Olaparib, Talazoparib (BMN 673), Niraparib (MK-4827) and Rucaparib were purchased from Medchem Express (MCE). Cisplatin and mitomycin C were obtained from Sigma-Aldrich.
Talazoparib
Manufactured by MedChemExpress
Sourced in United States
Talazoparib is a lab equipment product manufactured by MedChemExpress. It is a PARP inhibitor that functions by inhibiting the activity of Poly(ADP-ribose) polymerase (PARP) enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Olaparib, by MedChemExpress (5 mentions)
Niraparib, by MedChemExpress (4 mentions)
Rucaparib, by MedChemExpress (3 mentions)
Veliparib, by MedChemExpress (3 mentions)
Cisplatin, by Merck Group (2 mentions)
Cisplatin, by MedChemExpress (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mitomycin c, by Merck Group (1 mentions)
Glacial acetic acid, by Thermo Fisher Scientific (1 mentions)
Dextran sulfate sodium salt, by Merck Group (1 mentions)
Temozolomide, by MedChemExpress (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by American Type Culture Collection (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Cyanine7.5 carboxylic acid, by Lumiprobe (1 mentions)
Phosphate buffered saline 1x, by Corning (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
10 protocols using talazoparib
1
PARP Inhibitor Evaluation Protocol
2
Murine Cell Culture and Reagents
3
Induction and Inhibition of Stress Granules
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For oxidative stress induction, HeLa cells were treated with 300 μM or 1 mM hydrogen peroxide (H2O2) or 300 μM sodium arsenite for the indicated period of time.
For induction of SGs in nucleus, cells were treated with 4 μM actinomycin D (ActD) (Thermo Fisher Scientific, #11805017) for 1 h before treatment with 300 µM H2O2.
For induction of SGs in cytoplasm, cells were treated for 60 min with 300 µM sodium arsenite or 200 µM puromycin (Sigma, St. Louis, MO, USA, #P9620) for 1 h before treatment with 300 µM H2O2.
For inhibition of PARP activity, cells were pre-treated with 10 μM olaparib (Apexbio Technology, Houston, TX, USA, #A4154) or 1 μM talazoparib (MedChemexpress, #HY-108413) and diluted in DMSO for 1 h before and during indicated treatments.
To study the disassembly of SGs formed by treatment with sodium arsenite, cells were treated with 300 µM sodium arsenite for 60 min, then the culture medium was replaced with fresh medium containing DMSO (control), 300 µM H2O2 or 3 µM olaparib or 300 µM H2O2, as indicated, and then the cells were left alone for one hour for further fixation and analysis.
After incubation with the drugs at indicated time points, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 45 min at 37 °C, if another cell fixation method is not indicated.
For induction of SGs in nucleus, cells were treated with 4 μM actinomycin D (ActD) (Thermo Fisher Scientific, #11805017) for 1 h before treatment with 300 µM H2O2.
For induction of SGs in cytoplasm, cells were treated for 60 min with 300 µM sodium arsenite or 200 µM puromycin (Sigma, St. Louis, MO, USA, #P9620) for 1 h before treatment with 300 µM H2O2.
For inhibition of PARP activity, cells were pre-treated with 10 μM olaparib (Apexbio Technology, Houston, TX, USA, #A4154) or 1 μM talazoparib (MedChemexpress, #HY-108413) and diluted in DMSO for 1 h before and during indicated treatments.
To study the disassembly of SGs formed by treatment with sodium arsenite, cells were treated with 300 µM sodium arsenite for 60 min, then the culture medium was replaced with fresh medium containing DMSO (control), 300 µM H2O2 or 3 µM olaparib or 300 µM H2O2, as indicated, and then the cells were left alone for one hour for further fixation and analysis.
After incubation with the drugs at indicated time points, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 45 min at 37 °C, if another cell fixation method is not indicated.
4
Inhibition of PARP and NAMPT Enzymes
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PARP inhibitors olaparib (MedChemExpress, Monmouth Junction, NJ, USA), niraparib (Abmole Bioscience Inc., Houston, TX, USA), and talazoparib (MedChemExpress), as well as NAMPT inhibitors daporinad (MedChemExpress), OT-82 (SelleckChem Chemicals, Houson, TX, USA) and KPT-9274 (SelleckChem Chemicals), were dissolved in dimethyl sulfoxide (DMSO) (MilliporeSigma, Burlington, MA, USA). β-nicotoninamide mononucleotide (NMN) (Sigma-Aldrich, St-Louis, MO, USA) was dissolved in sterile water.
5
Renal Cell Carcinoma Cell Line Characterization
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Human RCC cell lines Caki-2 (representing ccRCC), ACHN (representing pRCC) and the normal human renal epithelial cell line RPTEC/TERT1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and cultured in McCoy’s 5a modified medium with 10% complete FBS; Eagle’s Minimum Essential Medium with 15% FBS; and DMEM: F12 medium supplemented with hTERT RPTEC Growth kit as recommended by ATCC, respectively. All experiments with cell lines were performed either within 6 months of receipt from ATCC or resuscitation after cryopreservation. ATCC uses Short Tandem Repeat (STR) profiling for testing and authentication of cell lines. Antibodies against Tubulin were from Thermo Fisher Scientific. Antibodies against cleaved caspase-3, cleaved caspase-7, cleaved caspase 9, cleaved PARP, whole caspase 3, whole caspase 7, whole caspase 9, and whole PARP were from Cell Signaling Technologies. PARP inhibitors (niraparib, olaparib, rucaparib, talazoparib, and veliparib) were obtained from MedChem Express. All other reagents were of analytical grade and obtained from local suppliers.
6
Inhibitors Targeting ALK, PARP, CDK9 in Cancer
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Anti-neoplastic agents used in the present study, including talazoparib (PARP inhibitor), lorlatinib (ALK inhibitor), ceritinib (ALK inhibitor), cisplatin and CDK9-IN-2 (CDK9 inhibitor), were purchased from MedChemExpress. The following antibodies were used for WB: anti-p-ALK (1:500), anti-CDK9 (1:2,000), anti-p-Thr186 CDK9, (1:2,000), anti-p-Rpb1 CTD (1:2,000), anti-cyclin T1 (1:1,000), anti-FLAG tag (1:2,000), anti-PARP (1:1,000), anti-BRD4 (1:2,000), anti-RAD50 (1:3,000), anti-Lamin B1 (1:3,000), anti-BRCA1, anti-CtIP (1:500), anti-p-Tyr (1:5,000) and anti-BRCA2 (1:1,000). The following antibodies were used for IF: anti-RAD51 (1:250) and anti-phospho-histone H2A.X (1:200). The following antibodies were used for IHC: anti-p-ALK (1:200), anti-RAD51 (1:1,000), anti-CtIP (1:10), anti-p-RPA32 (1:100) and anti-p-Tyr19-CDK9 (1:10). Mouse monoclonal antibody against the phosphorylation site of CDK9 at Tyr19 was produced by our lab with a synthetic phosphopeptide: DEVSKP-pY-EKLAKIGQTFGE.
7
Synthesis and Characterization of PARP Inhibitors
8
Optimizing Talazoparib-Loaded Nanoparticle Formulation
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For talazoparib-loaded nanoparticles (NP-Tal), synthesis parameters were initially varied for protocol optimization (Table 1 ). PLGA-PEG-COOH and PLGA-PEG-DBCO were obtained from PolySciTech, and talazoparib (Tal) was obtained from MedChemExpress. Polymer was first co-dissolved with talazoparib in 1 mL of acetone, then added to 10 mL of ultrapure water (Invitrogen) containing 0.1% Pluronic F-68 (Gibco). The solution was sonicated with a 120 Watt, 20 kHz probe sonicator at 20% amplitude for 3 min and acetone was evaporated at room temperature for 4–6 h under continuous stirring at 400 rpm. The obtained NP-Tal were filtered through 0.22 μm syringe filter units (Millipore) and concentrated in an Amicon centrifugal filter unit (30 kDa MWCO, Millipore Sigma). Next, PIC was conjugated to the DBCO-containing nanoparticles through copper-free click chemistry. For conjugation, PIC and nanoparticles were mixed overnight at volume ratios of 0.5:1, 1:1, 2:1, and 3:1, then purified via Sepharose CL-4B size exclusion chromatography.
Varying parameters in nanoparticle formulation
| Parameter | Values |
|---|---|
| PLGA-PEG-COOH (mg) | 10.7, 21.4, 42.8, 85.6 |
| talazoparib (mg) | 0, 0.107, 0.535, 1.07, 2.675 |
| PLGA-PEG-DBCO/total polymer (%) | 0, 25, 50, 100 |
9
Talazoparib and Lapatinib Quantification in HLMs
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Pooled HLMs (Product Number: M 0567, male human liver) was purchased from Sigma-Aldrich (USA) and stored at −70°C. HLMs consists of a mixture of liver microsomes pooled from various individual human donors. Standard powders are of analytical grade (AR) and organic solvents are HPLC grade. HPLC grade water (H2O) was arranged by in situ Milli-Q plus filtration system (USA). Talazoparib (99.89%) and lapatinib (99.83%) were procured from Med Chem. Express Company (USA). Acetonitrile, HLMs, pooled (M0567), formic acid (HCOOH) and ammonium formate (NH4COOH) were procured from Sigma-Aldrich company (USA).
10
Bladder Cancer Cell Line Characterization
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UM-UC-3, T-24 (human bladder cancer cell lines), and SV-HUC-1 (normal human bladder epithelial cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in EMEM, McCoy’s 5a, or F12K media respectively, supplemented with 10% FBS and penicillin/streptomycin. Cells were used within half a year after being received from ATCC or after thawing from cryopreservation. Short Tandem Repeat (STR) profiling is used by the ATCC for cell line authentication. The cell lines in culture were routinely tested for mycoplasma contamination every two months using the MycoFluorTM Mycoplasma detection kit (Thermo Fisher Scientific, Waltham, MA). Tubulin antibodies were obtained from Thermo Fisher Scientific, Waltham, MA. Cleaved and whole caspases 3, 7, and 9 antibodies were obtained from Cell Signaling Technology, Danvers, MA. Ki-67 antibodies were obtained from Neomarker, Fremont, CA. The PARP inhibitors, olaparib, niraparib, rucaparib, veliparib, and talazoparib, were obtained from MedChem Express, Monmouth Junction, NJ. Cisplatin was from Sigma Aldrich, St. Louis, MO. Other reagents were supplied by local suppliers such as Fisher Scientific and VWR International.
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