Ko143

Manufactured by Bio-Techne

Sourced in United Kingdom, United States

Ko143 is a small molecule inhibitor that targets the breast cancer resistance protein (BCRP/ABCG2). It is used for research purposes in laboratory settings.

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Lab products found in correlation

Heparin, by Leo pharma (7 mentions) Bovine serum albumin fraction 5, by Roche (6 mentions) D luciferin, by GoldBio (3 mentions) Gefitinib, by Cayman Chemical (3 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Calcein am ca am, by Thermo Fisher Scientific (2 mentions) Verapamil, by Merck Group (2 mentions) Pheophorbide a, by Frontier Scientific (2 mentions) Vincristine, by Cayman Chemical (2 mentions) Mitoxantrone, by Merck Group (2 mentions) Nilotinib, by Cayman Chemical (2 mentions) Zosuquidar, by Bio-Techne (2 mentions) Propidium iodide, by Merck Group (1 mentions) Celltiter glo, by Promega (1 mentions) Envision plate reader, by PerkinElmer (1 mentions) Tariquidar, by Merck Group (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions) Flx800 fluorescence reader, by Agilent Technologies (1 mentions) Mk571, by Bio-Techne (1 mentions) Topotecan, by Merck Group (1 mentions)

32 protocols using ko143

Hoechst 33342 Staining for Side Population Analysis

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The side population (SP) was assessed by staining cells according to Goodell's protocol [60 (link)]. Briefly, cells (density 1 × 10⁶/mL) were suspended in prewarmed phenol-free DMEM (Sigma-Aldrich) with 2% FBS. Hoechst 33342 dye (B2261, bisBenzimide HOE 33342; Sigma-Aldrich) was added to a final concentration of 5 μg/mL in the presence or absence of 1 μM Ko143, an ABCG2 extrusion pump inhibitor (Tocris). Cells were incubated at 37°C for 90 min, with intermittent shaking. They were then washed with phenol-free DMEM, centrifuged at 4°C, and resuspended in ice-cold PBS. Propidium iodide (Sigma-Aldrich) was added for the exclusion of dead cells by flow cytometry. The Hoechst 33342 dye was excited at 357 nm and its fluorescence was analyzed at two wavelengths (blue, 402–446 nm; red, 650–670 nm). We evaluated drug efflux activity, by incubating cells suspended in prewarmed DMEM (Sigma-Aldrich) with 2% FBS in polystyrene tubes for 30 min at 37°C with the fluorescent molecule mitoxantrone (0.01 μg/mL; 5 × 105 cells) in the presence or absence of Ko143 (1 μM; Tocris). The cells were then washed, resuspended in prewarmed DMEM with or without Ko143 and incubated for another hour. The accumulation of MIT was assessed by flow cytometry. The mitoxantrone fluorescence emitted was detected at 670/40 nm, after excitation at 633 nm.

Le Coz V., Zhu C., Devocelle A., Vazquez A., Boucheix C., Azzi S., Gallerne C., Eid P., Lecourt S, & Giron-Michel J. (2016). IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process. Oncotarget, 7(50), 82511-82527.

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Synthesis and Characterization of Biphenyl Ester

Cited 2 times

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Ko143 was purchased from Tocris (Bristol, United Kingdom). PSC-833 (Valspodar)
was from Sequoia Research Products (Pangbourne, UK). URB937 and analogs
1–7,9 were synthesized as previously described [12 (link),15 (link)].
Compound 8 (cyclohexylcarbamic acid
3′-carbamoyl-6-methylbiphenyl-3-yl ester) was prepared as reported [12 (link)]. 8: White crystals. Mp:
165–166 °C (EtOH). MS (ESI): 353 (M+H⁺).
¹H NMR (200 MHz, DMSO-d₆):
δ = 8.07 (s, 1H), 7.83–7.90 (m, 2H),
7.70–7.74 (m, 1H), 7.43–7.57 (m, 3H), 7.27–7.31 (m, 1H),
6.95–7.05 (m, 2H), 3.28–3.30 (m, 1H), 2.20 (s, 3H), 1.05–1.83 (m,
10H) ppm. IR (Nujol): ν = 3484, 3293, 3133, 1706
cm⁻¹. All the other chemicals were of analytical grade and were
available from commercial sources.

Moreno-Sanz G., Barrera B., Armirotti A., Bertozzi S.M., Scarpelli R., Bandiera T., Prieto J.G., Duranti A., Tarzia G., Merino G, & Piomelli D. (2014). Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain. Pharmacological research : the official journal of the Italian Pharmacological Society, 0, 87-93.

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Screening ABCG2 Inhibitors in Mouse G3 MB

Cited 1 time

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We performed a high throughput screen of twelve chemotherapeutic drugs that are known ABCG2 substrates on the mouse G3 MB Myc1 neurosphere, as previously described (22 (link)). Briefly, 1000 cells per well were plated in 384-well plates. After 24 hr, half of the plates were treated with 10 µM of FTC (Alexis Biochemical, San Diego, CA, USA), 10 µM of Ko143 (Tocris Bioscience, Bristol, UK), or 10 µM of Tariquidar (Sigma-Aldrich, USA), incubated for 30 minutes, and all plates were treated with 28 nl of compound in a dose-response (final drug concentration 9.3 µM to 0.5 nM). After 72 hr, the cell number was determined in each well using CellTiter-Glo^© (Promega). The luminescence signal was measured in an automated Envision plate reader (Perkin-Elmer). Luminescence data were normalized by log10-transformation prior to calculating the percentage of inhibition using the following equation: 100 × (negative control mean − compound value)/(negative control mean − positive control mean). Cells treated with 1% DMSO served as negative control with 0% inhibition. Cells treated with 35 µM cycloheximide were used as positive control with 100% inhibition.

Morfouace M., Cheepala S., Jackson S., Fukuda Y., Patel Y.T., Fatima S., Kawauchi D., Shelat A.A., Stewart C.F., Sorrentino B.P., Schuetz J.D, & Roussel M.F. (2015). ABCG2 transporter expression impacts Group3 Medulloblastoma response to chemotherapy. Cancer research, 75(18), 3879-3889.

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Functional Assays for MRP1 and BCRP

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Calcein AM (ThermoFisher, NY, USA) cellular accumulation assays were used for MRP1 function, while BCRP function was assessed with Hoechst 33342 (ThermoFisher, NY, USA). MK571 (Tocris, Bristol, UK) and KO143 (Tocris, Bristol, UK), specific MRP1 and BCRP inhibitors, respectively, were used in the functional assay. M1, M2, and unstimulated U937 cells were washed and resuspended in serum-free RPMI, and then seeded in 96-well Black Clear-Bottom Plates (Costar, Washington, DC, USA). Plates were incubated at 37°C with or without inhibitor (MK571, 10 min incubation; KO143, 2 h incubation). After incubation, 10 μM Calcein AM or 10 μM Hoechst 33342 was added to the plate. Plates were immediately placed in an FLx800 Fluorescence Reader (BioTek, Winooski, VT, USA) for 60 min, and read at 485/528 (ex/em). Cell viability was determined via trypan blue staining.

He H., Buckley M., Britton B., Mu Y., Warner K., Kumar S, & Cory T.J. (2018). Polarized macrophage subsets differentially express the drug efflux transporters MRP1 and BCRP, resulting in altered HIV production. Antiviral Chemistry & Chemotherapy, 26, 2040206617745168.

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In vivo Efficacy of ABCG2 Inhibitor in Glioblastoma

Cited 1 time

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1×10⁵ mouse Myc1-luc tumorspheres were implanted in the cortices of recipient CD-1 nude mice, as previously described (4 (link)). For drug efficacy studies, compounds were injected intraperitoneally (IP) in Myc1-luc-bearing mice 4 days post-tumor implantation, when the luminescence signal reached 5×10⁵ photons. Ko-143 (Tocris Bioscience, Bristol, UK) was dissolved in saline at a concentration of 2.5 mg/mL and injected IP at 10 mg/kg. Ko143 is the non-neurotoxic, but equally effective derivative of the specific ABCG2 inhibitor, FTC (23 (link)). Topotecan (Sigma, St Louis, MO, USA) was prepared in saline and injected IP at 0.75 mg/kg. Control animals received saline IP. Tumor growth was monitored by measuring bioluminescence, as previously described (4 (link)). Mice displaying signs of morbidity including head dome, slow moving, seizure, or >20% weight loss were euthanized, the tumor removed and fixed in 10% formalin. Histopathology was performed to determine tumor grade and presence of necrotic area. All animal studies were conducted according to the NIH guidelines and approved by the St. Jude Children’s Research Hospital Animal Care and Usage Committee.

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Cytotoxicity Assay with Pheophorbide a

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Dulbecco’s Modified Eagle’s Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), fetal bovine serum (FBS), PBS without Ca²⁺ and Mg²⁺ (PBS (−)), PBS containing Ca²⁺ and Mg²⁺ (PBS (+)), penicillin-streptomycin, and pheophorbide a (PhA), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ko143 was purchased from Tocris Bioscience (Bristol, UK). SN-38 was purchased from Wako Pure Chemical Industries (Tokyo, Japan). Irinotecan was purchased from Yakult Honsha (Tokyo, Japan). CellTiter 96 AQueous One Solution Reagent was purchased from Promega (Madison, WI, USA).

Kokubo S., Ohnuma S., Murakami M., Kikuchi H., Funayama S., Suzuki H., Kajiwara T., Yamamura A., Karasawa H., Sugisawa N., Ohsawa K., Kano K., Aoki J., Doi T., Naitoh T., Ambudkar S.V, & Unno M. (2021). A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo. International Journal of Molecular Sciences, 22(22), 12502.

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Preparation of Topotecan, FL118, and Analogues

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Topotecan (Selleckchem Chemicals, Houston, TX), FL118 (in house), and FL118 analogues (RTI International) were prepared as stocks at 1 mM in DMSO (Merck KGaA, Darmstadt, Germany). The synthesis of FL118 and FL118 analogues (7-methyl-FL118, 7-ethyl-FL118, 7-allyl-FL118, 7-bromomethyl-FL118, 7-chloromethyl-FL118 and 7-hydroxymethyl-FL118) were reported previously [21 (link),31 (link),32 (link)]. Stock SN-38 (Sigma-Aldrich Corporation, St. Louis, MO) was prepared at 2.5 mM in DMSO. Ko143 (Tocris Bioscience, Bristol, United Kingdom) was prepared as stock solutions at 10 mM in DMSO.

Westover D., Ling X., Lam H., Welch J., Jin C., Gongora C., Del Rio M., Wani M, & Li F. (2015). FL118, a novel camptothecin derivative, is insensitive to ABCG2 expression and shows improved efficacy in comparison with irinotecan in colon and lung cancer models with ABCG2-induced resistance. Molecular Cancer, 14, 92.

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ABCG2 Surface Expression Quantification

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Cell surface expression of ABCG2 was determined in the transfected HEK293 and HeLa cells 48 h after transfection. Trypsinized cells were incubated in small volumes (100 µL) and gently shaken for 40 min at 37 °C in 0.5% BSA/PBS with 1 µM Ko143 (Tocris Bioscience, Bristol, UK, cat. 3241) and the ABCG2-specific 5D3 mouse monoclonal antibody (gift of Bryan Sorrentino, Division of Experimental Hematology, Department of Hematology/Oncology, St. Jude Children’s Research Hospital). Ko143 was added to the samples because it has been shown to help the conformation-sensitive 5D3 antibody recognition. Cells were washed twice with 2 mL PBS. After washing, the cells were centrifuged and the supernatant discarded, then incubated for 30 min at 37 °C with the secondary antibody (goat anti-mouse IgG2b Alexa Fluor 647, cat. A-21242, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in 0.5% BSA/PBS. Cells were washed again with 2 mL PBS. Measurements were performed in the Attune NxT Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) using the RL1 detector. For data analysis, the Attune NxT Cytometer Software v3.1.2 was used.

Mózner O., Zámbó B., Bartos Z., Gergely A., Szabó K.S., Jezsó B., Telbisz Á., Várady G., Homolya L., Hegedűs T, & Sarkadi B. (2023). Expression, Function and Trafficking of the Human ABCG2 Multidrug Transporter Containing Mutations in an Unstructured Cytoplasmic Loop. Membranes, 13(10), 822.

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Selpercatinib Detection Assay Protocol

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Selpercatinib was purchased from Chemgood (Glen Allen, VA, USA). Zosuquidar and elacridar HCl were obtained from Sequoia Research Products (Pangbourne, UK). Ko143 was from Tocris Bioscience (Bristol, UK). Bovine Serum Albumin (BSA) Fraction V was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Glucose water 5% w/v was from B. Braun Medical Supplies, Inc. (Melsungen, Germany). Isoflurane was purchased from Pharmachemie (Haarlem, The Netherlands) and heparin (5000 IU mL⁻¹) was from Leo Pharma (Breda, The Netherlands). Other chemicals used in the selpercatinib detection assay were described before [40 (link)]. All other chemicals and reagents were obtained from Sigma-Aldrich (Steinheim, Germany).

Wang Y., Sparidans R.W., Potters S., Şentürk R., Lebre M.C., Beijnen J.H, & Schinkel A.H. (2021). P-Glycoprotein (ABCB1/MDR1) and BCRP (ABCG2) Limit Brain Accumulation and Cytochrome P450-3A (CYP3A) Restricts Oral Exposure of the RET Inhibitor Selpercatinib (RETEVMO). Pharmaceuticals, 14(11), 1087.

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APC Analogs for Cancer Imaging and Therapy

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The APC analogs CLR1404, CLR1501 and CLR1502 were provided by Cellectar Biosciences, Inc. (Madison, WI, USA). These APC analogs consist of a cancer-targeting alkylphosphocholine scaffold with an attached cancer imaging, visualization or therapeutic moiety (refs ^{17 , 18 (link), 25 (link), 28 (link)} and Figure 1): iodine isotopes for CLR1404; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (i.e. BODIPY, green fluorescence) for CLR1501; and 2-[2-[2-Chloro-3-[2-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,3,3-trimethyl-3H-indolium chloride (i.e. IR-775, near infrared fluorescence) for CLR1502. Pharmacological cellular efflux transporter inhibitors were purchased: PSC833 and Ko143 from Tocris Biosciences, and MK571 from Sigma-Aldrich (St. Louis, MO, USA). [¹⁴C]-sucrose and [³H]-diazepam were purchased from American Radiolabeled Chemicals (St Louis, MO, USA).

Clark P.A., Alahmad A.J., Qian T., Zhang R.R., Wilson H.K., Weichert J.P., Palecek S.P., Kuo J.S, & Shusta E.V. (2016). Analysis of Cancer-targeting Alkylphosphocholine Analog Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model. Molecular pharmaceutics, 13(9), 3341-3349.

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