IRE1 RNase (IC50 = 0.29 μM)
MKC-8866 specifically targets the RNase domain of IRE1α, a key sensor of ER stress. Upon ER stress, IRE1α oligomerizes and undergoes autophosphorylation, activating its endoribonuclease (RNase) activity. This RNase activity catalyzes the unconventional splicing of XBP1 mRNA, which produces the active transcription factor XBP1s. XBP1s then upregulates the expression of genes involved in protein folding, ER-associated degradation (ERAD), and other adaptive responses. By binding to and inhibiting the IRE1α RNase domain, MKC-8866 prevents the splicing of XBP1 mRNA. This blocks the downstream signaling of the IRE1α branch of the UPR, thereby sensitizing cancer cells to ER stress and promoting cell death. The compound is selective for mammalian IRE1α and does not inhibit RNase A, RNase L, or yeast IRE1.

All breast cancer cell lines' growth is inhibited by MKC8866 (20 μM; 6 days) [2]. Cells entering S phase are less frequently when MKC8866 (20 μM; 48 hours) is applied [2]. Under standard circumstances, MKC8866 (0.2-10 μM; 3 days) exhibits a dose-dependent inhibition of the viability of all four cell lines, with the greatest effect observed in LNCaP cells [1]. For a duration of 72 hours, MKC8866 (20 μM) is adequate to entirely prevent NSC 125973-induced XBP1 expression [1].
In vitro studies have demonstrated that MKC-8866 is a potent and selective inhibitor of the IRE1α RNase domain. In biochemical assays using recombinant human IRE1α protein, the compound inhibits the cleavage of a synthetic XBP1 RNA probe with an IC50 of 0.29 μM. It shows no activity against RNase A, RNase L, or yeast IRE1, confirming its selectivity. In cell-based assays, MKC-8866 inhibits IRE1 RNase activity in breast cancer cells, as measured by the reduction of XBP1 splicing and the expression of downstream UPR target genes. These in vitro findings provide strong evidence for the compound's mechanism of action and its potential as an anticancer agent.

When NSC 125973 is stopped, MKC8866 (oral; 300 mg/kg; for 28 days) decreases tumor regrowth [1].

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MKC8866, strongly inhibits prostate cancer (PCa) tumor growth as monotherapy in multiple preclinical models in mice and shows synergistic antitumor effects with current PCa drugs.[1]
MKC8866 strongly inhibited xenografted tumor growth in all PCa cell lines tested (Fig. 2a). XBP1s expression was significantly lower in MKC8866-treated tumors compared to controls, confirming that MKC8866 was active in mice harboring the tumors and that IRE1α activity was appropriately inhibited in vivo (Supplementary Fig. 4a). In addition, there was a decrease in PCNA expression and an increase in cleaved Caspase-3 levels, indicating that MKC8866 treatment resulted in decreased proliferation and increased apoptosis, respectively (Supplementary Fig. 4b). Removal of MKC8866 during the course of the treatment resulted in rebounding of XBP1s levels and enhanced tumor growth (Fig. 2b and Supplementary Fig. 4c), indicating the importance of sustained MKC8866 application for its growth inhibitory effects. These results show that pharmacological targeting of IRE1α exerts potent antitumor effects in preclinical mouse models of PCa.[1]

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There was strong synergy in tumor growth inhibition when MKC8866 was co-administered with enzalutamide. Co-administration of MKC8866 with abiraterone acetate and cabazitaxel also synergistically inhibited tumor growth. Taken together, these data demonstrate that in preclinical models MKC8866 synergizes with some of the central PCa drug regimens that are currently used in the clinic.[1]

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MKC8866 enhances the effectiveness of paclitaxel in vivo[2]
To determine the efficacy of MKC8866 treatment in vivo, MDA-MB-231 tumor xenografts were established in athymic nude mice. Once tumors had reached a palpable size (225–250 mm3), animals were randomized into treatment groups and treated with vehicle alone, 300 mg kg−1 MKC8866 alone, 10 mg kg−1 paclitaxel alone or a combination of paclitaxel and MKC8866. Treatments in all groups were administered until tumors reached maximal size (2000 mm3) or on day 60, whichever came first. MKC8866 was well tolerated after 60 consecutive oral doses and, based on pharmacokinetic allometric scaling, systemic exposures were well above anticipated clinical therapeutic levels. Treatment with MKC8866 alone did not attenuate tumor growth compared to vehicle-only controls (Fig. 7a). Analysis of percentage XBP1 mRNA splicing in those tumors treated with MKC8866 confirmed a reduction in IRE1 RNase activity verifying on-target effect (Fig. 7b). While paclitaxel treatment reduced tumor growth, combination with MKC8866 markedly enhanced the efficacy of paclitaxel. Significantly reduced tumor growth (P ≤ 0.0001) was observed throughout the 60-day experiment in animals receiving a paclitaxel-MKC8866 combination compared to paclitaxel alone (Fig. 7c). A similar synergistic effect was observed following a paclitaxel-MKC8866 combination starting on day 14 (or ~700 mm3 tumor volume) (P ≤ 0.001) or on day 28 (or ~1300 mm3 tumor volume) (P ≤ 0.05) when compared to paclitaxel alone (Fig. 7c). Examination of XBP1 splicing in tumors revealed paclitaxel treatment increased IRE1 RNase activity, which was reduced upon combination with MKC8866 (Fig. 7d, Supplementary Fig. 5). The decrease in tumor volume observed following a combination of paclitaxel and MKC8866 also translated to an increase in survival. Mice receiving daily MKC8866 administration in combination with paclitaxel from day 1 to 60, day 14 to 60, and day 28 to 60 displayed significantly longer survival compared to those treated with paclitaxel alone (Fig. 7e).
In vivo, MKC-8866 is orally bioavailable and has shown significant therapeutic activity in preclinical models of cancer. In mouse models of prostate cancer, oral administration of MKC-8866 has been shown to inhibit tumor growth. The compound's ability to inhibit the IRE1α pathway in vivo has been confirmed by the reduction of XBP1 splicing in tumor tissues. These results demonstrate that MKC-8866 can effectively engage its target in a living system and produce a therapeutic effect, supporting its potential as a novel cancer therapy.

Luciferase reporter assay[1]
LNCaP or 293T cells were cultured in six-well plates, transfected with 1 μg of pGL3-MYC luciferase reporter plasmid plus either empty vector (pCDNA3) or the pCDNA3-Flag-XBP1s plasmid of the indicated concentration for 24 h before harvest. Luciferase activity was determined using a luciferase assay system and a Wallac Victor2 1420 Multilabel counter.
The in vitro enzyme inhibition assay for MKC-8866 involves measuring the RNase activity of IRE1α. In this assay, recombinant human IRE1α protein is incubated with a synthetic RNA probe that mimics the XBP1 mRNA substrate. The cleavage of the probe by IRE1α is then quantified, typically using fluorescence-based detection or gel electrophoresis. MKC-8866 is added at various concentrations, and its ability to inhibit the RNase activity is measured, allowing for the calculation of the IC50. This biochemical assay is the primary method for characterizing the compound's potency and selectivity.

Cell proliferation assay[2]
Cell Types: MCF7, SKBR3, MDA-MB-231 and MCF10A Cell
Tested Concentrations: 20 μM
Incubation Duration: 6 days
Experimental Results: Proliferation was diminished in all breast cancer cell lines.

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Cell cycle analysis[2]
Cell Types: MDA-MB-231, MCF7 and SKBR3 Cell
Tested Concentrations: 20 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: diminished number of cells entering S phase.

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Cell cycle analysis[1]
Cell Types: LNCaP, VCaP, 22Rv1 and C4-2B Cell
Tested Concentrations: 0.2, 0.5, 1, 5, 10 μM
Incubation Duration: 3 days
Experimental Results: Inhibited viability of all four cell lines in one dose Dependence method.

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Cell cycle analysis [2]
Cell Types: MDA-MB-231 Cell
Tested Concentrations: 20 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Completely blocked NSC 125973-induced XBP1 expression.
Cellular assays for MKC-8866 are conducted to confirm its functional activity in a biological context. In these assays, cancer cell lines, such as breast or prostate cancer cells, are treated with MKC-8866. The inhibition of IRE1α activity is assessed by measuring the splicing of XBP1 mRNA using RT-PCR or by Western blotting for the spliced form of XBP1 (XBP1s). The expression of downstream UPR target genes, such as BiP/GRP78, is also measured. Cell viability and apoptosis assays are used to assess the compound's cytotoxic effects. These cell-based experiments are essential for confirming that the biochemical inhibition translates to functional effects in a cellular environment.

Animal/Disease Models: MDA-MB-231 tumor in female athymic nude mice [1]
Doses: 300 mg/kg
Route of Administration: oral; continued for 28 days
Experimental Results: NSC 125973 diminished tumor regeneration after drug withdrawal.

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In vivo MDA-MB-231 xenograft model[2]
Mice were administered 10 mg kg−1 paclitaxel weekly by intravenous injection. The IRE1 inhibitor, MKC8866, was administered at a dose volume of 10 ml kg−1 from a 30 mg ml−1 suspension in 1% microcrystalline cellulose in a simple sugar at 300 mg kg−1 daily by oral gavage (Vehicle 2). Treatment groups were as follows: For Group 1, the paclitaxel vehicle was administered intravenously weekly and the MKC8866 vehicle was administered orally daily throughout the course of the study. For Groups 2–6, paclitaxel was administered weekly throughout the course of the study. In combination with paclitaxel, MKC8866 was also administered orally daily from day 1 to 28 (Group 3), from day 14 to 60 (Group 4), from day 28 to 60 (Group 5), and from day 1 to 60 (Group 6). Treatments in all groups were administered until tumors reached maximal size or day 60, whichever came first.[2]
MKC8866 was administered daily for 28 days at a dose volume of 10 ml kg−1 from a 30 mg ml−1 suspension in 1% microcrystalline cellulose in a simple sugar at 300 mg kg−1 daily by oral gavage (Vehicle 2). Group 1 received paclitaxel (7.5 mg kg−1) alone while Group 2 received paclitaxel (7.5 mg kg−1) plus 300 mg kg−1 MKC8866.

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After palpable tumors appeared, the mice were randomly grouped and daily received either 300 mg kg−1 MKC8866 or vehicle (1% microcrystalline in 1 g ml−1 sucrose). For the combinatorial experiment of MKC8866 and enzalutamide, mice were assigned into four groups (n = 6 per group): oral gavage of 300 mg kg−1 MKC8866 every other day, oral gavage of 30 mg kg−1 enzalutamide every other day, a combination of daily gavage of either MKC8866 or enzalutamide, and daily gavage of either MKC8866 or 0.5% HP methyl cellulose with 0.1% Tween 20 as a vehicle control. The four groups (n = 6 per group) for the combination of MKC8866 and abiraterone acetate were: oral gavage of 300 mg kg−1 MKC8866 every other day, oral gavage of 20 mg kg−1 abiraterone acetate every other day, a combination of two treatments, and corresponding vehicles. The four groups (n = 6 per group) for the combination of MKC8866 and cabazitaxel were: oral gavage of 300 mg kg−1 MKC8866 every other day, intraperitoneal injections of 5 mg kg−1 cabazitaxel twice a week, a combination of two treatments, and corresponding vehicles. Tumor weight was measured in the end of experiment.[1]
In vivo animal experiments for MKC-8866 are conducted in mouse models of cancer, typically prostate or breast cancer xenografts. In these models, cancer cells are implanted subcutaneously into immunocompromised mice. When tumors reach a certain size, MKC-8866 is administered orally. Tumor growth is monitored over time, and at the end of the study, tumors are harvested and analyzed for biomarkers of IRE1α inhibition, such as XBP1 splicing. These in vivo studies are crucial for demonstrating the compound's therapeutic efficacy and for evaluating its pharmacokinetic and pharmacodynamic properties.

MKC-8866 has a molecular weight of 361.35 g/mol and a molecular formula of C18H19NO7. It is a small molecule that is orally bioavailable. The compound is typically stored as a powder at -20°C. Its physicochemical properties, such as solubility and permeability, support its oral administration.

The toxicity profile of MKC-8866 has been evaluated in preclinical studies. As an inhibitor of the UPR, its safety profile is being carefully assessed. Common side effects may include gastrointestinal effects, as the UPR plays a role in maintaining the health of secretory tissues. However, the compound's selectivity for IRE1α is a key feature for minimizing off-target toxicity.

[1]. IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling. Nat Commun. 2019 Jan 24;10(1):323.

[2]. Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy. Nat Commun. 2018 Aug 15;9(1):3267.

ORIN1001, an IRE1 RNase inhibitor, is an orally bioavailable serine/threonine protein kinase/ribonuclease inositol-requiring enzyme 1 (IRE1) inhibitor with potential immune-activating, chemosensitizing, and antitumor activities. After oral administration, ORIN1001 targets and binds to the RNase domain of IRE1, thereby inhibiting IRE1 activity. This prevents activation of the IRE1/X-box binding protein 1 (XBP1) pathway, inhibits unfolded protein response (UPR) stress adaptation, and prevents the production of pro-tumor factors. This may inhibit tumor growth induced by IRE1 overactivation. Furthermore, ORIN1001 eliminates the immunosuppressive tumor microenvironment (TME) by infiltrating cytotoxic T cells and clearing immunosuppressive myeloid-derived suppressor cells (MDSCs) from the TME. IRE1 is a transmembrane protein located in the endoplasmic reticulum (ER), containing an ER-mediated stress-sensing domain and a cytoplasmic RNase domain. It is a key sensor for the endoplasmic reticulum (UPR) and plays a crucial role in the response to and resolution of ER stress. IRE1 participates in ER stress-dependent signaling pathway-mediated protein phosphorylation, as well as mRNA processing and degradation. IRE1 is frequently co-amplified with the MYC oncogene.
MKC-8866 (ORIN1001) is a potent, selective, and orally bioavailable inhibitor of the IRE1α RNase domain that has been developed as a potential anticancer therapy. Its mechanism of action involves blocking the IRE1α branch of the UPR, a pathway that is often upregulated in cancer cells to manage ER stress. The compound has shown significant therapeutic activity in preclinical models of prostate and breast cancer and has advanced into clinical trials. As an investigational drug, MKC-8866 represents a novel approach to targeting cancer cell survival mechanisms and holds promise for the treatment of various cancers that are dependent on the UPR.

C18H19NO7

361.3460

361.12

C, 59.83; H, 5.30; N, 3.88; O, 30.99

1338934-59-0

89542346

Light yellow to yellow solid powder

0.5

1

7

4

26

611

0

IFDGMRMUJYGWQQ-UHFFFAOYSA-N

InChI=1S/C18H19NO7/c1-10-11-7-14(24-2)16(22)13(9-20)17(11)26-18(23)12(10)8-15(21)19-3-5-25-6-4-19/h7,9,22H,3-6,8H2,1-2H3

7-Hydroxy-6-methoxy-4-methyl-3-(2-morpholino-2-oxoethyl)-2-oxo-2H-chromene-8-carbaldehyde

ORIN1001; ORIN-1001; ORIN 1001; MKC-8866; MKC 8866; 2H-1-Benzopyran-8-carboxaldehyde, 7-hydroxy-6-methoxy-4-methyl-3-(2-(4-morpholinyl)-2-oxoethyl)-2-oxo-; UNII-1NZ0YBP9HB;MKC8866; IRE1-IN-8866; IRE1IN8866; IRE1; IN 8866; IRE1-IN8866; IRE1-IN 8866; IRE1IN-8866; IRE1IN 8866

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)

DMSO : ~6.67 mg/mL (~18.46 mM)

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.)