Pha 767491

Manufactured by Selleck Chemicals

Sourced in United States

PHA-767491 is a lab equipment product that serves as a protein phosphatase inhibitor. It is used for research purposes in biological and chemical applications.

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11 protocols using pha 767491

Preparation and Storage of Mithramycin and PHA-767491

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Mithramycin (Tocris Bioscience) was dissolved as a 1 mg/mL stock solution in PBS, and frozen in aliquots. PHA-767491 and other small molecule inhibitors (Selleck Chemicals) were dissolved in DMSO as stock solutions. Small molecule stocks were stored at −80 °C for storage or −20 °C for experimental use, minimizing freeze-thaw cycles. For in vivo experiments, a 30 mg/mL stock solution of PHA-767491 (Adooq Bioscience) was made by dissolving the powder in a solution of 1 mg/10mL D-α-Tocopherol polyethylene 1000 succinate (MilliporeSigma) for oral gavage.

Flores G., Everett J.H., Boguslawski E.A., Oswald B.M., Madaj Z.B., Beddows I., Dikalov S., Adams M., Klumpp-Thomas C.A., Kitchen-Goosen S.M., Martin S.E., Caplen N.J., Helman L.J, & Grohar P.J. (2020). CDK9 Blockade Exploits Context-Dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma. Molecular cancer therapeutics, 19(5), 1183-1196.

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Anticancer Drug Combination Protocol

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Dinaciclib, doxorubicin, etoposide, cytarabine, flavopiridol, SNS-032, and PHA-767491 were purchased from Selleck Chemicals (Houston, TX). ABT-199 was kindly provided by AbbVie Inc. (North Chicago, IL).

Li L., Pongtornpipat P., Tiutan T., Kendrick S.L., Park S., Persky D.O., Rimsza L.M., Puvvada S.D, & Schatz J.H. (2015). Synergistic Induction of Apoptosis in High-Risk DLBCL by BCL2 Inhibition with ABT-199 Combined With Pharmacologic Loss of MCL1. Leukemia, 29(8), 1702-1712.

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Antibody and Inhibitor Characterization for DNA Damage Response

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Antibodies were as follows: MCM4 (Cell Signaling, catalog no. 3228) (used at 1:1,000), CHK1 (Cell Signaling, catalog no. 2360) (used at 1:1,000), CHK1 pS345 (Cell Signaling, catalog no. 2348) (used at 1;1000), CHK1 pS296 (Cell Signaling, catalog no. 2349) (used at 1:1,000), GAPDH (Abcam, catalog no. 8245) (used at 1:10,000), IdU (Becton Dickinson, catalog no. 347580) [used 1:300 for immunofluorescence (IF)], CldU (Abcam, catalog no. 6326) (used 1:100 for IF), PCNA (Abcam, catalog no. ab201674) (used 1:1,000 for STORM), RIF1 [Bethyl, catalog no. A300-568A; used 1:1,000 for Western blot (WB) and 1:500 for PLA], PP1alpha (Thermo, catalog no. 43-8100, used 1:1,000 for PLA; Cell Signaling, catalog no. 2582, used 1:500 for WB). RIF1 pSer2205 antibody was custom-made by Bethyl by injecting rabbits with KVRRV(pS)FADPI peptide and subsequent purifications. Other materials included ATR inhibitor AZD6738 (AstraZeneca); CHK1 inhibitor UCN01 (Sigma); CHK1 inhibitor AZD7762 (AstraZeneca); CDC7 inhibitor PHA-767491, Aurora B inhibitor AZD1152, PKA inhibitor H89, CK2 inhibitor CX-4945, CDK1 inhibitor Ro-3306, and CDK1/4/9 inhibitor P276-00 (all from Selleckchem); CDK2i CVT-313 (Santa Cruz); EdU (Invitrogen); IdU (Sigma); and CldU (MP Biomedical).

Moiseeva T.N., Yin Y., Calderon M.J., Qian C., Schamus-Haynes S., Sugitani N., Osmanbeyoglu H.U., Rothenberg E., Watkins S.C, & Bakkenist C.J. (2019). An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication. Proceedings of the National Academy of Sciences of the United States of America, 116(27), 13374-13383.

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DNA Repair Pathway Inhibitors

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The following drugs were used in this study: HU, aphidicolin, nocodazole (Sigma-Aldrich), ATR inhibitor (ETP-46464; Selleckchem, Houston, TX), DNA-PK inhibitor (Nu7026; Selleckchem), CDC7 inhibitor (PHA-767491; Selleckchem), RAD51 inhibitor (B02; Merck Millipore, Burlington, MA), and MRE11 inhibitor (mirin; Sigma-Aldrich).

Park S.H., Kang N., Song E., Wie M., Lee E.A., Hwang S., Lee D., Ra J.S., Park I.B., Park J., Kang S., Park J.H., Hohng S., Lee K.Y, & Myung K. (2019). ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress. Nature Communications, 10, 5718.

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Comprehensive Pharmacological Screening Protocol

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PHA-767491, dinaciclib, ON123300, AMG925, KW2449, AZD2932, dasatinib, BGJ398, saracatinib, lapatinib and taselisib were purchased from Selleckchem, Houston, TX, USA. Doxorubicin was purchased from Cayman, Ann Arbor, MI, USA. All reagents and kits, including ethanol and dimethyl sulfoxide (DMSO), were purchased from Sigma Chemical Co. Jeddah, MK, KSA, unless otherwise reported.

Abdalla A.N., Abdallah M.E., Aslam A., Bader A., Vassallo A., Tommasi N.D., Malki W.H., Gouda A.M., Mukhtar M.H., El-Readi M.Z., Alkahtani H.M., Abdel-Aziz A.A, & El-Azab A.S. (2020). Synergistic Anti Leukemia Effect of a Novel Hsp90 and a Pan Cyclin Dependent Kinase Inhibitors. Molecules, 25(9), 2220.

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Multiparametric Imaging of DNA Damage Response

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DNA oligonucleotides used in Figure 5 are:
5’-TGGCGACGGCAGCGAGGCTTTTTTTTTTTTTTTTTTTT-iCy3-TTTTTTTTTTTT-3’-Cy5 5’-GCCTCGCTGCCGTCGCCA-3’-biotin
DNA oligonucleotides used in Figure S6 are:
5’-TGGCGACGGCAGCGAGGCTTTTTTTTTTTTTTTT-iCy3-TTTTTTTTTTTTTT-3’ Cy5–5’-GCCTCGCTGCCGTCGCCA-3’-biotin
For the inhibitors used in this study, cells were treated with inhibitors 3 hours before fixation. ATRi: VE-821 (Selleckchem), 2 μM, 3 hours incubation; CDC7i: PHA767491 (Selleckchem), 20μM, 3 hours incubation; Chk1i: UCN01 (Sigma-Aldrich), 100 nM, 1 hour incubation; Chk2i: PV1019 (Sigma-Aldrich), 5 μM, 3 hours incubation; For Hydroxyurea treatments (HU, Sigma-Aldrich), cells were treated 2 hours before fixation.
Antibodies against the imaged proteins are (ms = mouse; rb = rabbit; AF = AlexaFluor): ms-anti-PCNA (SCBT sc56, 1:1,000), rb-anti-MCM6 (AF568 conjugated, Abcam ab211916, 1:1,000), rb-anti-RPA1 (AF647 conjugated, Abcam ab199240, 1:10,000), rb-anti-pS4/S8RPA2 (Abcam ab87277, 1:1000). Secondary antibodies are: goat-anti-ms (AF488 conjugated, ThermoFisher, 1:10,000), goat-anti-rb (AF647 conjugated, ThermoFisher, 1:10,000).
Antibodies against the blotted proteins are:
Ms-anti-RPA2 (Millipore, MABE285, 1:1000), rb-anti-c-myc (Bethyl, A190–105A, 1:1000), ms-anti-β-tubulin (Sigma-Aldrich T8328, 1:1000), Ms-anti-HRP (Abcam, ab181708, 1: 10000), Rb-anti-HRP (Abcam, ab34885, 1:10000).

Yin Y., Lee W.T., Dipika G., Xue H., Tonzi P., Borowiec J.A., Huang T.T., Modesti M, & Rothenberg E. (2021). A Basal-Level Activity of ATR Links Replication Fork Surveillance and Stress Response. Molecular cell, 81(20), 4243-4257.e6.

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Cell Signaling Pathway Inhibitors

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Flavopiridol (FVP), PHA767491 (PHA), PD-0332991 (Palbociclib, Palbo) and Dinaciclib (Dina) were purchased from Selleck Chemicals. THZ1 was purchased from Apex Bio, MG132 was purchased from Calbiochem and Cycloheximide was purchased from Sigma. All the above drugs were dissolved in DMSO.

Sampathi S., Acharya P., Zhao Y., Wang J., Stengel K.R., Liu Q., Savona M.R, & Hiebert S.W. (2019). The CDK7 inhibitor THZ1 alters RNA polymerase dynamics at the 5′ and 3′ ends of genes. Nucleic Acids Research, 47(8), 3921-3936.

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BRCA2 Haploinsufficiency and Drug Sensitivity

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BRCA2 haploinsufficient Jurkat T-ALL cells or their Cas9-transduced BRCA2 wild-type controls were plated at 0.1 million cells/ml in 96 well plates, and treated with vehicle (DMSO or PBS) control and at the indicated concentrations of VE-821 (SelleckChem, S8007), Topotecan (SelleckChem, S1231), KU60019 (SelleckChem, S1570), Mitomycin C (Sigma, M4287), PF-477736 (SelleckChem, S2904), Olaparib (SelleckChem, S1060), CCT-241533 (Tocris, 4968), Etoposide (Sigma, E1383), C527 (ApexBio, A8693), PHA-767491 (SelleckChem, S2742), MK-1775 (SelleckChem, S1525) for 96 hours. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).
For 15-day treatment of BRCA2-haploinsufficient vs. wild-type controls, clone W4, clone W5, and parental Cas9 control cells were plated at 0.1 million cells/ml and split every 4 days at 1:9 in growth media (RPMI1640 with 10% FBS) in the presence of vehicle or drug. Cells were treated with VE821 (1 μM), AZD6738 (AstraZeneca, 0.25 μM), or vehicle (DMSO), and cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay when cells were split. Cell counts of drug-treated cells are normalized to those in vehicle-treated cells at that time point.

Pouliot G.P., Degar J., Hinze L., Kochupurakkal B., Vo C.D., Burns M.A., Moreau L., Ganesa C., Roderick J., Peirs S., Menten B., Loh M.L., Hunger S.P., Silverman L.B., Harris M.H., Stevenson K.E., Weinstock D.M., Weng A.P., Van Vlierberghe P., D’Andrea A.D, & Gutierrez A. (2019). Fanconi-BRCA pathway mutations in childhood T-cell acute lymphoblastic leukemia. PLoS ONE, 14(11), e0221288.

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Colorectal and Breast Cancer Cell Lines

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Colorectal RKO (ATCC), RKO^HIF1a+/+ and RKO^HIF1a−/− (28 (link)), HCT116, Breast MCF7, MDA-MB231, Bladder T24, VMCUB1 (provided by Prof Anne Kiltie, Oxford University) and HCT116 p53+/+ and p53−/− (provided by Prof Bert Vogelstein, Johns Hopkins Medicine) cells were grown in DMEM media. Non-tumorigenic breast MCF10A (provided by Prof Paul Span, Nijmegen University) cells were grown in MEGM media. Non-transformed lung MRC5 cells (ATCC) were grown in EMEM media. Esophageal OE21 cells (PHE) were grown in RPMI media. All cell media was supplemented with 10% FBS, except MEGM media which was supplemented with 20% FBS, and cells were maintained in an incubator set at 37°C and 5% CO₂. All cell lines were verified mycoplasma free using a HEK-Blue™ detection kit (Invivogen). Inhibitors/drugs used were: VX-970 (M6620) (MedChemExpress), AZD6738 (MedChemExpress), Gö6976 (Sigma Aldrich), MK-8776 (Selleckchem), Bay11-7085 (Tocris Biosciences), PHA-767491 (Selleckchem), N’ acetylcysteine (NAC) (Sigma Aldrich), and roscovitine (Ros) (Selleckchem). For siRNA-mediated knockdowns, RKO cells were transfected with siRNA to a final concentration of 50 nM using DharmaFECT 1 (T-2001-03; Horizon Discovery, Cambridge, UK) following manufacturer's protocols. Sequences of siRNAs used: siA3B (CCUGAUGGAUCCAGACACA[dT][dT]), sip53 (GUAAUCUACUGGGACGGAA[dT][dT]), and siATR (CAG GCA CTA ATT GTT CTT CAAd[T]d[T]).

Bader S.B., Ma T.S., Simpson C.J., Liang J., Maezono S.E., Olcina M.M., Buffa F.M, & Hammond E.M. (2021). Replication catastrophe induced by cyclic hypoxia leads to increased APOBEC3B activity. Nucleic Acids Research, 49(13), 7492-7506.

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Chemical Inhibitor Procurement Protocol

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The ATR inhibitor VE-821 (S8007), Chk1 inhibitor MK-8776 (S2735), Wee1 inhibitor MK-1775 (S1525), CDC7 inhibitor PHA-767491 (S2742), and CDK1 inhibitor RO-3306 (S7747) were purchased from Selleck Chemicals and solubilized in dimethylsulfoxide (DMSO). Chk1 inhibitor UCN-01 (U6508) was purchased from Sigma-Aldrich. These inhibitors were stored at −80 °C and employed in accordance with the manufacturer’s guidelines.

Chen S., Pan C., Huang J, & Liu T. (2024). ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability. The EMBO Journal, 43(7), 1301-1324.

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