Journal: Frontiers in Endocrinology

Article Title: CU Cilia – an application for image analysis by machine learning – reveals significance of cysteine cathepsin K activity for primary cilia of human thyroid epithelial cells

doi: 10.3389/fendo.2025.1588394

Figure Lengend Snippet: Detection of nuclei and primary cilia of human thyroid epithelial cells in vitro using CellProfiler™ pipelines. (A) Bar charts comparing the numbers of detected nuclei and primary cilia (a1) , cilia frequencies (a2) and cilia lengths (a3) as revealed by CellProfiler™ pipelines set up by non-expert (pink, left panels) and expert users (violet, right panels). Mean values ± standard deviations are displayed in the bar charts with individual data points indicated by circles or dots (a1–a3) . Statistical analysis was by Kruskal-Wallis multiple comparisons tests; levels of significance are indicated as **** for p<0.0001. Note that the ranges of measurements differed between non-expert and expert determinations, while the 1 h-treatments with broad-spectrum (E64d) or specific cysteine peptidase inhibitors (CA074me, Odanacatib, CathLi III) did not usually differ from controls (DMSO). (B) Merged channel confocal laser scanning micrographs depicting ARL13B-positive primary cilia ( B , green in b1–b5 ; black in b1’–b5’ ) and Draq5™-stained nuclei ( B , blue in b1–b5 ) of DMSO-treated controls (b1, b1’) or Nthy-ori 3–1 cell cultures treated with broad-spectrum (b2, b2’) or specific inhibitors of cathepsin B (b3, b3’) , cathepsin K (b4, b4’) or cathepsin L (b5, b5’) , respectively. Note that corresponding single channels of anti-ARL13B-positive primary cilia are shown in inverted contrast (b1’–b5’) for clarity. Scale bars represent 50 µm. Identical images were analyzed with the two different pipelines with n=9 technical replicates, except n=8 for Odanacatib-treated cells.

Article Snippet: However, in this study, cell cultures were incubated for 1 h and 24 h with 10 μM E64d (#BML-PI107, Enzo Life Sciences, Lörrach, Germany) for broad-spectrum inhibition of cysteine peptidase activities and with cysteine cathepsin B-, K-, and L-specific inhibitors, namely, 10 μM CA074 me (#BML-PI126, Enzo Life Sciences, Lörrach, Germany), 10 μM Odanacatib (#S1115, Selleck Chemicals GmbH, Cologne, Germany), and 10 μM cathepsin L inhibitor III (#219427, Merck KGaA, Darmstadt, Germany), respectively.

Techniques: In Vitro, Staining

Journal: Frontiers in Endocrinology

Article Title: CU Cilia – an application for image analysis by machine learning – reveals significance of cysteine cathepsin K activity for primary cilia of human thyroid epithelial cells

doi: 10.3389/fendo.2025.1588394

Figure Lengend Snippet: Elongation of primary cilia extending from human thyroid epithelial cells upon treatment with cysteine peptidase inhibitors for 24 h as revealed by CellProfiler™ and CU Cilia analyses. (A) Bar charts comparing the numbers of detected nuclei and primary cilia (a1) , cilia frequencies (a2) and cilia lengths (a3) as revealed by CellProfiler™ pipelines set up by an expert user (violet, left panels) and CU Cilia (green, right panels). Identical images (n=9) were analyzed with both approaches. Mean values ± standard deviations are displayed in the bar charts with individual data indicated by circles or dots (a1–a3) . Note that both approaches yielded comparable results. Cilia elongation was observed upon treatment with E46d and Odanacatib for 24 h (a3) . Statistical analysis was by Kruskal-Wallis and Dunn’s multiple comparisons tests; levels of significance are indicated as * for p<0.01, ** for p<0.001, and **** for p<0.0001. (B) Merged channel confocal laser scanning micrographs depicting ARL13B-positive primary cilia ( B , green in b1–b5 ; black in b1’–b5’ ) and Draq5™-stained nuclei ( B , blue in b1–b5 ) of DMSO-treated controls (b1, b1’) or Nthy-ori 3–1 cell cultures treated with broad-spectrum (b2, b2’) or specific inhibitors of cathepsin B (b3, b3’) , cathepsin K (b4, b4’) or cathepsin L (b5, b5’) , respectively. Note that corresponding single channels of anti-ARL13B-positive primary cilia are shown in inverted contrast (b1’–b5’) for clarity. Scale bars represent 50 µm. Cathepsin L inhibitor III treatment for 24 h was cytotoxic towards Nthy-ori 3–1 cells as obvious from fewer detected nuclei (a1) and more abundant apoptotic bodies (b5) .

Article Snippet: However, in this study, cell cultures were incubated for 1 h and 24 h with 10 μM E64d (#BML-PI107, Enzo Life Sciences, Lörrach, Germany) for broad-spectrum inhibition of cysteine peptidase activities and with cysteine cathepsin B-, K-, and L-specific inhibitors, namely, 10 μM CA074 me (#BML-PI126, Enzo Life Sciences, Lörrach, Germany), 10 μM Odanacatib (#S1115, Selleck Chemicals GmbH, Cologne, Germany), and 10 μM cathepsin L inhibitor III (#219427, Merck KGaA, Darmstadt, Germany), respectively.

Techniques: Staining

Journal: Frontiers in Endocrinology

Article Title: CU Cilia – an application for image analysis by machine learning – reveals significance of cysteine cathepsin K activity for primary cilia of human thyroid epithelial cells

doi: 10.3389/fendo.2025.1588394

Figure Lengend Snippet: Elongation and thinning of primary cilia extending from human thyroid epithelial cells upon treatment with general cysteine peptidase and cathepsin K-specific inhibitors for 24 h as revealed by advanced CU Cilia analyses. (A) Bar charts and box plots comparing the areas of primary cilia (a1) , major axis length (a2) , minor axis length (a3) , cilia perimeters (a4) , cilia skeleton lengths (a5) , mean distances of cilia to nearest center of mass (a6) or to nuclei boundaries (a7) as well as form factor (a8) or eccentricity of primary cilia (a9) as revealed by CU Cilia. Images (n=6) of DMSO-treated controls or Nthy-ori 3–1 cell cultures treated for 24 h with broad-spectrum or specific inhibitors of cathepsin B, cathepsin K, or cathepsin L, respectively, were analyzed and data is denoted as indicated. Mean values ± standard deviations are displayed in the bar charts with individual data indicated by circles or dots (a1–a5, a8–a9) , while box plots are displayed in a6 and a7 . Statistical analysis was conducted by Kruskal-Wallis and Dunn’s multiple comparisons tests; levels of significance are indicated as ** for p<0.01, *** for p<0.001, and **** for p<0.0001. Cilia elongation (a2, a4, a5) and cilia thinning (a3) was observed upon treatment with CA074me, E46d and Odanacatib for 24 h, while more elliptic cilia were observed in E64d- and Odanacatib-treated Nthy-ori 3–1 cells, only (a8, a9) . (B) Output images generated by CU Cilia consisting of merged channel confocal laser scanning micrographs depicting ARL13B-positive primary cilia ( B , white) and Draq5™-stained nuclei ( B , blue) with identified objects boxed ( B , orange) and object sizes indicated in white font (B) of Nthy-ori 3–1 control cell cultures. Schematic drawing summarizing the advanced features of CU Cilia measurements.

Article Snippet: However, in this study, cell cultures were incubated for 1 h and 24 h with 10 μM E64d (#BML-PI107, Enzo Life Sciences, Lörrach, Germany) for broad-spectrum inhibition of cysteine peptidase activities and with cysteine cathepsin B-, K-, and L-specific inhibitors, namely, 10 μM CA074 me (#BML-PI126, Enzo Life Sciences, Lörrach, Germany), 10 μM Odanacatib (#S1115, Selleck Chemicals GmbH, Cologne, Germany), and 10 μM cathepsin L inhibitor III (#219427, Merck KGaA, Darmstadt, Germany), respectively.

Techniques: Generated, Staining, Control

Journal: Studies in Natural Products Chemistry

Article Title: Natural Products as Cathepsin Inhibitors

doi: 10.1016/B978-0-444-63749-9.00006-2

Figure Lengend Snippet: Chemical structure of odanacatib (MK–0822).

Article Snippet: Odanacatib , a selective inhibitor of cathepsin K, was developed by Merck Frosst/Celera and is expected to be approved for postmenopausal osteoporosis treatment during this year.

Techniques:

Journal: Frontiers in Chemistry

Article Title: In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2

doi: 10.3389/fchem.2019.00534

Figure Lengend Snippet: Molecular structures of the hits selected in the prospective virtual screening campaign that were submitted to experimental confirmation. (A) methacycline; (B) benzthiazide; (C) bendroflumethiazide; (D) odanacatib.

Article Snippet: Odanacatib (99% HPLC) was acquired from AK Scientific (Y0388).

Techniques:

Journal: Frontiers in Chemistry

Article Title: In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2

doi: 10.3389/fchem.2019.00534

Figure Lengend Snippet: Dose-response curves of identified falcipain-2 inhibitors (open circles). For each compound, dotted line represents the best fit of experimental data to the four-parameter Hill equation. (A) E-64. (B) Odanacatib. (C) Methacycline. (D) Benzthiazide. (E) Bendroflumethiazide. For those compounds achieving data convergence, the resultant values for the parameters IC50, Hillslope and R2 are indicated. In all cases, equivalent volumes of DMSO vehicle were assayed in parallel (closed circles).

Article Snippet: Odanacatib (99% HPLC) was acquired from AK Scientific (Y0388).

Techniques:

Journal: Frontiers in Chemistry

Article Title: In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2

doi: 10.3389/fchem.2019.00534

Figure Lengend Snippet: Reversibility and time dependence of the inhibition of falcipain-2 by methacycline and odanacatib. Top panel: Product progress curves for the dissociation of E-I complex by rapid dilution (100-fold) of enzyme-inhibitor mix into substrate solution. (A) Methacycline. (B) Odanacatib. Bottom Panel: Product progress curves for the formation of E-I complex by rapid addition of enzyme to a substrate-inhibitor mix. (C) Methacycline. (D) Odanacatib.

Article Snippet: Odanacatib (99% HPLC) was acquired from AK Scientific (Y0388).

Techniques: Inhibition

Journal: Frontiers in Chemistry

Article Title: In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2

doi: 10.3389/fchem.2019.00534

Figure Lengend Snippet: (A) Dose-response curves for odanacatib at fixed substrate concentrations. Dotted lines represent the best fit of experimental data to the four-parameter Hill equation. (B) Effect of substrate concentration on the IC50 values of falcipain-2 inhibition by odanacatib. IC50 values increase linearly (>9-fold) with substrate concentration in the range 1.95–62.5 μM. Dotted line represents the best fit of data to linear equation. Y-axis intercept accounts for the Ki value. (C) Global fitting of kinetic data to the competitive inhibition model equation. (D) Global fit ting of kinetic data to the Morrison equation.

Article Snippet: Odanacatib (99% HPLC) was acquired from AK Scientific (Y0388).

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Chemistry

Article Title: In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2

doi: 10.3389/fchem.2019.00534

Figure Lengend Snippet: Effect of odanacatib and methacycline on the development of P. falciparum under culture. Cultures of erythrocytes infected (trophozoite stage) at 2% hematocrit and 0.5% parasitemia were incubated with increasing concentrations of odanacatib (ODA; 1, 10, or 100 μM) and methacycline (METHA; 10, 50, or 500 μM). E-64 (1, 5, or 25 μM) was used as positive control of falcipain-2 inhibition. DMSO was used as a vehicle control (V) and RPMI 1640 medium as a control (C). After 48 h, the number of infected erythrocytes (ring, trophozoite, and schizont-stages) was evaluated by light microscopy in stained blood smears. Data are the means ± SD of one experiment performed by triplicate. ( * ) p < 0.05 represents the differences between control and the treatments.

Article Snippet: Odanacatib (99% HPLC) was acquired from AK Scientific (Y0388).

Techniques: Infection, Incubation, Positive Control, Inhibition, Control, Light Microscopy, Staining

Journal: Frontiers in Chemistry

Article Title: In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2

doi: 10.3389/fchem.2019.00534

Figure Lengend Snippet: Best poses from the molecular docking experiments for odanacatib (Left) and methacycline (Right) . Interacting residues are shown in sticks.

Article Snippet: Odanacatib (99% HPLC) was acquired from AK Scientific (Y0388).

Techniques:

Journal: bioRxiv

Article Title: Effects of Parathyroid Hormone, Alendronate and Odanacatib on the mineralisation process in intracortical and endocortical Haversian bone of ovariectomized rabbits

doi: 10.1101/255703

Figure Lengend Snippet: Lumbar vertebral bone mineral density (BMD) (A) and histomorphometric analysis of the femoral diaphyseal periosteum (B) and endocortical surface (C-E) from sham-operated and ovariectomized (OVX) rabbits treated with vehicle, alendronate (ALN), odanacatib (ODN) or parathyroid hormone (PTH). PsMS/BS = periosteal mineralising surface, EcMS/BS = endocortical mineralising surface, EcMAR = endocortical mineral appositional rate (with imputation), EcBFR/BS = endocortical bone formation rate (with imputation). Data is mean ± SEM; n= 10-17 per group. *, p<0.05, **p<0.01 vs OVX+vehicle.

Article Snippet: At 6 months post-surgery, OVX rabbits were randomized by lumbar vertebrae 5-6 bone mineral density and body weight and treated for 10 months with alendronate (ALN, Merck, 100 μg/kg/2xweek, by subcutaneous injection), odanacatib (ODN, Merck, 7.5 μM•24 hr/day in Harlan Teklad 2030 and 2031 control diet) or human parathyroid hormone (1-34) (PTH, Bachem, 15 μg/kg/day, s.c. 5 times per week) ( ) .

Techniques:

Journal: bioRxiv

Article Title: Effects of Parathyroid Hormone, Alendronate and Odanacatib on the mineralisation process in intracortical and endocortical Haversian bone of ovariectomized rabbits

doi: 10.1101/255703

Figure Lengend Snippet: Mineral:matrix ratio in sham-operated and ovariectomized (OVX) rabbits treated with vehicle, alendronate (ALN), odanacatib (ODN) or parathyroid hormone (PTH) measured at endocortical surfaces without underlying tetracycline, and with underlying parallel tetracycline (B) and at intracortical surfaces without underlying tetracycline (C). Spectra were collected at calcein labels (C1 and C2), intermediate zone between the two calcein labels (I), tetracycline label (T) and deeper regions of bone, as defined in . *, p<0.05, **, p<0.01, ***, p<0.001 vs C2 or C1; +p<0.05, ++p<0.01, +++p<0.001 vs same region of OVX+vehicle; p values shown with square brackets indicate significant differences in slope of the curve. Data is mean ± SEM; n= 3-9 per group; where n<3, individual data points are shown.

Article Snippet: At 6 months post-surgery, OVX rabbits were randomized by lumbar vertebrae 5-6 bone mineral density and body weight and treated for 10 months with alendronate (ALN, Merck, 100 μg/kg/2xweek, by subcutaneous injection), odanacatib (ODN, Merck, 7.5 μM•24 hr/day in Harlan Teklad 2030 and 2031 control diet) or human parathyroid hormone (1-34) (PTH, Bachem, 15 μg/kg/day, s.c. 5 times per week) ( ) .

Techniques:

Journal: bioRxiv

Article Title: Effects of Parathyroid Hormone, Alendronate and Odanacatib on the mineralisation process in intracortical and endocortical Haversian bone of ovariectomized rabbits

doi: 10.1101/255703

Figure Lengend Snippet: Carbonate:mineral ratio in sham-operated and ovariectomized (OVX) rabbits treated with vehicle, alendronate (ALN), odanacatib (ODN) or parathyroid hormone (PTH) measured at endocortical surfaces without underlying tetracycline (F <4mo, A), and with underlying parallel tetracycline (B) and at intracortical surfaces without underlying tetracycline (C). Spectra were collected at calcein labels (C1 and C2), intermediate zone between the two calcein labels (I), tetracycline label (T) and deeper regions of bone, as defined in . *, p<0.05, **, p<0.01, ***, p<0.001 vs C2 or C1; +p<0.05, ++p<0.01, +++p<0.001 vs same region of OVX+vehicle; p values shown with square brackets indicate significant differences in slope of the curve. Data is mean ± SEM; n= 3-9 per group; where n<3, individual data points are shown.

Article Snippet: At 6 months post-surgery, OVX rabbits were randomized by lumbar vertebrae 5-6 bone mineral density and body weight and treated for 10 months with alendronate (ALN, Merck, 100 μg/kg/2xweek, by subcutaneous injection), odanacatib (ODN, Merck, 7.5 μM•24 hr/day in Harlan Teklad 2030 and 2031 control diet) or human parathyroid hormone (1-34) (PTH, Bachem, 15 μg/kg/day, s.c. 5 times per week) ( ) .

Techniques:

Journal: bioRxiv

Article Title: Effects of Parathyroid Hormone, Alendronate and Odanacatib on the mineralisation process in intracortical and endocortical Haversian bone of ovariectomized rabbits

doi: 10.1101/255703

Figure Lengend Snippet: Amide I:II ratio in sham-operated and ovariectomized (OVX) rabbits treated with vehicle, alendronate (ALN), odanacatib (ODN) or parathyroid hormone (PTH) measured at endocortical surfaces without underlying tetracycline (A), and with underlying parallel tetracycline (B) and at intracortical surfaces without underlying tetracycline (C). Spectra were collected at calcein labels (C1 and C2), intermediate zone between the two calcein labels (I), tetracycline label (T) and deeper regions of bone, as defined in . *, p<0.05, **, p<0.01, ***, p<0.001 vs C2 or C1; p values shown with square brackets indicate significant differences in slope of the curve. Data is mean ± SEM; n= 3-9 per group; where n<3, individual data points are shown.

Article Snippet: At 6 months post-surgery, OVX rabbits were randomized by lumbar vertebrae 5-6 bone mineral density and body weight and treated for 10 months with alendronate (ALN, Merck, 100 μg/kg/2xweek, by subcutaneous injection), odanacatib (ODN, Merck, 7.5 μM•24 hr/day in Harlan Teklad 2030 and 2031 control diet) or human parathyroid hormone (1-34) (PTH, Bachem, 15 μg/kg/day, s.c. 5 times per week) ( ) .

Techniques: