PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “target validation”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

Viruses as gene delivery vectors: application to gene function, target validation, and assay development.

A Biochemical Pharmacology Discussion Group Conference, was held at the headquarters of the New York Academy of Sciences on December 4, 2001 as part of an ongoing series designed to highlight and review areas important to modern drug development (Figure 1). Briefly introduced by Tom Kost (GlaxoSmithKline) and Michael Lotze (University of Pittsburgh), the focus was on the intersection of genomics, proteomics, and now "viromics." The latter term refers to the use of viruses and viral gene transfer to explore the complexity arising from the vast array of new targets available from the human and murine genomes. Indeed, access to large numbers of genes using viral vectors is a key tool for drug discovery and drug delivery. With 38,000 genes identified within the human genome, only 5000 are considered readily druggable. Generating tools such as these to validate targets represents a major part of the armamentarium of the postgenomic scientist. During the last 12 years alone, there have been over 26,000 publications on virus vectors. Many of them have been found useful in target validation, assay development, and evaluation in in vivo models and gene therapy. Thus, there is now an extensive knowledge base for several viral vectors, with unique attributes within each of them providing versatility, efficiency, and ease of use. The individual scientists presenting at the meeting illustrated many of the unique and useful characteristics of such vector systems including retrovirus, adenovirus, herpes virus, simbis virus, and baculovirus.

Animals↗

RNAi: for functional analysis and target validation.

The third annual conference on discovery on target, organised by the Cambridge Healthtech Institute was held on 19 - 20 October 2005, in Boston. More than 300 delegates from both academic and industrial institutes attended the meeting. The presentations provided insights into understanding the RNA interference technology as a useful tool to identify and validate new targets for therapeutic intervention. Discussions focused in the design of siRNA for effective gene silencing, RNAi screens to identify new targets, RNAi delivery and the in vivo validation of targets using this technology.

Animals↗

Fluorophore-assisted light inactivation: a high-throughput tool for direct target validation of proteins.

To exploit advances in proteomics for drug discovery, high-throughout methods for target validation that directly address the cellular roles of proteins are required. To do this, we have characterized fluorophore-assisted light inactivation (FALI) which uses coherent or diffuse light targeted by fluorescein-labeled probes to inactivate specific proteins. We have shown that it is spatially restricted and tested its efficacy in living cells. FALI is efficient using conventional antibodies and single chain variable fragment phage display antibodies (that are compatible with high-throughput applications). We have shown that singlet oxygen is one of the major components required for FALI-mediated damage. The half-maximal radius of damage is approximately 40 A. FALI causes the specific loss of function of beta 1 integrin in HT-1080 fibrosarcoma cells resulting in a reduction in invasiveness. The efficacy of diffuse light sources (such as a desk lamp) with FALI to inactivate many samples in parallel provides an inexpensive, high-throughput method of wide general applicability for functional proteomics.

Animals↗

Application of antisense oligonucleotides for gene functionalization and target validation.

The Human Genome Project (complete sequencing of the human genome) will be complete soon and the information made available to the biomedical community. Although the project is not yet complete, it has dramatically changed the practice of biomedical sciences. With enormous amounts of information available from sequencing efforts, increasing demands are being put on researchers to quickly determine the biochemical function of novel molecular targets and to validate them as appropriate for drug discovery endeavors. Antisense oligonucleotides are an ideal technology for gene functionalization and target validation. They are an efficient methodology for gene functionalization and target validation and are a proven technology. Antisense technology can answer questions with a high degree of precision and it is a versatile technology. In this review the use of antisense oligonucleotides as a research tool for gene functionalization and target validation is discussed.

Animals↗

Antisense oligonucleotides: a systematic high-throughput approach to target validation and gene function determination.

Antisense technology provides a high-throughput and systematic approach to drug target validation and gene function discovery. In combination with other emerging technologies (such as microarrays), this technology will enable efficient evaluation of the sequence data generated by the Human Genome Project. The authors review recent advances in the antisense field and discuss the potential use of antisense technology for functional genomics.

Journal Article↗

Target validation in silico: does the virtual patient cure the pharma pipeline?

Genomics has multiplied the number of targets for new therapeutic interventions, but this has not yet lead to a marked increase of pharma pipeline outputs. The complexity of protein function in higher order biological systems is often underestimated. Translation from in vitro and in vivo results to the human setting frequently fails due to unforeseen toxicity and efficacy issues. Biosimulation addresses these issues by capturing the complex dynamics of interacting molecules and cells in mechanistic, predictive models. A central concept is that of the virtual patient, an encapsulation of a specific pathophysiological behaviour in a biosimulation model. The authors describe how virtual patients are being used in target identification, target validation and clinical development, and discuss challenges for the acceptance of biosimulation methods.

Computer Simulation↗

Target validation of G-protein coupled receptors.

G-protein coupled receptors (GPCRs) represent possibly the most important target class of proteins for drug discovery. Over 30% of clinically marketed drugs are active at this receptor family. These drugs exhibit their activity at <10% of all known GPCRs. A major challenge for the pharmaceutical industry is to associate the many novel GPCRs with disease to identify the drugs of the future. This process consists of a collection of experimental paradigms that together can be loosely labelled 'target validation'.

Animals↗

Antisense oligonucleotides for target validation and gene function determination.

Antisense technology is attracting attention from the biotechnology and pharmaceutical industries because it provides a high-throughput and systematic approach to drug target validation and gene function discovery. Antisense represents a logical approach to gene function analysis and discovery as it is specific, broadly applicable, and can be designed with minimal information (ie, expressed sequence tags). This technology in combination with other emerging technologies (eg, microarray technology), will enable efficient 'mining' of the sequence data generated by the human genome project. This review addresses recent advances in the antisense field and discusses the potential use of antisense technology for functional genomics approaches.

Journal Article↗

Cross-species studies for target validation.

The completion of the genome sequences of several model organisms and the recent development of high throughput procedures to map genes, expression patterns and interactions is providing a steadily increasing number of candidate target genes. The function of most of these genes still remains unknown. Therefore, there is a growing demand in genetically tractable animal models in which the function of individual factors can be studied in large scale, particularly of those that are thought to segregate with human disorders. In this paper, current methods to validate target gene function and the advantages of different model organisms are compared.

Animals↗

Microchip-based systems for target validation and HTS.

Microarray and microfluidic device technologies for performing genetic and biochemical analyses are revolutionizing biological research. These technologies are now being applied to gene expression profiling and to primary screening for target validation and lead discovery in the pharmaceutical industry. In this article, we briefly review microchip technology and discuss future development trends.

Journal Article↗

Target validation and functional analyses using antisense oligonucleotides.

The human genome project (HGP) has been described as the single most important project in biology and the biomedical sciences to date. In February 2001, the efforts of the HGP resulted in the publication of a 'working draft' of the entire human genome and it is expected that final sequencing and annotation of the genome will be completed by 2003. Researchers are now focusing efforts on the identification of the function of the reported 30,000 human genes. During the past few years, antisense oligomers have been widely used as potent tools for functional genomics and drug target validation. This article describes the emerging and established antisense technologies that will be used to continue the efforts to unlock the function of the human genome and to discover novel drug targets for the treatment of human diseases.

Journal Article↗

Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.

With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.

Actins↗

Validating targets for antiparasite chemotherapy.

The enzymes and receptors in parasites that can be qualified as targets for antiparasite chemotherapy should perform essential functions in the parasites and demonstrate some feasibility for selective inhibition. They can be tentatively identified through detailed analysis of various aspects of metabolisms in the parasites or elucidation of the mechanisms of action among proven antiparasitic agents. Preliminary verifications of these putative targets can be indicated by in vitro antiparasite activity of an inhibitor of the target. However, before a major long-term effort to pursue in-depth structure-activity analysis of the target is to be committed for specific inhibitor design, further validations of the target are essential to insure that future studies are not misguided. One old-fashioned approach to validate a target in the pharmaceutical industry is by correlating target inhibitions with antiparasitic activities among large numbers of drug derivatives. The results are often indicative but hardly ever conclusive. Another method is by comparing the putative drug targets between the drug-sensitive and the drug-resistant parasites for potential discrepancies. Unfortunately, the latter often result from indirect causes, such as reduced drug transport, instead of an alteration of the drug target itself. The third experimental approach is by disrupting the gene encoding the putative target in parasite, which can provide the most conclusive evidence on whether the target plays an indispensible role in the parasite. But special conditions are needed for the gene knockout mutants to survive to exhibit their phenotypes and to allow genetic complementation studies for further verifications. Furthermore, gene knockout experiments are often difficult to perform on cells of multiple ploidy or genes of multiple copies, and are currently applicable only to a limited number of protozoan parasites. In the current article I have tried to take a cursory look at some eleven putative drug targets among various parasites, each supported by well-established antiparasitic agents identified as its inhibitors. I have also considered the evidence for validity of each of them and the potential means of further verifying their validity.

Animals↗

Target validation.

With the publication of draft maps of the human genome and an interim agreement that the human genome comprises approximately 21000 genes, there has been considerable anticipation that many novel disease-specific molecular targets will be rapidly identified and that these will form the basis of many new drug discovery programs. Genes associated with a given disease can thus be identified using genotyping and microarray approaches. However, transitioning from the identification to the subsequent validation and prioritization of their cognate proteins as bona fide drug targets using proteomic techniques--a process that could appropriately be termed targetomics--is still very much in its infancy, with expectations far exceeding present capabilities. The criteria for target validation have yet to be determined and the timing to success has been underestimated. Integrated pharmacological approaches that involve the use of the traditional null hypothesis approach and statistically validated replication have been largely overlooked in the enthusiasm to be the first to find new targets. Inevitably, the only useful measure of target validation occurs when a drug-like molecule, selective for the identified target, is advanced to the clinic where it can be shown to be efficacious in the appropriate human disease state.

Animals↗

Target validation for genomics using peptide-specific phage antibodies: a study of five gene products overexpressed in colorectal cancer.

Genomic approaches are providing a wealth of information on differential gene expression in cancer. To identify the most interesting genes amongst the many identified, high-throughput methods for analysis of genes at the translational level are required. We have used a rapid method for the in vitro selection of antibodies to peptide antigens for the generation of probes to 5 gene products that we have found to be overexpressed in colorectal cancer. The rationale of our study was to select a non-immune phage displayed human antibody library on peptides designed from the coding regions of the gene sequences and to verify whether such antibodies would be suitable probes for the parental protein in immunohistochemical and Western blot analysis. After the generation of a profile of genes overexpressed in primary colorectal cancer (CRC) we selected 5 genes, Ese-3b, Fls353, PBEF, SPARC and Smad5 for a more detailed analysis using phage display-derived antibodies. For these 5 antigens we designed 14-20 amino acid peptides predicted to be exposed on the surface of the parental protein. Selection of a large phage displayed antibody library resulted in specific antibodies for 6 of 8 different peptides with between 2 and 15 different antibodies isolated per peptide. Of 20 antibodies tested, 2 antibodies recognized the putative parental protein from primary CRC tissue. An antibody specific for a PBEF-derived peptide (Fab/PBEF-D4) was shown to recognize a protein product of the expected molecular weight in Western blotting and showed overexpression in n = 6/8 matched tumor/normal protein lysates. Furthermore, in immunohistochemistry this antibody showed restricted staining of the tumor stromal compartment with no detectable staining of epithelial cells. The discovery that PBEF is overexpressed in cancer is unexpected given that the normal function of PBEF is as a cytokine required for the maturation of B cell precursors. We also report on the isolation of an antibody (Fab/SMAD-50) specific for a Smad5-derived peptide that showed cytoplasmic staining of epithelial cells in both CRC tumor and matched normal mucosa. Fab/SMAD-50 also bound to a group of proteins in Western blotting with molecular weights consistent with belonging to the Smad family. These antibodies may be suitable probes for further investigation of the roles of PBEF and Smad5 in cancer. The amenability of phage display to automation suggests that this approach may be developed for implementation on a genomics scale. Indeed, the large-scale generation of antibody probes that can be used to study protein expression in situ would be of great value in target validation for functional genomics.

Amino Acid Sequence↗

Protein interaction mapping for target validation: the need for an integrated combinatory process involving complementary approaches.

Identification, selection, validation and prioritization of targets for therapeutic intervention requires understanding of the biological role of individual proteins in cellular pathways. Unraveling the ways in which proteins interact with each other appears to be crucial in achieving that goal. A number of recently described high-throughput approaches for analyzing cellular protein-protein interactions and previously proposed prediction procedures are compared in this review. The relative advantages of each method are discussed in relation to reproducibility, comprehensiveness and biological significance. It is concluded that only a combination of complementary biochemical technologies supported by reliable algorithms, will provide exhaustive maps of protein interactions for a cellular interactome.

Animals↗

Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

Animals↗