Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins.

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Site-Specific 5-Formyl Cytosine Mediated DNA-Histone Cross-Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η.

DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.

Opportunity knocks for uncovering the new function of an understudied nucleosome remodeling complex member, the bromodomain PHD finger transcription factor, BPTF.

Nucleosome remodeling provides access to genomic DNA for recruitment of the transcriptional machinery to mediate gene expression. The aberrant function of nucleosome remodeling complexes has been correlated to human cancer, making them emerging therapeutic targets. The bromodomain PHD finger transcription factor, BPTF, is the largest member of the human nucleosome remodeling factor NURF. Over the last five years, BPTF has become increasingly identified as a protumorigenic factor, prompting investigations into the molecular mechanisms associated with BPTF function. Despite a druggable bromodomain, small molecule discovery is at an early stage. Here we highlight recent investigations into the biology being discovered for BPTF, chemical biology approaches used to study its function, and small molecule inhibitors being designed as future chemical probes and therapeutics.

Computational-Based Mechanistic Study and Engineering of Cytochrome P450 MycG for Selective Oxidation of 16-Membered Macrolide Antibiotics.

MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide M-IV. The enzyme evolved to oxidize M-IV preferentially over M-III and M-VI, which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG. Density functional theory computations determined that epoxidation of the substrate hampers its ability to undergo C-H abstraction, primarily due to a loss of hyperconjugation in the transition state. Metadynamics and molecular dynamics simulations revealed a hydrophobic sugar-binding pocket that is responsible for substrate recognition/specificity and was not apparent in crystal structures of the enzyme/substrate complex. Computational results also led to the identification of other interactions between the enzyme and its substrates that had not previously been observed in the cocrystal structures. Site-directed mutagenesis was then employed to test the effects of mutations hypothesized to broaden the substrate scope and alter the product profile of MycG. The results of these experiments validated this complementary effort to engineer MycG variants with improved catalytic activity toward earlier stage mycinamicin substrates.

Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains.

As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed 19F (PrOF) NMR and 1H CPMG NMR are two fragment screening assays that have complementary advantages. Here, we sought to combine these two NMR-based assays into a new screening workflow. This combination of protein- and ligand-observed experiments allows for a time- and resource-efficient multiplexed screen of mixtures of fragments and proteins. PrOF NMR is first used to screen mixtures against two proteins. Hit mixtures for each protein are identified then deconvoluted using 1H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and Plasmodium falciparum (Pf) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, PfGCN5, and fragments binding both proteins, respectively. Select hits were characterized, revealing a broad range of affinities from low µM to mM dissociation constants. Follow-up experiments supported a low-affinity second binding site on PfGCN5. This approach can be used to bias fragment screens towards more selective hits at the onset of inhibitor development in a resource- and time-efficient manner.

New inhibitors for the BPTF bromodomain enabled by structural biology and biophysical assay development.

Bromodomain-containing proteins regulate transcription through protein-protein interactions with chromatin and serve as scaffolding proteins for recruiting essential members of the transcriptional machinery. One such protein is the bromodomain and PHD-containing transcription factor (BPTF), the largest member of the nucleosome remodeling complex, NURF. Despite an emerging role for BPTF in regulating a diverse set of cancers, small molecule development for inhibiting the BPTF bromodomain has been lacking. Here we cross-validate three complementary biophysical assays to further the discovery of BPTF bromodomain inhibitors for chemical probe development: two direct binding assays (protein-observed 19F (PrOF) NMR and surface plasmon resonance (SPR)) and a competitive inhibition assay (AlphaScreen). We first compare the assays using three small molecules and acetylated histone peptides with reported affinity for the BPTF bromodomain. Using SPR with both unlabeled and fluorinated BPTF, we further determine that there is a minimal effect of 19F incorporation on ligand binding for future PrOF NMR experiments. To guide medicinal chemistry efforts towards chemical probe development, we subsequently evaluate two new BPTF inhibitor scaffolds with our suite of biophysical assays and rank-order compound affinities which could not otherwise be determined by PrOF NMR. Finally, we cocrystallize a subset of small molecule inhibitors and present the first published small molecule-protein structures with the BPTF bromodomain. We envision the biophysical assays described here and the structural insights from the crystallography will guide researchers towards developing selective and potent BPTF bromodomain inhibitors.

NMR Analyses of Acetylated H2A.Z Isoforms Identify Differential Binding Interactions with the Bromodomain of the NURF Nucleosome Remodeling Complex.

Gene specific recruitment of bromodomain-containing proteins to chromatin is affected by post-translational acetylation of lysine on histones. Whereas interactions of the bromodomain with acetylation patterns of native histones (H2A, H2B, H3, and H4) have been well characterized, the motif for recognition for histone variants H2A.Z I and H2A.Z II by bromodomains has yet to be fully investigated. Elucidating these molecular mechanisms is crucial for understanding transcriptional regulation in cellular processes involved in both development and disease. Here, we have used protein-observed fluorine NMR to fully characterize the affinities of H2A.Z I and II acetylation patterns for BPTF's bromodomain and found the diacetylated mark of lysine 7 and 13 on H2A.Z II to have the strongest interaction with K7ac preferentially engaging the binding site. We further examined the selectivity of H2A.Z histones against a variety of bromodomains, revealing that the bromodomain of CECR2 binds with the highest affinity and specificity for acetylated H2A.Z I over isoform II. These results support a possible role for different H2A.Z transcriptional activation mechanisms that involve recruitment of chromatin remodeling complexes.

Nickel-Catalyzed Coupling of Azoles with Aromatic Nitriles.

This manuscript describes the Ni-catalyzed coupling of azoles with aromatic nitriles. The use of BPh3 promotes these arylations with electronically diverse azoles and benzonitriles. While the nickel catalyst is necessary for the arylations of phenyl oxazoles, arylation of benzoxazoles with some nitriles affords the arylated products even in the absence of the Ni catalyst albeit in lower yield than the catalyzed process. The Ni-catalyzed process exhibits higher rates and a broader scope than the uncatalyzed transformation.