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| Targets |
MKC9989 is a potent and selective inhibitor of the endoribonuclease (RNase) activity of IRE1α, an endoplasmic reticulum (ER) stress transducer.
Murine IRE1α RNase: IC₅₀ = 0.23 µM (range: 0.11 - 0.35 µM, n=6). Human IRE1α RNase: IC₅₀ = 0.52 µM (range: 0.26 - 0.78 µM, n=4). Yeast IRE1 RNase: IC₅₀ = 44 µM (n=6). [1] |
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| ln Vitro |
MKC9989 totally stopped basal and thapsigargin-induced XBP1 mRNA splicing at a dose of 10 μM. These results were seen even in cells that had received a thapsigargin pretreatment, suggesting that MKC9989 is capable of totally reversing the incidence of XPB1 splicing following the start of UPR. as MKC9989 and thapsigargin were administered together in a parallel investigation, the RIDD target CD59 mRNA was greatly stabilized, and the levels of CD59 mRNA in non-stressed cells were somewhat elevated as compared to thapsigargin treatment alone. This finding may indicate a suppression of baseline RIDD activity. Two hours after thapsigargin treatment, MKC9989 slightly stabilized CD59 levels in contrast to its effect on XBP1 splicing. Lastly, MKC9989's effectiveness against RNA cleavage in vitro was similar to its potency against XBP1 mRNA splicing (EC50=0.33 μM) [1].
MKC9989 potently inhibits the RNase activity of murine and human IRE1α in vitro with IC₅₀ values in the sub-micromolar range, while it is much less potent against yeast IRE1. In contrast, MKC9989 has a weak or negligible inhibitory effect on the auto-kinase activity of IRE1α at concentrations up to 10 µM, indicating high selectivity for the RNase domain. Direct binding measurements using microscale thermophoresis (MST) determined the dissociation constant (K_d) for MKC9989 binding to murine IRE1α as 0.84 µM, confirming its high affinity. Mutational analysis of the IRE1α RNase active site residues (e.g., Lys907Ala, Phe889Ala, Tyr892Ala, Asn906Leu, His910Ala) significantly impaired or abolished both RNase activity and MKC9989 binding, validating the binding site identified by X-ray crystallography. [1] |
| Enzyme Assay |
IRE1α RNase Activity Assay (Fluorescence-based): The assay utilized a single hairpin RNA substrate (sequence: 5′-CAU GUC CGC AGC GCA UG-3′) labeled with an Alexa Fluor 647 fluorophore at the 5′ end and a black hole quencher at the 3′ end. Reactions contained 10 nM IRE1α protein and 100 nM RNA substrate in a buffer consisting of 50 mM Tris pH 7.0, 0.5 mM MgCl₂, 10 mM KCl, 0.025% Tween-20, 0.063 mg/ml tRNA, and 2 mM DTT. Compounds were pre-incubated with IRE1α for 1 hour at room temperature before adding the substrate. RNA cleavage was monitored in real-time by measuring the increase in fluorescence intensity (excitation 651 nm, emission 672 nm) at two-minute intervals using a microplate reader.
IRE1α RNase Activity Assay (Gel-based): This assay used a fluorescein-labeled single hairpin RNA substrate (same sequence) at 45 nM concentration. Reactions were performed in a similar buffer with added BSA (0.09 mg/ml). After pre-incubation of IRE1α with inhibitor for 1 hour at room temperature, the RNA substrate was added and the reaction proceeded for 10-45 minutes at 37°C. Reactions were quenched with formamide/EDTA loading buffer, and cleavage products were separated and visualized using 20% urea-TBE PAGE and a fluorescence imager. IRE1α Kinase Activity Assay (Auto-phosphorylation): For trans-auto-phosphorylation assays, 150 nM murine IRE1α was incubated with 15 µM kinase-dead yeast IRE1 as a phospho-acceptor substrate in the presence of inhibitor for 1 hour at room temperature. The reaction was initiated by adding an ATP mixture containing [γ-³²P]ATP. Reactions were terminated with SDS-PAGE loading buffer, separated by electrophoresis, and phosphorylation was detected and quantified using phosphor imaging. [1] |
| Cell Assay |
XBP1 Splicing Analysis in Human RPMI 8226 Plasmacytoma Cells: Cells were cultured and treated with 1 µM thapsigargin (Tg, an ER stress inducer) and/or 10 µM MKC9989 according to specified time courses. In some experiments, MKC9989 was added 2 hours post-Tg treatment. Cells were harvested, and total RNA was extracted. The splicing status of XBP1 mRNA (unspliced XBP1u and spliced XBP1s) was analyzed by RT-PCR followed by PAGE separation and quantification. MKC9989 (10 µM) completely inhibited both basal and Tg-induced XBP1 splicing, even when added after Tg treatment.
CD59 mRNA Stability Analysis: Using the same RNA samples from the XBP1 splicing experiments, the mRNA levels of CD59, a known Regulated IRE1-Dependent Decay (RIDD) target, were measured. MKC9989 co-administered with Tg markedly stabilized CD59 mRNA levels compared to Tg treatment alone, and moderately increased CD59 levels in non-stressed cells, indicating inhibition of baseline RIDD activity. When administered 2 hours post-Tg, MKC9989 showed a moderate stabilization effect on CD59 mRNA. [1] |
| References | |
| Additional Infomation |
MKC9989 belongs to the hydroxyaryl aldehyde (HAA) class of IRE1 ribonuclease inhibitors. Its chemical structure is a coumarin backbone with specific substituents: formyl (aldehyde) at position 8, hydroxyl at position 7, methoxy at position 6, a flexible polyether chain at position 3, and a methyl group at position 4. X-ray co-crystal structure of mouse IRE1α and MKC9989 reveals its binding mode within the shallow pocket of the ribonuclease active site. Key interactions include: 1) a reversible Schiff base formed between its aldehyde group and the amino group of the Lys907 side chain; 2) a hydrogen bond formed between its hydroxyl group and Tyr892; 3) π-π stacking interactions with Phe889 and His910; and 4) additional hydrogen bonds and van der Waals forces formed between its coumarin core and polyether side chains and residues such as Glu913, Pro915, and Leu914.
MKC9989 As a non-competitive inhibitor, it acts on RNA substrates, binding via three of four conserved catalytic residues (Tyr892, Asn906, and His910). It inhibits two major downstream pathways of IRE1α: XBP1 mRNA splicing and RIDD (regulated IRE1-dependent decay). This study suggests that HAA drugs, including MKC9989, may have therapeutic potential because they are selective and the targets are difficult to induce resistance mutations without impairing their essential catalytic activity. [1] |
| Molecular Formula |
C17H20O7
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| Molecular Weight |
336.336505889893
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| Exact Mass |
336.12
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| CAS # |
1338934-20-5
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| PubChem CID |
89542001
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
512.2±50.0 °C at 760 mmHg
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| Flash Point |
184.5±23.6 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.561
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| LogP |
2.18
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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 |
8
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| Heavy Atom Count |
24
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| Complexity |
487
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
QSVDQIXSUDHAOF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H20O7/c1-10-11(4-5-23-7-6-21-2)17(20)24-16-12(10)8-14(22-3)15(19)13(16)9-18/h8-9,19H,4-7H2,1-3H3
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| Chemical Name |
7-hydroxy-6-methoxy-3-[2-(2-methoxyethoxy)ethyl]-4-methyl-2-oxochromene-8-carbaldehyde
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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 |
| 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 : ≥ 50 mg/mL (~148.66 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.43 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9732 mL | 14.8659 mL | 29.7318 mL | |
| 5 mM | 0.5946 mL | 2.9732 mL | 5.9464 mL | |
| 10 mM | 0.2973 mL | 1.4866 mL | 2.9732 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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