MDBNP may serve as a lead compound to identify further potent and selective inhibitors, and the structure information obtained in the study could be useful for optimizing drugs em in silico /em . ? Highlights ANT4 is a germ cell specific ATP transporter in mitochondria ANT4 is required Evista (Raloxifene HCl) for spermatogenesis and male fertility ANT4 has uniquely conserved structure which is different from somatic isoforms Molecular docking recognized a compound inhibiting ANT4 with modest selectivity The study provides a basis for developing novel male contraceptives Acknowledgments The authors thank Drs. ANT4 activity over the other ANTs. We used a structure-based method in which ANT4 was modeled then utilized as the basis for selection of compounds that interact with sites unique to ANT4. A large chemical library ( 100,000 small molecules) was screened by molecular docking and effects of these compounds on ADP/ATP exchange through ANT4 were examined using yeast mitochondria expressing human ANT4. Through this, we recognized one particular candidate compound, [2,2-methanediylbis(4-nitrophenol)], Evista (Raloxifene HCl) which inhibits ANT4 activity with a lower IC50 than the other ANTs (5.8 M, 4.1 M, 5.1 M and 1.4 M for ANT1, 2, 3 and 4). This newly identified active lead compound and its chemical structure are expected to Rabbit polyclonal to PAK1 provide new opportunities to optimize selective ANT4 inhibitors for contraceptive purposes. screening for the most favorable intermolecular conversation with this unique ANT4 binding pocket, and then subjected to a biological screening for inhibitory activity on ADP/ATP exchange of human ANT4 expressed in yeast mitochondria. Further, the specificity in ANT4 inhibition was tested by comparing the effect on other human ANTs. MATERIALS AND METHODS Molecular Docking We utilized SWISS-MODEL, an Automated Comparative Protein Modeling Server, to generate an atomic model of human ANT4 based on the most comparable solved structure, bovine Ant1/Adt1 (PDB 1okc)24. We utilized DOCK6.1 (UCSF) to carry out molecular docking simulations19; 22. We used ZINC15 to download the coordinates for 139,735 compounds representing the NCI plated 2007 molecules set, pH 6C8. The small molecules were docked into a structural feature of the modeled human ANT4 protein selected by the program SPHGEN_CPP, a altered form of the DOCK suite program Sphgen that permits input of large proteins (http://dock.compbio.ucsf.edu/Contributed_Code/sphgen_cpp.htm). We selected a structural pocket for molecular docking that contains an isoform specific amino acid at position 152 (Arg for ANT4). Spheres within 8 ? were selected for molecular docking. The scoring grid was set to extend 5 ? beyond the selected spheres. Each small molecule was positioned in Evista (Raloxifene HCl) the selected site in 1,000 orientations. DOCK6.1 was set to score two types of interactions: van der Waals contacts (nonpolar interactions) and electrostatic interactions (polar interactions). These grid-based scores were summed (for each compound) to generate overall energy scores. The scores were used to rank the selected compounds for functional screening. Distances were measured using Coot6. Chemical Compounds The compounds recognized through molecular docking were acquired from your NCI/DTP. The compounds were dissolved and diluted in DMSO for use in the ADP/ATP exchange assays. Atractyloside (ATR) was obtained by Sigma and dissolved in distilled water. Yeast Strains and Media Endogenous ANT (AAC) genes of were replaced with human ANT1, ANT2, ANT3 or ANT4 as we explained previously 11. For ANT4, an additional mutation (A30V) was required for successful expression in yeast mitochondria as explained. The strains used in this study were: yNhANT1 (ANT1, 2, 3 & 4, cow (mouse Ant4 and yeast (molecular docking and was identified as disulfoacetic acid. Disulfoacetic acid failed to completely dissolve in DMSO. A dose response analysis with six different concentrations of the compound, ranging from 5 10?10 M to 5 10?5 M, is shown in Fig. 4A with an IC50 of 3.5 M. However, the dose response curve showed an atypical artificial inhibition pattern likely caused by aggregation of the small molecule compound. Therefore, further analysis of this disulfoacetic acid was not pursued. Open in a separate window Physique 4 The dose response of disulfoacetic acid (A) and MDBNP (B). The effects of the compounds around the ADP/ATP exchange through human ANT4 were examined at the range of 5 10?10 M to 5 10?5 M. The red-dot collection indicates the concentration of 50% inhibition (IC50). In contrast, the dose response curve for compound #8 (NSC25062) showed a typical inhibition curve demonstrated in Fig. 4B with an IC50 of 0.62 M. Compound #8 was ranked 21 out of the 41 highest-scoring compounds and was identified as 2,2-methanediylbis(4-nitrophenol) (MDBNP), also known as bis(2-hydroxy-5-nitro-phenyl)methane. It has a chemical formula of C13H10N2O6 and a molecular excess weight of 290 g/mol. The chemical structure is shown in Fig. 5. The predicted interactions of MDBNP with ANT4 are depicted in Fig. 1A, B, and C. It should be noted that this IC50 value of MDBNP on ANT4 inhibition is usually more than 10 fold higher when compared to the known potent ANT inhibitor atractyloside (ATR) (IC50:.