Inhibition of the Na+/Dicarboxylate Cotransporter by Anthranilic Acid Derivatives

The Na⁺/dicarboxylate cotransporter NaDC1 absorbs citric acid cycle intermediates from the lumen of the small intestine and kidney proximal tubule. No effective inhibitor has been identified yet, although previous studies showed that the nonsteroidal anti-inflammatory drug, flufenamate, inhibits the human (h) NaDC1 with an IC₅₀ value of 2 mM. In the present study, we have tested compounds related in structure to flufenamate, all anthranilic acid derivatives, as potential inhibitors of hNaDC1. We found that N-(p-amylcinnamoyl) anthranilic acid (ACA) and 2-(p-amylcinnamoyl) amino-4-chloro benzoic acid (ONO-RS-082) are the most potent inhibitors with IC₅₀ values lower than 15 μM, followed by N-(9-fluorenylmethoxycarbonyl)-anthranilic acid (Fmoc-anthranilic acid) with an IC₅₀ value of ∼80 μM. The effects of ACA on NaDC1 are not mediated through a change in transporter protein abundance on the plasma membrane and seem to be independent of its effect on phospholipase A₂ activity. ACA acts as a slow inhibitor of NaDC1, with slow onset and slow reversibility. Both uptake activity and efflux are inhibited by ACA. Other Na⁺/dicarboxylate transporters from the SLC13 family, including hNaDC3 and rbNaDC1, were also inhibited by ACA, ONO-RS-082, and Fmoc-anthranilic acid, whereas the Na⁺/citrate transporter (hNaCT) is much less sensitive to these compounds. The endogenous sodium-dependent succinate transport in Caco-2 cells is also inhibited by ACA. In conclusion, ACA and ONO-RS-082 represent promising lead compounds for the development of specific inhibitors of the Na⁺/dicarboxylate cotransporters.