BI-01383298

sodium-citrate co-transporter (SLC13A5)

The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity [1].

Uptake measurement[1]
HepG2 cells were seeded at 4 × 105 cells per well on 24-well culture plates and cultured for 1–2 days. HEK293FT cells were seeded at 2 × 105 cells per well on 24-well culture plates, cultured for 1 day, and then transfected with human SLC13A5/pcDNA3.1(+) or mouse SLC13A5/pcDNA3.1(+) using Lipofectamine-3000 (Thermo Fisher Scientific), according to the manufacturer's recommendations. Uptake measurements were made in HepG2 cells 48 h after the initial seeding to monitor the activity of the constitutively expressed SLC13A5. In the case of HEK293FT cells, uptake measurements were made 48 h after the transfection to monitor the ectopically expressed human SLC13A5 or mouse Slc13a5. To initiate the uptake of citrate, the culture medium was removed by aspiration and then the cells were incubated in a NaCl buffer (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose, buffered with 25 mM Hepes/Tris, pH 7.5) with or without the NaCT inhibitor BI01383298 for the indicated time intervals. In some experiments with ectopically expressed human SLC13A5 in HEK293FT cells, the effects of the vehicle control (0.1% dimethylsulfoxide) or the inactive chemical analogue BI01372674 (10 µM in 0.1% dimethylsulfoxide) on the transporter activity were investigated. In the case of recovery experiments, the buffer with the inhibitor was removed from the wells, and the cells were washed at least twice with the NaCl buffer, and then incubated in the NaCl buffer or culture medium for the indicated time intervals. The transport function of SLC13A5 in these cells was monitored by measuring the uptake of [14C]-citrate (0.1 µCi; 4 µM citrate) using the NaCl-uptake buffer with or without 10 mM LiCl because Li+ is an activator of human SLC13A5. The transport function of mouse Slc13a5 was monitored in the NaCl buffer but without LiCl because Li+ is an inhibitor of murine NaCT. The uptake measurements were recorded using a 30 min period at 37°C. At the end of the incubation, the medium was removed, the cells washed with ice-cold uptake buffer, and the cells lysed to measure the radioactivity. To determine specifically the Na+-dependent citrate uptake, the uptake in NMDG (N-methyl-D-glucamine)-Cl buffer (i.e. Na+-free) was subtracted from the uptake in the NaCl buffer with or without 10 mM LiCl. The uptake values were normalized with the protein content of the cells, measured by a Pierce™ BCA Protein Assay Kit.
To estimate the kinetic parameters, the uptake velocity of citrate at each concentration (0.5–30 mM, 30 min incubation) was calculated by subtraction of non-saturable component from the total uptake velocity. The non-saturable component was estimated by subjecting the uptake data to a transport model consisting of a single saturable transport system with Michaelis–Menten saturable kinetic characteristics and a non-carrier-mediated, non-saturable, diffusional component. The uptake velocity for the saturable component was plotted and analyzed using the Michaelis–Menten equation by GraphPad Prism 7.01 software (GraphPad, San Diego, CA, U.S.A.). The linear form of the Michaelis–Menten equation was also used to calculate the kinetic constants (Eadie–Hofstee plot).

Colony formation assay [1]
Colony formation assay was done by seeding HepG2 cells at 5000 cells per well in six-well culture plates and culturing the cells in the normal medium with 0.1% dimethylsulfoxide or the medium with 5 or 10 µM BI01383298 (plus 0.1% dimethylsulfoxide). The medium was changed with fresh corresponding medium every other day for 10 days. At the end of the experiment, cells were washed with phosphate-buffered saline, fixed with methanol, and stained with KaryoMax Giemsa stain. The colonies were then photographed and then the stain was extracted and quantified.

J.Biol.Chem., 2017;292:13902

C19H19CL2FN2O3S

445.33

444.0477472

C, 51.24; H, 4.30; Cl, 15.92; F, 4.27; N, 6.29; O, 10.78; S, 7.20

2227549-00-8

White to off-white solid powder

3.7

74.9Ų

O=C(C1CCN(S(=O)(C2=CC(Cl)=CC(Cl)=C2)=O)CC1)NCC3=CC=C(F)C=C3

VUOYAALVGSMUHC-UHFFFAOYSA-N

InChI=1S/C19H19Cl2FN2O3S/c20-15-9-16(21)11-18(10-15)28(26,27)24-7-5-14(6-8-24)19(25)23-12-13-1-3-17(22)4-2-13/h1-4,9-11,14H,5-8,12H2,(H,23,25)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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