Membrane signalling complexes: implications for development of functionally selective ligands modulating heptahelical receptor signalling

Technological development has considerably changed the way in which we evaluate drug efficacy and has led to a conceptual revolution in pharmacological theory. In particular, molecular resolution assays have revealed that heptahelical receptors may adopt multiple active conformations with unique signalling properties. It is therefore becoming widely accepted that ligand ability to stabilize receptor conformations with distinct signalling profiles may allow to direct the stimulus generated by an activated receptor towards a specific signalling pathway. This capacity to induce only a subset of the ensemble of responses regulated by a given receptor has been termed "functional selectivity" (or "stimulus trafficking"), and provides the bases for a highly specific regulation of receptor signalling. Concomitant with these observations, heptahelical receptors have been shown to associate with G proteins and effectors to form multimeric arrays. These complexes are constitutively formed during protein synthesis and are targeted to the cell surface as integral signalling units. Herein we summarize evidence supporting the existence of such constitutive signalling arrays and analyze the possibility that they may constitute viable targets for developing ligands with "functional selectivity".     

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.     

Role of sex hormones in lung cancer

Lung cancer represents the world’s leading cause of cancer deaths. Sex differences in the incidence and mortality rates for various types of lung cancers have been identified, but the biological and endocrine mechanisms implicated in these disparities have not yet been determined. While some cancers such as lung adenocarcinoma are more commonly found among women than men, others like squamous cell carcinoma display the opposite pattern or show no sex differences. Associations of tobacco product use rates, susceptibility to carcinogens, occupational exposures, and indoor and outdoor air pollution have also been linked to differential rates of lung cancer occurrence and mortality between sexes. While roles for sex hormones in other types of cancers affecting women or men have been identified and described, little is known about the influence of sex hormones in lung cancer. One potential mechanism identified to date is the synergism between estrogen and some tobacco compounds, and oncogene mutations, in inducing the expression of metabolic enzymes, leading to enhanced formation of reactive oxygen species and DNA adducts, and subsequent lung carcinogenesis. In this review, we present the literature available regarding sex differences in cancer rates, associations of male and female sex hormones with lung cancer, the influence of exogenous hormone therapy in women, and potential mechanisms mediated by male and female sex hormone receptors in lung carcinogenesis. The influence of biological sex on lung disease has recently been established, thus new research incorporating this variable will shed light on the mechanisms behind the observed disparities in lung cancer rates, and potentially lead to the development of new therapeutics to treat this devastating disease.     

Synthesis and evaluation of cholecystokinin trimers: A multivalent approach to pancreatic cancer detection and treatment

In the quest for novel tools for early detection and treatment of cancer, we propose the use of multimers targeting overexpressed receptors at the cancer cell surface. Indeed, multimers are prone to create multivalent interactions, more potent and specific than their corresponding monovalent versions, thus enabling the potential for early detection. There is a lack of tools for early detection of pancreatic cancer, one of the deadliest forms of cancer, but CCK2-R overexpression on pancreatic cancer cells makes CCK based multimers potential markers for these cells. In this Letter, we describe the synthesis and evaluation of CCK trimers targeting overexpressed CCK2-R.     

A solid-phase method for synthesis of dimeric and trimeric ligands: Identification of potent bivalent ligands of 14-3-3σ

Multivalent protein-protein interactions including bivalent and trivalent interactions play a critical role in mediating a wide range of biological processes. Hence, there is a significant interest in developing molecules that can modulate those signaling pathways mediated by multivalent interactions. For example, multimeric molecules capable of binding to a receptor protein through a multivalent interaction could serve as modulators of such interactions. However, it is challenging to efficiently generate such multimeric ligands. Here, we have developed a facile solid-phase method that allows for the rapid generation of (homo- and hetero-) dimeric and trimeric protein ligands. The feasibility of this strategy was demonstrated by efficiently synthesizing fluorescently-labeled dimeric peptide ligands, which led to dramatically increased binding affinities (~400-fold improvement) relative to a monomeric 14-3-3σ protein ligand.     

Salen and tetrahydrosalen derivatives act as effective inhibitors of the tumor-associated carbonic anhydrase XII—A new scaffold for designing isoform-selective inhibitors

Salen and tetrahydrosalen derivatives possess metal-chelating properties and have been used as ligands in organic synthesis and as scaffolds for developing therapeutic agents. Fourteen such compounds were synthesized in order to explore their ability to inhibit the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Human (h) isoforms hCA I, hCA II, hCA IX and hCA XII were included in the investigation. Several aliphatic and aromatic spacers were introduced between the two chelating groups from salen/tetrahydrosalen in order to explore a diverse chemical space for designing CA inhibitors, which incorporate both phenol and polyamine fragments in their molecule. Some of these compounds showed CA inhibitory activity in the low micromolar–nanomolar range and a pronounced selectivity for inhibiting an isoform over-expressed in hypoxic tumors, hCA XII, over hCA I, II and IX.     

Development of a 3D pharmacophore for nonspecific ligand recognition of alpha1, alpha2, alpha3, alpha5, and alpha6 containing GABA(A)/benzodiazepine receptors

Transfected cells containing GABA(A) benzodiazepine receptors (BDZRs) have been utilized to systematically determine the affinity of ligands at alpha1, alpha2, alpha3, alpha5 and alpha6 subtypes in combination with beta2 and gamma2. All but a few of the ligands thus far studied have relatively high affinities for each of these alpha subtype receptors. Thus, these ligands must contain common stereochemical properties favorable for recognition by each of the subtype combinations. In the present work, such a common three-dimensional (3D) pharmacophore for recognition of alpha1, alpha2, alpha3, alpha5 and alpha6 containing GABA(A)/BDZRs types of receptors has been developed and assessed, using as a database receptor affinities measured in transfected cells for 27 diverse compounds. The 3D-recognition pharmacophore developed consists of three proton accepting groups, a hydrophobic group, and the centroid of an aromatic ring found in a common geometric arrangement in the 19 nonselective ligands used. Three tests were made to assess this pharmacophore: (i) Four low affinity compounds were used as negative controls, (ii) Four high affinity compounds, excluded from the pharmacophore development, were used as compounds for pharmacophore validation, (iii) The 3D pharmacophore was used to search 3D databases. The results of each of these types of assessments provided robust validation of the 3D pharmacophore. This 3D pharmacophore can now be used to discover novel nonselective ligands that could be activation selective at different behavioral end points. Additionally, it may serve as a guide in the design of more selective ligands, by determining if candidate ligands proposed for synthesis conform to this pharmacophore and selecting those that do not for further experimental assessment.     

A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.     

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro-vWF) that consists of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. Previously, we have defined the domains on the pro-vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel-Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full-length vWF cDNA, vWF-containing organelles are encountered in monkey kidney CV-1 cells that are morphologically similar to the endothelial-specific Weibel-Palade bodies. Expression in CV-1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense-cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a 'condensed', multimeric vWF is required for targeting to the Weibel-Palade body.     

Synthesis,antiproliferative and pro-apoptotic activity of 2-phenylindoles

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC₅₀ values of 2.71 μM and 1.86 μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86.     

Evaluation of cytotoxic,antifungal, and larvicidal activities of different bis-sulfonamide Schiff base compounds

Schiff bases (imines or azomethines) are versatile ligands synthesized from the condensation of amino compounds with active carbonyl groups and used for many pharmaceutical and medicinal applications. In our study, we aimed to determine the cytotoxic, antifungal and larvicidal activities of biologically potent bis-sulfonamide Schiff base derivatives that were re-synthesized by us. For this aim, 16 compounds were re-synthesized and tested for their cytotoxic, antifungal and larvicidal properties. Among this series, compounds A1B2 , A1B4 , A4B2 , A4B3 , and A4B4 were shown to have cytotoxic activity against tested cancer lung cell line (A549). The most potent antifungal activity was observed in compounds A2B1 and A2B2 against all fungi. A1B1 showed the strongest larvicidal effect at all concentrations at the 72nd h (100% mortality). These obtained results demonstrate that these type of bis-substituted compounds might be used as biologically potent pharmacophores against different types of diseases.     

ROS-Mediated Antitumor Activity,Apoptosis, and Molecular Docking Studies of Platinum(II) Coordination Complexes Bearing 2-(Diphenylphosphino)pyridine Ligands

Platinum-based drugs have been widely used in cancer treatment. However, their severe side effects have limited their use. So, researchers have been striving to find compounds with fewer side effects and greater efficacy, to overcome these drawbacks. Here, the cytotoxicity of platinum(II) complexes containing 2-(diphenylphosphino)pyridine ligands have been studied on human lung (A549), ovarian (SKOV3), breast (MCF-7) cancer, and normal breast (MCF-10A) cell lines. The most potent compound exhibits a marked cell growth-inhibitory effect against ovarian and lung cancer cells with IC₅₀ values of 9.41 and 5.58 μM, respectively, which were significantly better than that observed for cisplatin (19.02, and 8.64 μM). Additionally, all complexes achieved significantly lower cytotoxicity towards MCF-10A. To investigate the interaction of complexes with DNA, an electrophoresis mobility shift assay was conducted, which indicated that complexes bind to DNA and affect its electrophoretic mobility. An analysis of apoptosis in A549 cells supported the conclusion that they inhibits cell proliferation via induction of apoptosis in a concentration-dependent manner. Molecular docking was also used to investigate the interactions of compounds with different DNA structures. These compounds have the ability to be a suitable pharmaceutical compound with further investigations in the field of cancer research.     

Validation of HB-EGF and amphiregulin as targets for human cancer therapy

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.     

Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation

A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the protein's distribution within living cells.     

Role of extracellular molecular chaperones in the folding of oxidized proteins. Refolding of colloidal thyroglobulin by protein disulfide isomerase and immunoglobulin heavy chain-binding protein

The process of thyroid hormone synthesis, which occurs in the lumen of the thyroid follicles, results from an oxidative reaction leading, as side effects, to the multimerization of thyroglobulin (TG), the prothyroid hormone. Although hormone synthesis is a continuous process, the amount of Tg multimers is relatively constant. Here, we investigated the role of two molecular chaperones, protein disulfide isomerase (PDI) and immunoglobulin heavy chain-binding protein (BiP), present in the follicular lumen, on the multimerization process due to oxidation using both native Tg and its N-terminal domain (NTD). In vitro, PDI decreased multimerization of Tg and even suppressed the formation of NTD multimers. Under the same conditions, BiP was able to bind to Tg and NTD multimers but did not affect the process of multimerization. Associating BiP with PDI did not enhance the ability of PDI to limit the formation of multimers produced by oxidation. However, when BiP and PDI were reacted together with the multimeric forms and for a longer time (48 h), BiP greatly increased the efficiency of PDI. Accordingly, these two molecular chaperones probably act sequentially on the reduction of the intermolecular disulfide bridges. In the thyroid, a similar process may also be effective and participate in limiting the amount of Tg multimers present in the colloid. These results suggest that extracellular molecular chaperones play a similar role to that occurring in the endoplasmic reticulum and, furthermore, take part in the control of multimerization and aggregation of proteins formed by oxidation.     

Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast

Lung cancer is the leading cause of cancer related deaths worldwide. It is necessary to better understand the molecular mechanisms involved in lung cancer in order to develop more effective therapeutics for the treatment of this disease. Recent reports have shown that Wnt signaling pathway is important in a number of cancer types including lung cancer. However, the role of Frizzled-8 (Fzd-8), one of the Frizzled family of receptors for the Wnt ligands, in lung cancer still remains to be elucidated. Here in this study we showed that Fzd-8 was over-expressed in human lung cancer tissue samples and cell lines. To investigate the functional importance of the Fzd-8 over-expression in lung cancer, we used shRNA to knock down Fzd-8 mRNA in lung cancer cells expressing the gene. We observed that Fzd-8 shRNA inhibited cell proliferation along with decreased activity of Wnt pathway in vitro, and also significantly suppressed A549 xenograft model in vivo (p<0.05). Furthermore, we found that knocking down Fzd-8 by shRNA sensitized the lung cancer cells to chemotherapy Taxotere. These data suggest that Fzd-8 is a putative therapeutic target for human lung cancer and over-expression of Fzd-8 may be important for aberrant Wnt activation in lung cancer.     

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

Angio-associated migratory cell protein interacts with epidermal growth factor receptor and enhances proliferation and drug resistance in human non-small cell lung cancer cells

Angio-associated migratory cell protein (AAMP) is expressed in some human cancer cells. Previous studies have shown AAMP high expression predicted poor prognosis. But its biological role in non-small cell lung cancer (NSCLC) cells is still unknown. In our present study, we attempted to explore the functions of AAMP in NSCLC cells. According to our findings, AAMP knockdown inhibited lung cancer cell proliferation and inhibited lung cancer cell tumorigenesis in the mouse xenograft model. Epidermal growth factor receptor (EGFR) is a primary receptor tyrosine kinase (RTK) that promotes proliferation and plays an important role in cancer pathology. We found AAMP interacted with EGFR and enhanced its dimerization and phosphorylation at tyrosine 1173 which activated ERK1/2 in NSCLC cells. In addition, we showed AAMP conferred the lung cancer cells resistance to chemotherapeutic agents such as icotinib and doxorubicin. Taken together, our data indicate that loss of AAMP from NSCLC inhibits tumor growth and elevates drug sensitivity, and these findings have clinical implications to treat NSCLC cancers.     

Evidence for nonrandom alterations in a fraction of the highly repetitive DNA of a eukaryote.

Although the DNA of the red crab Geryon quinquedens has no patent satellites, a large fraction (approximately 40%) is highly repetitive. Treatment of total DNA by Hind III produces fragments comprising 5-6% of the genome. While the sizes of some of these fragments form an arithmetic series based on an 81 bp repeating unit, the amounts of the multimers differ significantly from distributions observed for multimeric series in the DNAs of other eukaryotes. In red crab DNA, the amounts of some of the multimers suggest that they may have undergone as much as four times the divergence as the others. Other data, however, are more compatible with the conclusion that there has been selective amplification of segments of highly repeated DNA which results in the enhanced amount of specific multimers. These results indicate the presence of a nonrandom process in the evolution of the highly repetitive DNA. Selective mutation alone seems insufficient to explain these results.