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Results for “target validation”

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PubMed2003

Development and assessment of a quantitative reverse transcription-PCR assay for simultaneous measurement of four amplicons.

BACKGROUND: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. METHODS: We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl(2), and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. RESULTS: The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log(10) cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of -3.322. CONCLUSIONS: This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.

DNA Repair↗

PubMed2007

Transgenic animal models in biomedical research.

Transgenic animals have become a key tool in functional genomics to generate models for human diseases and validate new drugs. Transgenesis includes the addition of foreign genetic information to animals and specific inhibition of endogenous gene expression. Recently, animal models provided novel insight and significantly improved our understanding of the initiation and perpetuation of human diseases. Moreover, they are an invaluable tool for target discovery, validation, and production of therapeutic proteins. However, despite the generation of several transgenic and knockout models, obtaining relevant models still faces several theoretical and technical challenges. Indeed, genes of interest are not always available and gene addition or inactivation sometimes does not allow clear conclusions because of the intrinsic complexity of living organisms or the redundancy of some metabolic pathways. In addition to homologous recombination, endogenous gene expression can be specifically inhibited using several mechanisms such as RNA interference. Here, some animal models are described to illustrate their importance in biomedical research. Moreover, guidelines for generation of these animals are presented.

Animals↗

PubMed1999

Telomerase as an anti-cancer target: current status and future prospects.

As is common with a newly discovered cancer-associated gene/protein, there is a lag between the elucidation of its cellular and molecular biology and appropriate therapeutic intervention. Telomerase represents an interesting and promising anticancer drug target but poses a particular drug discovery challenge. It is unclear at present what is the optimum means of targeting this complex ribonucleoprotein and associated telomeric DNA and binding proteins: various strategies are actively being explored. Some recent data (e.g. 2-5A antisense against telomeric RNA, targeting TRF2, introduction of dominant-negative hTERT into cells) has raised doubts over the previously presumption of a requirement for prolonged enzyme inhibition with gradual telomere erosion, especially in tumour cells with relatively short telomeres. Highly potent and selective in vivo inhibitors are required to validate the target and address these critical issues.

Animals↗

PubMed2003

Receptor activator of NF-kappaB ligand, macrophage inflammatory protein-1alpha, and the proteasome: novel therapeutic targets in myeloma.

BACKGROUND: The bone destruction in myeloma patients is largely responsible for the clinical features of the disease. However, only recently has attention focused on identifying and developing drugs targeted specifically at the osteolysis. Receptor activator of NF-kappaB ligand (RANKL), macrophage inflammatory protein (MIP)-1alpha, and proteasomal function have been implicated in the pathogenesis of myeloma and associated bone disease. We provide "proof of principle" in preclinical myeloma models that these are indeed valid molecular targets in development of novel therapeutics. METHODS: The efficacy of antagonists of RANKL and MIP-1alpha bioactivities (RANK.Fc and neutralizing monoclonal anti-MIP-1alpha antibody) in ameliorating osteolysis and reducing tumor burden was evaluated in a mouse model in which murine myeloma 5TGM1 cells are injected intravenously into syngeneic mice. In addition, the activity of a petidyl aldehyde proteasome inhibitor (proteasome inhibitor-1 [PSI]) on tumor growth was tested in a murine 5TGM1 plasmacytoma model and in mice intravenously inoculated with 5TGM1 cells. RESULTS: RANK.Fc and anti-MIP-1alpha antibody inhibited the development and progression of osteolytic lesions and significantly reduced tumor load assessed by serum monoclonal paraprotein titers. Intratumoral injections of PSI inhibited growth of 5TGM1 plasmacytomas and induced tumor regression in some cases. In addition, systemic administration of PSI significantly prolonged time to onset of paraplegia in tumor-bearing mice. CONCLUSIONS: The results highlight the critical roles of RANKL and MIP-1alpha in the development and progression of myeloma and provide a basis for future evaluation in myeloma patients of novel therapeutics that disrupt interactions of RANKL and MIP-1alpha with their cognate receptors. The data also suggest that further studies in preclincal myeloma models aimed at identifying other proteasome inhibitors with antitumor efficacy would be worthwhile.

Animals↗

PubMed2006

Antiviral drug discovery targeting to viral proteases.

Proteases fulfill multiple roles in health and disease, and considerable interest has been expressed in the design and development of synthetic inhibitors of disease-related proteases. Virus-encoded proteases have been shown to be involved in the replication of many viruses. The success of anti-HIV-1 therapy using specific and potent protease inhibitors suggests that viral proteases can be the valid molecular targets for the development of antiviral drugs against other viruses. Intensive genetic and biochemical studies have been conducted on viral proteases and new insights and results are rapidly emerging. This work reviews features of viral proteases with respective to the development of effective antiviral therapy.

Antiviral Agents↗

PubMed2006

The topoisomerase II-Hsp90 complex: a new chemotherapeutic target?

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIalpha-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

Adenocarcinoma↗

PubMed2026

Untargeted-targeted metabolomics: energy metabolism characteristics in heart failure staging and discovery of novel biomarkers.

BACKGROUND: Heart Failure represents the severe stage of various heart diseases. Its global morbidity and mortality are on the rise, making it a serious public health issue that imposes a heavy burden on patients' families and society. Currently, there are relatively few systematic studies on the changes in specific metabolites and pathways in different stages of heart failure, such as Stage A, Stage B and Stage C. AIMS: Using untargeted-targeted metabolomics to explore the metabolic characteristics of Heart Failure, and screen out serum metabolic markers with potential diagnostic and prognostic value. METHODS: This study is a cross-sectional study. A total of 210 heart failure patients from Xiyuan Hospital of China Academy of Chinese Medical Sciences were enrolled between October 2023 and October 2024. Among them, 60 patients were selected for targeted metabolomics analysis via stratified sampling. Serum samples of the patients were collected and pretreated with methanol, then metabolites were detected using untargeted and targeted LC-MS respectively. After the raw data were processed with MSDIAL, pattern recognition was performed using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Differential metabolites with variable importance in projection (VIP) > 1 and P < 0.05 were screened, and relevant pathways were analyzed via enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. RESULTS: Untargeted metabolomics revealed that, compared with patients in Stages A and B, those with heart failure in Stage C had decreased serum levels of alanine, creatine, and branched-chain amino acids, along with increased levels of citric acid, fumaric acid, and malic acid. The differential metabolites were primarily enriched in pathways including the citric acid cycle, central carbon metabolism, and amino acid metabolism, indicating that energy metabolism plays a crucial role in the occurrence and progression of HF. Targeted metabolomics validated the findings from untargeted metabolomics: compared with Stage A, the level of phosphoenolpyruvate in Stage B was reduced; and in comparison with patients in Stage A or B, patients in Stage C showed decreased serum levels of multiple energy metabolites (e.g., glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglyceric acid, AMP, ADP and ATP) as well as increased levels of malic acid, which is consistent with the characteristics of the "hypermetabolism-energy starvation" paradox. CONCLUSION: Stage C of heart failure is characterized by energy metabolism collapse (decreased ATP and TCA compensation), and differential metabolites (such as malic acid) may serve as potential candidate biomarkers pending longitudinal validation.

Humans↗

PubMed2000

Acetylcholine affects the spatial scale of attention: evidence from Alzheimer's disease.

Location precues were used to manipulate the spatial scale of attention in visual search for a target in an array of letters in patients with dementia of the Alzheimer type (DAT) and in age-matched older controls. Cue size varied in the amount of spatial precision conferred. Scopolamine, a muscarinic antagonist, decreased overall arousal and broadened spatial attention after a precise precue (small and valid) to target location for DAT patients but not for controls, suggesting a selective effect for attentional impairment induced by cholinergic blockade. In contrast, physostigmine, a cholinesterase inhibitor, did not alter the distribution of spatial attention relative to no-drug baseline testing for patients. Results support a differential role for cholinergic mechanisms in the modulation of the spatial scale of visual attention.

Acetylcholine↗

PubMed2004

The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing.

MicroRNAs (miRNAs) are 21-24 nucleotides long molecules processed from imperfect double-stranded RNAs (dsRNAs). They regulate gene expression by targeting complementary mRNA for cleavage or interfering with their translation. In Arabidopsis, point mutations in or short truncations of the nuclear DICER-LIKE1 (DCL1) or HEN1 protein reduce miRNA accumulation and increase uncleaved target mRNAs accumulation, resulting in developmental abnormalities. Here, we show that miRNA accumulation also depends on the activity of HYL1, a nuclear dsRNA binding protein. hyl1 mutants exhibit developmental defects overlapping with that of dcl1 and hen1 mutants, suggesting that DCL1, HEN1, and HYL1 act together in the nucleus. We validate additional target mRNAs and show that reduced miRNA accumulation in hyl1 correlates with an increased accumulation of uncleaved target mRNAs, including meristem- and auxin-related genes, providing clues for the developmental abnormalities of hyl1 and for the previous identification of hyl1 as a mutant with altered responses to phytohormones. Lastly, we show that posttranscriptional transgene silencing occurs in hyl1, suggesting that HYL1 has specialized function in the plant miRNA pathway, whereas the HYL1-related RDE-4 and R2D2 proteins associate with DICER in the cytoplasm and act in the RNAi pathway in C. elegans and Drosophila, respectively.

Arabidopsis↗

PubMed2003

The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen.

Aspergillus fumigatus causes invasive aspergillosis, a mycosis that is usually fatal in immunocompromised patients. Functional genomics in this fungus will aid the discovery of novel antifungal drugs to treat invasive aspergillosis. However, there is still a need for appropriate molecular genetic tools to facilitate such functional studies. Here, we describe the use of a conditional gene expression system allowing the identification of novel therapeutic targets through validation of essential genes in A. fumigatus. This system is based on the capacity of the Aspergillus nidulans alcA promoter (alcA(p)) to tightly regulate gene expression in this fungus. Conditionally regulated gene expression in A. fumigatus was demonstrated by transcriptional and phenotypic analyses of strains expressing a nuclear migration gene with a terminal phenotype, the A. fumigatus nudC gene, under control of this promoter. This conditional expression system, the first one described in A. fumigatus, will also be useful for investigating the function of essential genes by altering the threonine/glucose ratio in the growth medium.

Alcohol Dehydrogenase↗

PubMed2006

Targeting epidermal growth factor receptor signaling in the treatment of head and neck cancer.

In this review, key aspects of epidermal growth factor receptor (EGFR) biology and the fruitful translation of these fundamental findings into recent treatment advances in head and neck squamous cell cancer (HNSCC) are highlighted. In contrast to a number of contemporary reviews of the EGFR, many of which focus on colorectal and nonsmall cell lung cancer, this review discusses the EGFR as a validated therapeutic target in HNSCC. Recent data confirm a survival advantage for the addition of the anti-EGFR monoclonal antibody cetuximab to definitive radiation therapy in locoregionally advanced HNSCC patients, as well as palliative benefits for patients with incurable recurrent and metastatic HNSCC. Small-molecule EGFR tyrosine kinase inhibitors also show considerable promise in this disease, both alone and in combination with radiation and chemotherapy. Both classes of anti-EGFR agent are generally well tolerated, with side effects (notably skin rash) that are distinct from the toxicities of conventional chemotherapy. Ongoing clinical trials will more clearly define the role for EGFR inhibitors in all treatment phases of HNSCC.

Drug Delivery Systems↗

PubMed2005

Chromatin immunoprecipitation (ChIP) on chip experiments uncover a widespread distribution of NF-Y binding CCAAT sites outside of core promoters.

The CCAAT box is a prototypical promoter element, almost invariably found between -60 and -100 upstream of the major transcription start site. It is bound and activated by the histone fold trimer NF-Y. We performed chromatin immunoprecipitation (ChIP) on chip experiments on two different CpG islands arrays using chromatin from hepatic HepG2 and pre-B cell leukemia NALM-6 cell lines, with different protocols of probe preparation and labeling. We analyzed and classified 239 known or predicted targets; we validated several by conventional ChIPs with anti-YB and anti-YC antibodies, in vitro EMSAs, and ChIP scanning. The importance of NF-Y binding for gene expression was verified by the use of a dominant negative NF-YA mutant. All but four genes are new NF-Y targets, falling into different functional categories. This analysis reinforces the notion that NF-Y is an important regulator of cell growth, and novel unexpected findings emerged from this unbiased approach. (i) A remarkable proportion of NF-Y targets, 40%, are complex transcriptional units composed of divergent, convergent, and tandem promoters. (ii) 40-50% of NF-Y sites are not in core promoters but are in introns or at distant 3' or 5' locations. The abundance of "unorthodox" CCAAT positions highlights an unexpected complexity of the NF-Y-mediated transcriptional network.

Amino Acid Sequence↗

PubMed2002

Discovery of compounds that will prevent tau pathology.

Tau is certainly a reasonable target for the development of compounds to prevent neurofibrillary pathology, particularly in the fronto-temporal dementias. Although the mechanism of the filamentous accumulations remains unclear, sufficient knowledge is in place to move forward with high throughput screens. In fact, the development of compounds from such screens will ultimately be the only way to validate the target. The dichotomy for such screens is that in vitro screens are easier to design, but require more assumptions as to the mechanism, in contrast to cell-based screens that are more difficult to design, but make fewer assumptions about mechanism. We have designed a moderate throughput for tau binding that relies on fluorescence detection in living cells and an in vitro cdk5/p25 tau phosphorylation high throughput screen.

Animals↗

PubMed1999

Alternative strategies in drug development: clinical pharmacological aspects.

Due to the continuous increase in time and cost of drug development and the considerable amount of resources required by the traditional approach, companies can no longer afford to continue to late phase 3 with drugs which are unlikely to be therapeutically effective. The future challenge must be for the pharmaceutical industry to slash its research and development costs by achieving a significant cut in the attrition rate for drugs entering preclinical and clinical development, and to reduce the development time and to increase the probability of success in later clinical trials by streamlining the development processes. In the 100 years to 1995, the pharmaceutical industry worked on about 500 targets with a limited number of compounds, whereas now, using new technologies like genomics, high throughput screening and combinatorial chemistry, drug companies will see an explosion in the number of targets and leads it can explore. Therefore, a tough selection process for picking candidate compounds out of research and a quick kill process for the candidate, which does not measure up in advanced trials, is mandatory to avoid wasting time, energy and money. To improve the transition from research to development it is necessary to validate new targets, define success criteria for research, integrate bioinformation at every stage in drug discovery, define prerequisites for development, identify the "losers" and select the "winners" early and concentrate efforts on them, and to automate the research and development (R&D) process to optimize resource requirements versus time lines and to ensure effective flow of information from drug discovery to late phase of development. In drug development a deeper understanding of a drugs' action is necessary from animal models and phase I, IIa studies prior to taking the drug further in development. Instead of moving from discovery thorough development phases in sequential steps, drug development should be streamlined combining preclinical and early clinical development as an exploratory stage and phases IIb, III as a confirmatory stage. Preclinical and clinical-pharmacological studies in the exploratory stage of drug development should be designed for decision making in contrast to later clinical trials that require power for proof-of-safety and efficacy. Strategies to improve the quality of decisions in drug development are: the use and integration of new tools and technologies such as pharmacogenomics to improve our knowledge about the origin of the disease and to identify new therapeutic strategies; modelling and simulation of preclinical and clinical trials to bridge the gap between the early stages of the development of a new drug and its potential effects in humans; more sophisticated clinical pharmacokinetics to answer the question if the drug is present at the disease site for a sufficient time and to provide information on concentration-effect-relationships; selecting and evaluating surrogates/biomarkers for safety and efficacy; involvement of the target population as soon as possible; using information technologies to make better use of existing data. The more thorough and profound studies have been carried out during this exploratory stage of development, the earlier a decision can be made on the continuation or discontinuation of further development, thus saving development time and money and assessing and considerably reducing the risk for the patients and increasing the success-rate of the project in the later confirmatory effectiveness trial. Taking responsibility as the link between research and development gives clinical pharmacology a major opportunity to assume a pivotal role in research and development of new drugs. To reach this goal, clinical pharmacology must be fully integrated in the whole process from the candidate selection to its approval.

Chemistry, Pharmaceutical↗

PubMed2005

Mining nematode genome data for novel drug targets.

Expressed sequence tag projects have currently produced over 400 000 partial gene sequences from more than 30 nematode species and the full genomic sequences of selected nematodes are being determined. In addition, functional analyses in the model nematode Caenorhabditis elegans have addressed the role of almost all genes predicted by the genome sequence. This recent explosion in the amount of available nematode DNA sequences, coupled with new gene function data, provides an unprecedented opportunity to identify pre-validated drug targets through efficient mining of nematode genomic databases. This article describes the various information sources available and strategies that can expedite this process.

Animals↗

PubMed2004

A rational approach to maximize success rate in target discovery.

To overcome the problem of high attrition rates in the drug discovery process, an efficient strategy how to identify, select, characterize and validate the most suitable drug targets before embarking on the resource-intense steps of lead discovery and lead optimization is mandatory. We have implemented such an efficient strategy consisting of (i) Target Selection based on gene expression analyses of drugable target genes in clinical samples and relevant in vitro model systems, to identify candidate targets with a specific tissue distribution and presence in human disease; (ii) Target Assessment exploiting the three-dimensional structure of proteins for detailed binding site analysis, to estimate the drugability of the protein for small-molecule inhibitor binding as well as selectivity profiles; and (iii) Target Validation providing evidence for a functional role in in vitro model systems, thus corroborating the biological hypothesis underlying the therapeutic concept. This rational approach has led to the discovery of drug targets for Lead Discovery, maximizing the likelihood for achieving target-selective inhibition by small-molecule inhibitors with minimal in vivo side effects and a therapeutic effect based on a sound biological hypothesis.

Binding Sites↗

PubMed2006

Hypervigilance to learned pain signals: a componential analysis.

UNLABELLED: We report 2 experiments investigating hypervigilance to pain signals in healthy individuals. In order to allow a detailed analysis, we decomposed attention in 3 different processes: 1) initial shifting, 2) engagement, and 3) disengagement. We used a spatial cueing paradigm in which the location of targets is validly or invalidly predicted by spatial cues. In the first experiment, cues were differentially conditioned to create pain signals, allowing the investigation of engagement and disengagement of attention. Because this procedure does not allow an adequate isolation of the shift component, we introduced a new adaptation of the spatial cueing paradigm in the second experiment, in which targets instead of cues were differentially conditioned. We replicated previous findings, showing enhanced engagement to and retarded disengagement from pain signals compared to control signals. In addition, we demonstrated that participants were still hypervigilant to pain signals after extinction. We were not able to demonstrate speeded shifting to pain signals. Instead we found a generally faster detection of pain signals compared to control signals. We conclude that hypervilance to pain signals does not emerge as rapid initial shifting to the pain signal but rather as enhanced processing once it is detected. Theoretical and clinical implications of these findings are discussed. PERSPECTIVE: We investigated the fundamental processes associated with hypervigilance to pain. Our findings suggest that treatment approaches using attention techniques should not focus on preventing patients to shift their attention to the pain but rather on diminishing the threat value and learning to disengage from their pain.

Adolescent↗

PubMed2006

ADOA3R as a therapeutic target in experimental colitis: proof by validated high-density oligonucleotide microarray analysis.

Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation ( 90% of genes tested (33 of 37 genes). We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

Adenosine↗