Mitochondrial and Plasma Membrane Citrate Transporters: Discovery of Selective Inhibitors and Application to Structure/Function Analysis

Abstract

Cytoplasmic citrate is the prime carbon source for fatty acid, triacylglycerol, and cholesterol biosyntheses, and also regulates glucose metabolism via its allosteric inhibition of phosphofructokinase. It originates either via the efflux of citrate from the mitochondrial matrix on the inner membrane citrate transport protein (CTP) or via the influx of extracellular citrate on the plasma membrane citrate transporter (PMCT). Despite their common substrate, the two transport proteins share little sequence similarity and they transport citrate via fundamentally different mechanisms. We tested the ability of a set of previously identified CTP inhibitors, to inhibit the PMCT. We found that of the top 10 CTP inhibitors only one substantially inhibited the PMCT. Conversely, we identified two other inhibitors that inhibited the PMCT but had little effect on the CTP. All three identified PMCT inhibitors displayed a noncompetitive mechanism. Furthermore, models to explain inhibitor interactions with the CTP are proposed. As part of the present studies a PMCT homology model has been developed based on the crystal structure of the leucine transporter, and a possible citrate binding site has been identified and its composition compared with the two known citrate binding sites present within the CTP. The ability to selectively inhibit the PMCT may prove key to the pharmacologic amelioration of metabolic disorders resulting from the synthesis of excess lipid, cholesterol, and glucose, including human obesity, hyperlipidemia, hyper-cholesterolemia, and Type 2 diabetes.

Figure 1. Schematic depiction of the roles of the PMCT and the CTP in supplying citrate to fuel hepatic fatty acid, triacylglycerol, and sterol biosyntheses

Citrate can be transported from the blood across the hepatocyte plasma membrane into the cytoplasm on the PMCT, or it can be effluxed from the mitochondrial matrix across the mitochondrial inner membrane on the CTP. Cytoplasmic citrate derived from either source is then broken down to acetyl CoA by citrate lyase, and the resulting acetyl CoA provides all of the carbon precursor to fuel the fatty acid, triacylglycerol, and sterol biosynthetic pathways.

Figure 2. Structures tested as inhibitors of the PMCT and the CTP

Structures are identified by their ZINC database accession numbers.

Figure 3. Determination of the kinetic mechanism for ZINC compound 39396- and 4180643-mediated inhibition of the PMCT

Panels A and D depict the structures of ZINC compounds 39396 and 4180643, respectively. Percentage inhibition for PMCT is derived from panels B and E. The CTP inhibition numbers are from Table 1. Panels B and E illustrate the v versus [S] data obtained for the PMCT in the presence of several concentrations of 39396 and 4180643, respectively. Data were analyzed via nonlinear regression analysis. Panels C and F display Lineweaver-Burk plots depicting the effects of inhibitors 39396 and 4180643, respectively, on PMCT function.

Figure 4. Homology-based model of PMCT, with a citrate molecule docked into the transport path

The protein is shown as a backbone ribbon, with color shading from dark blue at the N-terminus to dark red at the C-terminus. Citrate is shown as a space-filling model, with carbons in green, oxygens in red, and hydrogens omitted for clarity. Panel A, View in the plane of the membrane, with the extracellular face at the top and the cytosolic face at the bottom. Panel B, View looking into the transport path from the extracellular face (rotated 90° around the x-axis relative to view A).

Figure 5. Citrate interactions with PMCT and CTP binding sites

Protein backbones are shown as green ribbons. Nitrogen atoms are blue, oxygen atoms red, sulfur atoms yellow; transporter carbon atoms are green, and citrate carbon atoms are magenta. Panel A, In PMCT, citrate interacts with R33 of TMD1, R103 and R104 of TMD3, R274 of TMD6, and C412 of TMD10. Panel B, In site 1 of CTP, citrate interacts with K83 and R87 of TMD2 and with R189 of TMD4. Panel C, In site 2 of CTP, citrate interacts with K37 of TMD1, R181 of TMD4, K239 of TMD5, and R276 and R279 of TMD6.

Figure 6. Inhibitors modeled in the CTP transport path

Panel A, Compound 4180643, a significant CTP inhibitor, interacts with K83 and R87 of TMD2, K134 of TMD3, and K239 of TMD5. Panel B, Compound 339393, a much less effective inhibitor of CTP, has a shorter substituent on the pyrrole-nitrogen; its carboxylate can still interact with K83, but is unable to reach R87 on TMD2.