BI01383298 A chemical probe for SLC13A5

This probe is available from Tocris, Sigma and opnMe.com.

For any inquiries please contact proberequests@thesgc.org.

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Overview

Biology of SLC13A5

SLC13A5 (NaCT, INDY) is a sodium-citrate co-transporter that is highly expressed in the liver. It is a member of the SLC13 family of which there are 4 other members. SLC13A5 transports citrate from the circulatory system into hepatocytes where it is used in the synthesis of sterols and fatty acids. SLC13A5 was first identified in Drosophila where the name I’m Not Dead Yet or INDY was coined. A study in 2000(1) showed that lower expression of SLC13A5 in Drosophila led to increased lifespan. Mouse models have demonstrated the potential of this protein as a target for obesity and diabetes with knockout mice protected from adiposity (2), reduced lipid concentrations in a siRNA study (3) and a substrate analogue used to lower blood glucose levels (4). More recently mutations in SLC13A5 have been linked to early-infantile epileptic encephalopathy (5) while silencing of the SLC13A5 gene inhibits proliferation of human hepatocarcinoma cells (6).

BI1383298 is a potent inhibitor of SLC13A5 which unlike previously published inhibitors (4,7) has no structural homology to the substrate (citrate). It is selective over other family members and other transporters. A chemically related negative control (BI01372674) is also provided.

BI01383298: a chemical probe for SLC13A5

BI01383298

BI01372674

In addition to the chemical probe (BI01383298) we also include a negative control (BI01372674) which is chemically analogous to the probe molecule.

The probe molecule (BI01383298) and control (BI01372674) are soluble, after visual inspection, at 10 µM and 200µM in the presence of 0.1% DMSO in media and assay buffer.

Aliquots of stock Solutions were prepared as follows: 10mM BI01383298 in DMSO (Stored -20oC), 5mM BI01383298 in DMSO (Stored -20oC, need agitation upon thaw and gentle warming to 30oC).

SLC13A5 engagement in vitro

BI01383298 but not BI01372674 demonstrates target engagement in vitro with purified human SLC13A5 protein as shown using a thermostabilisation assay with the Prometheus label-free system from Nanotemper (figure 2).

Figure 2: Thermostabilisation of human SLC13A5 by BI01383298 and BI01372674 between 0.1µM and 10µM. SLC13A5 assay concentration of 1µM. Data summarises 3 biological samples with between 4 and 8 replicates for each.

Properties

BI01383298

BI1372674
(negative control)

Physical and chemical properties for BI01383298
Molecular weight	444.0
Molecular formula	C₁₉H₁₉Cl₂FN₂O₃S
IUPAC name	(1-(3,5-dichloro-phenylsulfonyl)-piperidin-4-yl)-((4-fluoro-phenyl)-methylamino)-methanone
MollogP	4.64
PSA	56.3
No. of chiral centres	0
No. of rotatable bonds	6
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	1

Physical and chemical properties for BI1372674 (Negative Control)
Molecular weight	502.1
Molecular formula	C₂₁H₂₅Cl₂N₂O₄PS
IUPAC name
MollogP	3.54
PSA	69.5
No. of chiral centres	0
No. of rotatable bonds	7
No. of hydrogen bond acceptors	9
No. of hydrogen bond donors	1

SMILES:
BI01383298: C1CN(CCC1C(NCc1ccc(cc1)F)=O)S(c1cc(cc(c1)[Cl])[Cl])(=O)=O
BI1372674: C1(C=C(C=C(C=1)[Cl])S(N(CCC(C1)C(=O)NCC(=C2)C=CC(=C2)P(=O)(C)C)C1)(=O)=O)[Cl]

InChI:
BI01383298: 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)

BI1372674: InChI=1S/C21H25Cl2N2O4PS/c1-30(2,27)19-5-3-15(4-6-19)14-24-21(26)16-7-9-25(10-8-16)31(28,29)20-12-17(22)11-18(23)13-20/h3-6,11-13,16H,7-10,14H2,1-2H3,(H,24,26)

InChIKey:
BI01383298: VUOYAALVGSMUHC-UHFFFAOYSA-N
BI1372674: GSNNWZZIEKEEQF-UHFFFAOYSA-N

	Solvent	BI01383298	BI01372674
Solubility (200uM, 4% DMSO)	Assay Buffer*- 0hr	Soluble	Soluble
	Assay Buffer*- 12hrs	Soluble	Soluble
	Freestyle Media- 0hr	Soluble	Soluble
	Freestyle Media- 12hr	Soluble	Soluble
	Water- 0hrs	ppt. observed	ppt. observed
Solubility (10uM, 0.1% DMSO)	Assay Buffer*- 0hr	Soluble	Soluble
	Assay Buffer*- 12hrs	Soluble	Soluble
	Freestyle Media- 0hr	Soluble	Soluble
	Freestyle Media- 12hr	Soluble	Soluble
	Water- 0hrs	Soluble	Soluble

*50mM HEPES (pH 7.5), 200mM NaCl, 0.024% n-Dodecyl-β-D-Maltopyranoside, 0.0024% Cholesterol Hemisuccinate.

Selectivity Profile

Selectivity against SLC13 family members and other transporters in cellular assays

BI01383298 is more than 1000-fold selective (table 1) over the closest family members: human SLC13A2 / SLC13A3 that share physiological substrates citrate and succinate.

Table 1: Citrate uptake inhibition was measured for all citrate transporters and glycine uptake measured to GLYT2. Potency was assessed for the probe candidate and the negative control on uptake of ¹⁴C-citrate into cells over-expressing SLC13A5, SLC13A2, SLC13A3, mouse SLC13A5 and in HEPG2 cells.

	Substrate uptake inhibition IC₅₀ [nM]
	BI01383298	BI01372674
HEK cells- hSLC13A5	56	>100,000
HepG2 cells	24	>100,000
HEK cells- mSLC13A5	>100,000	88,000
HEK cells- hSLC13A2	>100,000	n.d.
HEK cells- hSLC13A3	>100,000	n.d.
HEK cells – GLYT2	>100,000	>100,000

Selectivity panel (% inhibition @ 10μM): 35/38 targets<50%; CB1(h): 78%; K(KOP): 81%; Na⁺channel: 52%

Cell-based Assay Data

Potency against SLC13A5 in cellular assays

BI01383298 is a potent inhibitor of SLC13A5 with an apparent IC50 value of 56nM in HEK cells overexpressing SLC13A5 and 24nM in HepG2 cell expressing endogenous SLC13A5. BI01383298 was not found to inhibit citrate transport in HEK cells over-expressing mouse SLC13A5 and thus we do not recommend this compound for use in mouse models. BI01372674 was not found to inhibit citrate uptake in a HEPG2 model expressing endogenous levels of SLC13A5 or in HEK cellular models over-expressing human SLC13A5, mouse SLC13A5 or GLYT2.

Figure 1: Measured IC₅₀ of hSLC13A5-mediated ¹⁴C-citrate in (A) overexpressed, HEK293-Flp-In-hSLC13A5 and (B) endogenous, HepG2 celluar models.

References

Rogina, B., et al. (2000). Science 290, 2137-2140
Birkenfeld, A. L., et al. (2011). Cell Metab 14, 184-195
Brachs, S., et al. (2016). Mol Metab 5, 1072-1082
Huard, K., et al. (2015). Sci Rep 5, 17391
Bainbridge, M. N., et al. (2017). Mol Genet Metab 121, 314-319
Li, Z., et al. (2017). The Journal of biological chemistry 292, 13890-13901
Huard, K., et al. (2016). Journal of medicinal chemistry 59, 1165-1175