Molecular Basis for Inhibition of the Na+/Citrate Transporter NaCT (SLC13A5) by Dicarboxylate Inhibitors.
The Na+/citrate transporter, NaCT (SLC13A5), is a therapeutic target for metabolic diseases. Citrate is an important signaling molecule that regulates the activity of lipid- and glucose-metabolizing enzymes in cells. Previous studies identified two compounds, PF-06649298 (compound 2: ) and PF-06678419 (compound 4: ), that inhibit human NaCT with high affinity, and one of the compounds demonstrated specificity relative to other SLC13 family members. Here we use molecular modeling and site-directed mutagenesis of hNaCT followed by transport characterization and cell-surface biotinylation to examine the residues involved in inhibitor binding and transport. The results indicate that residues located near the putative citrate binding site, G228, V231, V232, and G409, affect both citrate transport and inhibition of citrate uptake by compounds 2: and 4: V231 appears to distinguish between compounds 2: and 4: as inhibitors. Furthermore, residues located outside of the putative citrate binding site, Q77 and T86, may also play a role in NaCT inhibition by compounds 2: and 4: Our results provide new insight into the mechanism of transport and inhibition in NaCT and the SLC13 family. These findings should provide a basis for future drug design of SLC13 inhibitors.
University of California San Diego (A.M.P.), Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California; Cardiovascular, Metabolic and Endocrine Disease Research Unit (K.S., K.H., D.M.E.), Pfizer Worldwide Research and Development, Cambridge, Massachusetts; Center of Chemistry Innovation and Excellence (C.A.O., V.S.), Pfizer Worldwide Research and Development, Groton, Connecticut apajor@ucsd.edu.
Ana M
AM
Pajor
University of California San Diego (A.M.P.), Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California; Cardiovascular, Metabolic and Endocrine Disease Research Unit (K.S., K.H., D.M.E.), Pfizer Worldwide Research and Development, Cambridge, Massachusetts; Center of Chemistry Innovation and Excellence (C.A.O., V.S.), Pfizer Worldwide Research and Development, Groton, Connecticut.
Cesar A
CA
de Oliveira
University of California San Diego (A.M.P.), Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California; Cardiovascular, Metabolic and Endocrine Disease Research Unit (K.S., K.H., D.M.E.), Pfizer Worldwide Research and Development, Cambridge, Massachusetts; Center of Chemistry Innovation and Excellence (C.A.O., V.S.), Pfizer Worldwide Research and Development, Groton, Connecticut.
Kun
K
Song
University of California San Diego (A.M.P.), Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California; Cardiovascular, Metabolic and Endocrine Disease Research Unit (K.S., K.H., D.M.E.), Pfizer Worldwide Research and Development, Cambridge, Massachusetts; Center of Chemistry Innovation and Excellence (C.A.O., V.S.), Pfizer Worldwide Research and Development, Groton, Connecticut.
Kim
K
Huard
University of California San Diego (A.M.P.), Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California; Cardiovascular, Metabolic and Endocrine Disease Research Unit (K.S., K.H., D.M.E.), Pfizer Worldwide Research and Development, Cambridge, Massachusetts; Center of Chemistry Innovation and Excellence (C.A.O., V.S.), Pfizer Worldwide Research and Development, Groton, Connecticut.
Veerabahu
V
Shanmugasundaram
University of California San Diego (A.M.P.), Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California; Cardiovascular, Metabolic and Endocrine Disease Research Unit (K.S., K.H., D.M.E.), Pfizer Worldwide Research and Development, Cambridge, Massachusetts; Center of Chemistry Innovation and Excellence (C.A.O., V.S.), Pfizer Worldwide Research and Development, Groton, Connecticut.
Derek M
DM
Erion
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism; D003998:Dicarboxylic Acids / Q000737:chemistry / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D012964:Sodium / Q000494:pharmacology; D040681:Structural Homology, Protein; D014734:Vibrio cholerae / Q000378:metabolism
D002352:Carrier Proteins; D003998:Dicarboxylic Acids; D050505:Mutant Proteins; C020723:citrate-binding transport protein; D019343:Citric Acid; D012964:Sodium