Longdaysin is an inhibitor of CK1α and CK1δ (IC50s: 5.6/8.8 μM). It also can inhibit ERK2 (IC50: 52 μM).

CKIα:5.6 μM (cell free), ERK2:52 μM (cell free), CKIδ:8.8 μM (cell free), CDK7:29 μM (cell free)

Longdaysin对CKIδ、CKIα、ERK2和CDK7的活性产生了抑制作用（IC50分别为8.8、5.6、52和29 μM），而对p38α的影响则较小。CKIδ缺陷细胞的周期比野生型细胞长1.1小时。在CKIδ缺陷细胞和野生型细胞中，Longdaysin以剂量依赖性方式延长了周期[1]。在乳腺癌Hs578T和MDA-MB-231细胞中，micromolar浓度的Longdaysin减弱了LRP6和DVL2的磷酸化，并降低了活性β-catenin和总β-catenin的表达，从而导致Wnt靶基因Axin2、DKK1、LEF1和Survivin的下调[2]。

在MDA-MB-231乳腺癌异种移植模型中，longdaysin与Wnt/β-catenin信号传导的抑制相关，抑制了肿瘤生长[2]。

The CKIδ, CKIα, CDK7, and ERK2 kinase assays were performed on 384-well plates (10 μl volume). The reaction mixture was as follows: for CKIδ, 2 ng/μl CKIδ, 50 μM peptide substrate RKKKAEpSVASLTSQCSYSS corresponding to human PER2 Lys659-Ser674, and CKI buffer (40 mM Tris, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/ml BSA, pH 7.5); for CKIα, 1 ng/μl CKIα, 50 μM CKI peptide substrate, and CKI buffer; for CDK7, 5 ng/μl CDK7, 100 μM Cdk7/9 peptide substrate, and CKI buffer; for ERK2, 1.5 ng/μl ERK2, 0.8 μg/μl MBP, and ERK buffer (50 mM Tris, 10 mM MgCl2, 0.5 mM DTT, 1 mM EGTA, pH 7.5). Five hundred nl of compound was added to the mixture (final 5% DMSO), and the reaction was started by adding ATP (final 5 μM). After incubation at 30°C for 3h, 10 μl of Kinase-Glo Luminescent Kinase Assay reagent was added, and the luminescence was detected to determine the remaining ATP amount. All of the tested compounds did not inhibit luciferase activity directly [1].

2×10^5 cells were suspended in 100 μL serum-free medium containing the indicated concentrations of longdaysin, and then seeded in 24-transwell chambers with 8 μm pore membrane. The lower chamber contained medium with 20% FBS. After incubation at 37°C for 6 hours, the unmigrated cells on the upper side of membrane were removed by a cotton swab, and the migrated cells were stained with crystal violet and stained cells were photomicrographed. For invasion assays, the transwell chambers with 8 μm pore membranes were coated with Matrigel [2].

MDA-MB-231 cells were injected s.c. into the right flank of nude mice (1×10^7 cells per mouse), and tumor growth was closely observed and measured every 3 days. When the tumors reached approximately 50 mm3, the mice were randomly divided into two groups (eight mice per group) and i.p. injected with the vehicle (0.8% DMSO/12% Cremophor/8% ethanol in normal saline) or 5 mg/kg longdaysin in vehicle every 3 days. This longdaysin dosage was selected based on results from preliminary experiments, and was well tolerated in the mouse model. Subsequently, tumor volumes were measured with a caliper and calculated as follows: 0.523×(length)×(width)2. After treatment for 3 weeks, the mice were sacrificed and the tumor tissues were collected and weighed before being fixed in buffered formalin [2].