scholarly journals Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications

2021 ◽  
Vol 22 (16) ◽  
pp. 8552
Author(s):  
Ran Tohar ◽  
Tamar Ansbacher ◽  
Inbal Sher ◽  
Livnat Afriat-Jurnou ◽  
Evgeny Weinberg ◽  
...  
Keyword(s):  
Protein Design ◽  
High Throughput Screening ◽  
Collagenase Activity ◽  
Direct Evaluation ◽  
Deep Well ◽  
Support Medium ◽  
Starting Point ◽  
Major Interest ◽  
Rational Protein Design ◽  
Bacterial Lysates

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

Antimicrobial Activity of Formylchromones: Detection by a Micro-Scale Method

2011 ◽  
Vol 66 (11-12) ◽  
pp. 562-570
Author(s):  
Markku Lehtinen ◽  
Eila Pelttari ◽  
Hannu Elo
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput Screening ◽  
Test Substance ◽  
Minimal Inhibitory Concentrations ◽  
Micro Scale ◽  
Drug Candidates ◽  
Scale Method ◽  
Deep Well ◽  
Quinolone Antibiotics ◽  
Microbial Suspension

We report the antimicrobial activity of formylchromones. These compounds are remote structural analogues of nalidixic acid and quinolone antibiotics, and their activity was investigated by a simple micro-scale method designed for the determination of minimal inhibitory concentrations (MIC) of drug candidates and antibiotics against aerobic bacteria and yeasts. Minimal bactericidal and fungicidal concentrations (MBC and MFC, respectively) were also determined in connection with the MIC determinations. The results obtained were compared with those obtained using classical agar diffusion methodology. In the MIC method, deep-well micro-titration plates are used, covered by silicone sealing mats that allow diffusion of oxygen to the wells. The appropriate broth is pipetted into the wells, followed by a standardized microbial suspension (except for sterile controls) and a dilution series of the test substance or control antibiotic or a mere control solvent. The use of white nontransparent polypropylene plates allows easy visual inspection of microbial growth. For the MBC and MFC methods, samples are taken from all wells that contain a test substance or control antibiotic and do not display growth in the MIC test. The samples are streaked on agar plates, the liquid is allowed to absorb into the agar, and finally the microbes are spread all over the plate with a bent rod. Colony counts are compared with that of the untreated microbial suspension at the beginning of the MIC test. The MIC method is suitable for high-throughput screening

Lumen-oriented versus wall-oriented treatment strategies for intracranial aneurysms – A systematic review of suggested therapeutic concepts

2021 ◽  
pp. 0271678X2110574
Author(s):  
Basil E Grüter ◽  
Fabio von Faber-Castell ◽  
Serge Marbacher
Keyword(s):  
Systematic Review ◽  
Intracranial Aneurysms ◽  
Cell Injury ◽  
Treatment Strategies ◽  
Aneurysm Wall ◽  
Starting Point ◽  
Preclinical Treatment ◽  
Major Interest ◽  
Therapeutic Concepts ◽  
New Treatment

The development of new treatment strategies for intracranial aneurysms (IAs) has been and continues to be a major interest in neurovascular research. Initial treatment concepts were mainly based on a physical-mechanistic disease understanding for IA occlusion (lumen-oriented therapies). However, a growing body of literature indicates the important role of aneurysm wall biology (wall-oriented therapies) for complete IA obliteration. This systematic literature review identified studies that explored endovascular treatment strategies for aneurysm treatment in a preclinical setting. Of 5278 publications screened, 641 studies were included, categorized, and screened for eventual translation in a clinical trial. Lumen-oriented strategies included (1) enhanced intraluminal thrombus organization, (2) enhanced intraluminal packing, (3) bridging of the intraluminal space, and (4) other, alternative concepts. Wall-oriented strategies included (1) stimulation of proliferative response, (2) prevention of aneurysm wall cell injury, (3) inhibition of inflammation and oxidative stress, and (4) inhibition of extracellular matrix degradation. Overall, lumen-oriented strategies numerically still dominate over wall-oriented strategies. Among the plethora of suggested preclinical treatment strategies, only a small minority were translated into clinically applicable concepts (36 of 400 lumen-oriented and 6 of 241 wall-oriented). This systematic review provides a comprehensive overview that may provide a starting point for the development of new treatment strategies.

scholarly journals Natural Products as Modulators of Sirtuins

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3287 ◽  
Author(s):  
Berin Karaman Mayack ◽  
Wolfgang Sippl ◽  
Fidele Ntie-Kang
Keyword(s):  
Natural Products ◽  
Drug Discovery ◽  
Histone Deacetylases ◽  
High Throughput Screening ◽  
Chemical Space ◽  
Class Iii ◽  
Drug Candidates ◽  
Starting Point ◽  
Recent Developments ◽  
Potent Drug

Natural products have been used for the treatment of human diseases since ancient history. Over time, due to the lack of precise tools and techniques for the separation, purification, and structural elucidation of active constituents in natural resources there has been a decline in financial support and efforts in characterization of natural products. Advances in the design of chemical compounds and the understanding of their functions is of pharmacological importance for the biomedical field. However, natural products regained attention as sources of novel drug candidates upon recent developments and progress in technology. Natural compounds were shown to bear an inherent ability to bind to biomacromolecules and cover an unparalleled chemical space in comparison to most libraries used for high-throughput screening. Thus, natural products hold a great potential for the drug discovery of new scaffolds for therapeutic targets such as sirtuins. Sirtuins are Class III histone deacetylases that have been linked to many diseases such as Parkinson`s disease, Alzheimer’s disease, type II diabetes, and cancer linked to aging. In this review, we examine the revitalization of interest in natural products for drug discovery and discuss natural product modulators of sirtuins that could serve as a starting point for the development of isoform selective and highly potent drug-like compounds, as well as the potential application of naturally occurring sirtuin inhibitors in human health and those in clinical trials.

scholarly journals Probing active-site residues of pyranose 2-oxidase fromTrametes multicolorby semi-rational protein design

2009 ◽  
Vol 4 (4) ◽  
pp. 535-543 ◽  
Author(s):  
Clara Salaheddin ◽  
Oliver Spadiut ◽  
Roland Ludwig ◽  
Tien-Chye Tan ◽  
Christina Divne ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Active Site Residues ◽  
Rational Protein Design

scholarly journals Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)

Toxins ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 281 ◽  
Author(s):  
Jérôme Viallon ◽  
Mireille Chinain ◽  
Hélène Taiana Darius
Keyword(s):  
High Throughput Screening ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Neuroblastoma Cell ◽  
Consensus Protocol ◽  
Limit Of Quantification ◽  
Voltage Gated ◽  
Starting Point ◽  
Fish Samples ◽  
Seafood Products

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

Rational Protein Design of Paenibacillus barcinonensis Esterase EstA for Kinetic Resolution of Tertiary Alcohols

ChemCatChem ◽  
2010 ◽  
Vol 2 (8) ◽  
pp. 962-967 ◽  
Author(s):  
Arnau Bassegoda ◽  
Giang-Son Nguyen ◽  
Marlen Schmidt ◽  
Robert Kourist ◽  
Pilar Diaz ◽  
...  
Keyword(s):  
Protein Design ◽  
Kinetic Resolution ◽  
Tertiary Alcohols ◽  
Rational Protein Design ◽  
Paenibacillus Barcinonensis

scholarly journals Crystallization of interleukin-18 for structure-based inhibitor design

2015 ◽  
Vol 71 (6) ◽  
pp. 710-717
Author(s):  
Brian Krumm ◽  
Xiangzhi Meng ◽  
Yan Xiang ◽  
Junpeng Deng
Keyword(s):  
Protein Design ◽  
Immune Defense ◽  
Interleukin 18 ◽  
Structure Based Drug Design ◽  
Binding Interface ◽  
Naturally Occurring ◽  
Rational Protein Design ◽  
Entropy Reduction ◽  
Low Ionic Strength

Interleukin-18 (IL-18) is a pleiotropic pro-inflammatory cytokine belonging to the IL-1 superfamily. IL-18 plays an important role in host innate and acquired immune defense, with its activity being modulatedin vivoby its naturally occurring antagonist IL-18 binding protein (IL-18BP). Recent crystal structures of human IL-18 (hIL-18) in complex with its antagonist or cognate receptor(s) have revealed a conserved binding interface on hIL-18 representing a promising drug target. An important step in this process is obtaining crystals of apo hIL-18 or hIL-18 in complex with small-molecule inhibitors, preferably under low ionic strength conditions. In this study, surface-entropy reduction (SER) and rational protein design were employed to facilitate the crystallization of hIL-18. The results provide an excellent platform for structure-based drug design.

scholarly journals Using the BioAssay Ontology for Analyzing High-Throughput Screening Data

2014 ◽  
Vol 20 (3) ◽  
pp. 402-415 ◽  
Author(s):  
Linda Zander Balderud ◽  
David Murray ◽  
Niklas Larsson ◽  
Uma Vempati ◽  
Stephan C. Schürer ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Method Comparison ◽  
Formyl Peptide Receptor ◽  
The Public ◽  
Starting Point ◽  
Technology Gaps ◽  
Formyl Peptide ◽  
Assay Methods ◽  
Hit Identification

High-throughput screening (HTS) is the main starting point for hit identification in drug discovery programs. This has led to a rapid increase of available screening data both within pharmaceutical companies and the public domain. We have used the BioAssay Ontology (BAO) 2.0 for assay annotation within AstraZeneca to enable comparison with external HTS methods. The annotated assays have been analyzed to identify technology gaps, evaluate new methods, verify active hits, and compare compound activity between in-house and PubChem assays. As an example, the binding of a fluorescent ligand to formyl peptide receptor 1 (FPR1, involved in inflammation, for example) in an in-house HTS was measured by fluorescence intensity. In total, 155 active compounds were also tested in an external ligand binding flow cytometry assay, a method not used for in-house HTS detection. Twelve percent of the 155 compounds were found active in both assays. By the annotation of assay protocols using BAO terms, internal and external assays can easily be identified and method comparison facilitated. They can be used to evaluate the effectiveness of different assay methods, design appropriate confirmatory and counterassays, and analyze the activity of compounds for identification of technology artifacts.

PAINS: Relevance to Tool Compound Discovery and Fragment-Based Screening

2013 ◽  
Vol 66 (12) ◽  
pp. 1483 ◽  
Author(s):  
Jonathan B. Baell ◽  
Lori Ferrins ◽  
Hendrik Falk ◽  
George Nikolakopoulos
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Great Caution ◽  
Starting Point ◽  
Very High ◽  
Assay Interference

Pan assay interference compounds (PAINS) are readily discovered in any bioassay and can appear to give selective and optimisable hits. The most common PAINS can be readily recognised by their structure. However, there are compounds that closely resemble PAINS that are not specifically recognised by the PAINS filters. In addition, highly reactive compounds are not encoded for in the PAINS filters because they were excluded from the high-throughput screening (HTS) library used to develop the filters and so were never present to provide indicting data. A compounding complication in the area is that very occasionally a PAINS compound may serve as a viable starting point for progression. Despite such an occasional example, the literature is littered with an overwhelming number of examples of compounds that fail to progress and were probably not optimisable in the first place, nor useful tool compounds. Thus it is with great caution and diligence that compounds possessing a known PAINS core should be progressed through to medicinal chemistry optimisation, if at all, as the chances are very high that the hits will be found to be non-progressable, often after a significant waste of resources.

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