Limits...
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

Impact of citrate and compound 2 or 3 on cellular metabolism.(A) U-[13C]-citrate uptake and incorporation into malate, glutamate, and fumarate as a percentage of total cellular concentrations. No label was detected in cells treated with unlabeled citrate (n = 2). (B) Incorporation of [14C]-citric acid into triglycerides in human hepatocytes, treated with either compound 2 or 3 (n = 1). (C,D) Oxygen consumption and extracellular acidification rates in HEKNaCT treated with vehicle, compound 2 or 3 (***P < 0.001; one-way ANOVA - Tukey’s multiple comparison test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664966&req=5

f3: Impact of citrate and compound 2 or 3 on cellular metabolism.(A) U-[13C]-citrate uptake and incorporation into malate, glutamate, and fumarate as a percentage of total cellular concentrations. No label was detected in cells treated with unlabeled citrate (n = 2). (B) Incorporation of [14C]-citric acid into triglycerides in human hepatocytes, treated with either compound 2 or 3 (n = 1). (C,D) Oxygen consumption and extracellular acidification rates in HEKNaCT treated with vehicle, compound 2 or 3 (***P < 0.001; one-way ANOVA - Tukey’s multiple comparison test).

Mentions: In order to better understand the in vitro metabolic fate of citrate, human hepatocytes were incubated with 500 μM of uniformly labeled citrate (U-[13C]-citrate). Substantial uniform labeling of citrate, malate, glutamate, and fumarate was observed in cells treated with the labeled citrate, which was reduced in the presence of 30 μM compound 2 (Fig. 3A). In human hepatocytes treated with radiolabeled citrate for 24 hours, label incorporation in TAG decreased dose-dependently with 2 but not with inactive enantiomer 3 (Fig. 3B). To assess the roles of citrate in cellular metabolism and the consequences of limiting citrate uptake through inhibition of the NaCT transporter, HEKNaCT cells were incubated with 150 μM citrate and either vehicle, 2 or 3, and results were recorded using a Seahorse® apparatus. The presence of citrate alone acutely increased cellular oxygen consumption rate (OCR), while 30 μM of compound 2, but not 3, prevented citrate induced increase in mitochondrial respiration (Fig. 3C). Since citrate inhibits PFK, a key regulator of glycolytic flux, the extracellular acidification rate (ECAR) was measured as a marker of glycolytic flux. Treatment of HEKNaCT cells with citrate acutely suppressed ECAR by 50% which was restored by 30 μM of compound 2 but not by 3 (Fig. 3D). These data support a critical role for citrate in glycolysis and mitochondrial metabolism.


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)

Impact of citrate and compound 2 or 3 on cellular metabolism.(A) U-[13C]-citrate uptake and incorporation into malate, glutamate, and fumarate as a percentage of total cellular concentrations. No label was detected in cells treated with unlabeled citrate (n = 2). (B) Incorporation of [14C]-citric acid into triglycerides in human hepatocytes, treated with either compound 2 or 3 (n = 1). (C,D) Oxygen consumption and extracellular acidification rates in HEKNaCT treated with vehicle, compound 2 or 3 (***P < 0.001; one-way ANOVA - Tukey’s multiple comparison test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Impact of citrate and compound 2 or 3 on cellular metabolism.(A) U-[13C]-citrate uptake and incorporation into malate, glutamate, and fumarate as a percentage of total cellular concentrations. No label was detected in cells treated with unlabeled citrate (n = 2). (B) Incorporation of [14C]-citric acid into triglycerides in human hepatocytes, treated with either compound 2 or 3 (n = 1). (C,D) Oxygen consumption and extracellular acidification rates in HEKNaCT treated with vehicle, compound 2 or 3 (***P < 0.001; one-way ANOVA - Tukey’s multiple comparison test).
Mentions: In order to better understand the in vitro metabolic fate of citrate, human hepatocytes were incubated with 500 μM of uniformly labeled citrate (U-[13C]-citrate). Substantial uniform labeling of citrate, malate, glutamate, and fumarate was observed in cells treated with the labeled citrate, which was reduced in the presence of 30 μM compound 2 (Fig. 3A). In human hepatocytes treated with radiolabeled citrate for 24 hours, label incorporation in TAG decreased dose-dependently with 2 but not with inactive enantiomer 3 (Fig. 3B). To assess the roles of citrate in cellular metabolism and the consequences of limiting citrate uptake through inhibition of the NaCT transporter, HEKNaCT cells were incubated with 150 μM citrate and either vehicle, 2 or 3, and results were recorded using a Seahorse® apparatus. The presence of citrate alone acutely increased cellular oxygen consumption rate (OCR), while 30 μM of compound 2, but not 3, prevented citrate induced increase in mitochondrial respiration (Fig. 3C). Since citrate inhibits PFK, a key regulator of glycolytic flux, the extracellular acidification rate (ECAR) was measured as a marker of glycolytic flux. Treatment of HEKNaCT cells with citrate acutely suppressed ECAR by 50% which was restored by 30 μM of compound 2 but not by 3 (Fig. 3D). These data support a critical role for citrate in glycolysis and mitochondrial metabolism.

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