Ravindra K

Ravindra K. 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor. and plant genera and have a variety of biological activities (3). Our laboratory previously reported that one of the 1,4-naphthoquinones, plumbagin, is a potent inhibitor of the KAT p300. Plumbagin specifically inhibits p300-mediated p53 acetylation but not the p53 acetylation by the lysine acetyltransferase KAT2B (p300/CBP-associated factor) (4). This study described for the first time that a structural entity (a hydroxyl DRIP78 group at the 5th position of plumbagin) is required for the inhibition Sorafenib Tosylate (Nexavar) of acetyltransferase activity. However, naphthoquinone derivatives are relatively toxic molecules, and their efficacy and utility has been limited due to this characteristic (5,C8). The aim of the present study is to understand the mechanism of KAT inhibition as well as the chemical entity responsible for its cytotoxicity and, thus, to synthesize a non-toxic KAT inhibitor. Among the different small molecule KAT inhibitors known to date, Lys-CoA was the first to be discovered as a p300 acetyltransferase-specific inhibitor (9). The catalytic mechanisms of the enzyme have been investigated from the co-crystal structural analysis of the p300 KAT domain and Lys-CoA (10). Lys-CoA interacts extensively with the acetyltransferase domain, particularly in the hydrophobic tunnel. Lys-CoA-mediated inhibition supports a Theorell-Chance model rather than a standard ordered binding, ternary complex, or ping-pong mechanism. Based on the residues that Lys-CoA binds within the hydrophobic tunnel, a new enzyme-inhibitory scaffold, C646, has been synthesized by the same group (11). Over the years, we have discovered a few naturally occurring, small molecule KAT inhibitors (4, 12,C16). Our investigations have revealed that there are pockets in the p300 acetyltransferase KAT domain, other than the hydrophobic tunnel, where these small molecules may bind and cause enzyme inhibition (4, 17). These p300 inhibitors, such as garcinol, plumbagin, and the p300-specific garcinol derivative LTK14, have at least one binding site within the KAT domain (17). A docking analysis with plumbagin has shown that binding may not occur in the hydrophobic tunnel of the KAT domain, suggesting that other binding pockets might exist (4). Although the mechanisms of action for these small molecule inhibitors Sorafenib Tosylate (Nexavar) have been investigated in terms of enzyme binding and kinetics, the chemical nature of these small molecules has received much less attention. Notably, most KAT inhibitors consist of hydroxyl groups, leading to speculation that the -OH groups could facilitate enzyme-small molecule interactions and thereby KAT inhibition (4). In this respect, we have previously reported that the activity of plumbagin can be ascribed to the hydrogen bonding between the hydroxyl group and Sorafenib Tosylate (Nexavar) Lys-1358 in the KAT domain (4). However, plumbagin is known to react with free -SH (thiol) groups available in the intracellular milieu, including glutathione, and is also involved in redox cycling. These chemical properties of 1 1,4-naphthoquinones, such as plumbagin, may be the cause of their cytotoxicity and may influence their KAT-inhibitory activity. The toxicity also hampers their utility (5,C8). Therefore, we are interested in investigating the role of the chemical nature of plumbagin and other related 1,4-naphthoquinone analogs in KAT inhibition and cytotoxicity with the ultimate goal of synthesizing a non-toxic, reversible inhibitor. Our results suggest that the major mechanism of plumbagin-mediated KAT inhibition is through irreversible protein interactions. However, the cytotoxicity of plumbagin analogs is due to their ability to generate reactive oxygen species as well as their reactivity to thiols. The structure-function relationships of these 1,4-naphthaquinones lead us to the conclusion that the structural moieties responsible for KAT inhibition and those responsible for toxicity do not overlap and can be delineated. Based on these observations, we have synthesized a new molecule that does not Sorafenib Tosylate (Nexavar) have free thiol reactivity and has less redox cycling potential but retains KAT inhibitory activity. Thus, this molecule could potently reduce histone acetylation in cell-based assays with greatly decreased toxicity. EXPERIMENTAL PROCEDURES Cell Culture, Treatments, and Immunoblotting SHSY-5Y (human neuroblastoma) and HEK293 (human embryonic kidney) cells were cultured in Dulbecco’s modified Eagle’s.