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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

Characterization of the interaction between compound 2 and HEKNaCT cells.(A) Interaction of compound [3H]-2 (75 nM) with HEK-293 and HEKNaCT cells competed with increasing amounts of citric acid (n = 3). (B) Chemical structure of dicarboxylate 4, tritiated [3H]-2 and ester 5. (C) Using compound [3H]-2 (75 nM) from left to right, interaction after 2 hours of incubation in; (1) HEKNaCT, (2) parental HEK-293 cells, (3) HEKNaCT with sodium chloride replaced by choline chloride, and (4) following an additional 2 hours incubation with 100 μM of compound 2 (n = 3). (D) Inhibition of citrate uptake in human hepatocytes using the prodrug 5 with measurements of intracellular and extracellular conversion to compound 2 (n = 5).
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f2: Characterization of the interaction between compound 2 and HEKNaCT cells.(A) Interaction of compound [3H]-2 (75 nM) with HEK-293 and HEKNaCT cells competed with increasing amounts of citric acid (n = 3). (B) Chemical structure of dicarboxylate 4, tritiated [3H]-2 and ester 5. (C) Using compound [3H]-2 (75 nM) from left to right, interaction after 2 hours of incubation in; (1) HEKNaCT, (2) parental HEK-293 cells, (3) HEKNaCT with sodium chloride replaced by choline chloride, and (4) following an additional 2 hours incubation with 100 μM of compound 2 (n = 3). (D) Inhibition of citrate uptake in human hepatocytes using the prodrug 5 with measurements of intracellular and extracellular conversion to compound 2 (n = 5).

Mentions: In order to investigate the mode of action by which 2 inhibits the cellular transport of citrate, a radiolabeled analog was required. With this goal in mind, the iodo-substituted compound 4 (PF-06678419) was synthesized via a Sonogashira reaction with 1,4-diiodobenzene and terminal alkyne 12. The resulting alkyne 13 was reduced with p-tolylhydrazide followed by chiral separation and hydrolysis to afford diacid 4 (Fig. 2B and Scheme S2). Compound 4 showed inhibition of citrate uptake in the HEKNaCT cells with potency similar to that of 2 (IC50 = 0.44 μM). However, we did not pursue the preparation of a radiolabeled analog with compound 4 as it did not show complete selectivity for NaCT over NaDC1 and NaDC3 (Table 1). On the other hand, suitable specific activity was obtained with a tritiated version of 2 ([3H]-2, Fig. 2B). This compound was prepared by alkyne reduction of the chiral diacid 16 with palladium on carbon and tritium gas (Scheme S3), and evaluated in whole cell experiments where radioactivity was measured. An interaction between [3H]-2 and the NaCT-expressing cells was observed in whole cell experiments and this binding could be out-competed with increasing concentrations of citrate (Fig. 2A). The competitive nature of this interaction with citrate suggested a common binding site for 2 and citrate on NaCT. As opposed to the HEKNaCT cells, no significant interaction was observed with the parental HEK-293 cells (Fig. 2A,C). Since NaCT expression is not detected in qRT-PCR analysis of the parental HEK-293 cell (data not shown), these data support a specific interaction between [3H]-2 and NaCT. The presence of sodium in the cell media was required for this interaction as the entire signal observed with the HEKNaCT cells was abolished when sodium chloride was replaced with choline chloride (Fig. 2C). Transport of citrate via NaCT was previously shown to be a sodium-dependent process as well24. When HEKNaCT cells were incubated with a 75 nM solution of [3H]-2, washed with buffered saline and the media replaced with 100 μM of 2 for 2 hours, a large fraction of the total radioactive signal remained (Fig. 2C). This observation could be attributed to a slow dissociation or irreversible interaction; however, based on the overall data generated in these experiments, as well as the structural similarity of 2 with citrate, it was hypothesized to be the result of NaCT-mediated cellular uptake of the dicarboxylate.


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)

Characterization of the interaction between compound 2 and HEKNaCT cells.(A) Interaction of compound [3H]-2 (75 nM) with HEK-293 and HEKNaCT cells competed with increasing amounts of citric acid (n = 3). (B) Chemical structure of dicarboxylate 4, tritiated [3H]-2 and ester 5. (C) Using compound [3H]-2 (75 nM) from left to right, interaction after 2 hours of incubation in; (1) HEKNaCT, (2) parental HEK-293 cells, (3) HEKNaCT with sodium chloride replaced by choline chloride, and (4) following an additional 2 hours incubation with 100 μM of compound 2 (n = 3). (D) Inhibition of citrate uptake in human hepatocytes using the prodrug 5 with measurements of intracellular and extracellular conversion to compound 2 (n = 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Characterization of the interaction between compound 2 and HEKNaCT cells.(A) Interaction of compound [3H]-2 (75 nM) with HEK-293 and HEKNaCT cells competed with increasing amounts of citric acid (n = 3). (B) Chemical structure of dicarboxylate 4, tritiated [3H]-2 and ester 5. (C) Using compound [3H]-2 (75 nM) from left to right, interaction after 2 hours of incubation in; (1) HEKNaCT, (2) parental HEK-293 cells, (3) HEKNaCT with sodium chloride replaced by choline chloride, and (4) following an additional 2 hours incubation with 100 μM of compound 2 (n = 3). (D) Inhibition of citrate uptake in human hepatocytes using the prodrug 5 with measurements of intracellular and extracellular conversion to compound 2 (n = 5).
Mentions: In order to investigate the mode of action by which 2 inhibits the cellular transport of citrate, a radiolabeled analog was required. With this goal in mind, the iodo-substituted compound 4 (PF-06678419) was synthesized via a Sonogashira reaction with 1,4-diiodobenzene and terminal alkyne 12. The resulting alkyne 13 was reduced with p-tolylhydrazide followed by chiral separation and hydrolysis to afford diacid 4 (Fig. 2B and Scheme S2). Compound 4 showed inhibition of citrate uptake in the HEKNaCT cells with potency similar to that of 2 (IC50 = 0.44 μM). However, we did not pursue the preparation of a radiolabeled analog with compound 4 as it did not show complete selectivity for NaCT over NaDC1 and NaDC3 (Table 1). On the other hand, suitable specific activity was obtained with a tritiated version of 2 ([3H]-2, Fig. 2B). This compound was prepared by alkyne reduction of the chiral diacid 16 with palladium on carbon and tritium gas (Scheme S3), and evaluated in whole cell experiments where radioactivity was measured. An interaction between [3H]-2 and the NaCT-expressing cells was observed in whole cell experiments and this binding could be out-competed with increasing concentrations of citrate (Fig. 2A). The competitive nature of this interaction with citrate suggested a common binding site for 2 and citrate on NaCT. As opposed to the HEKNaCT cells, no significant interaction was observed with the parental HEK-293 cells (Fig. 2A,C). Since NaCT expression is not detected in qRT-PCR analysis of the parental HEK-293 cell (data not shown), these data support a specific interaction between [3H]-2 and NaCT. The presence of sodium in the cell media was required for this interaction as the entire signal observed with the HEKNaCT cells was abolished when sodium chloride was replaced with choline chloride (Fig. 2C). Transport of citrate via NaCT was previously shown to be a sodium-dependent process as well24. When HEKNaCT cells were incubated with a 75 nM solution of [3H]-2, washed with buffered saline and the media replaced with 100 μM of 2 for 2 hours, a large fraction of the total radioactive signal remained (Fig. 2C). This observation could be attributed to a slow dissociation or irreversible interaction; however, based on the overall data generated in these experiments, as well as the structural similarity of 2 with citrate, it was hypothesized to be the result of NaCT-mediated cellular uptake of the dicarboxylate.

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