Novel ligands that target the mitochondrial membrane protein mitoNEET

https://doi.org/10.1016/j.jmgm.2011.04.001Get rights and content

Abstract

Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET.

Highlights

► Determination of mitoNEET ligand binding sites. ► Discovery of novel ligands that target mitoNEET. ► In vitro fluorescence binding assay confirmed mitoNEET ligand binding.

Section snippets

Materials and methods

For the molecular docking study, protein structures were obtained from the RCSB Protein Data Bank (http://www.rcsb.org); the mitoNEET structure PDB ID was 2QD0, and the PPAR-γ PDB ID was 1ZGY. Ligand structures were obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov). If ligand structures were not available from PubChem, they were drawn using MarvinSketch 5.3.1 (ChemAxon). The UCSF Chimera program (http://www.cgl.ucsf.edu/chimera) was used to prepare the structures for input to

Validation of AutoDock Vina blind docking method

Blind docking (BD) was performed on the PPAR-γ receptor (PDB ID: 1ZGY) to validate its accuracy at reproducing the experimental binding mode of rosiglitazone. The results of BD indicate that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (Fig. 2). The root mean square deviation (RMSD) of the heavy atoms is only 1.45 Å (Fig. 2). This result, in addition to previous extensive validation [23], [25], [26], demonstrates the

Summary

Even though compounds 418 do not contain the TZD ring moiety, our molecular docking and in vitro binding studies predict that they bind with similar or greater affinity to mitoNEET, compared with the known TZD ligands, pioglitazone and rosiglitazone (Table 1). For the first time, these results indicate that it is possible to design novel ligands targeted for mitoNEET that are not structurally related to the TZDs, therefore having less of the undesirable side effects and toxicity associated

Acknowledgements

The project described was supported by Award Number R01DK059766 from the National Institute Of Diabetes And Digestive And Kidney Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute Of Diabetes And Digestive And Kidney Diseases or the National Institutes of Health. We wish to gratefully acknowledge Max R. Bieganski for creating graphical content and providing technical support, and Dr. Sandra Wiley,

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