Novel ligands that target the mitochondrial membrane protein mitoNEET

MitoNEET is a recently discovered iron–sulfur (2Fe–2S) outer mitochondrial integral membrane protein that binds the same ligands, specifically pioglitazone [1,2] and rosiglitazone [3], as the peroxisome proliferator-activated receptor gamma (PPAR-γ) nuclear receptor, and with similar binding affinity. Pioglitazone (1) and rosiglitazone (2) are members of the thiazolidinedione (TZD) class of compounds (Fig. 1), currently approved for treatment of type 2 diabetes. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of mitoNEET through a cross-linking study with a photoactive form of pioglitazone [1]. Moreover, accumulating evidence indicates that many of the clinical effects of TZDs may originate by binding to mitoNEET. A recent report [4] suggests that mitoNEET plays a key role in regulating electron transport and oxidative phosphorylation, as there is a decrease in the complex I-dependent oxygen consumption in the mitochondria isolated from mitoNEET-deficient mice heart. Fig. 1 Structures of 15 compounds predicted to bind to mitoNEET, as well as the structures of pioglitazone (1) and rosiglitazone (2). Pioglitazone was also reported to alter the oxidative capacity of mitochondria and exhibit negative regulatory effect on complex I activity in liver, muscle, and astroglioma cells [5,6], with positive regulatory effect in neuronal cells [7]. These correlations together with the mitochondrial localization of mitoNEET, strongly suggest that pioglitazone (and other TZDs) may exert their actions on mitochondria by regulating activities of the respiratory complexes via interaction with mitoNEET through yet unknown mechanism(s). In other studies, pioglitazone has been shown as a potential treatment for several neurodegenerative diseases, particularly Alzheimer disease (AD) [8], amyotrophic lateral sclerosis (ALS) [9,10], multiple sclerosis (MS) [11–13], and Parkinson’s disease (PD) [14]. Moreover, pioglitazone was shown to prevent LPS-induced activation of microglia, increased oxidative stress, and impaired mitochondrial function, all of which are specific in dopaminergic neurodegeneration [15,16], implicated in PD [14]. Published evidence indicates that increased oxidative stress [17] is implicated in PD, suggesting that pioglitazone may have anti-inflammatory and anti-oxidative properties. In addition, mitoNEET is likely to play a vital role in mitochondrial energy homeostasis and metabolism [4], transporting and sequestering iron, electron transfer, and oxidation of fatty acids [1]. Pioglitazone binding to mitoNEET was also found to stabilize the mitoNEET dimer formation, which correlates with decreased oxidative stress [18]. The fact that PPAR-γ ligands bind to mitoNEET suggests an alternate mode of action for these ligands, with the possibility of rationally designing novel targeted agents that bind to mitoNEET, and are structurally distinct from the TZD class of compounds, that have undesirable side effects and toxicity [19]. Currently, a major challenge is to understand the nature of TZD-mitoNEET binding at a much higher level of detail [3]. However, neither quantitative determination of association constants for TZD-mitoNEET binding, nor the precise location of the mitoNEET ligand binding site(s) (LBS) have yet been determined, in part because of the high insolubility of the TZD class of molecules in aqueous solutions, requiring drug solubilization with relatively high concentrations of DMSO, which denatures the structure of mitoNEET [3]. The present lack of structurally diverse and water-soluble ligands known to bind to mitoNEET (besides the TZDs) may likely be a key contributing factor for the absence of any X-ray and/or NMR structures reported to date that provide high resolution structure of mitoNEET complexed with a ligand, despite several reported X-ray crystal structures of the unbound mitoNEET (apo) structure [2,20,21]. Thus, the nature of the mitoNEET LBS remains unknown. Our discovery of potential mitoNEET ligands, with good water solubility and high binding affinity (e.g., molecules 5 and 11, Fig. 1) holds great promise for future high-resolution X-ray and NMR structural studies of the mitoNEET. The LBS reported in our study is roughly comparable to the major LBS reported in two previous publications [20,22]. In the more recent report [22], molecular docking software employing the SiteFinder module in MOE 2008.10 (Chemical Computing Group) found five potential binding pockets on mitoNEET, by probing the surface of the protein with alpha-spheres [22]. In contrast, AutoDock Vina [23] docks a flexible ligand against the protein using a sophisticated gradient optimization method [23]. The study also used a different crystal structure (PDB ID: 2QH7, personal communications), compared to the one we used (2QD0). The residues in one of the major binding sites identified [22] seem to agree with the rough location of a binding pocket that was first suggested in an earlier study [20], where the authors used AutoDock 3 (personal communications), but the same crystal structure [20] (2QD0) used in our study. 2QD0 has five additional residues on the N-terminus of chain A, while 2QH7 possesses additional lysine and glutamic acid residues on the N-terminus and C-terminus of chains A and B, respectively. Molecular docking is a powerful computational technique used in structure-based drug design. The availability of reliable structures of the target receptor is crucial for docking. Multiple receptor conformations can be used to incorporate receptor flexibility into the docking algorithm, to improve the accuracy of binding pose prediction. A recent computational study employing “ensemble docking” into multiple crystallographically derived protein structures [24], provides a good example of this approach. We are planning to include receptor flexibility in future studies. As there are no published structures of mitoNEET complexed with a ligand, we have used a technique called blind docking (BD) [25,26] to determine the potential binding site(s). BD is used for detecting possible binding sites and ligand conformations by scanning the entire surface of a protein target, and finding a location with the highest binding affinity on the protein. Based on previous studies [25,26], BD has been shown to reliably predict the binding location and mode of numerous known protein–ligand complexes, and can be used in the case where the binding site(s) on a protein is unknown. In this study, the program AutoDock Vina (v 1.0.2) [23] was used to perform the blind docking; this program was shown to be more accurate compared to previous versions of AutoDock used in BD studies [25,26]. Known mitoNEET ligands (pioglitazone and rosiglitazone) were screened against the protein surface, and a binding site was found on the surfaces of both mitoNEET protomers. Using the binding site information, a number of potential, structurally diverse novel ligands, unrelated to the TZDs, were discovered. Of these ligands, laetevirenol A (18) and troglitazone (3) were found to have the highest predicted binding affinity, and using an in vitro fluorescence binding assay, determined the binding affinity of a predicted mitoNEET ligand. The discovery of potential mitoNEET binding sites, and novel ligands, opens up the possibility for detailed structural investigation of mitoNEET–ligand complexes, as well as rational design of structurally diverse novel ligands specifically targeted for mitoNEET, and with fewer side effects and toxicity compared to the TZDs [19].