14 research outputs found
Protein formulations containing polysorbates
Proteins are prone to post-translational modifications at specific sites, which can affect their physicochemical properties, and consequently also their safety and efficacy. Sources of post-translational modifications include oxygen and reactive oxygen species. Additionally, catalytic amounts of Fe(II) or Cu(I) can promote increased activities of reactive oxygen species, and thus catalyse the production of particularly reactive hydroxyl radicals. When oxidative post-translational modifications are detected in the biopharmaceutical industry, it is common practice to add chelators to the formulation. However, the resultant complexes with metals can be even more damaging. Indeed, this is supported here using an ascorbate redox system assay and peptide mapping. Ethylenediaminetetraacetic acid (EDTA) addition strongly accelerated the formation of hydroxyl radicals in an iron-ascorbate system, while diethylenetriaminepentaacetic acid (DTPA) addition did not. When Fe(III) was substituted with Cu(II), EDTA addition almost stopped hydroxyl radical production, whereas DTPA addition showed continued production, but at a reduced rate. Further, EDTA accelerated metal-catalysed oxidation of proteins, and thus did not protect them from Fe-mediated oxidative damage. As every formulation is unique, justification for EDTA or DTPA addition should be based on experimental data and not common practice
Crystallographic Study of Peptidoglycan Biosynthesis Enzyme MurD: Domain Movement Revisited.
The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway
Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.
International audienceTremendous efforts have been dedicated to the development of effective therapeutics against Alzheimer's disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimer's disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aβ1-42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215nM), and also selectively complexes Cu(2+). Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimer's disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold
Superposition of <i>Intermediate Free MurD</i> (red) to known MurD structures with identified angles of rotation of the C-terminal domain.
<p>(A) open MurD (green) (PDB entry: 1e0d [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref007" target="_blank">7</a>]). (B) closed MurD (blue) (PDB entry: 3uag [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref006" target="_blank">6</a>]). (C) open MurD (cyan) (PDB entry: 1eeh [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref007" target="_blank">7</a>]).</p
X-ray diffraction data and structure refinement.
<p>X-ray diffraction data and structure refinement.</p
Review of <i>E</i>. <i>coli</i> MurD crystallization conditions and its conformations.
<p>Review of <i>E</i>. <i>coli</i> MurD crystallization conditions and its conformations.</p
Analysis of the superposition of the <i>Intermediate Free MurD</i> and <i>Intermediate Bound MurD</i>.
<p>Analysis of the superposition of the <i>Intermediate Free MurD</i> and <i>Intermediate Bound MurD</i>.</p
Reaction mechanism and known conformations of MurD.
<p>(A) Mechanism of reaction catalyzed by MurD. (B) Rotation of the C-terminal domain from Open UMA-bound (Magenta) (PDB entry: 1eeh [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref007" target="_blank">7</a>]) to Open (Red) (PDB entry: 1e0d [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref007" target="_blank">7</a>]) to Closed (Blue) (PDB entry: 3uag [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref006" target="_blank">6</a>]) with identified angles of rotation. For the sake of clarity N-terminal and central domains are colored in cyan in all conformations.</p
Orientation and LIGPLOT representations of Arg302 in MurD structures.
<p>(A) Open MurD (PDB entry: 1e0d [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref007" target="_blank">7</a>]). (B) Closed ADP- and UMA-bound MurD (PDB entry: 3uag [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref006" target="_blank">6</a>]). (C) Closed UMA-bound MurD (PDB entry 1uag [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref005" target="_blank">5</a>]) (D) Open UMA-bound MurD (PDB entry 1eeh [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152075#pone.0152075.ref007" target="_blank">7</a>]). (E) <i>Intermediate Free MurD</i>. (F) <i>Intermediate Bound MurD</i>.</p
Superposition of <i>Intermediate Free MurD</i> (red) and <i>Intermediate Bound MurD</i> (magenta).
<p>Superposition of <i>Intermediate Free MurD</i> (red) and <i>Intermediate Bound MurD</i> (magenta).</p