Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase.

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.

MitoNEET's Reactivity of Lys55 toward Pyridoxal Phosphate Demonstrates its Activity as a Transaminase Enzyme.

MitoNEET is a [2Fe-2S] redox active mitochondrial protein belonging to the CDGSH iron-sulfur domain (CISD) family of proteins. MitoNEET has been implicated as a potential target for drug development to treat various disorders, including type-2 diabetes, cancer, and Parkinson's disease. However, the specific cellular function(s) for mitoNEET still remains to be fully elucidated, and this presents a significant roadblock in rational drug development. Here, we show that mitoNEET binds the enzymatic cofactor pyridoxal phosphate (PLP) specifically at only one of its 11 lysine residues, Lys55. Lys55 is part of the soluble portion of the protein and is in a hydrogen-bonding network with the histidine residue that ligates the [2Fe-2S] cluster. In the presence of mitoNEET, PLP catalyzes the transamination reaction of the amino acid cysteine and the alpha-keto acid 2-oxoglutarate to form 3-mercaptopyruvate and glutamate. This work identifies, for the first time, mitoNEET as an enzyme with cysteine transaminase activity.

Binding of thiazolidinediones to the endoplasmic reticulum protein nutrient-deprivation autophagy factor-1.

Nutrient-deprivation autophagy factor-1 (NAF-1, miner1; gene cisd2) is part of the [2Fe-2S]-containing protein family which includes mitoNEET (gene cisd1) and MiNT (miner2; gene cisd3). These proteins are redox active and are thought to play an important role in cellular energy homeostasis with NAF-1 playing a critical role in calcium regulation and aging. To date, no studies have investigated potential ligand interaction with NAF-1. Here we show that the thiazolidinediones pioglitazone and rosiglitazone along with the mitoNEET ligand, NL-1, bind to NAF-1 with low micromolar affinities. Further, we show that overexpression of NAF-1 in hepatocellular carcinoma (HepG2) cells reduces inhibition of mitochondrial respiration by pioglitazone. Our findings support the need for further efforts of the rational design of selective NAF-1 ligands.

Crystal structure of the mitochondrial protein mitoNEET bound to a benze-sulfonide ligand.

MitoNEET (gene cisd1) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

Modulation of cellular energetics by galactose and pioglitazone.

The Warburg effect is ameliorated by culturing transformed cells in the presence of galactose instead of glucose as the primary carbon source. However, metabolic consequences may occur in addition to sensitizing the cells to mitochondrial toxins. The screening of pharmaceutical agents against transformed cells while using galactose must therefore be carefully evaluated. Pioglitazone is employed in clinical applications to treat type-2 diabetes but clearly has other off-target effects. Human hepatocellular carcinoma cells (HepG2) were cultured in glucose or galactose-containing medium to investigate the role of pioglitazone on cellular bioenergetics by calorimetry and respirometry. Compared with cells cultured in 10 mM glucose, HepG2 cells cultured in the presence of 10 mM galactose showed decreased metabolic activity as measured by cellular heat flow. Interestingly, cellular heat flow increased after the addition of pioglitazone for cells cultured in glucose, but not for cells cultured in galactose. Our calorimetric data indicated that a reduction in cellular capacity for glycolysis was the mechanism responsible for the increase in sensitivity to pioglitazone, and possibly to mitochondrial toxins in general, for cells cultured in galactose. Furthermore, oxygen consumption rates were decreased after the addition of pioglitazone to cells grown in glucose but remained unchanged for cells grown in the presence of galactose. We have demonstrated that pioglitazone induces a reduction in mitochondrial activity that is partially compensated via an increase in glycolysis in the presence of glucose.

Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics.

The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings.

The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster.

Rieske and Rieske-type proteins are electron transport proteins involved in key biological processes such as respiration, photosynthesis, and detoxification. They have a [2Fe-2S] cluster ligated by two cysteines and two histidines. A series of mutations, L135E, L135R, L135A, and Y158F, of the Rieske protein from Thermus thermophilus has been produced which probe the effects of the neighboring residues, in the second sphere, on the dynamics of cluster reduction and the reactivity of the ligating histidines. These properties were probed using titrations and modifications with diethyl pyrocarbonate (DEPC) at various pH values monitored using UV-Visible and circular dichroism spectrophotometry. These results, along with results from EPR studies, provide information on ligating histidine modification and rate of reduction of each of the mutant proteins. L135R, L135A, and Y158F react with DEPC similarly to wild type, resulting in modified protein with a reduced [2Fe-2S] cluster in <90 min, whereas L135E requires >15 h under the same conditions. Thus, the negative charge slows down the rate of reduction and provides an explanation as to why negatively charged residues are rarely, if ever, found in the equivalent position of other Rieske and Rieske-type proteins.

Identification of disulfide bond formation between MitoNEET and glutamate dehydrogenase 1.

MitoNEET is a protein that was identified as a drug target for diabetes, but its cellular function as well as its role in diabetes remains elusive. Protein pull-down experiments identified glutamate dehydrogenase 1 (GDH1) as a potential binding partner. GDH1 is a key metabolic enzyme with emerging roles in insulin regulation. MitoNEET forms a covalent complex with GDH1 through disulfide bond formation and acts as an activator. Proteomic analysis identified the specific cysteine residues that participate in the disulfide bond. This is the first report that effectively links mitoNEET to activation of the insulin regulator GDH1.

Chemical modification of the Rieske protein from Thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2FE-2S] cluster.

Rieske proteins are a class of electron transport proteins that are intricately involved in respiratory and photosynthetic processes. One unique property of Rieske proteins is that the reduction potential is pH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the two histidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibility of the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, the Thermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range of pH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies and the end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidines and surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was also found to reduce the metal cluster. The ability to control the redox state was examined by the addition of oxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterization of the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects of small molecules that inhibit the function of the bc(1) complex and that have also been shown to interact with the ligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.

Effects of pH on the Rieske protein from Thermus thermophilus: a spectroscopic and structural analysis.

The Rieske protein from Thermus thermophilus (TtRp) and a truncated version of the protein (truncTtRp), produced to achieve a low-pH crystallization condition, have been characterized using UV-visible and circular dichroism spectroscopies. TtRp and truncTtRp undergo a change in the UV-visible spectra with increasing pH. The LMCT band at 458 nm shifts to 436 nm and increases in intensity. The increase at 436 nm versus pH can be fit using the sum of two Henderson-Hasselbalch equations, yielding two pK(a) values for the oxidized protein. For TtRp, pK(ox1) = 7.48 +/- 0.12 and pK(ox2) = 10.07 +/- 0.17. For truncTtRp, pK(ox1) = 7.87 +/- 0.17 and pK(ox2) = 9.84 +/- 0.42. The shift to shorter wavelength and the increase in intensity for the LMCT band with increasing pH are consistent with deprotonation of the histidine ligands. A pH titration of truncTtRp monitored by circular dichroism also showed pH-dependent changes at 315 and 340 nm. At 340 nm, the fit gives pK(ox1) = 7.14 +/- 0.26 and pK(ox2) = 9.32 +/- 0.36. The change at 315 nm is best fit for a single deprotonation event, giving pK(ox1) = 7.82 +/- 0.10. The lower wavelength region of the CD spectra was unaffected by pH, indicating that the overall fold of the protein remains unchanged, which is consistent with crystallographic results of truncTtRp. The structure of truncTtRp crystallized at pH 6.2 is very similar to TtRp at pH 8.5 and contains only subtle changes localized at the [2Fe-2S] cluster. These titration and structural results further elucidate the histidine ligand characteristics and are consistent with important roles for these amino acids.

Indomethacin amides as a novel molecular scaffold for targeting Trypanosoma cruzi sterol 14alpha-demethylase.

Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14alpha-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51 inhibitors has been identified using optical high throughput screening followed by web-database search for similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-amides were found to bind to TCCYP51, inhibit its activity in vitro, and produce strong antiparasitic effects in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds is by inhibition of sterol biosynthesis in the parasite.