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Results for “target validation”

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PubMed2002

Participation of the thalamic CM-Pf complex in attentional orienting.

The centre médian-parafascicular (CM-Pf) complex is located at the posterior intralaminar nuclei of the thalamus and forms part of the nonspecific thalamocortical projection system and the internal circuit of the basal ganglia. However, the functional roles of this complex remain to be fully elucidated. Here we have examined whether the CM-Pf complex is involved in the process of covert attention. We trained two macaque monkeys to perform a task in which a visual target stimulus for button release appeared at either the same location as the preceding visual instruction cue (a "validly cued target") or a location on the opposite side (an "invalidly cued target"). Reaction times (RTs) to a validly cued target were significantly shorter than those to an invalidly cued target, leading to a "validity effect" of about 20 ms. We recorded the activity of 97 neurons in the CM-Pf while the monkeys performed the attention task with the hand that was contralateral to the neuronal recording. Seventy CM-Pf neurons showed task-related activity after the appearance of either the instruction cue or the target stimulus: 33 neurons responded with a prominent short-latency facilitation (SLF), whereas 37 responded with a short-latency suppression followed by a long-latency facilitation (LLF). Most of the SLF neurons responded preferentially to a cue appearing on the contralateral side (76%) and to an invalidly cued target appearing on the contralateral side (61%). In contrast, LLF neurons showed a short-latency suppression after the cue stimulus, regardless of whether the cue appeared on the contra- or ipsilateral side (84%). Inactivating the CM-Pf complex by local injection (1 microl) of the GABA(A) receptor agonist muscimol (1-5 microg/microl) resulted in a significant increase in the RT to a validly cued target presented on the contra- but not the ipsilateral side. In contrast, inactivating the CM-Pf complex did not affect RTs to invalidly cued targets on either the contra- or the ipsilateral side. Thus the validity effect was abolished only on the contralateral side. We conclude that the CM-Pf complex plays a specific and essential role in the process of attentional orienting to external events occurring on the contralateral side, probably through the projection of primary outputs to the striatum, which is involved in the action-selection mechanisms of the basal ganglia.

Animals↗

PubMed2005

HER2-mediated internalization of a targeted prodrug cytotoxic conjugate is dependent on the valency of the targeting ligand.

HER2 is a validated therapeutic target for cancer. There are no natural ligands, but monoclonal antibodies and peptides that bind HER2 act as artificial ligands, selectively affecting HER2-overexpressing tumors. One reported mechanism for this effect is receptor downregulation, but the expected correlation of ligand-dependent HER2 internalization and tumor inhibition remain poorly characterized. Moreover, HER2 ligands have limited therapeutic efficacy and often they require adjuvant treatment with the chemotherapeutic Taxol. Here, we generated a series of HER2 ligands (Anti-HER2/neu peptide ligands, AHNPmonovalent and AHNPbivalent) with different valency and correlated their internalization-promoting ability to biological potency. Since AHNPbivalent (but not AHNPmonovalent) induces rapid receptor internalization, we exploited this feature to deliver cytotoxic conjugates coupling AHNPbivalent and Taxol (Taxol . AHNPbivalent). The prodrug conjugate releases Taxol after receptor-mediated internalization, and cytotoxicity can be used as a marker of internalization. Taxol . AHNPbivalent is significantly more cytotoxic than free Taxol + free AHNPbivalent. Hence, the Taxol x AHNP(bivalent) prodrug binds to HER2, induces receptor internalization and downregulation, and the subsequent release of free Taxol inside the targeted cell results in synergistic toxicity, The effect is selective towards HER2- expressing cells. This work links HER2 receptor internalization and growth arrest, and the chemical conjugation strategy may yield improved and HER2 selective therapeutics.

Animals↗

PubMed2001

Protein kinases in drug discovery and development.

The Protein Kinases in Drug Discovery and Development meeting highlighted protein kinases as validated therapeutic targets in many types of disease, especially cancer. Drugs designed to inhibit protein kinase activity were widely tested in preclinical and clinical evaluation, demonstrating significant efficacy with acceptable toxicity profiles. More than 600 kinases are encoded by the human genome, some of which are already well established as validated targets due to extensive scientific research from both academic institutions and industrial companies. As demonstrated by several speakers during the meeting, different diseases exhibit mutations or over-expression of the same kinase. Therefore, a drug designed to inhibit these specific kinases can potentially be used as a therapeutic agent in the treatment of more then one disease. The major strategy employed by most companies today in drug discovery is using genomic information, together with clinical research, to identify novel potential targets. A drug discovery project is then established around the chosen target kinase, in order to identify, optimize and deliver novel potential drugs to clinical trials.

Journal Article↗

PubMed2003

Optical imaging and tumor angiogenesis.

Tumor angiogenesis is essential for tumor growth and progression. Therefore, targeting tumor blood vessels is a promising approach for cancer therapy. Angiogenesis, the formation of blood vessels, is a multistep process, and strongly influenced by the microenvironment. There are no in vitro assays that can resemble this dynamic process in vivo. For this reason, animal models and imaging technologies are critical for studying tumor angiogenesis, identifying therapeutic targets as well as validating the targets. Non-invasive molecular imaging in animal models presents an unprecedented opportunity and ability for us to perform repetitive observations and analysis of the biological processes underlying tumor angiogenesis and tumor progression in living animals in real time. As we gain a better understanding of the fundamental molecular nature of cancer, these techniques will be an important adjunct in translating the knowledge into clinical practice. This important information may elucidate how the tumor blood vessels behave and respond to certain treatments and therapies.

Angiogenesis Inhibitors↗

PubMed2005

Prediction and validation of microRNAs and their targets.

MicroRNAs are short non-coding RNAs that inhibit translation of target genes by binding to their mRNAs, and have been shown to play a central role in gene regulation in health and disease. Sophisticated computer-based prediction approaches of microRNAs and of their targets, and effective biological validation techniques for validating these predictions, now play a central role in discovery of microRNAs and elucidating their functions.

Algorithms↗

PubMed1996

Peripheral cuing of covert spatial attention before and after emotional conditioning of the cue.

Spatial cuing of attentional shifts were investigated before and after the visual cue had acquired emotional significance through a classical conditioning procedure. The study consisted of three phases; an attention preconditioning phase, the conditioning phase and an attention postconditioning (extinction) phase. In the attention phases, subjects participated in a trial-by-trial cuing task, in which the location of the target was validly or invalidly cued by either a frame-lit or a completely lit rectangle. During conditioning half the subjects (Conditioning group) had a 90 dB white noise unconditioned stimulus (UCS) presented together with one of the two attentional cues. This cue was, thus, turned into a conditioned stimulus (CS+), while the other cue became a CS-. The Control group received the noise uncontingent upon presentations of these stimuli. The Conditioning group showed greater skin conductance responses (SCRs) to the CS+ compared to the CS-, reflecting that a conditioned response was established. When the CS+ served as attentional cue, there was no difference in RTs between validly and invalidly cued targets, while responses to invalidly cued targets were delayed on all other trials. This suggests that the CS+ reduced the cognitive cost of shifting attention from the cued to the uncued location.

Adult↗

PubMed2004

Identification and validation of novel drug targets in tuberculosis.

There is an urgent need for new antimycobacterial drugs, and in particular for novel agents that will shorten the duration of tuberculosis chemotherapy, or overcome drug-resistant strains of the causative organism, Mycobacterium tuberculosis. Our knowledge of the tubercle bacillus and its complex interaction with the human host has improved dramatically in recent years, particularly with the determination of its complete genome sequence. New genome-scale tools are being applied to aid in drug target identification, alongside traditional approaches aimed at understanding the basic biology of M. tuberculosis. Many potential drug targets have been identified, but very few have been validated by showing that they are essential for growth or survival of the bacterium. In this review, the landscape of potential drug targets is surveyed.Structural Bioinformatic Approaches to the Discovery of New Antimycobacterial Drugs.

Animals↗

PubMed1990

Effects of physostigmine on spatial attention in patients with progressive supranuclear palsy.

We tested patients with progressive supranuclear palsy and control subjects on a task of visuopatial attention. Targets preceded by cues on the same side were termed validly cued; and those on the opposite side, invalidly cued. For all subjects, validly cued targets were responded to faster than those that were invalidly cued. The difference between reaction times for invalidly and validly cued targets, which is hypothesized to measure attentional movement, was significantly increased for the patients. The performance of the controls on certain neuropsychological tests was correlated with their attentional ability. These correlations were altered by progressive supranuclear palsy. Physostigmine treatment of the patients induced a speeding of responses to invalidly cued targets as a function of the duration of the disease. These studies show defects in cognition and attention in patients with progressive supranuclear palsy and demonstrate that physostigmine reduces some of the abnormal visual attentional performance.

Aged↗

PubMed2004

Genetically engineered mouse models for drug discovery: new chemical genetic approaches.

While standard transgenic and knockout mouse technologies have provided a wealth of information for target selection and validation, there have been great advances in using more sophisticated modeling techniques to achieve temporal and spatial regulation of individual genes in adult animals. Recent developments in RNA interference (RNAi) technology in in vivo models promise to further improve upon the static and irreversible features of gene knockouts. Chemical genetic approaches create novel functional alleles of targets and allow fine modulation of protein function in vivo by small molecules, providing the most pharmacologically relevant target validation. Using these advanced models, one can not only ask whether the function of the target is critical for the initiation and maintenance of the disease, but also whether therapies designed to alter the function of the target would be safe and efficacious. In this review, we describe various in vivo tools for target validation in mouse models, discuss advantages and disadvantages of each approach, and give examples of their impact on drug discovery.

Animals↗

PubMed2004

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference.

Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

Animals↗

PubMed2004

Identification and validation of cell surface antigens for antibody targeting in oncology.

Recent clinical successes with antibodies have reinvigorated interest in the identification and validation of new antigens for antibody therapy, including cell surface proteins for targeting in oncology, the focus of this review. Target identification commonly involves the search for differences between tumor and non-tumor cell lines and/or tissue at the DNA, mRNA, protein or antibody reactivity levels. The next stage, target validation, utilizes antibodies to profile the expression of antigen in normal and tumor tissue and to verify that the antigen is selectively expressed on the surface of tumor cells. Supportive evidence for protein expression is often sought by mRNA profiling and, sometimes, analysis for genomic defects. Unfortunately, concordance between mRNA and protein levels has been found in only about approximately 20% of cases and therefore must be evaluated for individual targets of interest. Antigens judged suitable for antibody targeting are then advanced to the next stage, namely, in vitro and then in vivo screening of antibodies for anti-tumor activities. Subsequent optimization of an antibody clinical lead for therapy is a desirable, if not obligatory, step to developing an antibody as an anti-cancer therapeutic. No single approach or even combination of methods has emerged as the preferred way to identify surface antigens suitable for targeting in oncology. Major options at each step in the process are reviewed here, including their strengths and limitations.

Animals↗

PubMed2002

Functional genomics approaches for the identification and validation of antifungal drug targets.

So far, antifungal drug discovery seems to have benefited little from the enormous advances in the field of genomics in the last decade. Although it has become clear that traditional drug screening is not delivering the long-awaited novel potent antifungals, little has been reported on efforts to use novel genome-based methodologies in the quest for new drugs acting on human pathogenic fungi. Although the market for a novel systemic and even topical broad-spectrum antifungal appears considerable, many large pharmaceutical companies have decided to scale back their activities in antifungal drug discovery. Here we report on some of the recent advances in genomics-based technologies that will allow us not only to identify and validate novel drug targets but hopefully also to discover active therapeutic agents. Novel drug targets have already been found by 'en masse' gene inactivation strategies (e.g. using antisense RNA inhibition). In addition, genome expression profiling using DNA microarrays helps to assign gene function but also to understand better the mechanism of action of known drugs (e.g. itraconazole) and to elucidate how new drug candidates work. No doubt, we have a long way to go just to catch up with the advances made in other therapeutic areas, but all tools are at hand to derive practical benefits from the genomics revolution. The next few years should prove a very exciting time in the history of antifungal drug discovery.

Animals↗

PubMed2004

Peripheral cuing by abrupt-onset cues: the influence of color in S-R corresponding conditions.

Research on visuospatial attention indicates that a peripheral abrupt-onset cue at target position (valid condition) facilitates processing of the target, whereas a cue at another position interferes. This validity effect seems to be contingent on a similarity of the cue's color to the set of target colors (cf. J. Exp. Psychol.: Human Percep. Perform. 18 (1992) 1030). In Experiments 1-3, we confirm this contingency with cues that have the potential to activate responses. Thus, attentional capture and response capture are apparently governed by the same principle. In Experiment 2, it is demonstrated that color priming is not responsible for the contingency. In Experiment 3, it is shown that a more efficient reallocation of attention after color-dissimilar cues than after color-similar cues might contribute to the contingency.

Adult↗

PubMed2006

Mid-IR biosensor: detection and fingerprinting of pathogens on gold island functionalized chalcogenide films.

Antibody (human IgG, anti-E. coli O157:H7, and anti-Salmonella) complexes on the surface of IR-transparent Ge-containing chalcogenide glass films were formed via thiol chemistry on 20-nm-thick gold islands. As a first step, the protocol was validated by monitoring fluorescently tagged targets to validate binding. FT-IR spectroscopy confirmed that the coating of the films with 20-nm gold did not have a significant effect on the propagation and penetration of IR evanescent waves through the film. The films functionalized with anti-E. coli O157:H7 and anti-Salmonella antibodies were used to detect E. coli O157:H7 and S. enteriditis through label-free IR fingerprinting. Highly selective detection of bacterial targets was achieved at both the species (E. coli vs. S. enteriditis) and strain level (E. coli O157:H7 vs E. coli K12). A mid-infrared approach could thus be used as a biosensor as well as a molecular fingerprinting tool.

Antibodies, Bacterial↗

PubMed2001

Molecular targets of lithium action.

Lithium is highly effective in the treatment of bipolar disorder and also has multiple effects on embryonic development, glycogen synthesis, hematopoiesis, and other processes. However, the mechanism of lithium action is still unclear. A number of enzymes have been proposed as potential targets of lithium action, including inositol monophosphatase, a family of structurally related phosphomonoesterases, and the protein kinase glycogen synthase kinase-3. These potential targets are widely expressed, require metal ions for catalysis, and are generally inhibited by lithium in an uncompetitive manner, most likely by displacing a divalent cation. Thus, the challenge is to determine which target, if any, is responsible for a given response to lithium in cells. Comparison of lithium effects with genetic disruption of putative target molecules has helped to validate these targets, and the use of alternative inhibitors of a given target can also lend strong support for or against a proposed mechanism of lithium action. In this review, lithium sensitive enzymes are discussed, and a number of criteria are proposed to evaluate which of these enzymes are involved in the response to lithium in a given setting.

Animals↗

PubMed2001

The human genome project: a historical perspective.

Efforts in genomics over the last decade have created a stream of opportunities for drug discovery. High-throughput DNA sequencing has forced a re-definition of the paradigm for identification and validation of targets for drug development. One purpose of this review is to delineate the different approaches to sequence data generation and to establish their various uses for the definition of gene function. There still remain crucial dilemmas for the pharmaceutical industry. The multitude of potential targets can each absorb enormous validation costs and the vast majority are likely to prove academically interesting but useless for drug development. An additional dimension arises from the importance of sequence variation between different individuals. These differences can determine response to therapy and must inform both the drug development process and healthcare delivery. This presents great challenges and opportunities for drug companies, their customers and society as a whole. I will review the technological aspects in some detail and give my view of the legal and social aspects. The field of bioinformatics is at the core of functional and pharmacogenomics and advances will depend on the continuing evolution of tools to interpret data. For the most part this evolution is reviewed in the context of specific application areas rather than as a discrete field, in recognition of its all-pervasive effects.

Animals↗

PubMed2003

Inhibition of return for the length of a line?

Inhibition of return is most often measured using an exogenous spatial cuing method. The experiments presented here follow up on a small number of studies that have examined whether a similar effect occurs for nonspatial stimulus attributes. In Experiments 1 and 2, the task was to identify a target line as either short or long. In this context, targets on valid trials were of the same length as that of a preceding cue, whereas targets on invalid trials were of a different length than that of a preceding cue. The results were similar to those in spatial orienting studies in that responses were slower for valid than for invalid targets only at stimulus onset asynchronies (SOAs) longer than 300 msec. In Experiment 3, the stimuli were the same but the task was to detect the onset of the target line. This task change resulted in slower responses for valid than for invalid targets at all SOAs. A similar result was observed in Experiment 4, in which validity was defined by color rather than line length, and the task was to identify the target color. The discussion centers on an opponent process approach to interpreting cuing effects, and consequent difficulties in distinguishing spatial and nonspatial cuing effects based on their time course.

Adult↗

PubMed2003

Identification of Drosophila MicroRNA targets.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, our study shows that valid targets can be identified from sequence alone.

3' Untranslated Regions↗