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opencc-by-4.0Oct 2021View details →

HDAC6 screening dataset using tau-based substrate in an enzymatic assay yields selective inhibitors and activators

Structure and information of the data file DATA SET; Contains the information to which data set this information belongs. There are four possibilities denoted 1 to 4. Data set1: Enzymatic assay of human HDAC6 with commercial peptide substrate. Data set2: Enzymatic assay of human HDAC6 with custom peptide substrate. Data set3: Hit confirmation of the active molecules of the enzymatic assay of human HDAC6 with custom peptide substrate. Data set4: Determination of IC50 values for inhibition of enzymatic assay of human HDAC6 with custom peptide substrate. INTERNAL NAME; An internal name which enables identification of the compound within data sets from Fraunhofer ITMP ScreeningPort. TYPE; Type of data. Either 'inhibition' for normalized inhibition values or 'IC50' for enzymatic IC50. RELATION; Relation between TYPE and VALUE, always '='. VALUE; Value of the normalized inhibition or the enzymatic IC50. UNITS; Unit of the value. Either '%' for the normalized inhibition or 'uM' for the enzymatic IC50. NAME; Trade name of the chemical compound. SMILES; The canonical Smile of the chemical compound.   Abstract Histone deacetylase 6 (HDAC6) and HDAC10 are unique among the other HDACs as they consist of two domains instead of one. Only in the case of HDAC6 both domains are active resulting in a number of unique deacetylase reactions. Interestingly, HDAC6 can regulate the microtubule network and plays a role in the degradation of misfolded and aggregated proteins. We therefore developed a substrate (Boc-Ile-Asp-(Dimethyl)Lys-(Ac)Lys-aminoluciferin) based on a critical acetylation site of misfolded human Tau, a hallmark of Alzheimer’s Disease. This substrate was used to screen a 5632 compound encompassing repurposing library at 10 µM in a coupled, luminescence based assay. The assay was miniaturised to 10 µL per enzymatic reaction. For comparison, a generic HDAC substrate (BOC-Gly-(Ac)Lys-aminoluciferin) was also used to screen the same library. Both substrates rely on a cascade of enzymatic reactions. First, HDAC6 deacetylates the substrate followed by cleavage of aminluciferin from the peptide by porcine Trypsin and conversion of the aminoluciferin using firefly Luciferase. Compounds with an activity of at least 75% inhibition against the custom human Tau based substrate were confirmed in triplicates at the screening concentration of 10 µM. Confirmed hits, activity of at least 75%, where analysed in 8 point or 15 point dose response curves, depending on their activity. The data presented here encompass both primary data sets including 5632 compounds as well as 249 values from hit confirmation screening against the hTau based substrate and 151 IC50 values from confirmed hits.   Methods of data generation Enzymatic assay of human HDAC6 with commercial peptide substrate. The assay using the commercial peptide substrate (BOC-Gly-(Ac)Lys-aminoluciferin) was obtained from Promega Inc.. In the beginning the assay buffer is thawed and the lyophilized substrate is dissolved according to the technical manual (Promega Inc.) to create the substrate reagent. HDAC6 (obtained from BPS Biosciences) is dissolved in assay buffer at 0.2 nM, which is twice the final assay concentration. Compounds and controls are added to the plates using acoustic dispensing to reach a final concentration of 10 µM in the assay followed by 5 µl enzyme solution per well. Plates are centrifuged shortly and incubated for 10 min at RT. Afterwards, 5 µL/well substrate solution are added to the wells, centrifuged shortly and incubated for 10 min prior detection of the luminescence signal on a multimode reader. Primary screening was done at one concentration (10 µM) in singlicates.   Enzymatic assay of human HDAC6 with custom peptide substrate. The assay was designed based on a commercial HDAC6 assay available from Promega Inc. This luminescence assay works by an aminoluciferin coupled HDAC6 peptide substrate. Upon deacetylation of the peptidic substrate by HDAC6 (obtained from BPS Biosciences) Trypsin (obtained from Sigma-Aldrich) can cleave the aminoluciferine from the peptide which can be converted by Luciferase (obtained from AAT Bioquest) to the detected signal. First, a twofold concentrated enzyme solution was generated, consisting of 4 nM HDAC6 and 0.1% BSA in HEPES buffer (25 mM HEPES, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl2, pH 7.0). Second, a twofold peptide solution was generated containing 100 µM custom made peptide (Boc-Ile-Asp-(Dimethyl)Lys-(Ac)Lys-aminoluciferin) in HEPES buffer. Compounds and controls are added to the plates using acoustic dispensing to reach a final concentration of 10 µM in the assay followed by 5 µl enzyme solution per well. Plates are centrifuged shortly and 5 µL/well peptide solution are added to the wells, centrifuged shortly and incubated for 30 min at RT. Afterwards, 5 µL detection reagent (0.067 mg/mL Luciferase, 133.3 µM ATP, 0.133 mg/mL Trypsin in HEPES buffer) were added to each well. Plates were centrifuged shortly and measured on a multimode reader after 30 min incubation at RT in the dark. Primary screening was done at one concentration (10 µM) in singlicates.   Hit confirmation of the active molecules of the enzymatic assay of human HDAC6 with custom peptide substrate The assay was designed based on a commercial HDAC6 assay available from Promega Inc. This luminescence assay works by an aminoluciferin coupled HDAC6 peptide substrate. Upon deacetylation of the peptidic substrate by HDAC6 (obtained from BPS Biosciences) Trypsin (obtained from Sigma-Aldrich) can cleave the aminoluciferine from the peptide which can be converted by Luciferase (obtained from AAT Bioquest) to the detected signal. First, a twofold concentrated enzyme solution was generated, consisting of 4 nM HDAC6 and 0.1% BSA in HEPES buffer (25 mM HEPES, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl2, pH 7.0). Second, a twofold peptide solution was generated containing 100 µM custom made peptide (Boc-Ile-Asp-(Dimethyl)Lys-(Ac)Lys-aminoluciferin) in HEPES buffer. Compounds and controls are added to the plates using acoustic dispensing to reach a final concentration of 10 µM in the assay followed by 5 µl enzyme solution per well. Plates are centrifuged shortly and 5 µL/well peptide solution are added to the wells, centrifuged shortly and incubated for 30 min at RT. Afterwards, 5 µL detection reagent (0.067 mg/mL Luciferase, 133.3 µM ATP, 0.133 mg/mL Trypsin in HEPES buffer) were added to each well. Plates were centrifuged shortly and measured on a multimode reader after 30 min incubation at RT in the dark. Hit confirmation was done at one concentration (10 µM) in triplicates.   Determination of IC50 values for inhibition of enzymatic assay of human HDAC6 with custom peptide substrate The assay was designed based on a commercial HDAC6 assay available from Promega Inc. This luminescence assay works by an aminoluciferin coupled HDAC6 peptide substrate. Upon deacetylation of the peptidic substrate by HDAC6 (obtained from BPS Biosciences) Trypsin (obtained from Sigma-Aldrich) can cleave the aminoluciferine from the peptide which can be converted by Luciferase (obtained from AAT Bioquest) to the detected signal. First, a twofold concentrated enzyme solution was generated, consisting of 4 nM HDAC6 and 0.1% BSA in HEPES buffer (25 mM HEPES, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl2, pH 7.0). Second, a twofold peptide solution was generated containing 100 µM custom made peptide (Boc-Ile-Asp-(Dimethyl)Lys-(Ac)Lys-aminoluciferin) in HEPES buffer. Compounds and controls are added to the plates using acoustic dispensing to reach a final concentration of 10 µM in the assay followed by 5 µl enzyme solution per well. Plates are centrifuged shortly and 5 µL/well peptide solution are added to the wells, centrifuged shortly and incubated for 30 min at RT. Afterwards, 5 µL detection reagent (0.067 mg/mL Luciferase, 133.3 µM ATP, 0.133 mg/mL Trypsin in HEPES buffer) were added to each well. Plates were centrifuged shortly and measured on a multimode reader after 30 min incubation at RT in the dark. IC50 values were determined using 7 point dose response curves (DRCs) between 20 µM and 312 nM. In case inhibition values were not below 50% additional 7 point DRCs were measured, starting at 312 nm with a dilution factor of 2. All DRCs were recorded in triplicates.

opencc-by-4.0Nov 2017View details →

Analog Series of Compounds with High Frequency of Activity in Screening Assays

A set of 6941 analog series and associated data are provided. These series exclusively consist of compounds that are most frequently active across public screening assays.

opencc-by-4.0Aug 2021View details →

Figure 1. Response curves for percent acetyl-coenzyme A in Detecting the effect of ACCase-targeting herbicides on ACCase activity utilizing a malachite green colorimetric functional assay

Figure 1. Response curves for percent acetyl-coenzyme A carboxylase (ACCase) activities of resistant and susceptible Digitaria ciliaris biotypes in response to the increasing concentrations of the ACCase-targeting herbicides, sethoxydim, clethodim,fluazifop-p-butyl, and pinoxaden.The response was modeled based on the log rate of ACCase-targeting herbicides to create equal spacing between rates using least-squares fit regression of ACCase activity to the non-treated check. Means are represented by differing symbols for each biotype, and regression equation models are represented by differing line types for each biotype. Vertical bars represent the standard errors of the means (n = 6). Digitaria ciliaris biotypes: R1 and R2, resistant; S, susceptible. The concentration of ACCase-targeting herbicides required to cause 50% inhibition of ACCase activity (IC50) was calculated from concentration-response curves. CI, confidence interval.

opencc-by-4.0Nov 2022View details →

A Multiplex Assay to Assess the Transaminase Activity toward Chemically Diverse Amine Donors

The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of hydroxy methyl furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.

opencc-by-4.0Dec 2019View details →

Tandem Tag Assay Optimized for Semi-automated in vivo Autophagic Activity Measurement in Arabidopsis thaliana roots

This is demo data set for AuTToFlux, the semi-automated assay for in vivo autophagic activity measurement in Arabidopsis thaliana roots. The protocol and software required for the assay are available here: <a href="https://github.com/jonasoh/AuTToFlux">https://github.com/jonasoh/AuTToFlux</a>

opencc-by-4.0Nov 2020View details →

Development and Optimization of an IQF USP5 Activity Assay

Development and optimization of a fluorescence based deubiquitinase (DUB) activity assay for USP5 with internally quenched fluorophore pair (IQF) substrates: di-ubiquitin lysine linked K48 and K63 (Ub2K48, Ub2K63). The inhibitory capability of USP5 zinc-finger ubiquitin binding domain (ZnF-UBD) ligands was investigated.

opencc-by-4.0Dec 2023View details →

Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity

Summary Parkinson’s disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve PD patient’s prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF/MS) based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.

opencc-by-4.0Nov 2018View details →

USP5 Expression and Purification & Development of a Mass Spectrometry USP5 Catalytic Activity Assay

Expression and purification of full length USP51-835 for optimization of a mass spectrometry (MS) based assay to detect polyubiquitin cleavage

dryad36/100

Primer sequences for H3N2 canine influenza virus full-length amplification, polymerase activity assay, and rescue

Influenza A viruses in animal reservoirs repeatedly cross species barriers to infect humans. Once an animal-borne virus with novel antigenicity acquired the efficient human-to-human transmissibility, it will become epidemic in the population. Dogs are the closest animal companions to humans, and canine respiratory tract expresses both SAα2,3-(avian type) and α2,6-Gal (human type) receptors. However, the role of dogs in the ecology of influenza viruses is unclear. H3N2 avian influenza viruses were transmitted to dogs around 2006 and have formed stable lineages. The long-term epidemic of avian-origin H3N2 virus in canines offers the best models to investigate the effect of dogs on the evolution of influenza viruses. Here, we carried out a systematic and comparative identification of the biological characteristics of H3N2 canine influenza viruses (CIVs) isolated in worldwide over 10 years. We found that during the adaptation of H3N2 CIVs to dogs, H3N2 CIVs became to recognize the human-like SAα2,6-Gal receptor, gradually increased HA acid stability and replication ability in human airway epithelial cells, and acquired a 100% transmission rate via respiratory droplet in ferret model, which were essential hallmarks of being adapted to humans. We also identified that the frequency of substitutions related to human adaptation has gradually increased in H3N2 CIVs and determined four cumulative molecular changes responsible for the increased airborne transmission ability in ferrets. Our results suggested that canines may serve as an intermediate for the adaptation of avian influenza virus to humans. Continuous surveillance coordinated with risk assessment for CIVs is necessary.

opencc-zeroApr 2023View details →

dryad36/100

Primer sequences for H3N2 canine influenza virus full-length amplification, polymerase activity assay, and rescue

Open the record for dataset details and reuse information.

publicApr 2023View details →

Diagnosis of Active and Latent TB Infection With IFN-y Assays in African Children

ClinicalTrials.gov study NCT00456469. IPD Sharing: Not stated. Countries: 1. Publications: 3.

Nanodisk-MS Assay for the Diagnosis of Active Pulmonary and Extrapulmonary Tuberculosis in Hospitalized Patients

ClinicalTrials.gov study NCT03271567. IPD Sharing: Not stated. Countries: 1. Publications: 2.

closedIPD-NOFeb 2026View details →

Active Surveillance SNEP Assay Registry Trial for Prostate Cancer

ClinicalTrials.gov study NCT04052048. IPD Sharing: NO. Countries: 1. Publications: 11.

closedIPD-NOFeb 2026View details →

Prognostic Value of Interferon Gamma Release Assays in Predicting Active Tuberculosis Among Individuals With, or at Risk of, Latent Tuberculosis Infection

ClinicalTrials.gov study NCT01162265. IPD Sharing: NO. Countries: 1. Publications: 5.

PNU-100480 In Newly Diagnosed, Drug Sensitive Patients With Pulmonary TB; Early Bactericidal Activity; Whole Blood Assay

ClinicalTrials.gov study NCT01225640. IPD Sharing: Not stated. Countries: 1. Publications: 1.

Effect of Activated Charcoal on Serum Osmolality, Osmolal Gap, and Enzymatic Ethylene Glycol Assay

ClinicalTrials.gov study NCT07220031. IPD Sharing: UNDECIDED. Countries: 1. Publications: 5.