The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.1 A resolution by X-ray crystallography. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein also reveals an L-shaped channel suitable for a peptidoglycan substrate. Analysis of the structural features of this enzyme provides clues to the mechanism of action of S. aureus FemA.
Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.
Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.egamycin, a natural product originally isolated from cultures of Streptomyces purpeofuscus, exhibits broad-spectrum antibacterial activity against key pathogens for which clinical treatment options are dwindling (1, 2). The chemical structure of negamycin, [2-[(3R, 5R)-3,6-diamino-5-hydroxyhexanoyl]-1-methylhydrazino]acetic acid, and synthetic routes for its synthesis were elucidated in the early 1970s ( Fig. 1A) (3,4). Early toxicity studies in dogs revealed that daily administration of negamycin led to the formation of N-methylhydrazinoacetic acid, an inhibitor of glutamate pyruvate transaminase, an outcome that caused reversible hepatic coma. Consequently, further clinical studies were not pursued (5). Although the antimicrobial activity and efficacy of negamycin have since been confirmed, analogs exhibiting an improved therapeutic window have yet to be found (6, 7). Progress on this front has been hampered, at least in part, by the fact that the molecular mechanisms of negamycin-induced cell growth inhibition have yet to be discerned conclusively.Negamycin decreases the viability of Escherichia coli by preferentially targeting protein synthesis (8). Early mechanistic investigations proposed that negamycin may act by inhibiting translation initiation (8), decreasing translational fidelity during the elongation phase of protein synthesis (9, 10), and disrupting proper translation termination (9,(11)(12)(13). Uncertainties regarding negamycin's inhibition mechanism, its highly polar physicochemical properties, and the lack of streptomycin cross-resistance led som...
The design and synthesis of a novel series of potent and cell permeable peptidomimetic inhibitors of the human beta-secretase (BACE) are described. These inhibitors feature a hydroxyethyl secondary amine isostere and a novel aromatic ring replacement for the C-terminus. The crystal structure of BACE in complex with this hydroxyethyl secondary amine isostere inhibitor is also presented.
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