Micro-X-ray Fluorescence as a General High-Throughput Screening Method for Catalyst Discovery and Small Molecule Recognition

2003 ◽  
Vol 5 (3) ◽  
pp. 245-252 ◽  
Author(s):  
Thomasin C. Miller ◽  
Grace Mann ◽  
George J. Havrilla ◽  
Cyndi A. Wells ◽  
Benjamin P. Warner ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
X Ray ◽  
Molecule Recognition

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.

Micro x-ray fluorescence as a high throughput screening method for metal chelating compounds

2005 ◽  
Author(s):  
Edel M. Minogue ◽  
George J. Havrilla ◽  
Tammy P. Taylor ◽  
Anthony K. Burrell ◽  
Benjamin P. Warner
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
X Ray ◽  
Metal Chelating ◽  
Chelating Compounds

scholarly journals A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

2011 ◽  
Vol 17 (3) ◽  
pp. 314-326 ◽  
Author(s):  
Xiaohu Tang ◽  
Kathleen I. Seyb ◽  
Mickey Huang ◽  
Eli R. Schuman ◽  
Ping Shi ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Small Molecule Inhibitors ◽  
Screening Method ◽  
Live Cells ◽  
Mutant Sod1 ◽  
Protein Protein Interaction

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

A High-Throughput Screening Method for Small-Molecule Pharmacologic Chaperones of Misfolded Rhodopsin

2008 ◽  
Vol 49 (7) ◽  
pp. 3224 ◽  
Author(s):  
Syed M. Noorwez ◽  
David A. Ostrov ◽  
J. Hugh McDowell ◽  
Mark P. Krebs ◽  
Shalesh Kaushal
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method

Micro X-Ray Fluorescence As A High Throughput Screening Method For Combinatorial Chemistry

2003 ◽  
Vol 804 ◽  
Author(s):  
George J. Havrilla ◽  
Thomasin Miller ◽  
Benjamin Warner ◽  
Cyndi Wells
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Degradation Products ◽  
Screening Method ◽  
Target Material ◽  
Chemical Weapon ◽  
Combinatorial Screening ◽  
X Ray ◽  
Binding Characteristics ◽  
Binding Event

ABSTRACTMicro X-ray fluorescence is used as a high-throughput screening method for combinatorial applications. These include chemical weapon degradation products, catalytic phosphate hydrolysis and radiologic dispersion device metals. In each application the intrinsic elemental signature was utilized to identify the lead hits for the combinatorial screening in locating peptide sequences exhibiting the desired binding characteristics. In addition to being nondestructive, rapid and non-perturbing to the binding event, this method can be used to quantify the amount of target material.

A High-Throughput Screening Method to Identify Small Molecule Inhibitors of Thyroid Hormone Receptor Coactivator Binding

2006 ◽  
Vol 2006 (341) ◽  
pp. pl3-pl3 ◽  
Author(s):  
L. A. Arnold ◽  
E. Estebanez-Perpina ◽  
M. Togashi ◽  
A. Shelat ◽  
C. A. Ocasio ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Small Molecule ◽  
High Throughput ◽  
Hormone Receptor ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Thyroid Hormone Receptor ◽  
Screening Method

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

Sign in / Sign up

Export Citation Format

Share Document