scholarly journals Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 43-56
Author(s):  
Hu Zhu ◽  
Olivia W. Lee ◽  
Pranav Shah ◽  
Ajit Jadhav ◽  
Xin Xu ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Cancer ◽  
Krebs Cycle ◽  
Fumarate Hydratase ◽  
Metabolic Enzyme ◽  
Oxidation Activity ◽  
Drug Candidates ◽  
Starting Point

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose–response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction–oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a “tough target” to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

scholarly journals Identification of activators of human fumarate hydratase by quantitative high-throughput screening

2019 ◽  
Author(s):  
Hu Zhu ◽  
Olivia W. Lee ◽  
Pranav Shah ◽  
Ajit Jadhav ◽  
Xin Xu ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Cancer ◽  
Krebs Cycle ◽  
Fumarate Hydratase ◽  
Metabolic Enzyme ◽  
Oxidation Activity ◽  
Drug Candidates ◽  
Starting Point

AbstractFumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs-cycle, and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis, renal cell cancer (HLRCC), and other diseases. Currently there are no high-quality small molecule probes for studying human fumarate hydratase. To address this, we developed a quantitative high throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human fumarate hydratase. These compounds were not substrates of fumarate hydratase, were inactive in a malate dehydrogenase counter screen, and showed no detectable reduction–oxidation activity. The binding of two compounds from the series to human fumarate hydratase was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a ‘tough target’ to modulate, and the small molecule activators of human fumarate hydratase reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

SuFEx-Enabled High-Throughput Medicinal Chemistry

2019 ◽  
Author(s):  
Seiya Kitamura ◽  
Qinheng Zheng ◽  
Jordan L. Woehl ◽  
angelo solan ◽  
Emily Chen ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Small Molecule ◽  
High Throughput ◽  
Cysteine Protease ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Biologically Active ◽  
Drug Candidates ◽  
Biological Probes ◽  
Small Molecule Probes

<p>Optimization of small-molecule probes or drugs is a lengthy, challenging and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible SuFEx click chemistry. A modest high-throughput screening hit against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN=S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products directly screened to yield drug-like inhibitors with 300-fold higher potency. We showed that the improved molecule is drug-like and biologically active in a bacteria-host coculture. Since these reactions can be performed on a picomole scale to conserve reagents, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.</p>

High-Throughput Screening for Small-Molecule Adiponectin Secretion Modulators

2011 ◽  
Vol 16 (6) ◽  
pp. 628-636 ◽  
Author(s):  
Kyosuke Hino ◽  
Hidetaka Nagata ◽  
Manabu Shimonishi ◽  
Motoharu Ido
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Drug Candidates ◽  
Insulin Resistant ◽  
Therapeutic Benefits ◽  
Pparγ Agonists ◽  
Agonistic Activity

Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.

SuFEx-Enabled High-Throughput Medicinal Chemistry

2019 ◽  
Author(s):  
Seiya Kitamura ◽  
Qinheng Zheng ◽  
Jordan L. Woehl ◽  
angelo solan ◽  
Emily Chen ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Small Molecule ◽  
High Throughput ◽  
Cysteine Protease ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Biologically Active ◽  
Drug Candidates ◽  
Biological Probes ◽  
Small Molecule Probes

<p>Optimization of small-molecule probes or drugs is a lengthy, challenging and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible SuFEx click chemistry. A modest high-throughput screening hit against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN=S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products directly screened to yield drug-like inhibitors with 300-fold higher potency. We showed that the improved molecule is drug-like and biologically active in a bacteria-host coculture. Since these reactions can be performed on a picomole scale to conserve reagents, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.</p>

Identification of Novel Small-Molecule Inhibitors for Human Transketolase by High-Throughput Screening with Fluorescent Intensity (FLINT) Assay

2004 ◽  
Vol 9 (5) ◽  
pp. 427-433 ◽  
Author(s):  
Mark X. Du ◽  
Janet Sim ◽  
Lijuan Fang ◽  
Zheng Yin ◽  
Sean Koh ◽  
...  
Keyword(s):  
Tumor Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Acid Synthesis ◽  
Pentose Phosphate ◽  
Metabolic Enzyme ◽  
Fluorescent Intensity ◽  
Novel Approach ◽  
Starting Point

The metabolic enzyme transketolase (TK) plays a crucial role in tumor cell nucleic acid synthesis, using glucose through the elevated nonoxidative pentose phosphate pathway (PPP). Identification of inhibitors specifically targeting TK and preventing the nonoxidative PPP from generating the RNA ribose precursor, ribose-5-phosphate, provides a novel approach for developing effective anticancer therapeutic agents. The full-length human transketolase gene was cloned and expressed in Escherichia coli and the recombinant human transketolase protein purified to homogeneity. A fluorescent intensity (FLINT) assay was developed and optimized. Library compounds were screened in a high-throughput screening (HTS) campaign using the FLINT assay. Fifty-four initial hits were identified. Among them, 2 scaffolds with high selectivity, ideal physiochemical properties, and low molecular weight were selected for lead optimization studies. These compounds specifically inhibited in vitro TK enzyme activity and suppressed tumor cell proliferation in at least 3 cancer cell lines: SW620, LS174T, and MIA PaCa-2. Identification of these active scaffolds represents a good starting point for development of drugs specifically targeting TK and the nonoxidative PPP for cancer therapy.

scholarly journals High-Throughput Screening Identifies Three Inhibitor Classes of the Telomere Resolvase from the Lyme Disease Spirochete

2009 ◽  
Vol 53 (10) ◽  
pp. 4441-4449 ◽  
Author(s):  
Georgia Lefas ◽  
George Chaconas
Keyword(s):  
Lyme Disease ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
Inhibitory Compounds ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Vector Borne ◽  
High Throughput Assay

ABSTRACT Lyme disease, the most common vector-borne zoonosis in North America, is caused by the spirochetal pathogen Borrelia burgdorferi. The telomere resolvase encoded by this organism (ResT) promotes the formation of covalently closed hairpin ends on the linear DNA molecules of B. burgdorferi through a two-step transesterification. ResT is essential for survival and is therefore an attractive target for the development of highly specific antiborrelial drugs. To identify ResT inhibitors, a novel fluorescence-based high-throughput assay was developed and used to screen a library of 27,520 small-molecule drug-like compounds. Six confirmed inhibitors of ResT, with 50% inhibitory concentrations between 2 and 10 μM, were identified. The inhibitors were characterized further and were grouped into three distinct classes based on their inhibitory features. The high-throughput screening assay developed in this paper, along with the six inhibitory compounds identified, provides a starting point for the future development of novel antiborrelial drugs as well as small-molecule inhibitors that will be helpful for the further dissection of the reaction mechanism.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

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.

scholarly journals A small molecule UPR modulator for diabetes identified by high throughput screening

2021 ◽  
Author(s):  
Valeria Marrocco ◽  
Tuan Tran ◽  
Siying Zhu ◽  
Seung Hyuk Choi ◽  
Ana M. Gamo ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening
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