Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: List of example PPD and TPD compounds: structure and activity in acetyl-tubulin ELISA Values for acetyl-tubulin change represent the fold change over control (DMSO)–treated cells; numbers in parentheses represent the fold change over 100 nM CNDR-51533–treated cells.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: High doses of prototype TPD and PPD compounds induce loss of post-translationally modified and total tubulin in QBI-293 cells. (A) ELISA measuring acetyl-tubulin levels in response to 4-hour treatment with TPD (CNDR-51555) or PPD (CNDR-51549). (B) ELISA measuring α-tubulin levels in response to 4-hour treatment with CNDR-51555 or CNDR-51549. (C) Representative immunoblot for acetyl-, glu-, α-, and β-tubulin levels in QBI-293 lysates after 4-hour treatment with CNDR-51555. (D) Quantification of tubulin levels from immunoblots of QBI-293 cell lysates after treatment with CNDR-51555. Loading controls GAPDH or β-actin were used for normalization. (E) Representative immunoblot for acetyl-, glu-, α-, and β-tubulin levels in QBI-293 lysates after 4-hour treatment with CNDR-51549. (F) Quantification of tubulin levels from immunoblots of QBI-293 cell lysates after treatment with CNDR-51549. Loading controls GAPDH or β-actin were used for normalization. Data are representative of three independent experiments (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle (DMSO)–treated controls. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tub, tubulin.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Modification, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: Inhibition of autophagy by CQ administration does not rescue TPD- and PPD-mediated loss of tubulin in QBI-293 cells. (A) ELISA measuring α-tubulin levels in response to 4-hour treatment with CNDR-51555 or CNDR-51549 in the absence or presence of 100 µM CQ. (B) Representative immunoblot and quantification of β-tubulin levels after 4-hour treatment with CNDR-51555 in the absence or presence of CQ. (C) Representative immunoblot and quantification of β-tubulin levels after 4-hour treatment with CNDR-51549 in the absence or presence of CQ. β-Tubulin levels were normalized to the loading control, β-actin. Data are representative of three independent experiments (n = 3 per group). *P < 0.05 compared with vehicle (DMSO)–treated controls; ***P < 0.001 compared with vehicle (DMSO)–treated controls; †P < 0.05 between indicated groups. Ctrl, control; Tub, tubulin.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: Inhibition of the proteasome by MG-132 administration rescues TPD- and PPD-mediated loss of tubulin in QBI-293 cells, with “rescued” tubulin largely redistributed to the RIPA-insoluble cellular fraction. (A) ELISA measuring α-tubulin levels in the RIPA-soluble fraction after 4-hour treatment with CNDR-51555 or CNDR-51549 in the absence or presence of 10 µM MG-132. (B) Immunoblot demonstrating accumulation of ubiquitinated proteins in response to MG-132 treatment. (C) Representative immunoblot of α- and β-tubulin within the RIPA-insoluble fraction after 4-hour treatment with CNDR-51555 in the absence or presence of MG-132. (D) Representative immunoblot of α- and β-tubulin within the RIPA-insoluble fraction after 4-hour treatment with CNDR-51549 in the absence or presence of MG-132. (E) Representative immunoblot of α- and β-tubulin in samples containing equal proportions of lysate from RIPA-soluble and SDS-soluble cellular fractions after 4-hour treatment with CNDR-51555 in the absence or presence of MG-132. (F) Quantification of tubulin levels after immunoblot analysis of lysates containing equal proportions of RIPA-soluble and SDS-soluble fractions after 4-hour treatment with CNDR-51555 in the absence or presence of MG-132. Tubulin levels were normalized to loading controls, GAPDH or β-actin. (G) Representative immunoblot of α- and β-tubulin in samples containing equal proportions of lysate from RIPA-soluble and SDS-soluble cellular fractions after 4-hour treatment with CNDR-51549 in the absence or presence of MG-132. (H) Quantification of tubulin levels after immunoblot analysis of lysates containing equal proportions of RIPA-soluble and SDS-soluble fractions after 4-hour treatment with CNDR-51549 in the absence or presence of MG-132. Tubulin levels were normalized to loading controls, GAPDH or β-actin. Data are representative of three independent experiments (n = 3 per group). **P < 0.01 compared with vehicle (DMSO)–treated controls; ***P < 0.001 compared with vehicle (DMSO)–treated controls; †††P < 0.001 between indicated groups. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MG, MG-132; Tub, tubulin.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: TPD+ compounds induce concentration-dependent increases in acetyl-tubulin levels without reducing α- and β-tubulin. (A) ELISA measuring acetyl- or α-tubulin levels in response to 4-hour treatment with CNDR-51597. (B) ELISA measuring acetyl- or α-tubulin levels in response to 4-hour treatment with CNDR-51657. (C) Representative immunoblot for acetyl-, glu-, α-, and β-tubulin levels in QBI-293 lysates after 4-hour treatment with CNDR-51657. (D) Quantification of tubulin levels after immunoblot analysis of QBI-293 cell lysates after treatment with CNDR-51657. Tubulin levels were normalized to loading controls, GAPDH or β-actin. Data are representative of three independent experiments (n = 3 per group). *P < 0.05; ***P < 0.001 compared with vehicle (DMSO)–treated controls. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tub, tubulin.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: The TPD+ compound, CNDR-51657, increases markers of stable MTs in rat cortical neurons, whereas other TPD and PPD compounds reduce total tubulin without increasing markers of stable MTs. (A) ELISA measuring acetyl-tubulin levels in rat cortical neurons after 24-hour treatment with CNDR-51555 or CNDR-51549. (B) ELISA measuring α-tubulin levels in rat cortical neurons after 24-hour treatment with CNDR-51555 or CNDR-51549. (C) ELISA measuring acetyl- and α-tubulin levels in rat cortical neurons after 24-hour treatment with CNDR-51657. (D) Representative immunoblot of acetyl-, glu-, α-, and β-tubulin levels in rat cortical neurons after 24-hour treatment with CNDR-51657. (E) Quantification of tubulin levels after immunoblot analysis of rat cortical neuron homogenates after 24-hour treatment with CNDR-51657. Tubulin levels were normalized to β-actin. Data are representative of three independent experiments (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle (DMSO)–treated controls. Ctrl, control; Tub, tubulin.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies

doi: 10.1124/jpet.115.231175

Figure Lengend Snippet: The TPD+ compound CNDR-51657 increases acetyl-tubulin levels in the cortex of wild-type mice. (A) ELISA analysis of acetyl- and α-tubulin levels in cortical tissue from mice treated with 1 or 5 mg/kg CNDR-51657. Mice received intraperitoneal injections of vehicle (DMSO) or compound twice in a 24-hour period, and brains were harvested 4 hours after the second dose. (B) ELISA analysis of acetyl- and α-tubulin levels in cortical tissue from mice treated with 1 mg/kg CNDR-51657 or 1 or 5 mg/kg of the TPD CNDR-51555. Mice received intraperitoneal injections of vehicle (DMSO) or compound twice in a 24-hour period, and brains were harvested 4 hours after the second dose. Bars represent average values normalized to vehicle-treated controls ± S.E.M. Results were analyzed by one-way analysis of variance with Dunnett’s post hoc testing where applicable (n = 3 per group for each experiment). *p<0.05; **p<0.01 compared to DMSO-treated animals Ctrl, control; Tub, tubulin.

Article Snippet: Briefly, 384-well plates were coated with 12G10 α -tubulin antibody (10 μ g/ml; Covance, Princeton, NJ) in 30 μ l cold 0.1 M bicarbonate buffer.

Techniques: Enzyme-linked Immunosorbent Assay, Control