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Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5).

Huard K, Brown J, Jones JC, Cabral S, Futatsugi K, Gorgoglione M, Lanba A, Vera NB, Zhu Y, Yan Q, Zhou Y, Vernochet C, Riccardi K, Wolford A, Pirman D, Niosi M, Aspnes G, Herr M, Genung NE, Magee TV, Uccello DP, Loria P, Di L, Gosset JR, Hepworth D, Rolph T, Pfefferkorn JA, Erion DM - Sci Rep (2015)

Bottom Line: NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions.Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT.The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

View Article: PubMed Central - PubMed

Affiliation: Worldwide Medicinal Chemistry, 610 Main street, Cambridge, MA 02139.

ABSTRACT
Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

No MeSH data available.


Related in: MedlinePlus

In vivo citrate metabolism in mice treated with compound 2.(A) Free liver exposure of compound 2 in mice after an oral dose of 250 mg/kg. (B) Uptake of radioactive [14C]-citric acid in liver, kidney and white adipose tissue in DIO mice treated with either vehicle, a single 250 mg/kg dose (‘acute’) of compound 2, or 3 days 250 mg/kg BID (‘sub-chronic’) of compound 2 (**P<0.01, One-way ANOVA – Dunnett’s post hoc test, n = 9–10). (C) Radioactive accumulation in urine over 24-hours of mice dosed with a radioactive bolus of [14C]-citric acid, and treated with either vehicle or compound 2 (*P = 0.07, Student’s t test, n = 5). (D) Incorporation of radioactive [14C]-citric acid into lipids in mice treated with either vehicle or compound 2 (**P < 0.01, Student’s t-test, n = 8). (E) Plasma glucose concentrations in DIO mice treated with either a single 250 mg/kg dose of compound 2, or 3 days 250 mg/kg BID with compound 2 (n = 9–10).
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f4: In vivo citrate metabolism in mice treated with compound 2.(A) Free liver exposure of compound 2 in mice after an oral dose of 250 mg/kg. (B) Uptake of radioactive [14C]-citric acid in liver, kidney and white adipose tissue in DIO mice treated with either vehicle, a single 250 mg/kg dose (‘acute’) of compound 2, or 3 days 250 mg/kg BID (‘sub-chronic’) of compound 2 (**P<0.01, One-way ANOVA – Dunnett’s post hoc test, n = 9–10). (C) Radioactive accumulation in urine over 24-hours of mice dosed with a radioactive bolus of [14C]-citric acid, and treated with either vehicle or compound 2 (*P = 0.07, Student’s t test, n = 5). (D) Incorporation of radioactive [14C]-citric acid into lipids in mice treated with either vehicle or compound 2 (**P < 0.01, Student’s t-test, n = 8). (E) Plasma glucose concentrations in DIO mice treated with either a single 250 mg/kg dose of compound 2, or 3 days 250 mg/kg BID with compound 2 (n = 9–10).

Mentions: Steady state intravenous infusion of 2 in rat demonstrates significant uptake of the compound in the liver and kidney, as compared to the plasma while exhibiting minimal brain penetration (Figure S4). In spite of its doubly-charged and polar nature, 2 achieved significant exposures in mice following oral dosing. When treated with 250 mg/kg po, the unbound concentration of 2 in liver tissue was over 4 fold higher than the IC50 observed in mouse hepatocyte (4.5 μM, Fig. 1) for a period of 8 hours (Fig. 4A). Thus, we were able to assess the effect of NaCT inhibition on citrate uptake into a variety of tissues or matrices (liver, urine, kidney and white adipose tissue) in vivo (Fig. 4B,C). Using the same 250 mg/kg po dose of 2, either acutely or sub-chronically (BID dosing for 3 days), labelled citrate uptake in liver was reduced by 33% relative to controls. Treatment with compound 2 increased the counts detected in urine collected over 24 hours after administration of a bolus of radioactive labeled citrate (Fig. 4C). Total kidney radioactivity increased although this is likely at least partially attributed to urine contamination (Fig. 4B). We selected white adipose tissue as one tissue with minimal SLC13a5 expression, and as anticipated there were no differences in the radioactive counts between drug-treated and control groups. Consistent with the reduction in hepatic citrate uptake, conversion of labelled citrate into hepatic lipids was also decreased (Fig. 4D), and furthermore, lower uptake of citrate by liver in treated mice was associated with modest but significant reductions in plasma glucose concentrations (Fig. 4E).


Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5).

Huard K, Brown J, Jones JC, Cabral S, Futatsugi K, Gorgoglione M, Lanba A, Vera NB, Zhu Y, Yan Q, Zhou Y, Vernochet C, Riccardi K, Wolford A, Pirman D, Niosi M, Aspnes G, Herr M, Genung NE, Magee TV, Uccello DP, Loria P, Di L, Gosset JR, Hepworth D, Rolph T, Pfefferkorn JA, Erion DM - Sci Rep (2015)

In vivo citrate metabolism in mice treated with compound 2.(A) Free liver exposure of compound 2 in mice after an oral dose of 250 mg/kg. (B) Uptake of radioactive [14C]-citric acid in liver, kidney and white adipose tissue in DIO mice treated with either vehicle, a single 250 mg/kg dose (‘acute’) of compound 2, or 3 days 250 mg/kg BID (‘sub-chronic’) of compound 2 (**P<0.01, One-way ANOVA – Dunnett’s post hoc test, n = 9–10). (C) Radioactive accumulation in urine over 24-hours of mice dosed with a radioactive bolus of [14C]-citric acid, and treated with either vehicle or compound 2 (*P = 0.07, Student’s t test, n = 5). (D) Incorporation of radioactive [14C]-citric acid into lipids in mice treated with either vehicle or compound 2 (**P < 0.01, Student’s t-test, n = 8). (E) Plasma glucose concentrations in DIO mice treated with either a single 250 mg/kg dose of compound 2, or 3 days 250 mg/kg BID with compound 2 (n = 9–10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664966&req=5

f4: In vivo citrate metabolism in mice treated with compound 2.(A) Free liver exposure of compound 2 in mice after an oral dose of 250 mg/kg. (B) Uptake of radioactive [14C]-citric acid in liver, kidney and white adipose tissue in DIO mice treated with either vehicle, a single 250 mg/kg dose (‘acute’) of compound 2, or 3 days 250 mg/kg BID (‘sub-chronic’) of compound 2 (**P<0.01, One-way ANOVA – Dunnett’s post hoc test, n = 9–10). (C) Radioactive accumulation in urine over 24-hours of mice dosed with a radioactive bolus of [14C]-citric acid, and treated with either vehicle or compound 2 (*P = 0.07, Student’s t test, n = 5). (D) Incorporation of radioactive [14C]-citric acid into lipids in mice treated with either vehicle or compound 2 (**P < 0.01, Student’s t-test, n = 8). (E) Plasma glucose concentrations in DIO mice treated with either a single 250 mg/kg dose of compound 2, or 3 days 250 mg/kg BID with compound 2 (n = 9–10).
Mentions: Steady state intravenous infusion of 2 in rat demonstrates significant uptake of the compound in the liver and kidney, as compared to the plasma while exhibiting minimal brain penetration (Figure S4). In spite of its doubly-charged and polar nature, 2 achieved significant exposures in mice following oral dosing. When treated with 250 mg/kg po, the unbound concentration of 2 in liver tissue was over 4 fold higher than the IC50 observed in mouse hepatocyte (4.5 μM, Fig. 1) for a period of 8 hours (Fig. 4A). Thus, we were able to assess the effect of NaCT inhibition on citrate uptake into a variety of tissues or matrices (liver, urine, kidney and white adipose tissue) in vivo (Fig. 4B,C). Using the same 250 mg/kg po dose of 2, either acutely or sub-chronically (BID dosing for 3 days), labelled citrate uptake in liver was reduced by 33% relative to controls. Treatment with compound 2 increased the counts detected in urine collected over 24 hours after administration of a bolus of radioactive labeled citrate (Fig. 4C). Total kidney radioactivity increased although this is likely at least partially attributed to urine contamination (Fig. 4B). We selected white adipose tissue as one tissue with minimal SLC13a5 expression, and as anticipated there were no differences in the radioactive counts between drug-treated and control groups. Consistent with the reduction in hepatic citrate uptake, conversion of labelled citrate into hepatic lipids was also decreased (Fig. 4D), and furthermore, lower uptake of citrate by liver in treated mice was associated with modest but significant reductions in plasma glucose concentrations (Fig. 4E).

Bottom Line: NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions.Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT.The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

View Article: PubMed Central - PubMed

Affiliation: Worldwide Medicinal Chemistry, 610 Main street, Cambridge, MA 02139.

ABSTRACT
Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

No MeSH data available.


Related in: MedlinePlus