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>

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>

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 High-Throughput Screening for Small-Molecule Inhibitors of Plasmodium falciparum Glucose-6-Phosphate Dehydrogenase 6-Phosphogluconolactonase

2012 ◽  
Vol 17 (6) ◽  
pp. 738-751 ◽  
Author(s):  
Janina Preuss ◽  
Michael Hedrick ◽  
Eduard Sergienko ◽  
Anthony Pinkerton ◽  
Arianna Mangravita-Novo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Antimalarial Drugs ◽  
Screening Assay ◽  
Compound Libraries ◽  
High Throughput Screening Assay ◽  
Glucose 6 Phosphate Dehydrogenase

Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites’ pentose phosphate pathway. In this study, we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries—namely, LOPAC (Sigma-Aldrich, 1280 compounds), Spectrum (MicroSource Discovery Systems, 1969 compounds), and DIVERSet (ChemBridge, 49 971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC50 values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.

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 Development of a High-Throughput Affinity Mass Spectrometry (AMS) Platform Using Laser Diode Thermal Desorption Ionization Coupled to Mass Spectrometry (LDTD-MS)

2020 ◽  
pp. 247255522097959
Author(s):  
Aniruddha Sahasrabuddhe ◽  
Dylan Oakley ◽  
Kui Chen ◽  
John D. McCarter
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
Thermal Desorption ◽  
Laser Diode ◽  
High Throughput ◽  
Solid Phase ◽  
Atmospheric Pressure Chemical Ionization ◽  
Ionization Source ◽  
Robust Detection ◽  
Compound Libraries

Affinity selection mass spectrometry (MS) or, simply, affinity mass spectrometry (AMS) is a label-free technology that has been used to identify high-affinity ligands of target proteins of interest by screening against small-molecule compound libraries and identifying molecules that are enriched in the presence of the target protein. We have previously applied Agilent Technology’s (Santa Clara, CA) RapidFire solid-phase extraction (SPE)-based high-throughput MS technology to screen small-molecule libraries using AMS. However, SPE-based technologies rely on fluidics for desalting and separation prior to mass analysis with attendant high solvent consumption, relatively high sample volume requirements, risk of sample carryover, and frequent maintenance. To address these challenges, we have established an AMS platform using a laser diode thermal desorption–atmospheric pressure chemical ionization (LDTD-APCI) ionization source (Phytronix, Quebec, Canada) coupled with a SCIEX 5600+ TripleTOF MS (Framingham, MA). We also validated a data-independent acquisition (DIA) Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) method for the robust detection and analysis of small-molecule affinity hits. An informatics platform developed in-house has resulted in a streamlined data analysis workflow for high-throughput AMS screening campaigns and reduced data processing time without compromising data quality. Finally, 68,000 compounds were screened in a single plate and affinity selected hits were confirmed in an orthogonal enzyme activity assay.

scholarly journals Phenylacetylene and Carbon Dioxide Activation by an Organometallic Samarium Complex

Inorganics ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 82 ◽  
Author(s):  
Violaine Goudy ◽  
Mathieu Xémard ◽  
Simon Karleskind ◽  
Marie Cordier ◽  
Carlos Alvarez Lamsfus ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Structure Analysis ◽  
Small Molecule ◽  
Chemical Structure ◽  
Theoretical Calculations ◽  
Small Molecule Activation ◽  
Subsequent Reaction ◽  
Samarium Complex ◽  
Low Valent ◽  
Carbon Dioxide Activation

Small molecule activation is a topic of growing importance and the use of low-valent f-elements to perform these reactions is nowadays well established. The complex Cptt2Sm(thf) (1, Cptt = 1,3-(tBu)2Cp) is shown to activate the alkyne C–H bond of phenylacetylene to form the Cptt2Sm(C≡C–Ph)(thf) complex. The subsequent reaction of this Sm(III) complex with CO2 leads to the CO2 insertion, yielding a dimeric [Cptt2Sm(O2C–C≡C–Ph)]2 complex (2), in which the carbon dioxide has been inserted in the Sm–C bond. Along with the experimental chemical structure analysis, theoretical calculations have been performed in order to rationalize the formation of 1 and 2.

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>

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