EC90: 26 nM (BCRP)
ABCG2 (IC50 for inhibition of ATPase activity = 9.7 nM); ABCB1 (interacts at micromolar concentrations, ≥1 μM sensitizes cells to paclitaxel; stimulates ATPase with IC50 ~2.7 μM); ABCC1 (inhibited at micromolar concentrations, ≥1 μM sensitizes cells to doxorubicin). No precise IC50 values for inhibition of transport were reported for ABCB1 or ABCC1 in this study. [1]
Ko-143 targets the breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) transporter that mediates multidrug resistance by effluxing chemotherapeutic agents out of cancer cells. By inhibiting BCRP, Ko-143 blocks drug efflux, increasing intracellular concentrations of chemotherapeutic agents and reversing drug resistance. Ko-143 displays >200-fold selectivity over P-gp and MRP-1 transporters.

Ko143 (10 nM) dramatically lowers the IC50 of MTX on mouse G2 and HEK G2 cells by a factor of 2.5. ABC transporter function is not inhibited by Ko143 (1-100 μM) metabolites [1]. Ko143, an FTC analog, overcomes drug resistance in human IGROV1/T8 cells and mouse MEF3.8/T6400 cells chosen with SKF 104864A. At zero, one, or eight times the EC90 of 25 nM, Ko143 can be utilized [2]. In Madin-Darby canine kidney (MDCK) 2-BCRP421CC (wild-type) and MDCK2-BCRP421AA (mutant) cells, Ko143 inhibits BCRP-mediated ZD 4522 trafficking [3].

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Ko143 potently inhibited ABCG2-mediated efflux of fluorescent substrates (MTX, PPA, Hoechst 33342, JC-1, P-18) in human and mouse cells at nanomolar concentrations, with significant species differences for MTX, Hoechst, and P-18 (Table 3). In cytotoxicity assays, Ko143 at 10 nM significantly sensitized human ABCG2 cells to MTX (2.5-fold reduction in IC50, P<0.01) and mouse ABCG2 cells (P<0.001). At ≥1 μM, Ko143 sensitized human ABCB1 cells to paclitaxel (P<0.0001) and human ABCC1 cells to doxorubicin (P<0.001), and mouse ABCB1 cells to paclitaxel (P<0.01). Flow cytometry showed that Ko143 at 20–100 μM significantly increased accumulation of rh123 in ABCB1 cells and CAM in ABCC1 cells, confirming inhibition of these transporters at high concentrations. ATPase assays revealed that Ko143 inhibited ABCG2 ATPase (IC50 = 9.7 nM) but stimulated ABCB1 ATPase activity in a biphasic manner (peak stimulation at 10 μM, IC50 for stimulation ~2.7 μM). Radiolabeled [³H]Ko143 showed 2-fold higher accumulation in ABCG2-expressing cells than in parental cells, which was displaced by FTC or cold Ko143, indicating specific binding to ABCG2; no such effect was seen for ABCB1 or ABCC1 cells. The hydrolytic metabolite (Ko143 acid) had negligible inhibitory activity on all three transporters except weak inhibition of ABCG2 at 100 μM. Concentrations of Ko143 above 5 μM were cytotoxic to HEK cells after 72 h. [1]
In vitro, Ko-143 is a potent and selective BCRP inhibitor with an EC90 of 26 nM. It reverses resistance of MTX and topotecan at 25 nM in human cells. Ko-143 displays >200-fold selectivity over P-gp and MRP-1 transporters. Its activity has been characterized in drug accumulation and efflux assays using BCRP-overexpressing cell lines.

In mice, Ko143 (10 mg/kg, po) raises SKF 104864A's oral availability [2]. Ko143 has a major impact on ZD 4522's pharmacokinetics in rats [3].
In Vivo: In rats, intravenous administration of Ko143 (dose not specified in the text, but given as a pretreatment for 7 consecutive days, once daily? Actually the protocol says "Ko143 (intravenously)" given for 7 consecutive days, two times per day? The text: "group four and five were firstly given 80mg/kg of ursolic acid (orally) and Ko143 (intravenously, inhibitor of BCRP) respectively for 7 consecutive days, two times per day" - but dose of Ko143 is not stated. However, the pharmacokinetic results showed that pretreatment with Ko143 significantly increased Cmax, AUC0-t, and AUC0-∞ of rosuvastatin, and decreased CLz/F compared to rosuvastatin alone (all P<0.05). No significant effect on Tmax or T1/2. The data are presented in Table 2: Cmax 4.18±0.18 vs 2.23±0.22 mg/L; AUC0-t 62.72±6.34 vs 31.99±1.93 mg·L⁻¹·min⁻¹; AUC0-∞ 80.17±9.56 vs 40.92±3.10; CLz/F 0.25±0.03 vs 0.49±0.04 L·h⁻¹·kg⁻¹. [3]
In vivo, Ko-143 is an orally bioavailable BCRP inhibitor. By inhibiting BCRP, it reverses multidrug resistance and enhances the efficacy of chemotherapeutic agents that are substrates of BCRP. The compound's in vivo activity has been demonstrated in preclinical models of drug-resistant tumors. However, specific protocols and detailed data are limited in publicly available sources.

ATPase assays were performed using crude membranes isolated from Hi-five insect cells expressing human ABCG2 or ABCB1. Membrane vesicles were incubated in ATPase assay buffer (50 mM MES-Tris pH 6.8, 50 mM KCl, 5 mM sodium azide, 1 mM EGTA, 1 mM ouabain, 10 mM MgCl₂, 2 mM dithiothreitol) with varying concentrations of Ko143 with or without beryllium fluoride (0.2 mM beryllium sulfate plus 2.5 mM NaF). ATP hydrolysis was measured by estimating inorganic phosphate release after incubation with 5 mM ATP, following a previously described method. Basal activity (without Ko143) was normalized to 100%, and percent change in activity was plotted against Ko143 concentration to determine IC50. For ABCG2, Ko143 decreased ATPase activity with an IC50 of 9.7 nM; for ABCB1, Ko143 stimulated ATPase activity with a biphasic response, reaching maximum stimulation at 10 μM (2-fold over basal) and an IC50 for stimulation of 2.7 μM. [1]
In non-cell-based biochemical assays, Ko-143's inhibition of BCRP is evaluated using membrane vesicle transport assays. Membrane vesicles prepared from cells expressing BCRP are incubated with a radiolabeled BCRP substrate (such as [³H]mitoxantrone or [³H]methotrexate) in the presence of varying concentrations of Ko-143. The accumulation of substrate inside the vesicles is measured, and IC50/EC90 values are determined from inhibition curves.

cells are plated at 400 or 1000/well in 96-well plates the night before addition of drugs. A concentration series of drug is applied along one plate axis and left for the duration of the assay. Plates are harvested after 4-5 days while untreated wells are still subconfluent. Relative cell proliferation is quantified with CyQuant or Sybr Green I fluorescent nucleic acid stains. Assays with human cell lines are performed in the presence of 0.1 μm PSC833 to inhibit confounding P-gp activity.

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Cytotoxicity assays: Cells were seeded at 4000 cells/well in 96-well plates. Serial dilutions of transporter-specific cytotoxic drugs (MTX for ABCG2, paclitaxel for ABCB1, doxorubicin for ABCC1) were added with or without Ko143 at various concentrations. After 72 h, cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay, and IC50 values for the cytotoxic drugs were calculated. Resistance ratios (RR) were determined by dividing IC50 of resistant cells by that of parental cells. Flow cytometry: Cells were suspended in media containing fluorescent substrates (MTX, PPA, Hoechst 33342, JC-1, or P-18 for ABCG2; rh123 for ABCB1; calcein-AM for ABCC1) with or without Ko143 or positive control inhibitors (FTC for ABCG2, cyclosporin A for ABCB1, MK571 for ABCC1). After incubation at 37°C for 30–45 min, cells were washed and analyzed on a flow cytometer; geometric mean fluorescence intensity was recorded for 10,000 cells per sample. Accumulation of [³H]Ko143: Cells were incubated with [³H]Ko143 for up to 45 min, with or without transporter inhibitors (FTC, Ko143, DCPQ, or MK571). After washing and trypsinization, radioactivity was measured by liquid scintillation counting and normalized to cell count. [1]
In vitro cellular assays for Ko-143 involve measuring its effects on drug accumulation and efflux in BCRP-overexpressing cancer cell lines. Cells are treated with Ko-143 in the presence of a fluorescent or radiolabeled BCRP substrate (such as mitoxantrone or doxorubicin). Intracellular accumulation of the substrate is measured using flow cytometry or scintillation counting. Ko-143's ability to reverse drug resistance is assessed by measuring cell viability in the presence of chemotherapeutic agents.

Animal Protocol: Sprague-Dawley rats (260-280 g) were randomly divided into groups (n=6). In the Ko143 treatment group, rats received Ko143 intravenously for 7 consecutive days, twice per day (exact dose not specified). On the 7th day, after Ko143 injection, rosuvastatin (50 mg/kg) was orally administered. Blood samples were collected from the femoral artery at 0.5, 1, 1.5, 3, 5, 8, 10, 12, and 24 hours post-dose. Plasma was separated and stored at -20°C until analysis. Rosuvastatin concentrations were determined by LC-MS with pitavastatin as internal standard. Pharmacokinetic parameters were calculated using DAS 2.0 software. [3]
In vivo animal studies for Ko-143 have been conducted in xenograft tumor models using BCRP-overexpressing cancer cells. The compound is typically administered orally, and its effects on tumor growth, drug accumulation, and survival are assessed in combination with chemotherapeutic agents. Ko-143 enhances the efficacy of BCRP substrate drugs by reversing multidrug resistance. However, specific protocols and data are limited.

In rat plasma, Ko143 was rapidly hydrolyzed with a half-life of approximately 12 minutes at room temperature, and no parent compound remained after 60 minutes. The addition of the esterase inhibitor sodium fluoride (NaF) completely prevented hydrolysis, indicating enzymatic degradation. Ko143 acid (the hydrolytic metabolite) showed negligible inhibitory activity on ABCG2, ABCB1, or ABCC1 except weak ABCG2 inhibition at 100 μM. Plasma protein binding of Ko143 is reported to be 92–95% (from literature, not measured in this study). [1]
Ko-143 has a molecular weight of 469.6 and a molecular formula of C26H35N3O5. It is a potent and selective BCRP inhibitor with an EC90 of 26 nM. It is orally bioavailable and displays >200-fold selectivity over P-gp and MRP-1 transporters. The compound is typically stored as a powder at -20°C for long-term stability.

Ko143 exhibited cytotoxicity at concentrations greater than 5 μM in HEK-293 cells after 72-hour incubation. No other toxicity data (e.g., in vivo) were reported. [1]
As a research compound, Ko-143 is not intended for human use and should be handled with appropriate safety precautions. Comprehensive toxicology studies have not been extensively published, but the compound is described as a nontoxic analog of fumitremorgin C. It should be stored and handled in accordance with standard laboratory safety guidelines.

[1]. The Inhibitor Ko143 Is Not Specific for ABCG2. J Pharmacol Exp Ther. 2015 Sep;354(3):384-93.

[2]. Potent and Specific Inhibition of the Breast Cancer Resistance Protein Multidrug Transporter in Vitro and in Mouse Intestine by a Novel Analogue of Fumitremorgin C. Mol. Cancer Ther. 2002, 1, 417-425.

[3]. Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of ZD 4522 In Vivo and Vitro. Chin Med Sci J. 2015 Dec;30(4):218-25.

[4]. Quantitative determination and pharmacokinetic study of the novel anti-Parkinson's disease candidate drug FLZ in rat brain by high performance liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 2012 Jul;66:232-9.

[5]. Metabolism of KO143, an ABCG2 inhibitor. Drug Metab Pharmacokinet. 2017 Aug;32(4):193-200.

LSM-6260 is a β-carboline compound, belonging to the tert-butyl ester class.

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Ko143 is a tert-butyl ester and is rapidly hydrolyzed by plasma esterases to its carboxylic acid metabolite, which is inactive. Despite being a potent ABCG2 inhibitor at nanomolar concentrations, it lacks specificity for ABCG2 at higher micromolar concentrations, also inhibiting ABCB1 and ABCC1. This non-selectivity should be considered when using Ko143 in both in vitro and in vivo experiments, especially for PET imaging studies where high local concentrations may occur. The study emphasizes the need for a specific ABCG2 substrate or inhibitor for reliable imaging of transporter function. [1]
Ko-143 is a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2) multidrug transporter with an EC90 of 26 nM. It is a nontoxic analog of the fungal toxin fumitremorgin C and displays >200-fold selectivity over P-gp and MRP-1 transporters. Ko-143 reverses resistance of MTX and topotecan at 25 nM in human cells. It is an orally bioavailable BCRP inhibitor and is a research compound, not an approved drug.

C26H35N3O5

469.582

469.257

461054-93-3

10322450

White to off-white solid powder

1.2±0.1 g/cm3

689.8±55.0 °C at 760 mmHg

147ºC

371.0±31.5 °C

0.0±2.2 mmHg at 25°C

1.597

2.42

2

5

8

34

794

3

CC(C)C[C@H]1C2=C(C[C@@H]3N1C(=O)[C@@H](NC3=O)CCC(=O)OC(C)(C)C)C4=C(N2)C=C(C=C4)OC

NXNRAECHCJZNRF-JBACZVJFSA-N

InChI=1S/C26H35N3O5/c1-14(2)11-20-23-17(16-8-7-15(33-6)12-19(16)27-23)13-21-24(31)28-18(25(32)29(20)21)9-10-22(30)34-26(3,4)5/h7-8,12,14,18,20-21,27H,9-11,13H2,1-6H3,(H,28,31)/t18-,20-,21-/m0/s1

tert-butyl 3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2'

Ko-143 Ko-143 Ko-143

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~212.96 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.32 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<50°C).
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (5.32 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)