Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains

This is the author accepted manuscript. The final version is available from American Chemical Society via the DOI in this recordBromodomains have been pursued intensively over the past several years as emerging targets for the devel-opment of anti-cancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected poly-pharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selec-tive target profile is desired. Here we report that benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones, versatile ATP-site di-rected kinase pharmacophores utilized in the development of inhibitors of multiple kinases including a number of previ-ously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity as well as how to di-rect selectivity towards inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first report-ed kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers rec-ognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid dock-ing studies.This work was supported by NIH (Grant No. U54HL127365, to N.S.G. and J.W.; No. NIH P50 GM107618, to X.X. and S.C.B.; Nos. NIH U54 HD093540 and P01 CA066996, to J.Q.), the Medical Research Council (No. MC_UU_12016/2, to D.R.A.), the Spanish Ministerio de Economia y Competitividad (MINECO) (Grant No. SAF2015-60268R, to J.M.L.), and Fondo Europeo de Desarrollo Regional (FEDER) funds (to J.M.L.). D.L.B. was supported as a Merck Fellow of Damon Runyon Cancer Research Foundation (No. DRG-2196-14)

Quantitative Interpretation of Protein Diffusion Coefficients in Mixed Protiated-Deuteriated Aqueous Solvents

Diffusion-ordered nuclear magnetic resonance (NMR) spectroscopy is widely used for the analysis of mixtures, dispersing the signals of different species in a two-dimensional spectrum according to their diffusion coefficients. However, interpretation of these diffusion coefficients is typically purely qualitative, for example, to deduce which species are bigger or smaller. In studies of proteins in solution, important questions concern the molecular weight of the proteins, the presence or absence of aggregation, and the degree of folding. The Stokes–Einstein Gierer–Wirtz estimation (SEGWE) method has been previously developed to simplify the complex relationship between diffusion coefficient and molecular mass, allowing the prediction of a species’ diffusion coefficient in a pure solvent based on its molecular weight. Here, we show that SEGWE can be extended to successfully predict both peptide and protein diffusion coefficients in mixed protiated–deuteriated water samples and, hence, distinguish effectively between globular and disordered proteins

[7-15N]guanosine-labeled oligonucleotides as a nuclear magnetic resonance probe for protein-nucleic acid interaction in the major groove

This article does not have an abstract

Palladium–peptide oxidative addition complexes for bioconjugation

Peptides bearing palladium oxidative addition complexes can be synthesized from the parent aryl halide containing substrates and react with thiol functional groups of small molecules, peptides, and proteins at low micromolar concentrations.</jats:p

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive <i>Escherichia coli</i> strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport