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Recently we reported that rapid determination of enthalpy of binding can be achieved for a large number of congeneric agents or in combinatorial libraries fairly efficiently. We show that using a thermodynamic Craig plot can be very useful in dissecting the enthalpy and entropy contribution of different substituents on a common scaffold, in order to design potent, selective, or pan-active compounds. In our implementation, the approach identified a critical Lys residue in the BIR3 domain of XIAP. We report for the first time that it is possible to target such residue covalently to attain potent and selective agents. Preliminary cellular studies in various models of leukemia, multiple myeloma, and pancreatic cancers suggest that the derived agents possess a potentially intriguing pattern of activity, especially for cell lines that are resistant to the pan-IAP antagonist and clinical candidate LCL161.

Industrial activities play a key role in the economic well-being of a country but they usually involve processes with a more or less profound environmental impact, including emission of pollutants. Among them, much attention has been given to airborne particulate matter (PM) whose exposure is ubiquitous and linked with several adverse health effects mainly due to its size and chemical composition. Therefore, there is a strong need to exploit monitoring systems for airborne PM able to provide accurate information on the potential health hazards and the specific emission sources for the implementation of adequate control strategies. The honey bee ( Apis mellifera , L.) is widely used as an indicator of environmental pollution: this social hymenopteran strongly interacts with vegetables, air, soil, and water surrounding the hive and, as a consequence, pollutants from these sources are translated to the insect and to the hive products. During the wide-ranging foraging activity, the forager bee is known to collect samples of the main airborne PM pollutants emitted from different sources and therefore it can be used as an efficient PM sampler. In the present research, PM contaminating forager bees living nearby a cement factory and several kilometers away from it has been analysed and characterised morphologically, dimensionally and chemically through SEM/EDX. This provided detailed information on the role of both the cement manufacturing activities and the vehicular traffic as sources of airborne PM. This may indeed help the implementation of appropriate preventive and corrective actions that would effectively minimize the environmental spread of pollutant PM not only in areas close to the plant, but also in more distant areas., Competing Interests: Ilaria Negri is an Academic Editor for PeerJ. Marco Pellecchia is an employee of Koiné—Environmental Consultancy, Montechiarugolo (Parma), Italy.

EphA2 overexpression has been associated with metastasis in multiple cancer types, including melanomas and ovarian, prostate, lung, and breast cancers. We have recently proposed the development of peptide-drug conjugates (PDCs) using agonistic EphA2-targeting agents, such as the YSA peptide or its optimized version, 123B9. Although our studies indicated that YSA- and 123B9-drug conjugates can selectively deliver cytotoxic drugs to cancer cells in vivo, the relatively low cellular agonistic activities (i.e., the high micromolar concentrations required) of the agents toward the EphA2 receptor remained a limiting factor to the further development of these PDCs in the clinic. Here, we report that a dimeric version of 123B9 can induce receptor activation at nanomolar concentrations. Furthermore, we demonstrated that the conjugation of dimeric 123B9 with paclitaxel is very effective at targeting circulating tumor cells and inhibiting lung metastasis in breast-cancer models. These studies represent an important step toward the development of effective EphA2-targeting PDCs.

We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme., Competing Interests: The authors declare no competing financial interest.

Cancer is a daunting global problem confronting the world's population. The most frequent therapeutic approaches include surgery, chemotherapy, radiotherapy, and more recently immunotherapy. In the case of chemotherapy, patients ultimately develop resistance to both single and multiple chemotherapeutic agents, which can culminate in metastatic disease which is a major cause of patient death from solid tumors. Chemoresistance, a primary cause of treatment failure, is attributed to multiple factors including decreased drug accumulation, reduced drug-target interactions, increased populations of cancer stem cells, enhanced autophagy activity, and reduced apoptosis in cancer cells. Reprogramming tumor cells to undergo drug-induced apoptosis provides a promising and powerful strategy for treating resistant and recurrent neoplastic diseases. This can be achieved by downregulating dysregulated antiapoptotic factors or activation of proapoptotic factors in tumor cells. A major target of dysregulation in cancer cells that can occur during chemoresistance involves altered expression of Bcl-2 family members. Bcl-2 antiapoptotic molecules (Bcl-2, Bcl-xL, and Mcl-1) are frequently upregulated in acquired chemoresistant cancer cells, which block drug-induced apoptosis. We presently overview the potential role of Bcl-2 antiapoptotic proteins in the development of cancer chemoresistance and overview the clinical approaches that use Bcl-2 inhibitors to restore cell death in chemoresistant and recurrent tumors., (© 2018 Elsevier Inc. All rights reserved.)

In modern drug discovery, the ability of biophysical methods, including nuclear magnetic resonance spectroscopy or surface plasmon resonance, to detect and characterize ligand-protein interactions accurately and unambiguously makes these approaches preferred versus conventional biochemical high-throughput screening of large collections of compounds. Nonetheless, ligand screening strategies that address simultaneously potency and selectivity have not yet been fully developed. In this work, we propose a novel method for screening large collections of combinatorial libraries using enthalpy measurements as a primary screening technique. We demonstrate that selecting binders that are driven by enthalpy (ΔH) results in agents that are not only potent but also more selective for a given target. This general and novel approach, we termed ΔH screening of fPOS (enthalpy screening of focused positional scanning library), combines the principles of focused combinatorial chemistry with rapid calorimetry measurements to efficiently identify potent and selective inhibitors.

Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown. Employing hRetn-expressing transgenic mice (h RETN Tg + ) and recombinant hRetn, we identify a therapeutic function for hRetn in lipopolysaccharide (LPS)-induced septic shock. hRetn promoted helminth-induced immunomodulation, with increased survival of Nippostrongylus brasiliensis ( Nb )-infected h RETN Tg + mice after a fatal LPS dose compared with naive mice or Nb -infected h RETN Tg - mice. Employing immunoprecipitation assays, h RETN Tg + Tlr4 -/- mice, and human immune cell culture, we demonstrate that hRetn binds the LPS receptor Toll-like receptor 4 (TLR4) through its N terminal and modulates STAT3 and TBK1 signaling, triggering a switch from proinflammatory to anti-inflammatory responses. Further, we generate hRetn N-terminal peptides that are able to block LPS proinflammatory function. Together, our studies identify a critical role for hRetn in blocking LPS function with important clinical significance in helminth-induced immunomodulation and sepsis., Competing Interests: The authors declare no conflict of interest.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Recent studies identified the receptor tyrosine kinase EphA4 as a disease-modifying gene that is critical for the progression of motor neuron degeneration. We report on the design and characterization of a family of EphA4 targeting agents that bind to its ligand binding domain with nanomolar affinity. The molecules exhibit excellent selectivity and display efficacy in a SOD1 mutant mouse model of ALS. Interestingly, the molecules appear to act as agonists for the receptor in certain surrogate cellular assays. While the exact mechanisms responsible for the therapeutic effect of the new agonists remain to be elucidated, we believe that the described agent represents both an invaluable pharmacological tool to further decipher the role of the EphA4 in ALS and potentially other human diseases, and a significant stepping stone for the development of novel treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Upregulation of antiapoptotic Bcl-2 proteins in certain tumors confers cancer cell resistance to chemotherapy or radiations. Members of the antiapoptotic Bcl-2 proteins, including Bcl-2, Mcl-1, Bcl-xL, Bcl-w, and Bfl-1, inhibit apoptosis by selectively binding to conserved α-helical regions, named BH3 domains, of pro-apoptotic proteins such as Bim, tBid, Bad, or NOXA. Five antiapoptotic proteins have been identified that interact with various selectivity with BH3 containing pro-apoptotic counterparts. Cancer cells present various and variable levels of these proteins, making the design of effective apoptosis based therapeutics challenging. Recently, BH3 profiling was introduced as a method to classify cancer cells based on their ability to resist apoptosis following exposure to selected BH3 peptides. However, these studies were based on binding affinities measured with model BH3 peptides and Bcl-2-proteins taken from mouse sequences. While the majority of these interactions are conserved between mice and humans, we found surprisingly that human NOXA binds to human Bfl-1 potently and covalently via conserved Cys residues, with over 2 orders of magnitude increased affinity over hMcl-1. Our data suggest that some assumptions of the original BH3 profiling need to be revisited and that perhaps further targeting efforts should be redirected toward Bfl-1, for which no suitable specific inhibitors or pharmacological tools have been reported. In this regard, we also describe the initial design and characterizations of novel covalent BH3-based agents that potently target Bfl-1. These molecules could provide a novel platform on which to design effective Bfl-1 targeting therapeutics.

Glioblastoma multiforme (GBM) is an intractable tumor despite therapeutic advances, principally because of its invasive properties. Radiation is a staple in therapeutic regimens, although cells surviving radiation can become more aggressive and invasive. Subtraction hybridization identified melanoma differentiation-associated gene 9 [MDA-9/Syntenin; syndecan-binding protein (SDCBP)] as a differentially regulated gene associated with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a highly conserved double-PDZ domain-containing scaffolding protein, is robustly expressed in human-derived GBM cell lines and patient samples, with expression increasing with tumor grade and correlating with shorter survival times and poorer response to radiotherapy. Knockdown of MDA-9/Syntenin sensitizes GBM cells to radiation, reducing postradiation invasion gains. Radiation induces Src and EGFRvIII signaling, which is abrogated through MDA-9/Syntenin down-regulation. A specific inhibitor of MDA-9/Syntenin activity, PDZ1i (113B7), identified through NMR-guided fragment-based drug design, inhibited MDA-9/Syntenin binding to EGFRvIII, which increased following radiation. Both genetic (shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin reduced invasion gains in GBM cells following radiation. Although not affecting normal astrocyte survival when combined with radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial GBM signaling involving FAK and mutant EGFR, EGFRvIII, and abrogated gains in secreted proteases, MMP-2 and MMP-9, following radiation. In an in vivo glioma model, PDZ1i resulted in smaller, less invasive tumors and enhanced survival. When combined with radiation, survival gains exceeded radiotherapy alone. MDA-9/Syntenin (SDCBP) provides a direct target for therapy of aggressive cancers such as GBM, and defined small-molecule inhibitors such as PDZ1i hold promise to advance targeted brain cancer therapy., Competing Interests: The authors have applied for patent protection for the small-molecule PDZ inhibitor reported in this paper.

Background: We examined racial/ethnic disparities in school-based behavioral health service use for children with psychiatric disorders., Methods: Medicaid claims data were used to compare the behavioral healthcare service use of 23,601 children aged 5-17 years by psychiatric disorder (autism, attention deficit hyperactivity disorder [ADHD], conduct/oppositional defiant disorder, and "other") and by race/ethnicity (African-American, Hispanic, white, and other). Logistic and generalized linear regression analyses were used., Results: Differences in service use by racial/ethnic group were identified within and across diagnostic groups, both for in-school service use and out-of-school service use. For all disorders, Hispanic children had significantly lower use of in-school services than white children. Among children with ADHD, African-American children were less likely to receive in-school services than white children; however, there were no differences in adjusted annual mean Medicaid expenditures for in-school services by race/ethnicity or psychiatric disorders. Statistically significant differences by race/ethnicity were found for out-of-school service use for children with ADHD and other psychiatric disorders. There were significant differences by race/ethnicity in out-of-school service use for each diagnostic group., Conclusions: Differences in the use of school-based behavioral health services by racial and ethnic groups suggest the need for culturally appropriate outreach and tailoring of services to improve service utilization., (© 2016, American School Health Association.)

Background: The number of children diagnosed with autism has rapidly outpaced the capacities of many public school systems to serve them, especially under-resourced, urban school districts. The intensive nature of evidence-based autism interventions, which rely heavily on one-to-one delivery, has caused schools to turn to computer-assisted interventions (CAI). There is little evidence regarding the feasibility, effectiveness, and implementation of CAI in public schools. While CAI has the potential to increase instructional time for students with autism, it may also result in unintended consequences such as reduction in the amount of interpersonal (as opposed to computerized) instruction students receive. The purpose of this study is to test the effectiveness of one such CAI-TeachTown-its implementation, and its effects on teachers' use of other evidence-based practices., Methods: This study protocol describes a type II hybrid cluster randomized effectiveness-implementation trial. We will train and coach 70 teachers in autism support classrooms in one large school district in the use of evidence-based practices for students with autism. Half of the teachers then will be randomly selected to receive training and access to TeachTown: Basics, a CAI for students with autism, for the students in their classrooms. The study examines: (1) the effectiveness of TeachTown for students with autism; (2) the extent to which teachers implement TeachTown the way it was designed (i.e., fidelity); and (3) whether its uptake increases or reduces the use of other evidence-based practices., Discussion: This study will examine the implementation of new technology for children with ASD in public schools and will be the first to measure the effectiveness of CAI. As importantly, the study will investigate whether adding a new technology on top of existing practices increases or decreases their use. This study presents a unique method to studying both the implementation and exnovation of evidence-based practices for children with autism in school settings., Trial Registration: NCT02695693 . Retrospectively registered on July 8, 2016.

Paneth cells are a highly specialized population of intestinal epithelial cells located in the crypt adjacent to Lgr5(+) stem cells, from which they differentiate through a process that requires downregulation of the Notch pathway. Their ability to store and release antimicrobial peptides protects the host from intestinal pathogens and controls intestinal inflammation. Here, we show that PKCλ/ι is required for Paneth cell differentiation at the level of Atoh1 and Gfi1, through the control of EZH2 stability by direct phosphorylation. The selective inactivation of PKCλ/ι in epithelial cells results in the loss of mature Paneth cells, increased apoptosis and inflammation, and enhanced tumorigenesis. Importantly, PKCλ/ι expression in human Paneth cells decreases with progression of Crohn's disease. Kaplan-Meier survival analysis of colorectal cancer (CRC) patients revealed that low PRKCI levels correlated with significantly worse patient survival rates. Therefore, PKCλ/ι is a negative regulator of intestinal inflammation and cancer through its role in Paneth cell homeostasis., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cell surface receptor CD44 is a glycoprotein belonging to the hyaluronan-binding proteins, termed hyaladherins. CD44 is expressed in a wide variety of isoforms in many cells and, in particular, is present on the surface of malignant cells where it is involved in the onset and progression of cancer. In a first attempt to identify novel CD44-binding agents, we first characterized, with NMR spectroscopic techniques, several agents that were reported to bind to human CD44 (hCD44). To our surprise, however, none of these putative CD44-binding agents, including a peptide that is in phase 2 clinical trials (A6 peptide) and a recently reported fragment hit, were found to interact significantly with recombinant hCD44(21-178). Nonetheless, we further report that a fragment-screening campaign, with solution NMR spectroscopy as the detection method, identified a viable fragment hit that bound in a potentially functional pocket on the surface of CD44, opposite to the hyaluronic acid binding site. We hypothesize that this pocket could be indirectly associated with the cellular and in vivo activity of the A6 peptide, which would provide a novel framework for the possible development of therapeutically viable CD44 antagonists., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently we described a novel approach, named high-throughput screening (HTS) by NMR that allows the identification, from large combinatorial peptide libraries, of potent and selective peptide mimetics against a given target. Here, we deployed the "HTS by NMR" approach for the design of novel peptoid sequences targeting the N-terminal domain of Yersinia outer protein H (YopH-NT), a bacterial toxin essential for the virulence of Yersinia pestis. We aimed at disrupting the protein-protein interactions between YopH-NT and its cellular substrates, with the goal of inhibiting indirectly YopH enzymatic function. These studies resulted in a novel agent of sequence Ac-F-pY-cPG-d-P-NH2 (pY=phosphotyrosine; cPG=cyclopentyl glycine) with a Kd value against YopH-NT of 310 nm. We demonstrated that such a pharmacological inhibitor of YopH-NT results in the inhibition of the dephosphorylation by full-length YopH of a cellular substrate. Hence, potentially this agent represents a valuable stepping stone for the development of novel therapeutics against Yersinia infections. The data reported further demonstrate the utility of the HTS by NMR approach in deriving novel peptide mimetics targeting protein-protein interactions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study examined the extent to which clinical and demographic characteristics predicted outcome for children with autism spectrum disorder. Participants included 152 students with autism spectrum disorder in 53 kindergarten-through-second-grade autism support classrooms in a large urban public school district. Associations between child characteristics (including age, language ability, autism severity, social skills, adaptive behavior, co-occurring psychological symptoms, and restrictive and repetitive behavior) and outcome, as measured by changes in cognitive ability following one academic year of an intervention standardized across the sample were evaluated using linear regression with random effects for classroom. While several scales and subscales had statistically significant bivariate associations with outcome, in adjusted analysis, only age and the presence of symptoms associated with social anxiety, such as social avoidance and social fearfulness, as measured through the Child Symptom Inventory-4, were associated with differences in outcome. The findings regarding the role of social anxiety are new and have important implications for treatment. Disentangling the construct of social anxiety to differentiate between social fearfulness and social motivation has important implications for shifting the focus of early treatment for children with autism spectrum disorder., (© The Author(s) 2015.)

Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

First line treatment for pancreatic cancer consists of surgical resection, if possible, and a subsequent course of chemotherapy using the nucleoside analogue gemcitabine. In some patients, an active transport mechanism allows gemcitabine to enter efficiently into the tumor cells, resulting in a significant clinical benefit. However, in most patients, low expression of gemcitabine transporters limits the efficacy of the drug to marginal levels, and patients need frequent administration of the drug at high doses, significantly increasing systemic drug toxicity. In this article we focus on a novel targeted delivery approach for gemcitabine consisting of conjugating the drug with an EphA2 targeting agent. We show that the EphA2 receptor is highly expressed in pancreatic cancers, and accordingly, the drug-conjugate is more effective than gemcitabine alone in targeting pancreatic tumors. Our preliminary observations suggest that this approach may provide a general benefit to pancreatic cancer patients and offers a comprehensive strategy for enhancing delivery of diverse therapeutic agents to a wide range of cancers overexpressing EphA2, thereby potentially reducing toxicity while enhancing therapeutic efficacy.

Background: The current extensive use of the domestic goat (Capra hircus) is the result of its medium size and high adaptability as multiple breeds. The extent to which its genetic variability was influenced by early domestication practices is largely unknown. A common standard by which to analyze maternally-inherited variability of livestock species is through complete sequencing of the entire mitogenome (mitochondrial DNA, mtDNA)., Results: We present the first extensive survey of goat mitogenomic variability based on 84 complete sequences selected from an initial collection of 758 samples that represent 60 different breeds of C. hircus, as well as its wild sister species, bezoar (Capra aegagrus) from Iran. Our phylogenetic analyses dated the most recent common ancestor of C. hircus to ~460,000 years (ka) ago and identified five distinctive domestic haplogroups (A, B1, C1a, D1 and G). More than 90 % of goats examined were in haplogroup A. These domestic lineages are predominantly nested within C. aegagrus branches, diverged concomitantly at the interface between the Epipaleolithic and early Neolithic periods, and underwent a dramatic expansion starting from ~12-10 ka ago., Conclusions: Domestic goat mitogenomes descended from a small number of founding haplotypes that underwent domestication after surviving the last glacial maximum in the Near Eastern refuges. All modern haplotypes A probably descended from a single (or at most a few closely related) female C. aegagrus. Zooarchaelogical data indicate that domestication first occurred in Southeastern Anatolia. Goats accompanying the first Neolithic migration waves into the Mediterranean were already characterized by two ancestral A and C variants. The ancient separation of the C branch (~130 ka ago) suggests a genetically distinct population that could have been involved in a second event of domestication. The novel diagnostic mutational motifs defined here, which distinguish wild and domestic haplogroups, could be used to understand phylogenetic relationships among modern breeds and ancient remains and to evaluate whether selection differentially affected mitochondrial genome variants during the development of economically important breeds.

Disrupting the eukaryotic translation initiation factor 4F (eIF4F) complex offers an appealing strategy to potentiate the effectiveness of existing cancer therapies and to overcome resistance to drugs such as BRAF inhibitors (BRAFi). Here, we identified and characterized the small molecule SBI-0640756 (SBI-756), a first-in-class inhibitor that targets eIF4G1 and disrupts the eIF4F complex. SBI-756 impaired the eIF4F complex assembly independently of mTOR and attenuated growth of BRAF-resistant and BRAF-independent melanomas. SBI-756 also suppressed AKT and NF-κB signaling, but small-molecule derivatives were identified that only marginally affected these pathways while still inhibiting eIF4F complex formation and melanoma growth, illustrating the potential for further structural and functional manipulation of SBI-756 as a drug lead. In the gene expression signature patterns elicited by SBI-756, DNA damage, and cell-cycle regulatory factors were prominent, with mutations in melanoma cells affecting these pathways conferring drug resistance. SBI-756 inhibited the growth of NRAS, BRAF, and NF1-mutant melanomas in vitro and delayed the onset and reduced the incidence of Nras/Ink4a melanomas in vivo. Furthermore, combining SBI-756 and a BRAFi attenuated the formation of BRAFi-resistant human tumors. Taken together, our findings show how SBI-756 abrogates the growth of BRAF-independent and BRAFi-resistant melanomas, offering a preclinical rationale to evaluate its antitumor effects in other cancers., (©2015 American Association for Cancer Research.)

The emergence of drug-resistant strains of influenza virus makes exploring new classes of inhibitors that target universally conserved viral targets a highly important goal. The influenza A viral genome is made up of eight single-stranded RNA-negative segments. The RNA promoter, consisting of the conserved sequences at the 3' and 5' end of each RNA genomic segment, is universally conserved among influenza A virus strains and in all segments. Previously, we reported on the identification and NMR structure of DPQ (6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) (compound 1) in complex with the RNA promoter. Here, we report on additional screening and SAR studies with compound 1, including ex vivo anti-influenza activity assays, resulted in improved cellular activity against influenza A virus in the micromolar range., (© 2015 John Wiley & Sons A/S.)

Cancer-selective viral replication and delivery of a therapeutic immunomodulating, cancer-selective killing cytokine ( mda-7/IL-24 ) by means of a new Cancer Terminator Virus ( CTV ) combined with a small molecule BH3 mimetic holds promise for treating both primary and metastatic hormone refractory prostate cancer (CaP).

This study evaluated the association of fidelity to each of the components of the Strategies for Teaching based on Autism Research (STAR) program, a comprehensive treatment package for children with autism that includes discrete trial training, pivotal response training, and teaching in functional routines, on outcomes for 191 students ages 5-8 years in a large public school district. Fidelity to all components was relatively low, despite considerable training and support, suggesting the need to develop new implementation strategies. Fidelity to pivotal response training, but not discrete trial training or functional routines, was positively associated with gains in cognitive ability despite low levels of fidelity, and may be an effective intervention choice in under-resourced settings.

The development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this study, we used a combination of nuclear magnetic resonance-guided structure-activity relationships along with biochemical and cellular studies to derive a novel tumor-homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand-binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan-Bcl-2 inhibitor (-)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (-)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan-Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (-)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (-)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan-Bcl-2 inhibition by an Mcl-1-targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment., (© 2015 by The American Society of Hematology.)

Honey bees (Apis mellifera L.) are bioindicators of environmental pollution levels. During their wide-ranging foraging activity, these hymenopterans are exposed to pollutants, thus becoming a useful tool to trace the environmental contaminants as heavy metals, pesticides, radionuclides and volatile organic compounds. In the present work we demonstrate that bees can also be used as active samplers of airborne particulate matter. Worker bees were collected from hives located in a polluted postmining area in South West Sardinia (Italy) that is also exposed to dust emissions from industrial plants. The area is included in an official list of sites of national interest for environmental remediation, and has been characterized for the effects of pollutants on the health of the resident population. The head, wings, hind legs and alimentary canal of the bees were investigated with Scanning Electron Microscopy coupled with X-ray spectroscopy (SEM-EDX). The analyses pointed to specific morphological and chemical features of the particulate, and resulted into the identification of three categories of particles: industry-, postmining-, and soil-derived. With the exception of the gut, all the analyzed body districts displayed inorganic particles, mostly concentrated in specific areas of the body (i.e. along the costal margin of the fore wings, the medial plane of the head, and the inner surface of the hind legs). The role of both past mining activities and the industrial activity close to the study area as sources of the particulate matter is also discussed. We conclude that honey bees are able to collect samples of the main airborne particles emitted from different sources, therefore could be an ideal tool for monitoring such a kind of pollutants.

Oral and oropharyngeal cancers are the sixth most common cancers worldwide. Despite intensive investigation, oral squamous cell carcinomas (OSCC) represent a clinical challenge resulting in significant morbidity and mortality. Resistance to cell death is common in OSCC and is often mediated by the Bcl-2 family proteins. Among all anti-apoptotic Bcl-2 family members, Mcl-1 functions as a major survival factor, particularly in solid cancers. Despite the confirmed importance of Mcl-1 in several neoplasms, the role of Mcl-1 in OSCC survival has yet to be explored. In this study, we found that knocking down Mcl-1 sensitized OSCC cells to ABT-737, which binds to Bcl-2/Bcl-xL but not Mcl-1. We report for the first time that a BH3 mimetic, Sabutoclax, which functions as an inhibitor of all anti-apoptotic Bcl-2 proteins, induced cancer-specific cell death in an Mcl-1-dependent manner through both apoptosis and toxic mitophagy. In vivo studies demonstrated that Sabutoclax alone decreased tumor growth in a carcinogen-induced tongue OSCC mouse model. In a combination regimen, Sabutoclax and COX-2 inhibitor, Celecoxib, synergistically inhibited the growth of OSCC in vitro and also significantly reduced OSCC tumor growth in vivo. Overall, these results identify Mcl-1 as a therapeutic prospective target in OSCC.

Improved treatments for pancreatic cancer remain a clinical imperative. Sabutoclax, a small-molecule BH3 mimetic, inhibits the function of antiapoptotic Bcl-2 proteins. Minocycline, a synthetic tetracycline, displays antitumor activity. Here, we offer evidence of the combinatorial antitumor potency of these agents in several preclinical models of pancreatic cancer. Sabutoclax induced growth arrest and apoptosis in pancreatic cancer cells and synergized with minocycline to yield a robust mitochondria-mediated caspase-dependent cytotoxicity. This combinatorial property relied upon loss of phosphorylated Stat3 insofar as reintroduction of activated Stat3-rescued cells from toxicity. Tumor growth was inhibited potently in both immune-deficient and immune-competent models with evidence of extended survival. Overall, our results showed that the combination of sabutoclax and minocycline was highly cytotoxic to pancreatic cancer cells and safely efficacious in vivo., (©2015 American Association for Cancer Research.)

Despite recent advances, treatment options for advanced prostate cancer (CaP) remain limited. We are pioneering approaches to treat advanced CaP that employ conditionally replication-competent oncolytic adenoviruses that simultaneously produce a systemically active cancer-specific therapeutic cytokine, mda-7/IL-24, Cancer Terminator Viruses (CTV). A truncated version of the CCN1/CYR61 gene promoter, tCCN1-Prom, was more active than progression elevated gene-3 promoter (PEG-Prom) in regulating transformation-selective transgene expression in CaP and oncogene-transformed rat embryo cells. Accordingly, we developed a new CTV, Ad.tCCN1-CTV-m7, which displayed dose-dependent killing of CaP without harming normal prostate epithelial cells in vitro with significant anti-cancer activity in vivo in both nude mouse CaP xenograft and transgenic Hi-Myc mice (using ultrasound-targeted microbubble (MB)-destruction, UTMD, with decorated MBs). Resistance to mda-7/IL-24-induced cell death correlated with overexpression of Bcl-2 family proteins. Inhibiting Mcl-1 using an enhanced BH3 mimetic, BI-97D6, sensitized CaP cell lines to mda-7/IL-24-induced apoptosis. Combining BI-97D6 with Ads expressing mda-7/IL-24 promoted ER stress, decreased anti-apoptotic Mcl-1 expression and enhanced mda-7/IL-24 expression through mRNA stabilization selectively in CaP cells. In Hi-myc mice, the combination induced enhanced apoptosis and tumor growth suppression. These studies highlight therapeutic efficacy of combining a BH3 mimetic with a novel CTV, supporting potential clinical applications for treating advanced CaP.

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment., (©2015 American Association for Cancer Research.)

An ultrahigh-throughput screen was performed to identify novel small molecule inhibitors of influenza virus replication. The screen employed a recombinant influenza A/WSN/33 virus expressing Renilla luciferase and yielded a hit rate of 0.5%, of which the vast majority showed little cytotoxicity at the inhibitory concentration. One of the top hits from this screen, designated S20, inhibits HA-mediated membrane fusion. S20 shows potent antiviral activity (IC 50 = 80 nM) and low toxicity (CC 50 = 40 μM), yielding a selectivity index of 500 and functionality against all of the group 1 influenza A viruses tested in this study, including the pandemic H1N1 and avian H5N1 viruses. Mechanism of action studies proved a direct S20-HA interaction and showed that S20 inhibits fusion by stabilizing the prefusion conformation of HA. In silico docking studies were performed, and the predicted binding site in HA2 corresponds with the area where resistance mutations occurred and correlates with the known role of this region in fusion. This high-throughput screen has yielded many promising new lead compounds, including S20, which will potentially shed light on the molecular mechanisms of viral infection and serve as research tools or be developed for clinical use as antivirals.

mda-9/Syntenin (melanoma differentiation-associated gene 9) is a PDZ domain containing, cancer invasion-related protein. In this study, we employed multiple integrated bioinformatic approaches to identify the probable epigenetic factors, molecular pathways, and functionalities associated with mda-9 dysregulation during cancer progression. Analyses of publicly available genomic data (e.g., expression, copy number, methylation) from TCGA, GEO, ENCODE, and Human Protein Atlas projects led to the following observations: (a) mda-9 expression correlates with both copy number and methylation level of an intronic CpG site (cg1719774) located downstream of the CpG island, (b) cg1719774 methylation is a likely prognostic marker in glioma, (c) among 22 cancer types, melanoma exhibits the highest mda-9 level, and lowest level of methylation at cg1719774, (d) cg1719774 hypomethylation is also associated with histone modifications (at the mda-9 locus) indicative of more active transcription, (e) using Gene Set Enrichment Analysis (GSEA), and the Virtual Gene Overexpression or Repression (VIGOR) analytical scheme, we were able to predict mda-9's association with extracellular matrix organization (e.g., MMPs, collagen, integrins), IGFBP2 and NF-κB signaling pathways, phospholipid metabolism, cytokines (e.g., interleukins), CTLA-4, and components of complement cascade pathways. Indeed, previous publications have shown that many of the aforementioned genes and pathways are associated with mda-9's functionality., (© 2015 Elsevier Inc. All rights reserved.)

In recent years the chaperone HSC-70 has become a target for drug design with a strong focus in anticancer therapies. In our study of possible inhibitors of HSC-70 enzymatic activity we screened compounds by NMR as well as X-ray crystallography. As part of our screening efforts we crystallized the human HSC-70 ATP binding domain and obtained novel crystal forms in addition to known structures. The new crystal structures highlight the mobility of the entire domain previously described by NMR, which was linked to its chaperone activity but not yet demonstrated by X-ray crystallography. Conformational changes across the entire molecule have been elucidated in response to the binding of small molecule ligands and show a pattern of mobility consistent with postulated signal transduction modes between the nucleotide binding domain (NBD) and the substrate binding domain (SBD). In addition, two crystal structures contained glycerol bound at a new site. Binding studies performed with glycerol analogs proved inhibitory properties of the site, which were further characterized by isothermal calorimetry and in silico docking studies. The presence of two binding pockets enabled us to explore a novel method of inhibition by compounds that bridge the adjacent phosphate and glycerol binding sites. Finally, an example of such a bridged inhibitor is proposed., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications.

With therapies that date back to the 1950s, and few newly approved treatments in the last 20 years, pancreatic cancer remains a significant challenge for the development of novel therapeutics. Current regimens have successfully extended patient survival, although they still lead to prognoses measured in months rather than years. The genetic diversity inherent in pancreatic tumors forms the roadblocks that must be overcome in future therapeutics. Recent insight into the genetic patterns found in tumor cells may provide clues leading to better understanding of the challenges hindering the development of treatments. Here, we review currently used drugs and established combination therapies that comprise the standard of care for a highly recalcitrant disease. Novel approaches can improve upon current therapies in a variety of ways. Enhancing specificity, such that growth inhibition and cytotoxic effects act preferentially on tumor cells, is one approach to advance treatments. This can be accomplished through the targeting of extracellular markers specific to cancer cells. Additionally, enlisting natural defenses and overcoming tumor-driven immune suppression could prove to be a useful tactic. Recent studies utilizing these approaches have yielded promising results and could contribute to an ongoing effort battling a particularly difficult cancer., (© 2015 Elsevier Inc. All rights reserved.)

Introduction: Melanoma differentiation-associated gene - 9 (MDA-9)/Syntenin has become an increasingly popular focus for investigation in numerous cancertypes. Originally implicated in melanoma metastasis, it has diverse cellular roles and is consistently identified as a regulator of tumor invasion and angiogenesis. As a potential target for inhibiting some of the most lethal aspects of cancer progression, further insight into the function of MDA-9/Syntenin is mandatory., Areas Covered: Recent literature and seminal articles were reviewed to summarize the latest collective understanding of MDA-9/Syntenin's role in normal and cancerous settings. Insights into its participation in developmental processes are included, as is the functional significance of the N- and C-terminals and PDZ domains of MDA-9/Syntenin. Current reports highlight the clinical significance of MDA-9/Syntenin expression level in a variety of cancers, often correlating directly with reduced patient survival. Also presented are assessments of roles of MDA-9/Syntenin in cancer progression as well as its functions as an intracellular adapter molecule., Expert Opinion: Multiple studies demonstrate the importance of MDA-9/Syntenin in tumor invasion and progression. Through the use of novel drug design approaches, this protein may provide a worthwhile therapeutic target. As many conventional therapies do not address, or even enhance, tumor invasion, an anti-invasive approach would be a worthwhile addition in cancer therapy.

The lipid phosphatase Ship2 is a protein that intervenes in several diseases such as diabetes, cancer, neurodegeneration, and atherosclerosis. It is made up of a catalytic domain and several protein docking modules such as a C-terminal Sam (Sterile alpha motif) domain. The Sam domain of Ship2 (Ship2-Sam) binds to the Sam domains of the EphA2 receptor (EphA2-Sam) and the PI3K effector protein Arap3 (Arap3-Sam). These heterotypic Sam-Sam interactions occur through formation of dimers presenting the canonical "Mid Loop/End Helix" binding mode. The central region of Ship2-Sam, spanning the C-terminal end of α2, the α3 and α4 helices together with the α2α3 and α3α4 interhelical loops, forms the Mid Loop surface that is needed to bind partners Sam domains. A peptide encompassing most of the Ship2-Sam Mid Loop interface (Shiptide) capable of binding to both EphA2-Sam and Arap3-Sam, was previously identified. Here we investigated the conformational features of this peptide, through solution CD and NMR studies in different conditions. These studies reveal that the peptide is highly flexible in aqueous buffer, while it adopts a helical conformation in presence of 2,2,2-trifluoroethanol. The discovered structural insights and in particular the identification of a helical motif, may lead to the design of more constrained and possibly cell permeable Shiptide analogs that could work as efficient antagonists of Ship2-Sam heterotypic interactions and embrace therapeutic applications., (© 2014 Wiley Periodicals, Inc.)

Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of Braf(V600E)::Pten(-/-) or Braf(V600E)::Cdkn2a(-/-)::Pten(-/-) mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in Braf(V600E)::Pten(-/-) mice. Pdk1(-/-) melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1(-/-) melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

Background: Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS., Methods: The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment., Results: BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity., Conclusions: BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.

Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2-targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long-lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate-limiting degradation sites on the peptide-drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide-drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Through screening by NMR spectroscopy, we discovered a novel scaffold (DPQ: 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) that binds specifically to the influenza A virus RNA promoter. The solution structure of the RNA-DPQ complex reported here demonstrates that the internal loop is the binding site of DPQ. The scaffold exhibits antiviral activity against influenza viruses.

Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting., (© 2013 Wiley Periodicals, Inc.)

Background: The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis., Materials: We assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo., Results: MDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo., Conclusions: Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.

The protein Kinase B alpha (AKT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up-regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI-69A11) as inhibitor of the AKT and the NF-κB pathways. Here, we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast, and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model., (© 2013 John Wiley & Sons A/S.)

The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Owing to the high levels of antiapoptotic B-cell lymphoma 2 (BCL-2) family members observed in several cancers, there has been a major effort to develop inhibitors of the BCL2-family as chemotherapeutic agents. Of the different members in the BCL-2 family, myeloid cell leukemia sequence 1 (MCL-1) is commonly amplified in human tumors and is associated with their relapse and chemoresistance. As a result, specific inhibitors of MCL-1 are being designed to treat resistant tumors. However, there is increasing evidence for other nonapoptotic roles of the BCL-2 family, ranging from ionic homeostasis and autophagy to the regulation of fission-fusion dynamics in subcellular organelles, including the endoplasmic reticulum and mitochondria. In this study, we characterize the specificity of two novel putative MCL-1 inhibitors, BI97C1 (Sabutoclax) and BI112D1, in inducing apoptosis in a BAX/BAK-dependent manner and in an MCL-1-dependent system. In addition to their being proapoptotic, these inhibitors also cause enhanced mitochondrial fragmentation that accompanies a time-dependent loss of optic atrophy 1 (OPA1), suggesting an impairment of mitochondrial fusion. This mitochondrial fragmentation occurs independently of dynamin-related protein 1 (DRP1)-mediated fission activity and, unlike most apoptotic stimuli, occurs upstream of and/or independent of BAX, BAK, and other BH3-only proteins. Furthermore, this mitochondrial fragmentation occurred rapidly and preceded other hallmarks of apoptosis, including the loss in mitochondrial membrane potential and the release of cytochrome c. Although such mitochondrial fragmentation did not deplete total cellular adenosine triphosphate (ATP) or alter other mitochondrial complexes, there was significant accumulation of reactive oxygen species.