hdac6 fluorogenic assay  (BPS Bioscience)

Journal: Cell reports

Article Title: Modeling spastic paraplegia 4 with corticospinal motor neuron-enriched cortical organoids reveals genotype-phenotype and HDAC6-targetable pathology

doi: 10.1016/j.celrep.2026.117036

Figure Lengend Snippet: (A) Representative immunofluorescence images of 3- and 6-month CSMN-COs derived from SPAST WT/WT , SPAST WT/S245X , and SPAST WT/C448Y lines, untreated or treated with the HDAC6 inhibitor Tub A, stained for acetylated tubulin and βIII-tubulin. (B and C) Quantification of the acetylated tubulin-to-βIII-tubulin ratio in 3-month (B) and 6-month (C) CSMN-COs ( n = 3–6). Tub A significantly increased tubulin acetylation in mutant CSMN-COs. (D) Representative immunofluorescence images of 3- and 6-month-old CSMN-COs from SPAST WT/WT , SPAST WT/S245X , and SPAST WT/C448Y lines, untreated or treated with Tub A, and stained for SMI32 (a marker of early axonal degeneration) and βIII-tubulin. (E–F) Quantification of the SMI32-to-βIII-tubulin ratio in individual axons from 3-month ( n = 8, (E)) and 6-month ( n = 10, (F)) CSMN-COs. Tub A treatment significantly reduced SMI32 levels in mutant organoids. (G–H) Quantification of HDAC6 enzymatic activity in 3-month ( n = 3, (G)) and 6-month ( n = 3, (H)) CSMN-COs from SPAST WT/WT , SPAST WT/S245X , and SPAST WT/C448Y lines, with or without Tub A treatment. HDAC6 activity is expressed as units (U) per mg of total protein, where one unit is defined as the amount of enzyme required to deacetylate 1 pmol of a synthetic acetylated peptide substrate per minute. Elevated HDAC6 activity in mutant organoids is significantly reduced by Tub A treatment at both time points. (I–J) WB (I) and quantification (J) of HDAC6 (black arrow) protein levels in 3-month CSMN-COs derived from SPAST WT/WT , SPAST WT/S245X , and SPAST WT/C448Y lines. HDAC6 expression, normalized to GAPDH and shown relative to SPAST WT/WT , revealed no significant differences across genotypes ( n = 4). One-way ANOVA with Tukey post hoc analysis. * p < 0.05, ** p < 0.002, *** p < 0.001, **** p < 0.0001, and unpaired t test. ## p < 0.005, ### p < 0.001. All data shown as mean ± SD; see and for details.

Article Snippet: HDAC6 Fluorogenic Assay , BPS Biosciences , 50076–1.

Techniques: Immunofluorescence, Derivative Assay, Staining, Mutagenesis, Marker, Activity Assay, Expressing

Journal: Cell reports

Article Title: Modeling spastic paraplegia 4 with corticospinal motor neuron-enriched cortical organoids reveals genotype-phenotype and HDAC6-targetable pathology

doi: 10.1016/j.celrep.2026.117036

Figure Lengend Snippet: (A and B) Co-immunoprecipitation (coIP) in HEK293T cells co-expressing HDAC6-mCherry and Flag-tagged spastin isoforms revealed that HDAC6 binds selectively to M1-spastin, but not M87-spastin (A). Quantification confirmed robust enrichment of M1-spastin isoforms in the HDAC6-containing immunoprecipitated complex, with significantly increased interaction observed for mutant forms (B). (C–E) Reciprocal coIP using anti-Flag antibody was performed to isolate spastin isoforms, followed by WB with anti-HDAC6 and anti-phospho-HDAC6 anti-bodies, showing that M1-spastin C448Y -Flag immunoprecipitated more phosphorylated HDAC6 (C). Quantification also confirmed that mutant M1-spastin isoforms pulled down greater amounts of HDAC6 compared to controls (D) and that M1-spastinC448Y-Flag immunoprecipitated more phosphorylated HDAC6 (E). (F and G) Western blot analysis of 293T lysates showing levels of acetylated tubulin and GAPDH in Con255- and M1-C448Y-overexpressing cells treated with HDAC6 siRNA (siHDAC6) or control siRNA (siCtrl) (F). C448Y overexpression results in a notable decrease in acetylated tubulin, while siHDAC6 treatment significantly increases acetylated tubulin levels in C448Y-overexpressing cells (G). GAPDH levels remain unchanged across groups and serve as a loading control ( n = 3 per group). (H) HDAC6 activity assays in SH-SY5Y cells expressing various M1- and M87-spastin mutants showed that only M1 mutations led to significantly elevated HDAC6 enzymatic activity, indicating an isoform-specific gain-of-function effect ( n = 3). One-way ANOVA with Tukey post hoc analysis. * p < 0.05, ** p < 0.002, *** p < 0.001, **** p < 0.0001. All data shown as mean ± SD; see and for details.

Article Snippet: HDAC6 Fluorogenic Assay , BPS Biosciences , 50076–1.

Techniques: Immunoprecipitation, Expressing, Mutagenesis, Western Blot, Control, Over Expression, Activity Assay

Journal: Cell reports

Article Title: Modeling spastic paraplegia 4 with corticospinal motor neuron-enriched cortical organoids reveals genotype-phenotype and HDAC6-targetable pathology

doi: 10.1016/j.celrep.2026.117036

Figure Lengend Snippet: (A) Experimental timeline of daily Tub A or vehicle treatment and CatWalk analysis in WT and dHet mice. (B) Representative CatWalk print view showing paw positioning and parameters analyzed, including print length, width, area, and position. (C–F) Quantitative gait metrics across experimental groups: print length (C), print width (D), print area (E), and print position (F) (hind paw placement relative to the ipsilateral front paw). Data represent classified runs from 6 animals per group. (G) HDAC6 enzymatic activity measured from spinal cord lysates across groups ( n = 3 mice per group). (H–J) WB analysis of lumbar spinal cord lysates showing levels of acetylated tubulin and βIII-tubulin in WT and dHet mice treated with Tub A or vehicle. Tub A treatment significantly increases acetylated tubulin levels in dHet mice. βIII-tubulin levels remain unchanged across groups and serve as a loading control ( n = 3 per group). (K) Representative toluidine blue-stained cross-sections of the dorsal column in lumbar spinal cords from treated and untreated WT and dHet mice. (L) Quantitative analyses of CST axons: total axon count per μm 2 (L), average axonal perimeter (M), and number of morphologically normal versus swollen axons (based on shape classification as normal: regular, swollen: irregular (N). Axons with a perimeter of ≥10 μm were considered swollen. Data represent cumulative axon counts ( n = 120–500) from 3 mice per group. One-way ANOVA with Tukey post hoc analysis. * p < 0.05, ** p < 0.002, *** p < 0.001, **** p < 0.0001, and unpaired t test. # p < 0.05, ## p < 0.005. All data shown as mean ± SD; see for details.

Article Snippet: HDAC6 Fluorogenic Assay , BPS Biosciences , 50076–1.

Techniques: Activity Assay, Control, Staining

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: ( A ) Ligand-based pharmacophore model derived from a diverse set of known HDAC6 inhibitors, highlighting key pharmacophoric features: hydrogen bond donors (green spheres), hydrogen bond acceptors (red spheres), hydrophobic regions (yellow sphere), and ionizable groups (red arrows). ( B ) Receiver Operating Characteristic (ROC) curve generated by automated analysis in LigandScout , demonstrating the model’s ability to discriminate between active and inactive compounds.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Derivative Assay, Generated

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: Workflow of the virtual screening process for discovering novel HDAC6 inhibitors, accompanied by the 2D structures of four selected compounds prioritized for experimental validation.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Biomarker Discovery

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: Three-dimensional representations of the best docking poses for the four hit compounds selected from the virtual screening. The binding conformations within the HDAC6 active site are shown for: Compound 7 (AKOS030496586), Compound 8 (AKOS000531201), Compound 9 (AKOS030461429) and Compound 10 (AKOS030273637). Ligands are represented as yellow carbon sticks, the protein is depicted as a mauve ribbon, and the catalytic zinc ion is shown as a gray sphere. Key interacting residues are highlighted to illustrate binding interactions.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: MDs and MM-GBSA analysis of Compound 8 , Compound 10 , Compound 9 , Compound 7 , and Trichostatin A (used as control) in complex with HDAC6. ( A ) RMSD of ligand heavy atoms over the simulation, expressed in Å; ( B ) Protein-ligand interaction profile showing contacts between ligand atoms and HDAC6 residues. Only interactions occurring for more than 30% of the 200 ns simulation time are displayed; ( C ) Binding free energy profiles calculated using the MM-GBSA method throughout the simulation.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Control, Binding Assay

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: ( A ) Ligand-based pharmacophore model derived from a diverse set of known HDAC6 inhibitors, highlighting key pharmacophoric features: hydrogen bond donors (green spheres), hydrogen bond acceptors (red spheres), hydrophobic regions (yellow sphere), and ionizable groups (red arrows). ( B ) Receiver Operating Characteristic (ROC) curve generated by automated analysis in LigandScout , demonstrating the model’s ability to discriminate between active and inactive compounds.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Derivative Assay, Generated

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: Workflow of the virtual screening process for discovering novel HDAC6 inhibitors, accompanied by the 2D structures of four selected compounds prioritized for experimental validation.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Biomarker Discovery

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: Three-dimensional representations of the best docking poses for the four hit compounds selected from the virtual screening. The binding conformations within the HDAC6 active site are shown for: Compound 7 (AKOS030496586), Compound 8 (AKOS000531201), Compound 9 (AKOS030461429) and Compound 10 (AKOS030273637). Ligands are represented as yellow carbon sticks, the protein is depicted as a mauve ribbon, and the catalytic zinc ion is shown as a gray sphere. Key interacting residues are highlighted to illustrate binding interactions.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: MDs and MM-GBSA analysis of Compound 8 , Compound 10 , Compound 9 , Compound 7 , and Trichostatin A (used as control) in complex with HDAC6. ( A ) RMSD of ligand heavy atoms over the simulation, expressed in Å; ( B ) Protein-ligand interaction profile showing contacts between ligand atoms and HDAC6 residues. Only interactions occurring for more than 30% of the 200 ns simulation time are displayed; ( C ) Binding free energy profiles calculated using the MM-GBSA method throughout the simulation.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Control, Binding Assay

Journal: bioRxiv

Article Title: Genotype–Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

doi: 10.1101/2025.07.15.664947

Figure Lengend Snippet: A. Representative immunofluorescent images of 3- and 6-month MCOs derived from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, untreated or treated with the HDAC6 inhibitor Tub A, stained for acetylated tubulin and βIII-tubulin. B-C. Quantification of the acetylated tubulin to βIII-tubulin ratio in 3-month (B) and 6-month (C) MCOs (n=3-6). Tub A significantly increased tubulin acetylation in mutant MCOs. D. Representative immunofluorescent images of 3- and 6-month-old MCOs from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, untreated or treated with TubA, stained for SMI32 (a marker of early axonal degeneration) and βIII-tubulin. E-F. Quantification of the SMI32 to βIII-tubulin ratio in individual axons from 3-month (n=8, E) and 6-month (n=10, F) MCOs. Tub A treatment significantly reduced SMI32 levels in mutant organoids. G-H. Quantification of HDAC6 enzymatic activity in 3-month (n=3, G) and 6-month (n=3, H) MCOs from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, with or without Tub A treatment. HDAC6 activity is expressed as units (U) per mg of total protein, where one unit is defined as the amount of enzyme required to deacetylate 1pmol of a synthetic acetylated peptide substrate per minute. Elevated HDAC6 activity in mutant organoids is significantly reduced by Tub A treatment at both time points. I-J. WB (I) and quantification (J) of HDAC6 (black arrow) protein levels in 3-month MCOs derived from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines. HDAC6 expression, normalized to GAPDH and shown relative to SPAST WT/WT , revealed no significant differences across genotypes (n = 4). One-way ANOVA with Tukey post hoc analysis. *p<0.05, **p<0.002, ***p<0.001, ****p<0.0001. All data shown as mean ± SD, see Supplementary Tables 3-4 for details.

Article Snippet: HDAC6 activity assays were performed according to the manufacturer’s instructions (BPS Biosciences, 50076-1) and as previously described., Briefly, treated and untreated organoid samples, as well as spinal cord tissue samples, were lysed in HDCA6 lysis buffer and centrifuged at 16,000g for 10 mins at 4°C.

Techniques: Derivative Assay, Staining, Mutagenesis, Marker, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Genotype–Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

doi: 10.1101/2025.07.15.664947

Figure Lengend Snippet: A-B. Co-immunoprecipitation (Co-IP) in HEK293T cells co-expressing HDAC6-mCherry and Flag-tagged spastin isoforms revealed that HDAC6 binds selectively to M1-spastin, but not M87-spastin (A). Quantification confirmed robust enrichment of M1-spastin isoforms, in the HDAC6-containing immunoprecipitated complex, with significantly increased interaction observed for mutant forms (B). C-D. Reciprocal Co-IP using anti-Flag antibody was performed to isolate spastin isoforms, followed by WB with anti-HDAC6 and anti-phospho-HDAC6 antibodies, showing M1-Spastin C448Y -Flag immunoprecipitated more phosphorylated HDAC6. (C). Quantification also confirmed that mutant M1-spastin isoforms pulled down greater amounts of HDAC6 compared to controls (D). E. HDAC6 activity assays in SH-SY5Y cells expressing various M1 and M87 spastin mutants showed that only M1 mutations led to significantly elevated HDAC6 enzymatic activity, indicating an isoform-specific gain-of-function effect. One-way ANOVA with Tukey post hoc analysis. **p<0.002, ***p<0.001, and Unpaired t-test. ##p<0.005, ###p<0.001. All data shown as mean ± SD, see Supplementary Tables 5-6 for details.

Article Snippet: HDAC6 activity assays were performed according to the manufacturer’s instructions (BPS Biosciences, 50076-1) and as previously described., Briefly, treated and untreated organoid samples, as well as spinal cord tissue samples, were lysed in HDCA6 lysis buffer and centrifuged at 16,000g for 10 mins at 4°C.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Activity Assay

Journal: bioRxiv

Article Title: Genotype–Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

doi: 10.1101/2025.07.15.664947

Figure Lengend Snippet: A. Experimental timeline of daily Tub A or vehicle treatment and CatWalk analysis in WT and dHet mice. B. Representative CatWalk print view showing paw positioning and parameters analyzed, including print length, width, area, and position. C–F. Quantitative gait metrics across experimental groups: Print length (C), Print width (D), Print area (E), and Print position (F) (hind paw placement relative to the ipsilateral front paw). Data represent classified runs from 6 animals per group. G. HDAC6 enzymatic activity measured from spinal cord lysates across groups (n=3 mice per group). H-J. WB analysis of lumbar spinal cord lysates showing levels of acetylated tubulin and βIII-tubulin in WT and dHet mice treated with Tub A or vehicle. Tub A treatment significantly increases acetylated tubulin levels in dHet mice. βIII-tubulin levels remain unchanged across groups and serve as a loading control (n=3 per group). K. Representative toluidine blue-stained cross-sections of the dorsal column in lumbar spinal cords from treated and untreated WT and dHet mice. L. Quantitative analyses of CST axons: total axon count per μm² (L), average axonal perimeter (M), and number of morphologically normal versus swollen axons (based on shape classification as normal: regular, swollen: irregular (N). Axons with a perimeter of ≥ 10µm was considered swollen). Data represents cumulative axon counts (n=120-500) from 3 mice per group. One-way ANOVA with Tukey post hoc analysis. *p<0.05, **p<0.002, ***p<0.001, ****p<0.0001 and Unpaired t-test. #p<0.05, ##p<0.005. All data shown as mean ± SD, see Supplementary Table 8 for details.

Article Snippet: HDAC6 activity assays were performed according to the manufacturer’s instructions (BPS Biosciences, 50076-1) and as previously described., Briefly, treated and untreated organoid samples, as well as spinal cord tissue samples, were lysed in HDCA6 lysis buffer and centrifuged at 16,000g for 10 mins at 4°C.

Techniques: Activity Assay, Control, Staining