Degradation of polysorbates 20 and 80 catalysed by histidine chloride buffer.

Polysorbates are amphiphilic, non-ionic surfactants, and they represent one of the key components of biopharmaceuticals. They serve as stabilisers, and their degradation can cause particle formation, which has been an industry-wide issue over the past decade. To determine the influence of the buffers most frequently used in biopharmaceuticals on polysorbate degradation, an accelerated stability study was carried out using placebo formulations containing 0.

Protein Formulations Containing Polysorbates: Are Metal Chelators Needed at All?

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.

Rational design to biologics development: The polysorbates point of view.

Formulation development is an essential part of any biopharmaceuticals development programme, and this will affect quality, safety and efficacy of the final drug product. The vast majority of biopharmaceuticals on the market are therapeutic proteins; however, these are less stable compared to conventional pharmaceuticals. To counter aggregation, denaturation and surface adsorption of proteins in solution, surfactants are added to the formulations; however, the choice of the best formulation is a challenge that is faced during formulation development.

Comparison of the NMR and the Acid Value Determination Methods for Quality Control of Input Polysorbates.

Polysorbates (PS) are the most common non-ionic surfactants used in protein formulations. Their degradation has been studied intensively in recent years. Ester bond hydrolysis is one of many pathways of PS degradation that can lead to accumulation of free fatty acids (FFAs) and particle formation.

The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity.

The enzymatic activity of butyrylcholinesterase (BChE) in the brain increases with the progression of Alzheimer's disease, thus classifying BChE as a promising drug target in advanced Alzheimer's disease. We used structure-based drug discovery approaches to develop potent, selective, and reversible human BChE inhibitors. The most potent, compound 3, had a picomolar inhibition constant versus BChE due to strong cation-π interactions, as revealed by the solved crystal structure of its complex with human BChE.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer's disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD.

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.

New direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold.

Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship study was initiated and a focused library of analogs was synthesized and evaluated. This yielded compound 42 with improved antimycobacterial activity and low cytotoxicity.

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.

Tremendous 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.

Design, synthesis, and evaluation of new thiadiazole-based direct inhibitors of enoyl acyl carrier protein reductase (InhA) for the treatment of tuberculosis.

Mycobacterial enoyl acyl carrier protein reductase (InhA) is a clinically validated target for the treatment of tuberculosis infections, a disease that still causes the death of at least a million people annually. A known class of potent, direct, and competitive InhA inhibitors based on a tetracyclic thiadiazole structure has been shown to have in vivo activity in murine models of tuberculosis infection. On the basis of this template, we have here explored the medicinal chemistry of truncated analogues that have only three aromatic rings.

MurD enzymes: some recent developments.

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents.

Inhibitors of the peptidoglycan biosynthesis enzymes MurA-F.

The widespread emergence of resistant bacterial strains is becoming a serious threat to public health. This thus signifies the need for the development of new antibacterial agents with novel mechanisms of action. Continuous efforts in the design of novel antibacterials remain one of the biggest challenges in drug development.

Dual Inhibitor of MurD and MurE Ligases from Escherichia coli and Staphylococcus aureus.

MurD and MurE ligases, consecutive enzymes participating in the intracellular steps of bacterial peptidoglycan biosynthesis, are important targets for antibacterial drug discovery. We have designed, synthesized, and evaluated the first d-glutamic acid-containing dual inhibitor of MurD and MurE ligases from Escherichia coli and Staphylococcus aureus (IC50 values between 6.4 and 180 μM) possessing antibacterial activity against Gram-positive S.

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: design, synthesis, crystal structures, and biological evaluation.

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S.

Structure-based design of a new series of D-glutamic acid based inhibitors of bacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD).

MurD ligase is one of the key enzymes participating in the intracellular steps of peptidoglycan biosynthesis and constitutes a viable target in the search for novel antibacterial drugs to combat bacterial drug-resistance. We have designed, synthesized, and evaluated a new series of D-glutamic acid-based Escherichia coli MurD inhibitors incorporating the 5-benzylidenethiazolidin-4-one scaffold. The crystal structure of 16 in the MurD active site has provided a good starting point for the design of structurally optimized inhibitors 73-75 endowed with improved MurD inhibitory potency (IC(50) between 3 and 7 μM).

Second-generation sulfonamide inhibitors of D-glutamic acid-adding enzyme: activity optimisation with conformationally rigid analogues of D-glutamic acid.

D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors.

False positives in the early stages of drug discovery.

High-throughput screening (HTS) is one of the most powerful approaches available for identifying new lead compounds for the growing catalogue of validated drug targets. However, just as virtual and experimental HTS have accelerated lead identification and changed drug discovery, they have also introduced a large number of peculiar molecules. Some of these have turned out to be interesting for further optimization, others to be dead ends when attempts are made to optimize their activity, typically after a great deal of time and resources have been devoted.

Discovery of novel 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD ligase.

We have designed, synthesized, and evaluated 5-benzylidenerhodanine- and 5-benzylidenethiazolidine-2,4-dione-based compounds as inhibitors of bacterial enzyme MurD with E. coli IC(50) in the range 45-206 μM. The high-resolution crystal structure of MurD in complex with (R,Z)-2-(3-[{4-([2,4-dioxothiazolidin-5-ylidene]methyl)phenylamino}methyl)benzamido)pentanedioic acid [(R)-32] revealed details of the binding mode of the inhibitor within the active site and provides a good foundation for structure-based design of a novel generation of MurD inhibitors.

Synthesis and biological evaluation of N-acylhydrazones as inhibitors of MurC and MurD ligases.

The Mur ligases have an essential role in the intracellular biosynthesis of bacterial peptidoglycan, and they represent attractive targets for the design of novel antibacterials. A series of compounds with an N-acylhydrazone scaffold were synthesized and screened for inhibition of the MurC and MurD enzymes from Escherichia coli. Compounds with micromolar inhibitory activities against both MurC and MurD were identified, and some of them also showed antibacterial activity.