SUMMARYTranscription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-tomesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma as-says. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.

SUMMARY Lysine acetylation regulates gene expression through modulating protein-protein interactions in chromatin. Chemical inhibition of acetyl-lysine binding bromodomains of the major chromatin regulators BET (bromodomain and extra-terminal domain) proteins, has been shown to effectively block cell proliferation in cancer and inflammation. However, whether selective inhibition of individual BET bromodomains has distinctive functional consequences, remains only partially understood. In this study, we show that selective chemical inhibition of the first bromodomain of BET proteins using our newly designed small molecule inhibitor, Olinone, accelerated the progression of mouse primary oligodendrocyte progenitors towards differentiation, while inhibition of both bromodomains of BET proteins hindered differentiation. This effect was target-specific, as it was not detected in cells treated with inactive analogues and independent of any effect on proliferation. Therefore, selective chemical modulation of individual bromodomains, rather than use of broad-based inhibitors may enhance regenerative strategies in disorders characterized by myelin loss such as aging and neurodegeneration.

Histone lysine acetylation is central to epigenetic control of gene transcription. Bromodomains of chromosomal proteins function as acetyl-lysine (Kac) binding domains. However, how bromodomains recognize site-specific histones remains unanswered. Here, we report three three-dimensional solution structures of the bromodomains of the human transcriptional coactivators CREB-binding protein (CBP) and p300/CBP-associated factor (PCAF) bound to peptides derived from histone acetylation sites at lysines 36 and 9 in H3, and lysine 20 in H4. From structural and biochemical binding analyses, we determine consensus histone recognition by the bromodomains of PCAF and CBP, which represent two different subgroups of the bromodomain family. Through bromodomain residues in the ZA and BC loops, PCAF prefers acetylation sites with a hydrophobic residue at (Kac+2) position and a positively charged or aromatic residue at (Kac+3), whereas CBP favors bulky hydrophobic residues at (Kac+1) and (Kac+2), a positively charged residue at (Kac-1), and an aromatic residue at (Kac-2).

As a master transcription factor in cellular responses to external stress, tumor suppressor p53 is tightly regulated. Excessive p53 activity during myocardial ischemia causes irreversible cellular injury and cardiomyocyte death. p53 activation is dependent on lysine acetylation by the lysine acetyltransferase and transcriptional co-activator CBP (CREB-binding protein) and on acetylation-directed CBP recruitment for p53 target gene expression. Here, we report a small molecule ischemin, developed with a structure-guided approach to inhibit the acetyl-lysine binding activity of the bromodomain of CBP. We show that ischemin alters post-translational modifications on p53 and histones, inhibits p53 interaction with CBP and transcriptional activity in cells, and prevents apoptosis in ischemic cardiomyocytes. Our study suggests small molecule modulation of acetylation-mediated interactions in gene transcription as a new approach to therapeutic interventions of human disorders such as myocardial ischemia.

The CREB binding protein (CBP) is a human transcriptional co-activator consisting of several conserved functional modules, which interacts with distinct transcription factors including nuclear receptors, CREB, and STAT proteins. Despite the importance of CBP in transcriptional regulation, many questions regarding the role of its particular domains in CBP functions remain unanswered. Therefore, developing small molecules capable of selectively modulating a single domain of CBP is of invaluable aid at unraveling its prominent activities. Here we report the design, synthesis and biological evaluation of conformationally restricted peptides as novel modulators for the acetyllysine binding bromodomain (BRD) of CBP. Utilizing a target structure-guided and computeraided rational design approach, we developed a series of cyclic peptides with affinity for CBP BRD significantly greater than those of its biological ligands, including lysine-acetylated histones and tumor suppressor p53. The best cyclopeptide of the series exhibited a K d of 8.0 µM, representing a 24-fold improvement in affinity over that of the linear lysine 382-acetylated p53 peptide. This lead peptide is highly selective for CBP BRD over BRDs from other transcriptional proteins. Cell-based functional assays carried out in colorectal carcinoma HCT116 cells further demonstrated the efficacy of this compound to modulate p53 stability and function in response to DNA damage. Our results strongly argue that these CBP modulators can effectively inhibit p53 transcriptional activity by blocking p53K382ac binding to CBP BRD and promoting p53 instability by changes of its post-translational modification states, a different mechanism to that of the p53 inhibitors reported to date.Human transcriptional co-activator CREB binding protein (CBP) functions to physically bridge many DNA-binding transcription factors to the basal transcription machinery 1 .Correspondence to: Ming-Ming Zhou. Supporting Information Available: Schemes S1 to S3, Figures S1 to S4, Table S1, experimental procedures for the peptide synthesis, computational protocols, binding and cell-based assays. This material is available free of charge via the internet at http://pubs.acs.org. Recent studies show that upon DNA damage CBP is recruited by the tumor suppressor p53 to modify chromatin and aid transcription activation of p53 target genes. This co-activator recruitment process is facilitated by the bromodomain (BRD) of CBP binding to p53 at the C-terminal acetylated lysine 382 (K382ac)4 , 5. The molecular basis of this CBP BRD/p53 recognition was defined by the three-dimensional solution structure of CBP BRD bound to a lysine 382-acetylated p53 peptide (p53-K382ac)5. Using this complex structure and following a target-structure guided design, we have identified two cyclic peptides that selectively inhibit CBP's acetylated p53 binding activity in cells under stress conditions. These cyclopeptide ligands represent the most potent CBP BRD chemical ligands reported to date (Fig. 1A). NIH Public Acc...

SUMMARY Many molecules that could manipulate cellular function are not practical due to their large size and concomitant undesirable pharmocokinetic properties. Here we describe a bioorthogonal, highly stable boronate ester (HiSBE) synthesis and use this reaction to synthesize a bioogically active molecule from smaller precursors in a physiological context. The rapid rate of HiSBE synthesis suggests that it may be useful for assembling a wide variety of biologically active molecules in physiological solutions.

A systematic study on the base-assisted intramolecular alkylation of N-benzyl-N-chloroacetyl amino acid derivatives is described. This study resulted in the first concise and versatile route to the preparation of 3-unsubstituted 4-alkyl-4-carboxy-2-azetidinones, to be included into the scarce family of beta-lactams with quaternary centers at the C(4) position. Particularly noteworthy is that the intramolecular N(alpha)-C(alpha)-cyclization of Phe and Leu derivatives afforded the corresponding beta-lactam derivatives with moderate enantioselectivity (up to 56%). It is suggested that, in these particular cases, the cyclization reaction proceeds by way of planar enolate intermediates, which possess dynamic chirality. The described sequence of reactions, that is compatible with commonly used protecting moieties for the alpha-carboxy group, cannot be applied to dipeptides, since the cyclization to the six-membered 2,5-diketopiperazine ring occurs preferentially.

Bromodomains (BrDs), a conserved structural module in chromatin‐associated proteins, are well known for recognizing ε‐N‐acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem BrD containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow‐up of our previous work, we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydropyrido indole‐based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations, and conformational analyses of the apo‐BRD4‐BrD1|2 and BRD4‐BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Val49 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl‐lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity toward BrD1 over BrD2. Altogether, our computer‐aided molecular design techniques can effectively guide the development of small‐molecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically improved derivatives.