MicroRNAs: the novel targets for Ebola drugs

<正>The re-emergence of Ebola viral disease(EVD)in West Africa has caused an international alarm,even panic.As of August 27th,2014,the Ebola virus(EBOV)has infected3069 people with 1552 death cases starting from December2013(http://www.who.int/csr/don/2014_08_28_ebola/en/).Recently,an outbreak of Ebola hemorrhagic fever in the north of Congo(Democratic Republic)has been confirmed     

Regulation of TRPC6 channels by non-steroidal anti-inflammatory drugs

Family focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis and hyalinosis of particular loops of glomeruli and is one of the causes of the nephrotic syndrome. Certain mutations in the structure of TRPC6 channels are the genetic impetus for FSGS development resulting in podocytes functional abnormalities and various nephropathies. We have recently demonstrated that non-steroid antiinflammatory drugs (NSAID) ibuprofen and diclofenac decrease the activity of endogenous TRPC-like calcium channels in the podocytes of the freshly isolated rat glomeruli. It has also been shown that TRPC6 channels are expressed in the podocytes. In the current study we have functionally reconstituted TRPC6 channels in mammalian cells to investigate the effects of diclofenac on the activity of wild type TRPC6 channel and TRPC6^P112Q channel containing a mutation in the N-terminus that was described in FSGS patients. Intracellular calcium level measurements in transfected cells revealed a more intensive carbachol-induced increase of calcium concentration in HEK-293 cells expressing TRPC6^P112Q versus the cells expressing wild-type TRPC6. We also performed patch-clamp experiments to study TRPC6 channels reconstituted in Chinese hamster ovary (CHO) cell line and found that application of diclofenac (500 μM) acutely reduced single channel activity. Preincubation with diclofenac (100 μM) also decreased the whole-cell current in CHO cells overexpressing TRPC6^P112Q. Therefore, our previously published data on the effects of NSAID on TRPC-like channels in the isolated rat glomeruli, along with this current investigation on the cultured overexpressed mammalian cells, allows hypothesizing that TRPC6 channels may be a target for NSAID that can be important in the treatment of FSGS.     

Molecular targets for existing and novel immunosuppressive drugs

Anti-neoplastic cytostatic antiproliferative agents, such as methotrexate, 6-mercaptopurine and cyclophosphamide, were originally used as immunosuppressive drugs. Although these agents induced only modest anti-rejection activity, they caused serious non-specific bone marrow suppression, impairing host resistance and increasing the incidence of infections. Unlike these non-selective agents, cyclosporine A, tacrolimus and sirolimus act more selectively on different stages of the T-lymphocyte (T-cell) and B-lymphocyte (B-cell) activation cycles; however, cyclosporine and tacrolimus are nephrotoxic, whereas sirolimus causes hypertriglyceridaemia. Thus, despite this progress, continued efforts must be made to develop and test new, potentially very selective agents. The agent 15-deoxyspergualin moderately inhibits both mitogen-stimulated T-cell proliferation and the generation of cytotoxic T lymphocytes (CTLs) but does not affect the production of interleukin 2 (IL-2). Another drug, FTY720, has a unique action to prevent rejection, by altering the homing of lymphocytes to the lymphoid compartments. The newest members of the family of antiproliferative agents, namely mycophenolate mofetil, leflunomide and brequinar, are potentially more selective than their predecessors. However, the most promising agents are produced using antisense technology. This approach involves the design of antisense oligodeoxynucleotides; these novel drugs are designed to block allograft rejection by blocking selected messenger RNA (mRNA). This review outlines the mechanisms of action, the limitations of application and the molecular or cellular targets of traditional agents, newly developed drugs and also antisense technology, which is an example of a new application of molecular medicine.     

Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs

Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions. Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase. Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase. Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr. Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE₂)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE₂. Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacinblocked cultures, indicating a PGE₂-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems. In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase. Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE₂ synthesis differed. Dexamethasone inhibited PGE₂ synthesis, which resulted in the suppression of collagenase. However, PGE₂ production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE₂ levels. Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE₂ or phospholipase A₂. These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.     

Comparative genomic assessment of novel broad-spectrum targets for antibacterial drugs

Single and multiple resistance to antibacterial drugs currently in use is spreading, since they act against only a very small number of molecular targets; finding novel targets for anti-infectives is therefore of great importance. All protein sequences from three pathogens (Staphylococcus aureus, Mycobacterium tuberculosis and Escherichia coli O157:H7 EDL993) were assessed via comparative genomics methods for their suitability as antibacterial targets according to a number of criteria, including the essentiality of the protein, its level of sequence conservation, and its distribution in pathogens, bacteria and eukaryotes (especially humans). Each protein was scored and ranked based on weighted variants of these criteria in order to prioritize proteins as potential novel broad-spectrum targets for antibacterial drugs. A number of proteins proved to score highly in all three species and were robust to variations in the scoring system used. Sensitivity analysis indicated the quantitative contribution of each metric to the overall score. After further analysis of these targets, tRNA methyltransferase (trmD) and translation initiation factor IF-1 (infA) emerged as potential and novel antimicrobial targets very worthy of further investigation. The scoring strategy used might be of value in other areas of post-genomic drug discovery.     

Sphingosine kinase signalling in immune cells: potential as novel therapeutic targets

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.     

Signaling protein networks as targets of new antineoplastic drugs

In-depth analysis of molecular regulatory networks in cancer holds the promise of improved knowledge of the pathophysiology of tumor cells so that it will become possible to design a detailed molecular tumor taxonomy. This knowledge will also offer new opportunities for the identification and validation of key molecular tumor targets to be exploited for novel therapeutic approaches. Some signaling proteins have already been identified as such, e.g. c-Myc, Cyclin D1, Bcl-XL, kinases and some nuclear receptors. This has led to the successful development of a few function-modulatory drugs (Glivec, SERM, Iressa), providing proof-of-principle of the validity of this approach. Further developments are likely to derive from "-omic" approaches, aimed at the understanding of signaling networks and of the mechanism of action of newfound lead molecules. High-throughput screening of small drug-like molecules from combinatorial chemical libraries or from microbial extracts will identify novel, "intelligent" drug candidates. An additional medicinal chemistry strategy (via 40-50 unit rosary-bead chains) has the potential to be much more effective than small molecules in interfering with protein-protein interactions. This may lead to considerably higher selectivity and effectiveness compared with historical approaches in drug discovery.     

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.     

A computational approach to finding novel targets for existing drugs

Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery. We have developed a computational drug repositioning pipeline to perform large-scale molecular docking of small molecule drugs against protein drug targets, in order to map the drug-target interaction space and find novel interactions. Our method emphasizes removing false positive interaction predictions using criteria from known interaction docking, consensus scoring, and specificity. In all, our database contains 252 human protein drug targets that we classify as reliable-for-docking as well as 4621 approved and experimental small molecule drugs from DrugBank. These were cross-docked, then filtered through stringent scoring criteria to select top drug-target interactions. In particular, we used MAPK14 and the kinase inhibitor BIM-8 as examples where our stringent thresholds enriched the predicted drug-target interactions with known interactions up to 20 times compared to standard score thresholds. We validated nilotinib as a potent MAPK14 inhibitor in vitro (IC50 40 nM), suggesting a potential use for this drug in treating inflammatory diseases. The published literature indicated experimental evidence for 31 of the top predicted interactions, highlighting the promising nature of our approach. Novel interactions discovered may lead to the drug being repositioned as a therapeutic treatment for its off-target's associated disease, added insight into the drug's mechanism of action, and added insight into the drug's side effects.     

The thiol-based redox networks of pathogens: unexploited targets in the search for new drugs

Hydroperoxide metabolism in diverse pathogens is reviewed under consideration of involved enzymes as potential drug targets. The common denominator of the peroxidase systems of Trypanosoma, Leishmania, Plasmodium, and Mycobacterium species is the use of NAD(P)H to reduce hydroperoxides including peroxynitrite via a flavin-containing disulfide reductase, a thioredoxin (Trx)-related protein and a peroxidase that operates with thiol catalysis. In Plasmodium falciparum, thioredoxin- and glutathione dependent systems appear to be linked via glutaredoxin and plasmoredoxin to terminal thioredoxin peroxidases belonging to both, the peroxiredoxin (Prx) and glutathione peroxidase (GPx) family. In Mycobacterium tuberculosis, a catalase-type peroxidase is complemented by the typical 2-C-Prx AhpC that, in contrast to most bacteria, is reduced by TrxC, and an atypical 2-C-Prx reduced by TrxB or C. A most complex variation of the scheme is found in trypanosomatids, where the unique redox metabolite trypanothione reduces the thioredoxin-related tryparedoxin, which fuels Prx- and GPx-type peroxidases as well as ribonucleotide reductase. In Trypanosoma brucei and Leishmania donovani the system has been shown to be essential for viability and virulence by inversed genetics. It is concluded that optimum efficacy can be expected from inhibitors of the most upstream components of the redox cascades. For trypanosomatids attractive validated drug targets are trypanothione reductase and trypanothione synthetase; for mycobacteria thioredoxin reductase appears most appealing, while in Plasmodium simultaneous inhibition of both the thioredoxin and the glutathione pathway appears advisable to avoid mutual substitution in co-substrate supply to the peroxidases. Financial and organisational needs to translate the scientific progress into applicable drugs are discussed under consideration of the socio-economic impact of the addressed diseases.     

Cell-surface proteomics for the identification of novel therapeutic targets in cancer

Introduction: Cancer is the second most common cause of death worldwide and its heterogeneity complicates therapy. Standard cytotoxic regiments disrupt rapidly dividing cells, regardless of their neoplastic status. The introduction of less toxic targeted therapies has partially contributed to the observed decrease in cancer-related mortality. Cell-surface proteins represent attractive targets for therapy, due to their easily-accessible localization and their involvement in essential signaling pathways, often dysregulated in cancer. Despite their clinical appeal, cell-surface proteins are often underrepresented in standard proteomic data sets, due to their poor solubility and lower expression levels compared to intracellular proteins.

Areas covered: This review will summarize some of the available techniques for enriching the cell-surface proteome, and discuss their advantages, limitations and applicability to clinical sample-testing. Moreover, we discuss currently available strategies for the development of novel targeted therapies in cancer.

Expert commentary: The interest in elucidating the cancer-associated surfaceome is growing and will likely benefit from recent advancements in instrument sensitivity, method development, and a growing body of high-quality proteomics databases. Multiomics studies, in combination with functional validations (e.g. dropout screens), and evaluation of the healthy surfaceome, will likely aid in the selection of relevant targets for future therapy development.     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

泛素化途径与细胞周期的关系

泛素化途径(the ubiquitin pathway)是一种有高度选择性的蛋白水解途径,是细胞周期调控的基础。本文主要论述了依赖SCF(skp-cullin-F-boxprotein)和APC／C(anaphase-promoting complexor cyclosome)的两种泛素化途径对细胞周期不同时期的调控作用及其研究进展。