Therapeutic proteins as biopharmaceuticals have emerged as a very important class of drugs for the treatment of many diseases. However, they are less stable compared to conventional pharmaceuticals. ...Their long-term stability in solid forms, which is critical for product performance, depends heavily on the retention of the native protein structure during the lyophilization (freeze-drying) process and, thereafter, in the solid state. Indeed, the biological function of proteins is directly related to the tertiary and secondary structure. Besides physical stability and biological activity, conformational stability (three-dimensional structure) is another important aspect when dealing with protein pharmaceuticals. Moreover, denaturation as loss of higher order structure is often a precursor to aggregation or chemical instability. Careful study of the physical and chemical properties of proteins in the dried state is therefore critical during biopharmaceutical drug development to deliver a final drug product with built-in quality that is safe, high-quality, efficient, and affordable for patients. This review provides an overview of common analytical techniques suitable for characterizing pharmaceutical protein powders, providing structural, and conformational information, as well as insights into dynamics. Such information can be very useful in formulation development, where selecting the best formulation for the drug can be quite a challenge.
The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner ...side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) d-glutamic acid and dipeptide d-alanyl- d-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.
Butyrylcholinesterase (BChE) is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer's disease. To discover novel BChE inhibitors, we ...used a hierarchical virtual screening protocol followed by biochemical evaluation of 40 highest scoring hit compounds. Three of the compounds identified showed significant inhibitory activities against BChE. The most potent, compound 1 (IC50 = 21.3 nM), was resynthesized and resolved into its pure enantiomers. A high degree of stereoselective activity was revealed, and a dissociation constant of 2.7 nM was determined for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for potential optimization. Additionally, compound 1 inhibited amyloid β(1-42) peptide self-induced aggregation into fibrils (by 61.7% at 10 μM) and protected cultured SH-SY5Y cells against amyloid-β-induced toxicity. These data suggest that compound 1 represents a promising candidate for hit-to-lead follow-up in the drug-discovery process against Alzheimer's disease.
Neurodegenerative diseases are severely debilitating conditions characterized primarily by progressive neuronal loss and impairment of the nervous system. Alzheimer’s and Parkinson’s diseases are the ...most common neurodegenerative disorders, and their impact is increasing as average life expectancy increases worldwide. Although the underlying mechanisms of both progressive diseases have been extensively studied, we still lack a comprehensive understanding of the molecular basis of both diseases. Current therapeutic options do not slow the progression of the diseases and only provide symptom relief. Cell models that resemble the characteristics of the disease in question are important in drug discovery projects because they provide information about the therapeutic benefits of drugs under development. Here, we review current in vitro cell models used to study the molecular basis of Alzheimer’s and Parkinson’s disease focusing on their potential for discovering of disease-modifying therapeutics to combat neurodegenerative diseases. We discuss phenotypic screening as an important approach for identifying novel therapeutic molecules. Advances in the development of cell-based assays for drug discovery are discussed, ranging from simple monoculture cell models to high-throughput three-dimensional cell models. Finally, we critically present the limitations of cell models and the caveats encountered in drug discovery to find effective treatment for neurodegenerative diseases.
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•Treatments that effectively slow the progression of neurodegeneration are lacking.•Cell-based models of neurodegeneration are pivotal in drug discovery projects.•Phenotypic screening is useful approach to screen for novel therapeutic molecules.•Functional assays of neuronal death can be used to screen therapeutic compounds.
Open innovation initiatives provide opportunities for collaboration and sharing of knowledge and experience between industry, academia, and government institutions. Through open innovation, Merck is ...offering a Mini Library of 80 carefully selected compounds from previous research and development projects to a broader scientific community for testing in academic drug discovery projects. These compounds are predominantly drug-like and cover a broad range of molecular targets. They could potentially interact with other enzymes, receptors, transporters, and ion channels of interest. The Mini Library was tested on seven in-house enzymes (bacterial MurA, MurC ligase, and DdlB enzyme, human MAO-A/B, human BChE, and murine AChE), and several hits were identified. A follow-up series of structural analogues provided by Merck gave a more detailed insight into the accessibility and the quality of the hit compounds. For example, sartan derivatives were moderate inhibitors of MurC, whereas bisarylureas were potent, selective, nanomolar inhibitors of hMAO-B. Importantly, 3-n-butyl-substituted indoles were identified as low nanomolar selective inhibitors of hBChE. All in all, the hit derivatives provide new starting points for the further exploration of the chemical space of high-quality enzyme inhibitors.
Monocyclic ß-lactams (azetidin-2-ones) exhibit a wide range of biological activities, the most important of which are antibacterial, anticancer, and cholesterol absorption inhibitory activities. The ...synthesis of decorated monocyclic ß-lactams is challenging because their ring is highly constrained and consequently reactive, which is also an important determinant of their biological activity. We present the optimized synthesis of orthogonally protected 3-amino-4-substituted monocyclic ß-lactams. Among several possible synthetic approaches, Staudinger cycloaddition proved to be the most promising method for initial ring formation, yielding monocyclic ß-lactams with different substituents at the C-4 position, a phthalimido-protected 3-amino group, and a (dimethoxy)benzyl protected ring nitrogen. Challenging deprotection methods were then investigated. Oxidative cleavage with cerium ammonium nitrate and ammonia-free Birch reduction was found to be most effective for selective removal of ring nitrogen protection. Hydrazine hydrate was used for deprotection of the phthalimido group, and the procedure had to be modified by the addition of HCl in the case of aromatic substituents at the C-4 position. The presented methods and the synthesized 3-amino-4-substituted monocyclic ß-lactam derivatives are an important step toward new ß-lactams with potential pharmacological activities.
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•Synthesis and extensive SAR of a series of quinazolinone-based inhibitors of MurA.•Potent MurA inhibitors with IC50 in low micromolar range were discovered.•Some quinazolinone-based ...MurA inhibitors possess antibacterial activity.•Important compounds in the development of novel antimicrobial agents.
MurA is an intracellular bacterial enzyme that is essential for peptidoglycan biosynthesis, and is therefore an important target for antibacterial drug discovery. We report the synthesis, in silico studies and extensive structure–activity relationships of a series of quinazolinone-based inhibitors of MurA from Escherichia coli. 3-Benzyloxyphenylquinazolinones showed promising inhibitory potencies against MurA, in the low micromolar range, with an IC50 of 8µM for the most potent derivative (58). Furthermore, furan-substituted quinazolinones (38, 46) showed promising antibacterial activities, with MICs from 1µg/mL to 8µg/mL, concomitant with their MurA inhibitory potencies. These data represent an important step towards the development of novel antimicrobial agents to combat increasing bacterial resistance.
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. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.
Canine cognitive dysfunction (CCD) is common in aged dogs and has many similarities with Alzheimer's disease. Unfortunately, like Alzheimer's disease, CCD cannot be cured. In the present study, we ...treated dogs with CCD with our newly developed and characterized butyrylcholinesterase inhibitor (BChEi). Seventeen dogs were randomized into two groups (treated with BChEi and untreated) and followed for 6 months at regular check-ups. The dogs' cognitive status was determined by a Canine Dementia Scale (CADES) questionnaire and two cognitive tests. In dogs with moderate cognitive impairment, treatment caused significant improvement in the clinical rating of cognitive abilities and the performance-based tests of cognitive functioning when compared to the untreated group (p < 0.001). Dogs treated with BChEi showed markedly improved cognitive function with enhanced quality of life. No side effects were observed in the treated dogs with moderate cognitive impairment. According to the results of this preliminary study, there is an indication that novel BChEi may be a promising drug for the treatment of CCD in dogs and may be an interesting candidate for the treatment of Alzheimer's disease in humans. However, further clinical studies are needed to confirm this.
Predicting the endocrine disruption potential of compounds is a daunting but essential task. Here we report a new tool for this purpose that we have termed Endocrine Disruptome. It is a free and ...simple-to-use Web service that runs on an open source platform called Docking interface for Target Systems (DoTS). The molecular docking is handled via AutoDock Vina. Compounds are docked to 18 integrated and well-validated crystal structures of 14 different human nuclear receptors: androgen receptor; estrogen receptors α and β; glucocorticoid receptor; liver X receptors α and β; mineralocorticoid receptor; peroxisome proliferator activated receptors α, β/δ, and γ; progesterone receptor; retinoid X receptor α; and thyroid receptors α and β. Endocrine Disruptome is free of charge and available at http://endocrinedisruptome.ki.si.
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