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. ; This data set is uploaded in preparation of a manuscript which will descibe assay and results in more detail.