Overexpression of coactivator associated arginine methyltransferase 1 (CARM1), a protein arginine N-methyltransferase (PRMT) family enzyme, is associated with various diseases including cancers. with S-adenosyl-L-homocysteine (AdoHcy, SAH) as a coproduct . There are nine PRMTs identified so far, which can be classified into three categories: type I (PRMT1, 2, 3, 4, 6, and 8), type II (PRMT5 and 9) and type III (PRM7) . Type I PRMTs catalyze mono- and asymmetric dimethylation of arginine residues, whereas type II PRMTs catalyze mono- and symmetric dimethylation of arginine residues . PRMT7 is the only known type III PRMT, which catalyzes monomethylation of arginine . PRMT4, also known as CARM1 (coactivator associated arginine methyltransferase 1) methylates a wide Resveratrol variety of histone and nonhistone substrates including H3R17, H3R26 , SRC-3 , CBP/p300 , NCOA2 , PABP1 , and SmB . Consequently, CARM1 participates in many cellular processes by impacting chromatin architecture and transcriptional initiation [9, 13], RNA processing and stability , and RNA splicing . Overexpression of CARM1 has been observed in multiple cancer types including myelocytic leukemia  and breast , prostate , lung , and colorectal carcinomas , making it a potential target for anticancer therapy. Due to essential roles of CARM1 in the regulation of cellular functions as well as tumorigenesis, discovery of CARM1 inhibitors has recently attracted much attention. To date, a number of CARM1 inhibitors have been reported [19C27] (see Physique S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/7086390). According to the chemical structures, these inhibitors can be divided into several categories: (i) 3,5-bis(bromohydroxybenzylidene) piperidin-4-one inhibitors (compounds 1-2 in Physique S1), (ii) pyrazole inhibitors (compounds 3C10 in Physique S1), (iii) benzo[in silicoscreening . Residues within a distance of 6?? around indole inhibitor were defined as binding pocket, which contains the binding site of AdoMet and the arginine substrate. The Specs database (http://www.specs.net/), containing ~287,000 compounds, was utilized for the virtual screening. To refine the database, we filtered it by Lipinski’s rule of five and removed pan-assay interference compounds (PAINS) [36C38] Lum with Pipeline Pilot, version 7.5 (Accelrys Inc., San Diego, CA, USA) , yielding a database of around 180,000 small-molecule compounds, which were subsequently docked and ranked with different score functions. The top-ranked 10500 candidates selected using energy scoring function of DOCK4.0  were subsequently evaluated and ranked by the AutoDock4.0 program , yielding a list of 1500 compounds. Then, the program Glide 5.5 (XP mode)  was chosen to calculate the free energy of binding between these 1500 compounds and CARM1 protein. According to the docking scores, the top-ranked 300 were clustered using Pipeline Pilot to ensure the scaffold diversity in the primary hits. The clustered molecules were cherry-picked by visual inspection based on the following considerations. (1) At least one compound Resveratrol was selected in each clustered group. (2) The binding modes were affordable and molecules not occupying the SAM or substrate binding pocket were not chosen. (3) Among a group of similar molecules, compounds with lower molecular weight were preferred. Finally, 57 compounds were purchased for further biochemical validation. 3.2. Resveratrol Enzyme Inhibition and Selectivity Assay All of the selected 57 candidate molecules were tested for CARM1 inhibition to determine their biochemical activities. Here, AlphaLISA assay, which is a powerful and versatile platform, was performed to test the inhibitory activities of the compounds. The enzyme solution and compounds or assay buffer were transferred to assay plates, which was incubated at RT. Then 5?in vivoin vitro. in vitroin vitroand in cellular environment. Open in a separate window Physique 2 Antiproliferative effect of DC_C11 and DC_C66 on several cancer cell lines. (aCc) Time-dependent and dose-dependent inhibitory effect of DC_C11 on HELA, K562, and MCF7 within 24?hrs, 48?hrs, and 72?hrs, respectively. (dCf) Time-dependent and dose-dependent inhibitory effect of DC_C66 on HELA, K562, and MCF7 within 24?hrs, 48?hrs, and 72?hrs, respectively. (gCi) Time-dependent and dose-dependent inhibitory effect of Sinefungin on HELA, K562, and MCF7 within 24?hrs, 48?hrs, and 72?hrs, respectively. 3.4. Binding-Mode Analysis To further understand the possible binding mode of DC_C11 and DC_C66 with CARM1, molecular docking study was performed with Glide in XP mode. As shown in.