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Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to those already known (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

Cancer cells are dependent on protein quality‐control mechanisms, including protein chaperones, the ubiquitin‐proteasome system, and autophagy. The p62 receptor is a classical, ubiquitously expressed receptor, involved in many signal transduction pathways. Upregulation and/or reduced degradation of p62 have been implicated in tumor formation and resistance to therapy. PTX80 is a first‐in‐class novel inhibitor of protein degradation, developed by Pi Therapeutics for the treatment of cancer. PTX80 binds to p62, inducing a decrease in soluble p62 and formation of insoluble p62 aggregates, and failure of polyubiquitinated proteins to colocalize with p62. PTX80 induces proteotoxic stress and activation of unfolded protein response, which, in turn, leads to apoptosis. Targeting p62, which is a major protein degradation hub, may serve as a novel and beneficial strategy for the treatment of cancer. [ABSTRACT FROM AUTHOR]

Background: Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330.Methods: Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays.Results: Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in >100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental and resistant cells, the extent of response was more robust in the parental cells.Conclusions: These results suggest that SINE resistance is conferred by alterations in signaling pathways downstream of XPO1 inhibition. Modulation of these pathways could potentially overcome the resistance to nuclear export inhibitors. [ABSTRACT FROM AUTHOR]

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets. Proteins 2004. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact withβ-strands S3/S9/S10, consistent with docking simulations. Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics. [ABSTRACT FROM AUTHOR]

Chromosome region maintenance 1/Exportin-1 (CRM1) is a key nuclear export protein whose inhibition leads to nuclear accumulation of tumor suppressor (TSP) and growth regulatory proteins (GRP). Through CRM1 inhibition, nuclear localization of these proteins restores cell cycle checkpoints and genome surveying functions culminating in apoptosis of tumor cells. Conversely, CRM1 inhibition of normal cells induces reversible cell cycle arrest. We developed a structural model of CRM1 suited for binding diverse small molecules and used it to guide the discovery and development of KPT-SINE (selective inhibitors of nuclear export). The model predicted that a switching motion of Met545 and Met583 in CRM1 was required for inhibitor accommodation. We found that published CRM1 inhibitors display efficient mimicry of cargo Nuclear Export Signals (NES) and a thiol reactive warhead proximal to Cys528 in CRM1. This was further confirmed by Yuh Min Chook who solved the X-ray structures of CRM1. In silico screening yielded 402 hits (from a library of ~250,000 drug-like compounds) with 19 molecules active in vitro (IC50<30μM) and guided the optimization of KPT-SINE. Lead KPT-SINE irreversibly bound and inhibited CRM1 nuclear export and were cytotoxic to a broad range of tumor cells and xenografts. Through comparisons of molecular and cellular markers in both cancer and normal cells we concluded that KPT-SINE treatment ultimately induced cancer cell death. Methods: CRM1 Model: The CRM1 model was developed using the novel consensus Induced Fit Docking approach (cIFD). In vitro/vivo evaluation: Compounds were tested in a cell-based microscopy assay to confirm CRM1-mediated nuclear export inhibition. Cells transfected with mutant CRM1-ys528Ser were treated with KPT-SINE in order to determine covalent binding to Cys528. Cytotoxic IC50s of KPT-SINE were determined on a panel of cancer cell lines. Multiple KPT-SINE derivatives were tested for: 1) Nuclear localization of TSPs, 2) Effects on cell cycle and viability, 3) Effects on TSP protein expression, and 4) Mouse xenograft model efficacy. Results: KPT-SINE, potent inhibitors in the cell-based microscopy assay (EC50=20-130nM), were robust inhibitors of cancer cell proliferation and inducers of apoptosis. KPT-SINE inhibition of CRM1 started within 30 minutes and reached a maximum in 8 hrs. Additionally, KPT-SINE effects were blocked by transient transfection of mutant CRM1-Cys528Ser, supporting the role of Cys528. We also identified nuclear accumulation of p53, FOXO1a, FOXO3a, APC, IκB, p27, PP2Aα, and p21, which was followed by cell cycle arrest and death of cancer cells while normal cells undergo a reversible block. Both solid (MDA-MB-468) and hematological (Z-138) mouse xenografts treated with KPT-SINE at 25 mg/kg QoDx3 PO weekly for 4 weeks inhibited tumor growth by more than 70%. Immunohistochemical analysis of the tumor molecular markers from KPT-SINE treated animals exhibited nuclear localization of TSPs, a decrease in proliferation (Ki-67), and an increase in apoptosis (TUNEL). Conclusions: We described the correlative effects of KPT-SINE from the accumulation of nuclear TSPs at the molecular level through to potent in vivo efficacy in xenograft models. KPT-SINE, which were discovered and optimized using a structure-based approach, are due for Phase I clinical trials Mid-2012. [ABSTRACT FROM AUTHOR]

We present a fast and effective covalent docking approach suitable for large-scale virtual screening (VS). We applied this method to four targets (HCV NS3 protease, Cathepsin K, EGFR, and XPO1) with known crystal structures and known covalent inhibitors. We implemented a customized "VS mode" of the Schrödinger Covalent Docking algorithm (CovDock), which we refer to as CovDock-VS. Known actives and target-specific sets of decoys were docked to selected X-ray structures, and poses were filtered based on noncovalent protein-ligand interactions known to be important for activity. We were able to retrieve 71%, 72%, and 77% of the known actives for Cathepsin K, HCV NS3 protease, and EGFR within 5% of the decoy library, respectively. With the more challenging XPO1 target, where no specific interactions with the protein could be used for postprocessing of the docking results, we were able to retrieve 95% of the actives within 30% of the decoy library and achieved an early enrichment factor (EF1%) of 33. The poses of the known actives bound to existing crystal structures of 4 targets were predicted with an average RMSD of 1.9 Å. To the best of our knowledge, CovDock-VS is the first fully automated tool for efficient virtual screening of covalent inhibitors. Importantly, CovDock-VS can handle multiple chemical reactions within the same library, only requiring a generic SMARTS-based predefinition of the reaction. CovDock-VS provides a fast and accurate way of differentiating actives from decoys without significantly deteriorating the accuracy of the predicted poses for covalent protein-ligand complexes. Therefore, we propose CovDock-VS as an efficient structure-based virtual screening method for discovery of novel and diverse covalent ligands.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter, functioning as a chloride channel critical for fluid homeostasis in multiple organs. Disruption of CFTR function is associated with cystic fibrosis making it an attractive therapeutic target. In addition, CFTR blockers are being developed as potential antidiarrheals. CFTR drug discovery is hampered by the lack of high resolution structural data, and considerable efforts have been invested in modeling the channel structure. Although previously published CFTR models that have been made publicly available mostly agree with experimental data relating to the overall structure, they present the channel in an outward-facing conformation that does not agree with expected properties of a "channel-like" structure. Here, we make available a model of CFTR in such a "channel-like" conformation, derived by a unique modeling approach combining restrained homology modeling and ROSETTA refinement. In contrast to others, the present model is in agreement with expected channel properties such as pore shape, dimensions, solvent accessibility, and experimentally derived distances. We have used the model to explore the interaction of open channel blockers within the pore, revealing a common binding mode and ionic interaction with K95, in agreement with experimental data. The binding-site was further validated using a virtual screening enrichment experiment, suggesting the model might be suitable for drug discovery. In addition, we subjected the model to a molecular dynamics simulation, revealing previously unaddressed salt-bridge interactions that may be important for structure stability and pore-lining residues that may take part in Cl(-) conductance.

P2Y nucleotide receptors (P2Y-Rs) play important physiological roles. However, most of the P2Y-R subtypes are still lacking potent and selective agonists and antagonists. Based on data mining analysis of binding interactions in 44 protein-uridine nucleos(t)ides complexes, we designed uracil nucleotides, substituted at the C5/C6 position. All C6-substituted derivatives were inactive at the P2Y(2,4,6)-Rs, while out of the C5-substituted analogues, only 5-OMe-UD(T)P showed activity. To rationalize the data, the ionization and conformation of these analogues were evaluated. The pK(a) values of most analogues substituted at the C5/C6 positions were unaltered compared to UTP (pK(a) 9.42), except for 5-F-UTP nucleotide (pK(a) 7.85). C6-substituted analogues adopt the syn or high-syn conformations, which are disfavored by the receptors, while 5-OMe-UD(T)P adopt the favored anti conformation. Furthermore, 5-OMe-UDP adopts the S sugar puckering, which is the conformation preferred by the P2Y(6)-R, but not the P2Y(2)- or P2Y(4)-Rs. 5-OMe-UDP fulfills the conformational and H-bonding requirements of P2Y(6)-R, thus, making a potent P2Y(6)-R agonist (EC(50) 0.08 microM), more than UDP (EC(50) 0.14 microM).

MDM2 is a key regulator of the p53 tumor-suppressor protein. Here we study the effect of modifications of a p53 N-terminal fragment on its binding to MDM2, using implicit-solvent MD and MM-GB/SA calculations. We provide interpretation of existing experimental data and predict the effect of mutations on binding. Notably 1) We analyze the effect of regulatory phosphorylations at Ser/Thr residues and suggest that a balance between favorable electrostatics and desolvation penalties determines the effect of phosphorylation; 2) We compare the helical stability in solution of p53 alanine mutants and propose a helix stabilizing role for several residues involved in hydrogen bonding and hydrophobic packing; 3) We obtain good correlations between calculated and experimental affinities for a set of peptidomimetic inhibitors, both alone and in combination with p53 analogues, demonstrating potential applicability to drug design. From the technical aspect, protocol optimization and selection of simulation tools are addressed in detail. To the best of our knowledge this is the first published example of MM-GB/SA calculations utilizing a conformational ensemble generated with implicit solvent MD. Our results suggest that this highly efficient variant of classical explicit-solvent MM-GB/SA may be used for studying protein-protein interactions and for the design of peptidomimetic drugs.