Of note, the enzymatic inhibitory activity of ATA against DNMT1 was greater than the activity of SAH and RG108 and it was comparable with the activity of EGCG under the current assay conditions. known inactive DNMT1 inhibitors. As part of our efforts to identify novel inhibitors of DNMT1, we conducted the experimental characterization of aurintricarboxylic acid (ATA) that in preliminary docking studies showed promising activity. ATA had a submicromolar inhibition (IC50?=?0.68?M) against DNMT1. ATA was also evaluated for Dnmt3a inhibition showing an IC50?=?1.4?M. This chapter illustrates the synergy from integrating molecular modeling and experimental methods to further advance the discovery of novel candidates for epigenetic therapies. methyltransferases (DNMT3A and DNMT3B) and the maintenance methyltransferase (DNMT1), which is generally the most abundant and active of the three (Robertson, 2001, Yokochi and Robertson, 2002, Goll and Bestor, 2005). DNMT1 is responsible for duplicating patterns of DNA methylation during replication and is essential for mammalian development and cancer cell growth (Chen et al., 2007). These enzymes are key regulators of gene transcription, and their roles in carcinogenesis have been the subject of considerable interest over the past decade (Robertson, 2001, Jones and Baylin, 2007). Therefore, specific inhibition of DNA methylation is an attractive and novel approach for cancer therapy (Robertson, 2001, Lyko and Brown, 2005, Kelly et al., 2010, Portela and Esteller, 2010). DNA methylation inhibitors have also emerged as a promising strategy for the treatment of immunodeficiency and brain disorders (Zawia et al., 2009, Miller et al., 2010). Human DNMT1 is a protein with 1616 amino acids whose structure can be divided into an N-terminal regulatory domain and a C-terminal catalytic domain (Cheng Rabbit Polyclonal to NCAPG2 and Blumenthal, 2008, Lan et al., 2010, Jurkowska et al., 2011). The mechanism of DNA cytosine-C5 methylation is schematically depicted in Fig. 1 (Vilkaitis et al., 2001, Schermelleh et al., 2005, Sippl and Jung, 2009). DNMT forms a complex with DNA and the cytosine which will be methylated flips out from the DNA. The thiol of the catalytic cysteine acts as a nucleophile that attacks the 6-position of the target cytosine to generate a covalent intermediate. The 5-position of the cytosine is activated and conducts a nucleophilic attack on the cofactor negative ionizable (N), hydrogen bond acceptor (A), hydrogen bond donors (D), and aromatic ring (R). Selected amino acid residues in the catalytic site are schematically depicted for reference. Aldicarb sulfone Comparison between the binding mode and pharmacophore hypothesis for representative DNMT inhibitors: (B) 5-azacytidine, (C) zebularine, (D) hydralazine, (E) ATA. Despite the fact that hydralazine has a small structure, it matches the aromatic ring (R), donor (D), and acceptor (A) features that are close to Glu1266, Arg1310, and Arg1312 (Fig. 7D). This is in agreement with our previous findings of the modeling of hydralazine with homology models of DNMT1 (Singh et al., 2009, Yoo and Medina-Franco, 2011b). Based on these results, it is expected that hydralazine analogues that match the top-ranked negative charge feature (N) will have improved potency, as we have suggested previously (Yoo and Medina-Franco, 2011a). Interestingly, RG108 and RG108-1, both with a carboxylate functional group, matched the negative feature (N). The indole ring of both structures satisfied the ring feature (R). EGCG also matched with four pharmacophoric features; the hydroxyl group of ring B matched with the ring feature (R), acceptor (A) and donor (D) features close to glutamate and arginine residues; the ketone of the gallate moiety matched with bad feature (N). Curcumin and mahanine matched with the aromatic ring (R) and donor feature (D) nearby Arg1310 and Arg1312. Although parthenolide matched only the acceptor feature (A), it is possible the -methylene lactone alkylates the catalytic Cys1226, as previously suggested (Liu et al., 2009). These results are similar to the insights previously reported having a homology model of the catalytic website of DNMT1. In contrast, the 19 compounds, inactive or previously used as decoys (Fig. 5B), did not match with any of the pharmacophore features or happy only one feature. Actually if most of these molecules possess a carboxylate group like known inhibitors, they did not match the bad feature (N) and the donor feature (D) interacting with Glu1266. Taken Aldicarb sulfone together, results of the structure-based pharmacophore modeling with the revised crystal structure were in good agreement with the docking studies explained above and previously reported docking and pharmacophore modeling having a Aldicarb sulfone homology model of DNMT1. V.?Experimental and Molecular Modeling Studies of a Novel Inhibitor of Human being DNMT1 It is well recognized that integration of computational and experimental approaches boosts the design of novel enzyme and additional molecular target inhibitors and further advances drug candidates (Lpez-Vallejo et al., 2011). This has been shown in the area of inhibitors of DNMT1 (Medina-Franco and.
Of note, the enzymatic inhibitory activity of ATA against DNMT1 was greater than the activity of SAH and RG108 and it was comparable with the activity of EGCG under the current assay conditions