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Results for “target validation”

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PubMed2006

Molecular biology of renal cell cancer and the identification of therapeutic targets.

Renal cell cancer (RCC) is a heterogeneous disease consisting of different histologic types. Major advances have been accomplished during the last 15 years in our understanding of the genetic events that initiate RCC. These advances were greatly facilitated by meticulous clinical description and registration of patients with familial predisposition to RCC. The cloning of the susceptibility genes that underline familial predisposition to RCC has offered entry points into the signaling pathways that are also deregulated in sporadic RCC. Biochemical studies of these signaling pathways and target validation experiments have already culminated in the discovery and clinical application of small molecules with promising activity in RCC. In this article, we highlight the molecular genetic features of RCC that are more directly related to identification and validation of promising targets for molecular therapy.

Carcinoma, Renal Cell↗

PubMed2007

Analysis of gene networks for drug target discovery and validation.

Understanding responses of the cellular system for a dosing molecule is one of the most important problems in pharmacogenomics. In this chapter, we describe computational methods for identifying and validating drug target genes based on the gene networks estimated from microarray gene expression data. We use two types of microarray gene expression data: gene disruptant microarray data and time-course drug response microarray data. For this purpose, the information of gene networks plays an essential role and is unattainable from clustering methods, which are the standard for gene expression analysis. The gene network is estimated from disruptant microarray data by the Bayesian network model, and then the proposed method automatically identifies sets of genes or gene regulatory pathways affected by the drug. We use an actual example from analysis of Saccharomyces cerevisiae gene expression profile data to express a concrete strategy for the application of gene network information toward drug target discovery.

Antifungal Agents↗

PubMed1998

Functional Genomics Conference: From Identifying Proteins to Faster Drug Discovery. March 10-11, 1998, Washington DC, USA.

The massive effort to sequence the human, mouse, rat, nematode (Caenorhabditis elegans), fruit fly (Drosophila), zebra fish, yeast (Saccharomyces cerevisiae), fungal (Candida albicans and Aspergillus fumigatus) and several bacterial genomes has produced a flood of sequence data. Of the more than 100,000 human genes and thousands from other organisms, many partial sequences and several completed microbial genomes are available in both public and private databases. However, elucidation of function has been achieved for only a very small portion and an even smaller percentage have been validated as drug targets. Many companies interested in identifying new drug targets also see this bounty of opportunity as a major challenge. The raw sequence data say little about the importance of the gene and nothing about its potential as a target for drug discovery. Since 1994, a new term, 'functional genomics', has entered our lexicon. Functional genomics, which in effect is 'high-throughput biology', was originally focused on understanding gene function by studying the genes of simpler organisms, such as the nematode, C. elegans. As the genes from a number of organisms are highly conserved across species, it is believed that studying these basic systems can yield valuable insights for drug companies interested in targeting therapeutics for the higher organisms. More recently, the approach to functional genomics has expanded to include study of gene function in organisms to be targeted for therapeutic intervention. This new approach was the theme of the Functional Genomics Conference: From Identifying Proteins to Faster Drug Discovery held in Washington DC on March 10 and 11, 1998. The organisers (NMHCC) hoped that the breadth of the conference topics would reflect the complexities of the modern drug discovery process and covered technologies from gene chips, bioinformatics, disease models, protein discovery and expression, target validation, high-throughput screening for genes of unknown function, to integration of the drug discovery process. The two day conference placed emphasis on cutting edge technology solutions and the development of high-throughput tools to address the emerging opportunities in genome-based drug discovery.

Journal Article↗

PubMed2003

Zebrafish: from disease modeling to drug discovery.

The study of zebrafish, a leading model organism for developmental biology, is rapidly expanding to include human disease. Zebrafish models based on known disease mechanisms have been developed in several therapeutic areas, including blood diseases, diabetes, muscular dystrophy, neurodegenerative disease, angiogenesis and lipid metabolism. This review summarizes recent progress in disease model development, and outlines the potential of zebrafish to contribute to drug discovery through the identification of novel drug targets, validation of those targets and screening for new therapeutic compounds.

Animals↗

PubMed2005

Validation of molecular targets in prostate cancer.

As prostate cancer is not a single disease, it is important to identify the pivotal pathway in the patient being treated. The molecular environment is the site of current oncological research to define new therapeutic targets for hormone-refractory disease, offering the potential to eventually individualize treatment through stratification of pathways. Targets may be validated either phenotypically (e.g. androgen receptor, cadherin) or functionally (e.g. prostate cancer-specific genes). In addition, several other candidates are potentially suitable, while others await discovery. Important initial steps have been made in the search for prostate cancer stem cells; identifying stem cells and the stromal, hormonal, and other signalling molecules that influence their behaviour would have important implications for managing prostate cancer. Although individual therapeutic pathways might be ineffective in a particular molecular environment, combinations of approaches might be capable of producing synergistic effects. A multimodal approach thus might be the best solution. Determining where best to search for a molecular target, and validating whether the target is associated with a sufficiently aggressive malignant process to justify further study is difficult, but the potential benefits are enormous.

Androgens↗

PubMed2003

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.

Adenoviridae↗

PubMed2006

Riboswitches as antibacterial drug targets.

New validated cellular targets are needed to reinvigorate antibacterial drug discovery. This need could potentially be filled by riboswitches-messenger RNA (mRNA) structures that regulate gene expression in bacteria. Riboswitches are unique among RNAs that serve as drug targets in that they have evolved to form structured and highly selective receptors for small drug-like metabolites. In most cases, metabolite binding to the receptor represses the expression of the gene(s) encoded by the mRNA. If a new metabolite analog were designed that binds to the receptor, the gene(s) regulated by that riboswitch could be repressed, with a potentially lethal effect to the bacteria. Recent work suggests that certain antibacterial compounds discovered decades ago function at least in part by targeting riboswitches. Herein we will summarize the experiments validating riboswitches as drug targets, describe the existing technology for riboswitch drug discovery and discuss the challenges that may face riboswitch drug discoverers.

Anti-Bacterial Agents↗

PubMed2003

Validation of GABA(A) receptor subtypes as potential drug targets by using genetically modified mice.

A key issue for drug discovery in the post genomic era is target validation. This is particularly important when considering the CNS, where currently the majority of drug targets are neurotransmitter receptors that are known to exist as multi-gene families. The GABAergic system, which is the major inhibitory neurotransmitter system in the CNS, is no exception in that respect. The GABA(A) receptors, which are the site of action of a number of clinically used drugs such as benzodiazepines and barbiturates, exist in a large gene family. Existing drugs mediating their effects through the GABA(A) receptor are generally non-selective, i.e. will act at several subtypes of that receptor family. Thus, if we are both to refine existing therapeutic approaches, and develop novel approaches, a key question is to define which subtype(s) of the GABA(A) receptor family we should target; which will mediate the beneficial effects of a drug, and which could be responsible for unwanted side effects? One of the tools, which has been developed over the last decade to elucidate the function of a given gene, is the generation and analysis of gene-targeted mice. This review will summarize progress on identifying individual GABA(A) receptor subtypes as potential drug targets by using genetically modified mice.

Anesthetics↗

PubMed2005

Functional proteomic screens in therapeutic protein drug discovery.

Biopharmaceuticals, mainly protein-based therapeutics, are rapidly being developed for several disease indications. However, most biopharmaceuticals are 'me-too' drugs or are being developed against the same handful of targets. Thus, the potential of biopharmaceuticals is not being fully exploited. The bottleneck is still the lack of validated targets and the means of implementing the appropriate target validation technology. Functional proteomic screens provide a rapid route to accelerate the discovery and development of the next generation of biopharmaceuticals.

Antibodies↗

PubMed2026

Prospective clinical validation of targeted long-read sequencing for preimplantation genetic testing of α-thalassaemia.

BACKGROUND: Preimplantation genetic testing for monogenic disorders (PGT-M) can prevent transmission of severe α-thalassaemia, but conventional workflows remain limited by family-specific assay design for direct variant detection, dependence on additional family samples for haplotype construction, and labour-intensive multi-step procedures across several platforms. Targeted long-read sequencing-based PGT-M for α-thalassaemia (tlrPGT-α-thal) integrates direct variant detection and haplotype linkage analysis within a single assay, but prospective clinical validation is lacking. METHODS: This prospective clinical study enrolled 103 families at high risk of transmitting α-thalassaemia at a reproductive medicine centre between August 2024 and March 2025. All families underwent blinded parallel analysis using both conventional NGS-based PGT-M (comparator) and tlrPGT-α-thal. RESULTS: In the primary concordance analysis, tlrPGT-α-thal was fully concordant with conventional NGS-based PGT-M (507/507, 100.0%; exact 95% CI, 99.3-100.0). Direct variant detection was successful in 501/507 embryos (98.82%; 95% CI, 97.4-99.6), haplotype linkage was established in 505/507 embryos (99.61%; 95% CI, 98.6-100.0), and one meiotic recombination event was identified. Among 93 families proceeding to embryo transfer, 57 pregnancies underwent invasive prenatal diagnosis, and all were concordant with the corresponding tlrPGT-α-thal results. Of the 26 comparator-inconclusive embryos, tlrPGT-α-thal resolved 6 complex cases, including cases with incomplete pedigrees or insufficient informative SNPs. Among the remaining 20 embryos with HBA-region aneuploidies, genotype and parental origin could be determined in 12. CONCLUSIONS: The findings show that tlrPGT-α-thal enables direct detection of diverse α-thalassaemia-causing variants together with efficient haplotype linkage analysis within a single workflow, without requiring family-specific assay design or additional family samples. The method demonstrated high diagnostic accuracy while providing added value in complex scenarios. Taken together, tlrPGT-α-thal represents a simplified and broadly applicable strategy for α-thalassaemia PGT-M.

Humans↗

PubMed2000

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes.

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.

Agar↗

PubMed2004

Setting sights on the treatment of ocular angiogenesis using antisense oligonucleotides.

The application of antisense technology to study physiological and disease processes continues to mature. Antisense approaches are among the most direct means to use genomic sequence information. When developing therapeutics, applications range from early target validation in discovery to the therapeutic product. In this review, we describe the application of antisense oligonucleotides (ASOs) to identify genes that are important in controlling angiogenesis. High-throughput assays in vitro have been used to evaluate many gene targets. Genes that appear to be important in angiogenesis are then evaluated further in animal models of ocular angiogenesis. The ability of ASOs to reduce target-gene expression in the appropriate cells in the eye raises the possibility that this class of compounds could be used for target validation in vivo, and also be developed as a novel class of therapeutics in their own right.

Angiogenesis Inhibitors↗

PubMed2006

RNA Silencing in the struggle against disease.

Numerous acquired and hereditary diseases are caused by aberrant cellular or microbial gene expression. As a result of sequencing of the human genome and the genomes of various human pathogens, researchers have gained access to a large number of genes with residual functions. For functional validation of unknown genes, their functions can be specifically inhibited by antisense nucleic acids or small interfering RNAs (siRNAs) and the consequences of the functional loss, that is, the resulting phenotypes, can be analyzed. While antisense nucleic acids block the translation stoichiometrically by docking on the mRNA, siRNAs induce a highly effective cellular mechanism that causes catalytic destruction of several mRNA molecules by a single siRNA molecule. This mechanism, called RNA interference (RNAi), is only intrinsic to eukaryotic cells. Consequently, only eukaryotic target validation is pushed by RNAi whereas time-consuming conventional knockout techniques or the less efficient antisense strategies have to be applied for prokaryotic target validation. We succeeded in triggering gene silencing by siRNA in prokaryotic cells. This opens promising perspectives regarding validation of prokaryotic gene functions.

Genome, Bacterial↗

PubMed2004

Predicting and validating microRNA targets.

Given that microRNAs select their targets by nucleotide base-pairing, it follows that it should be possible to find microRNA targets computationally. There has been considerable progress, but assessing success and biological significance requires a move into the 'wet' lab.

Animals↗

PubMed2006

siRNA-based approaches in cancer therapy.

The availability of the human genome sequence has revolutionized the strategy of employing nucleic acids with sequences complementary to specific target genes to improve drug discovery and target validation. Development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences offers the possibility of rational design with high specificity, lacking in many current drug treatments for various diseases including cancer, at relatively inexpensive costs. Antisense technology is one such example that has shown promising results and boasts of yielding the only approved drug to date in the genomics field. However, in vivo delivery issues have yet to be completely overcome for widespread clinical applications. In contrast to antisense oligonucleotides, the mechanism of silencing an endogenous gene by the introduction of a homologous double-stranded RNA (dsRNA), transgene or virus is called post-transcriptional gene silencing (PTGS) or RNA interference. PTGS is a natural mechanism whereby metazoan cells suppress expansion of genes when they come across dsRNA molecules with the same sequence. Short interfering RNA is currently the fastest growing sector of this antigene field for target validation and therapeutic applications. Although, in theory, the development of genomics-based agents to inhibit gene expression is simple and straightforward, the fundamental concern relies upon the capacity of the oligonucleotide to gain access to the target RNA. This paper summarizes the advances in the last decade in the field of PTGS using RNA interference approaches and provides relevant comparisons with other oligonucleotide-based approaches with a specific focus on oncology applications.

Genetic Therapy↗

PubMed2006

Sensorimotor adaptation to inertial forces in a multi-force environment does not depend on the number of targets: indirect validation of the altered-proprioception hypothesis.

The ability of our sensorimotor system to adapt to changing and complex environmental demands has been under experimental scrutiny for more than a century. Previous works have shown that aimed arm movements adapt quickly and completely to Coriolis force, but incompletely to the combination of Coriolis and centrifugal forces without visual cues. Two hypotheses may be advanced to explain this discrepancy: the workspace-exploration hypothesis, and the degraded-proprioception hypothesis. The aim of this study was to distinguish between the above two alternatives by comparing adaptive improvement during off-axis rotation in subjects pointing at one, three or seven different targets in complete darkness. Two main results emerge: (a) off-axis rotation led initially to errors in the direction of Coriolis force and in the opposite direction of the centrifugal force; (b) the size of the visited workspace has no effect on the way the subjects adapt to a multi-force environment. The lack of a target-number effect and the persistence of lateral errors in the pointing movements performed during rotation of the platform, support the degraded-proprioception rather than the workspace-exploration hypothesis of adaptation to a multi-force environment.

Acclimatization↗

PubMed2005

Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB.

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-kappaB-dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-kappaB p65 subunit, and the NF-kappaB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-kappaB-dependent secretion of factors that activate NF-kappaB through cell-surface receptors, establishing an autocrine loop.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Identification of novel inhibitors of UDP-Glc 4'-epimerase, a validated drug target for african sleeping sickness.

Novel inhibitors of Trypanosoma brucei and mammalian UDP-Glc 4'-epimerase were identified by screening a small library of natural products and commercially available drug-like molecules. The inhibitors possess low micromolar potency against the T. brucei and human enzymes in vitro, display a degree of selectivity between the two enzymes, and are cytotoxic to cultured T. brucei and mammalian cells.

Animals↗