Ciliobrevin A (HPI-4) is an inhibitor of hedgehog signaling pathway with an IC50 <10 μM.

Hedgehog:>10 μM.

经Ciliobrevin A培养的Shh-EGFPFLAG-Gli2细胞表现出原生纤毛的缩短，并且在一大部分经Ciliobrevin A处理的细胞中，该细胞器缺失。Ciliobrevin A扰乱Shh-LIGHT2FLAG-Gli1细胞中原生纤毛的形成，并促进FLAG-Gli1在此细胞器远端积累。Ciliobrevin A显著抑制这些神经前体细胞的增殖，通过组蛋白H3磷酸化水平(pH3)测量，并减少细胞内cyclin D1蛋白以及Gli1、Gli2和N-Myc转录本的水平。Ciliobrevin A能够阻止表达SmoM2的CGNPs的增殖，并应对缺乏Su(fu)功能的CGNPs同样有效，而Smo抑制剂Cyclopamine对这两种致癌损伤均无效。Ciliobrevin A阻止了Shh刺激下FLAG-Gli2全长/抑制剂比率的增加，但HPI-2和HPI-3无显著影响。Ciliobrevin A增加了纤毛中FLAG-Gli2的水平，这种增加与其对总FLAG-Gli2水平的影响不成比例[1]。

Smo-binding assays are conducted with BODIPY-cyclopamine and Smo-overexpressing HEK 293T cells, using a CMVpromoter-based SV40 origin-containing expression construct for Smo-Myc3 (murine Smo containing three consecutive Myc epitopes at the C terminus). HEK 293T cells are seeded into eight-well chambered coverslips (80,000 cells/well) and cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells are cultured until they reached 55 to 65% confluency (14-18 h), after which they are transfected with the Smo-Myc3 expression construct and Transit-LT1. Twenty-four hours after transfection, the cells are washed with PBS and cultured in DMEM containing 0.5% FBS, 5 nM BODIPY-cyclopamine, and various concentrations of either cyclopamine or individual HPIs. After 30 min, 10 μM Hoescht 33342 is added to each well, and the HPIs are incubated with the cells for an additional 30 min. The cells are then washed two times with PBS buffer, once with phenol red-free DMEM containing 0.5% FBS, and immediately imaged using a DMI6000B compound microscope. Images are background-substracted using ImageJ software with a rolling ball size of 75 pixels, and BODIPY-cyclopamine intensity is then determined using Metamorph software. Circular regions with a diameter of 300 pixels are placed over regions containing uniformly confluent cells, and the pixel intensities of approximately 20 regions from four independent images is used to determine the average BODIPY-cyclopamine levels for each experimental condition[1].

Using MTS-8 assay to measure cell proliferation and the toxicity of this drug. 2000 cells were plated in 96-well plates per well. HPI-4 was added to cells at concentrations of 0, 5 and 10 μM in 100 μl DMEM/F12 with 10% FBS and incubated for 0, 1, 3, 6 and 9 days. Then, 10 μl MST-8 was added to the media in each well and incubated in an environment without light for 90 min. The absorbance value was measured using an enzyme microplate reader at 450 nm wavelength. The relative viability of cells was expressed by OD value.(Only for Reference)