of AML cells induced caspase-dependent apoptosis, increased cleaved PARP and caspase-3, down-regulated pro-survival Bcl-2 family members (Bcl-2 and Bcl-xl), up-regulated the pro-apoptotic protein Bim, increased G1 phase regulators (p21cip1, p27kip1), and decreased cyclin D1 levels. [1]
HNA treatment increased mRNA and protein levels of the chaperone CHOP, while down-regulating other chaperone genes (Calnexin, HERPUD1, DNAJC3, DNAJB9, EDEM). [1]
Combination of HNA with either bortezomib or arsenic trioxide (AS2O3) synergistically inhibited the growth of AML cells (NB4 and primary sample #19). [1]

MKC-3946 significantly inhibits the growth of MM cells in a model of in vivo ER stress by inhibiting XBP1 splicing[1].

For three hours, RPMI 8226 cells are either treated with or without Tm (5 μg/mL) in addition to MKC-3946 (0-10μM). After extracting total RNA, XBP1 and β-actin mRNA are assessed via RT-PCR.

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Cell Viability (MTT assay): Cells (e.g., 10,000 per well) were seeded into 96-well plates followed by exposure to various concentrations of HNA. After 72 hours, cell viability was examined using the MTT assay. [1]
Soft Agar Clonogenic Assay: AML patient samples were exposed to HNA in soft agar to assess clonogenic growth inhibition. [1]
Annexin V/Propidium Iodide Apoptosis Assay: AML cells were treated with HNA (e.g., 25 μM, 50 μM) for 24 hours, stained with Annexin V and PI, and analyzed by flow cytometry to evaluate apoptosis. [1]
Cell Cycle Analysis: AML cells were treated with HNA (e.g., 25 μM, 50 μM) for 24 hours, stained with PI, and analyzed by flow cytometry to determine cell cycle distribution. [1]
Western Blotting: Treated cells (e.g., NB4 cells with HNA at 25 μM or 50 μM for 24 or 48 hours) were lysed, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against targets such as cleaved PARP, cleaved caspase-3, Bcl-2 family proteins, cell cycle regulators, and chaperone proteins. β-actin was used as a loading control. [1]
Quantitative RT-PCR (QRT-PCR): RNA was isolated from cells treated with compounds (e.g., HNA, tunicamycin). cDNA was synthesized and used for QRT-PCR to measure mRNA levels of genes like XBP1 (unspliced and spliced forms), chaperone genes (CHOP, Calnexin, HERPUD1, DNAJC3, DNAJB9, EDEM), and microRNAs (pre-miR-17, -21, -34a, -96, -147, -150). GAPDH was used as an internal control for normalization. [1]
XBP1 Splicing Assay (RT-PCR & Gel Electrophoresis): Cells were treated with tunicamycin with or without IRE1α inhibitors. RNA was isolated, reverse transcribed, and PCR was performed using primers flanking the XBP1 splice site. The PCR products (unspliced XBP1u and spliced XBP1s) were separated by gel electrophoresis to visualize inhibition of splicing. [1]
MicroRNA Antagonist Transfection: Small RNA antagonist against miR-34a or control RNA was transiently transfected into AML cell lines (K562, NB4, U937). Knockdown efficiency was validated by QRT-PCR at the pre-miR and mature miR levels. Transfected cells were then treated with HNA and cell viability was assessed by MTT assay. [1]

CB17 SCID mice (48-54 days old) are treated for 21 days beginning on day 1 after receiving a subcutaneous injection of 1×107 RPMI 8226 cells mixed with Matrigel on day 0. Four groups (n=8) of mice are allocated to receive daily intraperitoneal injections of 100 mg/kg MKC-3946; twice-weekly intravenous injections of 0.15 mg/kg bortezomib; a combination of intraperitoneal injections of MKC-3946 and intravenous injections of bortezomib; and intraperitoneal injections of 10% HPBCD with normal saline as a vehicle control. Mice are euthanized when tumors grow to a length of 1.5 cm. Tumor volume is estimated from caliper measurements every 3–4 days. From the moment of treatment initiation until death, survival is assessed.

[1]. Inhibition of IRE1α-driven pro-survival pathways is a promising therapeutic application in acute myeloid leukemia. Oncotarget. 2016 Apr 5;7(14):18736-49.

[2]. IRE1 inhibition perturbs the unfolded protein response in a pancreatic β-cell line expressing mutant proinsulin, but does not sensitize the cells to apoptosis. BMC Cell Biol. 2014 Jul 10;15:29.

[3]. Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma. Blood. 2012 Jun 14;119(24):5772-81.

MKC3946 is an IRE1α RNase inhibitor that was initially found to have potent antiproliferative activity against multiple myeloma. [1] Its hydrolytic precursor HNA is a stable and well-active compound, which was used in this study to investigate the function of IRE1α in acute myeloid leukemia (AML). [1] These compounds induce endoplasmic reticulum stress and promote apoptosis in AML cells by inhibiting IRE1α RNase activity, blocking the pro-survival pathway of the unfolded protein response (UPR). [1] Their anti-leukemic effect is achieved at least in part by preventing IRE1α from cleaving tumor suppressor microRNAs (e.g., miR-34a), leading to the accumulation of these microRNAs and ultimately downregulating the expression of oncogenic targets. [1] This study suggests that targeting the IRE1α-driven pro-survival pathway is a promising treatment for acute myeloid leukemia. [1]

C21H20N2O3S

380.46

380.119

C, 66.30; H, 5.30; N, 7.36; O, 12.62; S, 8.43

1093119-54-0

59599728

White to yellow solid powder

1.3±0.1 g/cm3

591.6±50.0 °C at 760 mmHg

311.6±30.1 °C

0.0±1.7 mmHg at 25°C

1.695

3.17

1

5

3

27

552

0

S1C(=C([H])C([H])=C1C(N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])=O)C1C([H])=C([H])C2C(C([H])=O)=C(C([H])=C([H])C=2C=1[H])O[H]

IVQVBMWPWPTSNO-UHFFFAOYSA-N

InChI=1S/C21H20N2O3S/c1-22-8-10-23(11-9-22)21(26)20-7-6-19(27-20)15-2-4-16-14(12-15)3-5-18(25)17(16)13-24/h2-7,12-13,25H,8-11H2,1H3

2-hydroxy-6-[5-(4-methylpiperazine-1-carbonyl)thiophen-2-yl]naphthalene-1-carbaldehyde

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 mg/mL (5.26 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 20.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)