MKC3946 is an effective and soluble IRE1α inhibitor which triggered modest growth inhibition in multiple myeloma cell lines.

MKC-3946通过阻断XBP1 mRNA剪接，并对AML细胞展现细胞毒性。MKC-3946抑制由tunicamycin (TM)在NB4细胞(B)及患者AML样本[1]中诱导的XBP1S表达。MKC-3946阻止因突变前胰岛素产生造成的内质网应激响应中XBP1 mRNA的剪接[2]。作为IRE1α核糖核酸酶领域抑制剂，MKC-3946阻断XBP1 mRNA剪接，在多发性骨髓瘤（MM）细胞中触发适度的生长抑制。MKC-3946以剂量依赖方式抑制由Tm诱导的XBP1s表达，但不影响IRE1α的磷酸化。MKC-3946阻断XBP1剪接，并增强由bortezomib或17-AAG诱导的细胞毒性。MKC-3946（10μM）增强了由bortezomib或17-AAG引起的内质网应激介导的凋亡，并即使在BMSCs或外源性IL-6存在的情况下，也增强了内质网应激剂的细胞毒性[3]。

MKC-3946（100 mg/kg，i.p.）在体内ER应激模型中抑制XBP1剪接，与显著的MM细胞生长抑制相关，无论是单独使用还是与博腾珠单抗联用。MKC-3946显著降低了治疗组相比对照组的MM肿瘤生长。通过MKC-3946抑制XBP1剪接与体内MM生长减少相关，无论是单独使用还是与博腾珠单抗联用[3]。

For each assay, the various numbers of cells (1,000 for cell proliferation and 10,000 for cell viability assays) are seeded in 96-well plates, followed by either vehicle (DMSO) or increasing concentrations of the drug. For detection of relative numbers of living cells, 10 μL of MTT (5 mg/mL) is added to each well, placed in an incubator for four hours, followed by centrifugation (1,000 rpm, 5 min); 100 μL of supernatant media from each well are carefully removed and 100 μL of SDS buffer (20% in water) is added to dissolve the crystals. Results are further read on the spectrophotometer machine at 570 nM wavelength. Half maximal inhibitory concentration (IC50) is calculated using the GraphPad Prism 5. A synergy of combination of two drugs is determined using the CalcuSyn software. The extent of drug interaction between the two drugs is determined using the combination index (CI) for mutually exclusive drugs. Different CI values are obtained when solving the equation for different concentrations of drugs. A CI of 1 indicates an additive effect, whereas a CI of <1denotes synergy. All experiments are repeated at least three times [1].

CB17 SCID mice (48-54 days old) are injected subcutaneously with 1×10^7 RPMI 8226 cells mixed with Matrigel on day 0, and receive treatment for 21 days starting on day1. Mice are assigned into 4 groups (n=8): daily intraperitoneal injections of 100 mg/kg MKC-3946; intravenous injections of 0.15 mg/kg bortezomib twice a week; a combination of MKC-3946 intraperitoneally with bortezomib intravenously; and 10% HPBCD intraperitoneally with normal saline intravenously as vehicle control. Tumor volume is calculated from caliper measurements every 3 to 4 days; mice are killed when tumors reached 1.5 cm in length. Survival is evaluated from the first day of treatment until death [3].