Inhibitor of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. late-stage preclinical development or human clinical screening as novel malignancy therapeutics (Nikolovska-Coleska et al., 2004, 2008; Sun et al., 2007; Lu et al., 2008; Flygare et al., 2012; buy 142998-47-8 Peng et al., 2012). For example, Birinapant (TL32711; TetraLogic Pharmaceuticals, Malvern, PA), now in Phase II study, can effectively suppress cIAP1 and XIAP at well-tolerated doses and promises antitumor activity either as a single agent or in combination with standard-of-care chemotherapeutic drugs in adult patients with advanced solid tumors or lymphoma (Krepler et al., 2013). However, no IAP inhibitors have been approved by the U.S. Food and Drug Administration as of today, and there are limitations with many existing IAP inhibitors. For example, YM155 is usually a well-known survivin inhibitor that has gone through clinical trials, but it has been shown to be a substrate for the P-glycoprotein (P-gp) drug efflux pump (Iwai et al., 2011), suggesting that it could suffer from multidrug resistance (MDR) in its eventual clinical use. Thus, exploring novel scaffolds to develop potent buy 142998-47-8 and selective IAP antagonists is usually still much needed. Because all IAP proteins share the signature baculoviral IAP repeat (BIR) domain name (Fulda and Vucic, 2012), which interacts with SMAC, shape-based virtual testing will be helpful in identifying potential small-molecule SMAC mimetics for regulating apoptosis in malignancy cells. In this statement, we describe our efforts to identify novel small-molecule SMAC mimetics through an integrated virtual testing and biologic affirmation approach. Their efficiency in inhibiting IAPs, especially XIAP and survivin (BIRC5), and inducing apoptosis in malignancy cells was further validated in serial biologic studies both in vitro and in vivo. These compounds represent novel scaffolds for IAP inhibition and can be further optimized to serve as a potential targeted agent for numerous types of cancers. Materials and Methods Shape-Based Virtual Screening. The University or college of Cincinnatis Drug Finding Center Library (contains 362,910 compounds) was used to conduct the shape-based virtual buy 142998-47-8 screening. All structures were first prepared using the LigPrep module in Maestro Collection 2012 (Schrodinger, LLC, New York, NY) to generate conformers and charged says. We used the phase_shape program in Canvas (version 1.4; Schrodinger, LLC). Conformers with a shape similarity below 0.7 were filtered out, and hits with a similarity value above this threshold were selected for subsequent molecular docking process. Molecule Docking. Crystal structures of SMAC bound to XIAP BIR3 domain name [Protein Data Lender codes 1G73 (Wu et al., 2000) and 1TW6 (Vucic et al., 2005)] were processed with the Protein Preparation Wizard, and the grid of AVPI binding site was defined by Glide (version 5.7; Schrodinger, LLC). One thousand hits with top-ranked similarity value were docked into the AVPI binding site in each individual complex. The best docking complexes were subject to restricted molecular mechanics to release any stresses by using the Macromodel module with OPLS-2005 force-field. The ligand and its surrounding residues within 15 ? were allowed to move freely, whereas residues outside the 15-? radius were kept rigid. Cell Culture and Reagents. Human melanoma A375 cell collection and buy 142998-47-8 human prostate PC-3 and DU145 cell lines were acquired from American Type Culture Collection (Manassas, VA). Human melanoma M14 cell collection was kindly provided by Dr. Robert Clarke (Georgetown University or college, Washington, DC). Human keratinocyte Hacat cell collection was a gift from Dr. Andrzej T. Slominski (University or college of Tennessee Health Science Center, Memphis, TN). Human dermal fibroblast adult cells (HDFa) cells were purchased from Life Technologies (Thermo Fisher Scientific Inc., Waltham, MA). All cell lines were authenticated prior to use for this study. Malignancy cells were cultured CCNA1 in Dulbeccos altered Eagles medium (DMEM; for melanoma cells) or RPMI 1640 (for prostate malignancy cells) medium (Mediatech, Inc., Manassas, VA), supplemented by 10% fetal bovine serum (FBS, Metro atlanta Biologicals, Lawrenceville, GA), 1% antibiotic/antimycotic combination (Sigma-Aldrich, St. Louis, MO), and 5 = 3) were synchronized through 24-hour buy 142998-47-8 starvation in growth media made up of only 0.1% FBS. The cells then were treated with 0, 1, or 4 = 3) decided the estimated MTD to be above 200 mg/kg with 1-week continuous treatment. To make sure a large security margin during the 3-week treatment and considering the practical doses in clinical, we scaled down the dose to 20 and 40 mg/kg in the xenograft model study. Seven- to eight-week-old male nude mice were purchased from Charles Water Laboratories World, Inc. (Wilmington, MA). Right before use, A375.