Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub)(1). Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates(2). By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate(3,4). Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism(5). Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity

Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle

The vicious cycle established between bone-associated tumours and bone resorption is the central problem with therapeutic strategies against primary bone tumours and bone metastasis. Here we report data to support inhibition of BET bromodomain proteins as a promising therapeutic strategy that target simultaneously the three partners of the vicious cycle. Treatment with JQ1, a BET bromodomain inhibitor, reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation both in vitro and in vivo. These effects are associated with transcriptional silencing of MYC and RUNX2, resulting from the depletion of BRD4 from their respective loci. Moreover, JQ1 also inhibits osteoclast differentiation by interfering with BRD4-dependent RANKL activation of NFATC1 transcription. Collectively, our data indicate that JQ1 is a potent inhibitor of osteoblast and osteoclast differentiation as well as bone tumour development

The Synthesis and Crystal Structures of Two Hydrogen-Bonded N-Oxides

Abstract: (RS,RS,RS,RS)-3-(N-4′-Methoxyphenyl-N-methylamino)cyclohexane-1,2-diol N-oxide 10 and (1R,2S,3S,4R,7R,7aS,1′R)-1,2-dihydroxy-1,2-O-isopropylidene-3-(1′-hydroxy-2′-tert-butoxy-2′-oxoethyl)-7-(phenylthio)hexahydro-1H-pyrrolizine N(4)-oxide 14 were synthesised and studied by X-ray diffraction. The structure of 10·0.5CHCl3 crystallizes in the monoclinic space group P21/c with cell parameters of a = 22.8016(5) Å, b = 11.9666(2) Å, c = 11.6835(2) Å, β = 99.8698(9)°, V = 3140.75(10) Å3 and Z = 8, whereas N-oxide 14 crystallizes in the orthorhombic space group P212121 with cell parameters of a = 5.8296(1) Å, b = 16.0362(1) Å, c = 23.7167(2) Å, V = 2217.147(19) Å3 and Z = 4. These studies served to establish unambiguously the configurations within these substrates. In both cases, adjacent molecules are linked by a series of O–H···O–N hydrogen-bonds, with the N-oxide moieties participating as hydrogen-bond acceptors, to form tape-like arrangements

The Synthesis and Crystal Structures of Two Hydrogen-Bonded N-Oxides

Abstract: (RS,RS,RS,RS)-3-(N-4′-Methoxyphenyl-N-methylamino)cyclohexane-1,2-diol N-oxide 10 and (1R,2S,3S,4R,7R,7aS,1′R)-1,2-dihydroxy-1,2-O-isopropylidene-3-(1′-hydroxy-2′-tert-butoxy-2′-oxoethyl)-7-(phenylthio)hexahydro-1H-pyrrolizine N(4)-oxide 14 were synthesised and studied by X-ray diffraction. The structure of 10·0.5CHCl3 crystallizes in the monoclinic space group P21/c with cell parameters of a = 22.8016(5) Å, b = 11.9666(2) Å, c = 11.6835(2) Å, β = 99.8698(9)°, V = 3140.75(10) Å3 and Z = 8, whereas N-oxide 14 crystallizes in the orthorhombic space group P212121 with cell parameters of a = 5.8296(1) Å, b = 16.0362(1) Å, c = 23.7167(2) Å, V = 2217.147(19) Å3 and Z = 4. These studies served to establish unambiguously the configurations within these substrates. In both cases, adjacent molecules are linked by a series of O–H···O–N hydrogen-bonds, with the N-oxide moieties participating as hydrogen-bond acceptors, to form tape-like arrangements

Synthesis and Crystal Structures of (RS,RS,RS)- and (1RS,2RS,3SR)-3-(N-Methylamino)cyclohexane-1,2-diol

3-(N-Methylamino)cyclohexane-1,2-diols (RS,RS,RS)-25 and (1RS,2RS,3SR)-26 were synthesised and their crystal structures were studied by X-ray diffraction. Compound 25 co-crystallizes with chloroform in the orthorhombic space group Pna2 1 with cell parameters of a = 11.8550(2) Å, b = 19.8293(4) Å, c = 10.6163(2) Å, V = 2495.64(8) Å3 and Z = 8. Compound 26 crystallizes in the triclinic space group P-1 with cell parameters of a = 13.1019(3) Å, b = 15.0122(3) Å, c = 18.5562(5) Å, α = 95.3082(8), β = 90.3738(10), γ = 115.2775(9), V = 3281.87(14) Å3 and Z = 16. Both structures were characterised by arrays of hydrogen bonding interactions. Graphical Abstract: (RS,RS,RS)-3-(N-Methylamino)cyclohexane-1,2-diol and (1RS,2RS,3SR)-3-(N- Methylamino)cyclohexane-1,2-diol were synthesised and their structures were studied by X-ray diffraction.[Figure not available: see fulltext.] © 2013 Springer Science+Business Media New York

Progress in the development and application of small molecule inhibitors of bromodomain-acetyl-lysine interactions.

Bromodomains, protein modules that recognize and bind to acetylated lysine, are emerging as important components of cellular machinery. These acetyl-lysine (KAc) "reader" domains are part of the write-read-erase concept that has been linked with the transfer of epigenetic information. By reading KAc marks on histones, bromodomains mediate protein-protein interactions between a diverse array of partners. There has been intense activity in developing potent and selective small molecule probes that disrupt the interaction between a given bromodomain and KAc. Rapid success has been achieved with the BET family of bromodomains, and a number of potent and selective probes have been reported. These compounds have enabled linking of the BET bromodomains with diseases, including cancer and inflammation, suggesting that bromodomains are druggable targets. Herein, we review the biology of the bromodomains and discuss the SAR for the existing small molecule probes. The biology that has been enabled by these compounds is summarized

Synthesis and Crystal Structures of N-Aryl-N-methylaminocyclohexanols

N-Aryl-N-methylaminocyclohexanols 10-12 were synthesised and their crystal structures were studied by X-ray diffraction. Compound 10 crystallizes in the monoclinic space group Cc with cell parameters of a = 13.7366(2) Å, b = 13.7234(2) Å, c = 26.9057(5) Å, β = 93.0900(5), V = 5064.70(14) Å3 and Z = 16. Compound 11 crystallizes in the trigonal space group P 3 ̄ with cell parameters of a = b = 25.4429(4) Å, c = 9.6417(2) Å, V = 5405.27(16) Å3 and Z = 18. Compound 12 crystallizes in the monoclinic space group P21/n with cell parameters of a = 9.9511(2) Å, b = 11.5777(2) Å, c = 20.5968(4) Å, β = 95.5037(9), V = 2362.04(8) Å3 and Z = 8. All three structures were characterised by arrays of hydrogen bonding interactions and these crystallographic studies revealed conformations of 10-12 which gave valuable information into the mechanism of the reaction during their formation. Graphical Abstract: Crystals of (RS,RS,RS)-3-[N-methyl-N-(4'-methoxyphenyl) amino]cyclohexane-1,2-diol, (RS,RS,RS)-3-(N-methyl-N-phenylamino)cyclohexane-1, 2-diol and (RS,RS,RS)-2-fluoro-6-(N-methyl-N-phenylamino)cyclohexan-1-ol were synthesised and their structures were studied by X-ray diffraction.[Figure not available: see fulltext.]. © 2013 Springer Science+Business Media New York

Synthesis and Crystal Structures of N-Aryl-N-methylaminocyclohexanols

N-Aryl-N-methylaminocyclohexanols 10-12 were synthesised and their crystal structures were studied by X-ray diffraction. Compound 10 crystallizes in the monoclinic space group Cc with cell parameters of a = 13.7366(2) Å, b = 13.7234(2) Å, c = 26.9057(5) Å, β = 93.0900(5), V = 5064.70(14) Å3 and Z = 16. Compound 11 crystallizes in the trigonal space group P 3 ̄ with cell parameters of a = b = 25.4429(4) Å, c = 9.6417(2) Å, V = 5405.27(16) Å3 and Z = 18. Compound 12 crystallizes in the monoclinic space group P21/n with cell parameters of a = 9.9511(2) Å, b = 11.5777(2) Å, c = 20.5968(4) Å, β = 95.5037(9), V = 2362.04(8) Å3 and Z = 8. All three structures were characterised by arrays of hydrogen bonding interactions and these crystallographic studies revealed conformations of 10-12 which gave valuable information into the mechanism of the reaction during their formation. Graphical Abstract: Crystals of (RS,RS,RS)-3-[N-methyl-N-(4'-methoxyphenyl) amino]cyclohexane-1,2-diol, (RS,RS,RS)-3-(N-methyl-N-phenylamino)cyclohexane-1, 2-diol and (RS,RS,RS)-2-fluoro-6-(N-methyl-N-phenylamino)cyclohexan-1-ol were synthesised and their structures were studied by X-ray diffraction.[Figure not available: see fulltext.]. © 2013 Springer Science+Business Media New York

Ammonium-directed olefinic epoxidation: kinetic and mechanistic insights.

The ammonium-directed olefinic epoxidations of a range of differentially N-substituted cyclic allylic and homoallylic amines (derived from cyclopentene, cyclohexene, and cycloheptene) have been investigated, and the reaction kinetics have been analyzed. The results of these studies suggest that both the ring size and the identity of the substituents on nitrogen are important in determining both the overall rate and the stereochemical outcome of the epoxidation reaction. In general, secondary amines or tertiary amines with nonsterically demanding substituents on nitrogen are superior to tertiary amines with sterically demanding substituents on nitrogen in their ability to promote the oxidation reaction. Furthermore, in all cases examined, the ability of the (in situ formed) ammonium substituent to direct the stereochemical course of the epoxidation reaction is either comparable or superior to that of the analogous hydroxyl substituent. Much slower rates of ring-opening of the intermediate epoxides are observed in cyclopentene-derived and cycloheptene-derived allylic amines as compared with their cyclohexene-derived allylic and homoallylic amine counterparts, allowing for isolation of these intermediates in both of the former cases