Our results indicate that in complex mixtures the electrophilicity of C646 limits its KAT inhibitor activity and likely results in the modification of high abundance heat shock proteins, glycolytic enzymes, and tubulins. disease.1 For example, the past five years have seen a transformation in our knowledge of how BET bromodomains impact cancer and other pathologies, primarily due to the development of tool compounds that can be used to probe the function of these proteins in cell and animal models.2,3 Lysine acetyltransferases (KATs) are an epigenetic enzyme family that has been comparatively recalcitrant to probe development. Senicapoc (ICA-17043) The most potent, useful, and broadly applied probe of KAT activity is the p300/CBP inhibitor C646 (Physique ?Physique11). C646 was identified by Cole and co-workers using a virtual ligand screening approach. These studies found that C646 inhibts p300 selectively in vitro over other KATs and metabolic acetyltransferase enzymes. 4 Substantial evidence indicates C646 can also inhibit p300 activity in cells. For example, Andrews and co-workers recently showed that the biochemical effects of C646 on p300-catalyzed histone acetylation accurately predict the manner in which patterns of histone acetylation are affected by C646 in cells.5 C646 can be used to inhibit the p300/CBP-dependent histone modification H3K27Ac, which has been used in imaging studies to establish a causal role for histone acetylation in transcription in living cells.6 Furthermore, C646 can selectively induce cell death in leukemia cells containing the AML-ETO gene fusion, which encodes a transcription factor whose activity is dependent on p300/CBP KAT function.7,8 Open in a separate window Figure 1 Design of a clickable C646 analogue. (a) Structure of C646 and C646-yne 1. (b) Docking of C646 and C646-yne to a structure of p300 (PDB: 3BIY) suggests the two molecules can Senicapoc (ICA-17043) adopt a similar conformation in the KAT active site. C646, blue; C646-yne 1, gold. (c) Relative in vitro inhibition of p300-catalyzed acetylation by C646 and clickable analogue C646-yne (1). Recently there has been an increased interest in understanding the mechanisms and liabilities of pan-assay interference compounds (commonly referred to as PAINS).9?11 C646 is a unique molecule because, while it has proven effects on p300/CBP-mediated phenotypes in cells, it also has several chemical features that would render it a PAIN.12,13 These include a conjugated pyrazolone-furan, which is potentially reactive with cellular nucleophiles, as well as an aromatic nitro group, which could form reactive intermediates upon metabolic reduction. Initial structureCactivity relationships found that the nitroaromatic group of C646 Rabbit Polyclonal to NFE2L3 could be replaced by more metabolically benign functionalities with little loss in potency.4 In contrast, the electrophilic conjugated pyrazolone-furan of C646 was observed to be essential for p300 inhibition. This was hypothesized to be due to a requirement for planarity for the molecule to bind the p300 active-site, supported by modeling studies.3 Enzyme activity analyses suggest C646 does not covalently modify p300. However, whether C646 possesses covalent targets in a cellular context was not determined. This inspired us to develop a chemical proteomic approach to identify the covalent targets of the C646 pyrazolone-furan chemotype, in order to better understand the cellular activity of this KAT inhibitor and also gain insights into the major liabilities of electrophilic tool compounds in cells. Since a structure of the p300-C646 complex has not yet been reported, we performed docking studies to facilitate the design of our chemical proteomic probe (Figure ?Figure11b). The C646 carboxylate represents the most straightforward route for derivatization of the parent molecule, and docking analyses suggested its modification would weaken p300CC646 interactions (deleting a hydrogen bond between the ligand and enzyme) but still enable the molecule to adapt its putative binding conformation in the KAT active site. This is also consistent with structureCactivity analyses of C646 performed by Cole and co-workers.4 Therefore, we targeted the carboxylate of C646 for modification with a propargylamide moiety, providing a latent affinity handle to enable click chemistry-based enrichment and visualization (Figure ?Figure11).14 Initial.Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. chromatin-modifying enzymes in disease.1 For example, the past five years have seen a transformation in our knowledge of how BET bromodomains impact cancer and other pathologies, primarily due to the development of tool compounds that can be used to probe the function of these proteins in cell and animal models.2,3 Lysine acetyltransferases (KATs) are an epigenetic enzyme family that has been comparatively recalcitrant to probe development. The most potent, useful, and broadly applied probe of KAT activity is the p300/CBP inhibitor C646 (Figure ?Figure11). C646 was identified by Cole and co-workers using a virtual ligand screening approach. These studies found that C646 inhibts p300 selectively in vitro over other KATs and metabolic acetyltransferase enzymes.4 Substantial evidence indicates C646 can also inhibit p300 activity in cells. For example, Andrews and co-workers recently showed that the biochemical effects of C646 Senicapoc (ICA-17043) on p300-catalyzed histone acetylation accurately predict the manner in which patterns of histone acetylation are affected by C646 in cells.5 C646 can be used to inhibit the p300/CBP-dependent histone modification H3K27Ac, which has been used in imaging studies to establish a causal role for histone acetylation in transcription in living cells.6 Furthermore, C646 can selectively induce cell death in leukemia cells containing the AML-ETO gene fusion, which encodes a transcription factor whose activity is dependent on p300/CBP KAT function.7,8 Open in a separate window Figure 1 Design of a clickable C646 analogue. (a) Structure of C646 and C646-yne 1. (b) Docking of C646 and C646-yne to a structure of p300 (PDB: 3BIY) suggests the two molecules can Senicapoc (ICA-17043) adopt a similar conformation in the KAT active site. C646, blue; C646-yne 1, gold. (c) Relative in vitro inhibition of p300-catalyzed acetylation by C646 and clickable analogue C646-yne (1). Recently there has been an increased interest in understanding the mechanisms and liabilities of pan-assay interference compounds (commonly referred to as PAINS).9?11 C646 is a unique molecule because, while it has proven effects on p300/CBP-mediated phenotypes in cells, it also has several chemical features that would render it a PAIN.12,13 These include a conjugated pyrazolone-furan, which is potentially reactive with cellular nucleophiles, as well as an aromatic nitro group, which could form reactive intermediates upon metabolic reduction. Initial structureCactivity relationships found that the nitroaromatic group of C646 could be replaced by more metabolically benign functionalities with little loss in potency.4 In contrast, the electrophilic conjugated pyrazolone-furan of C646 was observed to be essential for p300 inhibition. This was hypothesized to be due to a requirement for planarity for the molecule to bind the p300 active-site, supported by modeling studies.3 Enzyme activity analyses suggest C646 does not covalently modify p300. However, whether C646 possesses covalent targets in a cellular context was not determined. This inspired us to develop a chemical proteomic approach to identify the covalent targets of the C646 pyrazolone-furan chemotype, in order to better understand the cellular activity of this KAT inhibitor and also gain insights into the major liabilities of electrophilic tool compounds in cells. Since a structure of the p300-C646 complex has not yet been reported, we performed docking studies to facilitate the design of our chemical proteomic probe (Figure ?Figure11b). The C646 carboxylate represents the most straightforward route for derivatization of the parent molecule, and docking analyses suggested its modification would weaken p300CC646 interactions (deleting a hydrogen bond between the ligand and enzyme) but still enable the molecule to adapt its putative binding conformation in the KAT active site. This is also consistent with structureCactivity analyses of C646 performed by Cole and co-workers.4 Therefore, we targeted the carboxylate of C646 for modification with a propargylamide moiety, providing a latent affinity handle to enable click chemistry-based enrichment and visualization (Figure ?Figure11).14 Initial studies found that little or no product was formed when attempting to directly couple C646 and propargylamine using traditional coupling reagents (EDC, PyBOP). However, in situ formation of the C646 mixed anhydride, using isobutylchloroformate/ em N /em -methylmorpholine, followed by.