Label-free screening small-molecule compound libraries for protein-ligands using a high-throughput optical scanning microscope

2009 ◽  
Author(s):  
Y. Y. Fei ◽  
J. P. Landry ◽  
Y. S. Sun ◽  
X. D. Zhu ◽  
X. B. Wang ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Label Free ◽  
Scanning Microscope ◽  
Optical Scanning ◽  
Protein Ligands ◽  
Compound Libraries ◽  
Small Molecule Compound

scholarly journals A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays

2008 ◽  
Vol 79 (1) ◽  
pp. 013708 ◽  
Author(s):  
Y. Y. Fei ◽  
J. P. Landry ◽  
Y. S. Sun ◽  
X. D. Zhu ◽  
J. T. Luo ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Label Free ◽  
Scanning Microscope ◽  
Label Free Detection

scholarly journals High Throughput in Silico Identification of Novel Phytochemical Inhibitors for the Master Regulator of Inflammation (TNFα)

2021 ◽  
Author(s):  
Pratap Kumar Parida ◽  
Dipak Paul ◽  
Debamitra Chakravorty
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
In Silico ◽  
Small Molecule Inhibitors ◽  
Binding Free Energy ◽  
Natural Compounds ◽  
Free Energy Calculations ◽  
Compound Libraries ◽  
Relative Binding

<p><a>The over expression of Tumor necrosis factor-α (TNFα) has been implicated in a variety of disease and is classified as a therapeutic target for inflammatory diseases (Crohn disease, psoriasis, psoriatic arthritis, rheumatoid arthritis).Commercially available therapeutics are biologics which are associated with several risks and limitations. Small molecule inhibitors and natural compounds (saponins) were identified by researchers as lead molecules against TNFα, however, </a>they were often associated with high IC50 values which can lead to their failure in clinical trials. This warrants research related to identification of better small molecule inhibitors by screening of large compound libraries. Recent developments have demonstrated power of natural compounds as safe therapeutics, hence, in this work, we have identified TNFα phytochemical inhibitors using high throughput <i>in silico </i>screening approaches of 6000 phytochemicals followed by 200 ns molecular dynamics simulations and relative binding free energy calculations. The work yielded potent hits that bind to TNFα at its dimer interface. The mechanism targeted was inhibition of oligomerization of TNFα upon phytochemical binding to restrict its interaction with TNF-R1 receptor. MD simulation analysis resulted in identification of two phytochemicals that showed stable protein-ligand conformations over time. The two compounds were triterpenoids: Momordicilin and Nimbolin A with relative binding energy- calculated by MM/PBSA to be -190.5 kJ/Mol and -188.03 kJ/Mol respectively. Therefore, through this work it is being suggested that these phytochemicals can be used for further <i>in vitro</i> analysis to confirm their inhibitory action against TNFα or can be used as scaffolds to arrive at better drug candidates.</p>

scholarly journals Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

2020 ◽  
Vol 48 (20) ◽  
pp. e120-e120 ◽  
Author(s):  
Obtin Alkhamis ◽  
Weijuan Yang ◽  
Rifat Farhana ◽  
Haixiang Yu ◽  
Yi Xiao
Keyword(s):  
Ligand Binding ◽  
Small Molecule ◽  
High Throughput ◽  
Dna Aptamer ◽  
Label Free ◽  
General Applicability ◽  
Gold Standard Method ◽  
Characterization Methods ◽  
Increased Risk

Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.

scholarly journals High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

2021 ◽  
Author(s):  
◽  
Liam D P Sampson
Keyword(s):  
Structure Analysis ◽  
Small Molecule ◽  
High Throughput ◽  
Gene Networks ◽  
Chemical Structure ◽  
Resistance Mutation ◽  
Synthetic Genetic Array ◽  
Compound Libraries ◽  
Drug Mechanism ◽  
A Genome

<p>The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation.</p>

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