ß-amyloid model core peptides: Effects of hydrophobes and disulfides.

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of ß-amyloid (Aß21-30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aß, and variants of these either cyclized or with an N-terminal Cys. While Aß21-30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aß16-34 led to formation of typical amyloid fibrils. NMR showed no long-range nuclear overhauser effect (nOes) in Aß21-30, Aß16-34, or their variants, however. Serial 1 H-15 N-heteronuclear single quantum coherence spectroscopy, 1 H-1 H nuclear overhauser effect spectroscopy, and 1 H-1 H total correlational spectroscopy spectra were used to follow aggregation of Aß16-34 and Cys-Aß16-34 at a site-specific level. The addition of an N-terminal Cys residue (in Cys-Aß16-34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aß16-34 and Cys-Aß16-34, according to which Cys-Aß16-34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen.

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules.

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 µM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.