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Conditional mutagenesis of G-protein coupled receptors and G-proteins.

The G-protein-coupled receptor signaling system, consisting of a huge variety of receptors as well as of many G-proteins and effectors, operates in every cell and is involved in many physiological and pathological processes. The versatility of this system and the involvement of specific components makes G-protein-coupled receptors and their signaling pathways ideal targets for pharmacological interventions. Classical mouse knockout models have often provided important preliminary insights into the biological roles of individual receptors and signaling pathways and they are routinely used in the process of target validation. The recent development of efficient conditional mutagenesis techniques now allows a much more detailed analysis of G-protein-mediated signaling transduction processes. This review summarizes some of the areas in which progress has recently been made by applying conditional mutagenesis of genes coding for G-proteins and G-protein-coupled receptors.

Animals↗

Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions.

Gangliosides GD3, GD2 and GM2, which are the major gangliosides expressed on most human cancers of neuroectodermal and epithelial origin, have been focused on as effective targets for passive immunotherapy with monoclonal antibodies. We previously developed a chimeric anti-GD3 mAb, KM871, and a humanized anti-GM2 mAb, KM8969, which specifically bound to the respective antigen with high affinity and showed potent immune effector functions. Humanization of anti-ganglioside antibody is expected to enhance its use for human cancer therapy. In the present study, we generated a chimeric anti-GD2 mAb, KM1138, and further developed the humanized form of anti-GD2 and anti-GD3 mAbs by the complementarity-determining regions grafting method. The resultant humanized anti-GD2 mAb, KM8138, and anti-GD3 mAb, KM8871, showed binding affinity and specificity similar to those of their chimeric counterparts. In addition, both humanized mAbs had functional potency comparable to the chimeric mAbs in mediating the immune effector functions, consisting of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The production of these humanized anti-ganglioside mAbs, with potent effector functions and low immunogenicity, precedes the evaluation of the therapeutic value of anti-ganglioside mAbs in passive immunotherapy and the target validation for ganglioside-based vaccine therapy.

Amino Acid Sequence↗

Transforming growth factor-beta stimulates IL-1beta-induced monocyte chemoattractant protein-1 expression in human synovial cells via the ERK/AP-1 pathway.

OBJECTIVE AND DESIGN: Transforming growth factor- beta (TGF-beta) has not only a fibrogenic role, but also monocyte/ macrophage chemotactic properties in a synovial joint. However, little is known about the effects of TGF-beta on monocyte chemoattractant protein-1 (MCP-1) expression in human synovial cells under inflammatory status. The aim of this study was to determine whether TGF-modulates MCP-1 production under the chronic inflammation, and to elucidate the cell signaling mechanism involved. MATERIALS AND METHODS: Human synovial cells were exposed to IL-1beta, which mimics the environment of chronic inflammation. Production of MCP-1 protein and expression of MCP-1 mRNA were determined by ELISA and real-time PCR. RESULTS: TGF-beta upregulated the expression of MCP-1 mRNA and protein with or without IL-1beta. TGF-beta and IL-1beta synergistically enhanced MCP-1 gene expression, and an AP-1 binding site was involved in the signal transduction. In addition, MEK inhibitor completely suppressed TGF-beta-induced MCP-1 production. CONCLUSIONS: TGF-beta and IL-1beta synergistically enhance MCP-1 gene expression through the activation of the MEK/ERK1/2 pathways, which leads to AP-1 activation. The impairment of MCP-1 regulation by TGF-beta in resident synovial cells might represent an important mechanism of chronic inflammation and tissue fibrosis in a synovial joint. MCP-1 should be considered a valid target for therapeutic intervention.

Cells, Cultured↗

Aptamers--basic research, drug development, and clinical applications.

Since its discovery in the early 1990s, aptamer technology has progressed tremendously. Automated selection procedures now allow rapid identification of DNA and RNA sequences that can target a broad range of extra- and intracellular proteins with nanomolar affinities and high specificities. The unique binding properties of nucleic acids, which are amenable to various modifications, make aptamers perfectly suitable for different areas of biotechnology. Moreover, the approval of an aptamer for vascular endothelial growth factor by the US Food and Drug Administration highlights the potential of aptamers for therapeutic applications. This review summarizes recent developments and demonstrates that aptamers are valuable tools for diagnostics, purification processes, target validation, drug discovery, and even therapeutic approaches.

Animals↗

Disordered metabolic response with cancer and its management.

The metabolic changes in cancer cachexia appear to be mediated by a complex network of proinflammatory cytokines, neuroendocrine hormones, neurotransmitters, eicosanoids, and tumor-derived factors produced by the body in response to the tumor and by the tumor itself. The relative importance of these various mediator pathways in relation to either the anorexia or hypermetabolism associated with weight loss in cancer remains to be fully elucidated. Our current understanding suggests that proinflammatory mediators may indeed be a valid target for therapeutic intervention. Trials attempting to modify the inflammatory response to cancer with the hope of improving appetite and reversing catabolism and perhaps thereby improving qualityof life and survival are currently underway.

Animals↗

Cytotoxic activity of novel human monoclonal antibody MT201 against primary ovarian tumor cells.

PURPOSE: The epithelial cell adhesion molecule (Ep-CAM) is a clinically validated target for antibody-based therapy of cancer. The aim of this work was to evaluate the specific cytotoxic activity of a novel fully human Ep-CAM-specific IgG1 antibody, called MT201, against primary ovarian tumor cells and an ovarian tumor cell line. METHODS: The anti-tumor efficacy of MT201 was examined both in coculture of the ovarian cancer cell line OvCAR-3 and peripheral blood mononuclear cells (PBMCs) from healthy donors, and in primary metastatic tumor specimens freshly dissected from 21 patients with ovarian cancer using only the tumor-resident autologous effector cells. The extent of tumor cell depletion was determined by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM labeling, both combined with propidium iodide uptake as cell lysis marker. RESULTS: MT201 at sub- micro g/ml concentrations effectively eliminated OvCar-3 cells in the presence of PBMC. In freshly dissected tumor specimen, endogenous autologous immune cells could lyse, in a MT201-dependent fashion, Ep-CAM-positive tumor cells in 17 out of 21 patients showing an ex vivo response rate of 81%. In certain samples, up to 80% lysis of Ep-CAM-positive tumor cells by MT201 were observed after 16-30 h of incubation. CONCLUSIONS: These data indicate that MT201 can effectively redirect tumor-resident effector cells against Ep-CAM-positive ovarian cancer cells and may therefore offer an effective therapy for ovarian cancer.

Antibodies, Monoclonal↗

A novel multiplex RT-PCR system detects human endogenous retrovirus-K in breast cancer.

Human endogenous retrovirus HERV-K like-sequences have been implicated in certain cancers. We developed a novel multiplex RT-PCR system for HERV-K that yielded a 533 bp product together with a smaller sized product (319 bp) of the house keeping gene, histidyl tRNA synthetase (HtRNAS). The latter spanned an intron that also served to validate target cDNA. PCR amplicons of HERV-K and HtRNAS were visualised using a gel documentation system and the pixel intensity used to derive semi-quantitative levels of viral expression. Our data showed that HERV-K10 was significantly elevated in MCF-7 cells treated with estrogen. Interestingly, HERV-K expression was higher in MCF-7 cells selected with adriamycin. RT-PCR combined with Southern blotting also detected HERV-K from breast cancer tissue using laser capture microscopy. This study highlights the presence of HERV-K in the breast cancer cell lines MCF-7 and MCF-7 ADR and confirms HERV-K10 transcripts in the cell line T47D. We believe this study to be a novel approach in determining levels of HERV-K expression and for detecting this virus in cancer cell lines and tissues.

Breast Neoplasms↗

Visual attention in Huntington's disease: the effect of cueing on saccade latencies and manual reaction times.

Studies of eye movements in patients with Huntington's disease (HD) have suggested that frontal lobe-basal ganglia structures are more involved in HD than the parietal lobes. To test this hypothesis further we compared the ability of HD patients and normal subjects to direct "covert" visual attention, using saccade latency and thumb press reaction time tasks that have been shown to be sensitive to parietal lobe dysfunction. Subjects were instructed to move their eyes or to press a button when a peripheral target was illuminated. The peripheral stimulus appeared at various intervals after the appearance of a central arrow(s) that pointed in the direction of the target (valid cue), in the opposite direction (invalid cue), or pointed simultaneously in both directions (neutral cue). For both saccade and thumb press paradigms, the difference in the latencies for trials with invalid and valid cues was the same in HD patients and normals. These findings suggest that the ability to direct visual attention is normal in HD and are compatible with the hypothesis that in HD, frontal-basal-ganglia circuits are more affected than parietal lobe pathways.

Attention↗

The advantage of using positron emission tomography in drug research.

A large problem in drug discovery is to find relevant in vitro or in vivo animal models and to be able to extrapolate the results obtained to humans. Drug research now benefits from the fast development of imaging technologies that trace radiolabelled molecules directly in the human brain. Positron emission tomography (PET) and allied techniques use molecules that are labelled with short-lived radioisotopes and injected intravenously. The most straightforward approach is to radiolabel a new potential drug and then to trace its anatomical distribution and binding in the brain. An indirect approach is to study how the unlabelled drug inhibits specific radioligand binding. The demonstration of quantitative relationships between drug binding in vivo and drug effects in patients is used to validate targets for drug action and to optimize clinical treatment.

Antipsychotic Agents↗

Sphingolipid players in the leukemia arena.

Sphingolipids function as bioactive mediators of different cellular processes, mostly proliferation, survival, differentiation and apoptosis, besides being structural components of cellular membranes. Involvement of sphingolipid metabolism in cancerogenesis was demonstrated in solid tumors as well as in hematological malignancies. Herein, we describe the main biological and clinical aspects of leukemias and summarize data regarding sphingolipids as mediators of apoptosis triggered in response to anti-leukemic agents and synthetic analogs as inducers of cell death as well. We also report the contribution of molecules that modulate sphingolipid metabolism to development of encouraging strategies for leukemia treatment. Finally we address how deregulation of sphingolipid metabolism is associated to occurrence of therapy resistance both in vitro and in vivo. Sphingolipids can be considered promising therapeutic tools alone or in combination with other compounds, as well as valid targets in the attempt to eradicate leukemia and overcome drug resistance.

Antineoplastic Agents↗

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active.

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.

Binding Sites↗

Performance of F2 B6x129 hybrid mice in the Morris water maze, latent inhibition and prepulse inhibition paradigms: Comparison with C57Bl/6J and 129sv inbred mice.

Assessment of cognition and information processing in mice is an important tool in preclinical research that focuses on the development of cognitive enhancing drugs. Analysis of transgenic (TG) and knockout (KO) mice is usually performed on a F2 B6x 129 background. In the present study, we have compared performance of F2 B6x 129 hybrid mice (F2 mice) with that of the two parental inbred strains (C57Bl/6J and 129sv mice), and a wild-type (WT) strain (with a combined B6x 129 background) in three cognitive/information processing paradigms. It was found that the F2 mice outperformed either of the parental strains and provide a control sample with good baseline performance in the Morris water maze (MWM). Reliable deficits could be obtained in learning and memory in this paradigm following injections with scopolamine (0.16 mg/kg) in the F2 mice, which can potentially be used to test effects of reference and novel compounds in order to develop cognitive enhancing drugs. Furthermore, it was shown that the four genotypes showed normal latent inhibition (LI) using the conditioned taste aversion (CTA) paradigm and exhibited no differences in prepulse inhibition (PPI) levels. Following the setup of these procedures in mice, we are now able to compare the effects of gene knockout/mutations used for target validation with results in the present study as a frame of reference.

Animals↗

Cationic liposome-mediated delivery of siRNAs in adult mice.

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.

Animals↗

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates.

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.

Amidophosphoribosyltransferase↗

Mining the NCI antiviral compounds for HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is an essential enzyme for effective viral replication and is a validated target for the development of antiretroviral drugs. Currently, there are no approved drugs targeting this enzyme. In this study, we have identified 11 structurally diverse small-molecule inhibitors of IN. These compounds have been selected by mining the moderately active antiviral molecules from a collection of 90,000 compounds screened by the National Cancer Institute (NCI) Antiviral Program. These compounds, which were screened at the NCI during the past 20 years, resulted in approximately 4000 compounds labeled as 'moderately active.' In our study, chalcone 11 shows the most potent activity with an IC(50) of 2+/-1 microM against purified IN in the presence of both Mn(2+) and Mg(2+) as cofactors. Docking simulations using the 11 identified inhibitors as a training set have elucidated two unique binding areas within the active site: the first encompasses the conserved D64-D116-E152 motif, while the other involves the flexible loop region formed by amino acid residues 140-149. The tested inhibitors exhibit favorable interactions with important amino acid residues through van der Waals and H-bonding contacts.

Anti-HIV Agents↗

Synthesis of selective SRPK-1 inhibitors: novel tricyclic quinoxaline derivatives.

SR protein-specific kinase-1 (SRPK-1) has been identified as a validated target for hepatitis B virus (HBV). A series of novel tricyclic quinoxaline derivatives was designed and synthesised as potential kinase inhibitory antiviral agents and was found to be active and selective for SRPK-1 kinase. Most of these novel compounds have drug-like properties according to experimentally determined LogP and LogS values.

Antiviral Agents↗

In silico fragment-based discovery of DPP-IV S1 pocket binders.

Dipeptidyl peptidase IV is a clinically validated target for type-2 diabetes and belongs to a family of peptidases with a quite unique post-proline cleavage specificity. Known inhibitors contain a limited number of molecular anchors occupying the small prototypical S1 pocket. A virtual screening approach for such S1-binding fragments was carried out using FlexX docking to evaluate its potential to confirm known and find novel compounds. Several low molecular weight inhibitors exhibiting activities in the micromolar range could be identified as starting points for structure-based design.

Binding Sites↗

Triketoacid inhibitors of HIV-integrase: a new chemotype useful for probing the integrase pharmacophore.

Integrase is one of three enzymes expressed by HIV and represents a validated target for therapy. This study reports on the discovery of a new triketoacid-based chemotype that selectively inhibits the strand transfer reaction of HIV-integrase. SAR studies showed that the template binds to integrase in a manner similar to the diketoacid-based inhibitors. Moreover, comparison of the new chemotype to two different diketoacid templates led us to propose two aryl-binding domains in the inhibitor binding site. This information was used to design a new diketoacid template with improved activity against the enzyme.

HIV Integrase↗