DOI: 10.5061/dryad.stqjq2c2x
title: Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

description: Objective: The mitochondrial pyruvate carrier (MPC) has emerged as a
 promising drug target for metabolic disorders, including non-alcoholic
 steatohepatitis and diabetes, metabolically dependent cancers and
 neurodegenerative diseases. A range of structurally diverse small molecule
 inhibitors have been proposed but the nature of their interaction with MPC
 is not understood. Moreover, the composition of the functional
 human MPC is still debated. The goal of this study was to
 characterize the human MPC protein in vitro, to understand the
 chemical features that determine binding of structurally diverse
 inhibitors and to develop novel higher affinity ones. Results: We have
 determined that the functional unit of human MPC is a hetero-dimer. We
 have compared all different classes of MPC inhibitors to find that three
 closely arranged hydrogen bond acceptors followed by an aromatic ring are
 shared characteristics of all inhibitors and represent the minimal
 requirement for high potency. We also demonstrate that high
 affinity binding is not attributed to covalent bond formation with MPC
 cysteines, as previously proposed. Following the basic pharmacophore
 properties, we identify 14 new inhibitors of MPC, one outperforming
 compound UK5099 by tenfold.  Two of them are the
 commonly prescribed drugs entacapone and nitrofurantoin,
 suggesting an off-target mechanism associated with
 their adverse effects.  Conclusion: This work defines
 the composition of human MPC and the essential MPC inhibitor
 characteristics. In combination with the functional assays we describe,
 this new understanding will accelerate the development of
 clinically relevant MPC modulators.
description: 1. Mass determination by
 SEC-MALLS SEC-MALLS analysis was performed with a
 Superdex 200 10/300 GL column on an ÄKTA Explorer coupled in-line with a
 light scattering detector (Dawn HELEOSII, Wyatt Technologies) and a
 refractometer (Optilab T-rREX, Wyatt Technologies). The MPC complex was
 applied onto the Superdex 200 10/300 GL column equilibrated with 20 mM
 Tris-HCl, pH 7.4, 150 mM NaCl, 0.005% (w/v) LMNG, 0.005 mg/ml TOCL at 0.3
 ml/min. All data were recorded and analysed with ASTRA 6.03 (Wyatt
 Technologies). Molecular weight calculations were performed using the
 protein-conjugate method with the dn/dc value for protein of 0.185 ml/g
 and dn/dc value for LMNG-TOCL of 0.1675 ml/g. The contributions of each
 protein to the overall protein-detergent-lipid complex were determined
 from the extinction coefficients εA<sub>280</sub>, derived
 from the amino-acid sequence using the ProtParam tool on the ExPaSy
 server. 2. Thermostability shift
 analysis The assessment of ligand binding was performed
 via shifts in protein thermostability upon binding, using thermal
 denaturation on a rotary quantitative PCR (qPCR) instrument (Qiagen
 Rotor-Gene Q 2plex HRM, Venlo, Netherlands). In this method, cysteine
 residues, buried within the protein structure, become solvent exposed
 during denaturation in a temperature ramp and react with
 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) to form
 fluorescent-adducts. A CPM working solution was prepared by diluting the
 CPM stock (5 mg/ml in dimethyl sulfoxide) 50-fold into assay buffer (20 mM
 Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% (w/v) LMNG, 0.1 mg/ml TOCL) and
 incubated for 10 min at room temperature. In each analysis, performed in
 duplicates or triplicates, as indicated, 3 μg of purified MPC was diluted
 into assay buffer containing the desired concentration of inhibitors,
 incubated for 10 min on ice and then 5 μL of the CPM working solution were
 added. Samples were incubated on ice for a further 10 min and then
 subjected to a temperature ramp of 5.6 °C/min. The fluorescence increase
 was monitored with the HRM channel of the instrument (excitation at
 440–480 nm, emission at 505–515 nm). Unfolding profiles were analyzed with
 the Rotor-Gene Q software 2.3 and the peaks of their derivatives were used
 to determine the apparent melting temperature. The
 second method to assess ligand binding was dye-free nano differential
 scanning fluorimetry (nano-DSF), which monitors fluorescence changes due
 to altered environments of tryptophan and tyrosine residues during
 unfolding. Protein samples in buffer containing 20 mM Tris-HCl pH 7.4, 150
 mM NaCl, 0.1% (w/v) LMNG, 0.1 mg/mL TOCL, in the presence or absence of
 the indicated concentrations of small molecule inhibitors, were loaded
 into capillary tubes, and a temperature ramp of 5 °C/min was applied. The
 intrinsic fluorescence was measured using the NanoTemper Prometheus NT.48
 (NanoTemper, München, Germany). 3.
 Pyruvate transport assays  Transport was
 initiated at room temperature by diluting proteoliposomes 200-fold into
 external buffer, 20 mM MES, pH 6.4, 50 mM NaCl, containing 50 μM
 [<sup>14</sup>C]-pyruvate (500,000 dpm, Perkin Elmer, MA,
 USA). The reaction (0–30 s) was terminated by rapid dilution into 8
 volumes of ice-cold buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl), internal
 to proteoliposomes, followed by rapid filtration through cellulose nitrate
 0.45 μm filters (Millipore, Gillingham, UK) and washing with an additional
 8 volumes of buffer. The filters were dissolved in Ultima Gold
 scintillation liquid (Perkin Elmer, MA, USA) and the radioactivity was
 counted with a Perkin Elmer Tri-Carb 2800 RT liquid scintillation counter.
 For inhibition of pyruvate transport, various concentrations of compounds
 were added to the liposomes simultaneously with 50 μM radioactive
 substrate. The data analysis was performed with non-linear regression
 fittings using GraphPad Prism 7.0d ([Inhibitor] vs response, variable
 slope). The specific uptake rates were calculated based on the amount of
 protein used in reconstitutions, as estimated from bicinchoninic acid
 assay. The biological repeats represent independent proteoliposome
 preparations using protein from independent purifications.