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Results for “target validation”

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At least 397 records · Page 22Linked to original sources

PubMed2003

Functional analysis of expressed peptides that bind yeast STE proteins.

Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.

GTP-Binding Protein gamma Subunits↗

PubMedDate not supplied

Conducting the initiation of protein synthesis: the role of eIF4G.

The eukaryotic initiation factor eIF4G is a large modular protein which serves as a docking site for initiation factors and proteins involved in RNA translation. Together with eIF4E and eIF4A, eIF4G constitutes the eIF4F complex which is a key component in promoting ribosome binding to the mRNA. Thus, the central role of eIF4G in initiation makes it a valid target for events aimed at modulating translation. Such events occur during viral infection by picornaviruses and lentiviruses and result in the hijack of the translational machinery through cleavage of eIF4G. Proteolysis of eIF4G is also mediated by caspases during the onset of apoptosis causing inhibition of protein synthesis. We will review the role of eIF4G and protein partners as well as the cellular and viral events that modulate eIF4G activity in the initiation of translation.

Animals↗

PubMed2002

Global high-throughput screens for cellular function.

There is a critical need for global methods that allow for high-throughput assessment of cellular function for clinical and basic scientists working in both academia and the pharmaceutical industry. These methods typically couple systematic inactivation strategies with high-throughput cell-based assays that facilitate rapid target validation. We present here a survey of these technologies and their applications. We discuss their promise and limitations in addressing the vast number of candidate molecules of disease relevance that are emerging from genomics and proteomics.

Animals↗

PubMed2003

The epidermal growth factor receptor mediates radioresistance.

PURPOSE: The epidermal growth factor (EGF) receptor is frequently overexpressed in malignant tumors, and its level is correlated with increased cellular resistance to ionizing radiation. However, no precedent studies have investigated whether expression of EGF receptor would by itself confer on cancer cells resistance to radiation. The current study is aimed to address this question. METHODS AND MATERIALS: A full-length human EGF receptor expression vector was transfected into the OCA-I murine ovarian carcinoma cells for stable clones expressing various levels of EGF receptors. Apoptosis and cell clonogenic survival assays were used to evaluate the sensitivity of the resulting cell clones to ionizing radiation. RESULTS: OCA-I cell clones expressing various levels of EGF receptor (OCA-I EGFR) were obtained. These clones showed an EGF receptor level-dependent increase in resistance to ionizing radiation, measured by apoptosis and cell clonogenic survival assays. Compared with the results for parental OCA-I and control vector-transfected OCA-I cells at the 10% cell survival level, the radioresistance was increased by a factor of 1.60 for EGFR-C5 (high level of EGF receptor expression), 1.37 for EGFR-C3 (intermediate level of EGF receptor expression), and 1.28 for EGFR-C1 (low level of EGF receptor expression). Treatment of the OCA-I EGF receptor transfectants with the anti-EGF receptor monoclonal antibody C225 downregulated the levels of EGF receptor, reduced the phosphorylation levels of EGF receptor downstream substrates (such as Akt and MAPK), and reversed the cellular radioresistance. CONCLUSION: Our results demonstrate that overexpression of the EGF receptor conferred cellular resistance to ionizing radiation. The EGF receptor is thus a valid target for potential radiosensitization.

Animals↗

PubMed2004

Ear advantage and attention: an ERP study of auditory cued attention.

The neurophysiological manifestations of left ear advantage to tonal stimuli and its interaction with attention have rarely been studied. Cued attention is a behavioral paradigm to assess the behavioral benefits and costs of allocating attention. In this task a cue predicts the location of a subsequent target to which the subject responds. In most cases the cue correctly predicts the target (valid cues) but at times it does not (invalid cues). Cued attention is a spatial paradigm with stimuli presented to either side. The objectives of this study were: (1) to find the neurophysiological correlates of the ear advantage phenomenon and (2) to assess the interaction of the stimulated side (right vs. left ear advantage) with attention, in a cued attention task. Significant effects on event-related potentials (ERPs) in the cued attention task indicated left ear and right hemisphere advantage. Effects were mostly confined to the right hemisphere. The results indicate interactions among left ear advantage, attention and dominant hand utilization. Ear advantage and attention may involve the same neural mechanisms. In spite of the left ear advantage effect on ERP components, hand dominance determines the final behavioral results (reaction times).

Acoustic Stimulation↗

PubMed2002

Targeting intracellular signal transduction. A new paradigm for a brave new world of molecularly targeted therapeutics.

Significant advances in the field of molecular biology over the past decade have led to a new era in cancer therapeutics, with an explosion of rationally designed therapeutic strategies directed against selective molecular targets. The complex array of aberrant signal transduction proteins involved in carcinogenesis has been the focus of target-based anticancer agents. Inhibitors of intracellular signal transduction represent a unique approach in that they inhibit critical downstream regulatory proteins, which are vital to the process of cellular communication. Although these agents are in early-phase evaluations, the preliminary data suggest that they are well tolerated and capable of target inhibition in surrogate and tumor tissue. Although the primary therapeutic benefit of these agents is expected to be decreased tumor growth, evidence suggests that objective tumor responses may also be achieved. There are many unresolved questions pertaining to the development of this class of compounds, including selection of optimal dose and schedule, determination of relevant endpoints, methods for target validation, and strategies for combination with cytotoxic agents. However, despite the numerous unresolved issues, the emergence of this class of compounds has resulted in an undeniable impact on the present and future of cancer therapeutics.

Adult↗

PubMed2001

The novel long PDE4A10 cyclic AMP phosphodiesterase shows a pattern of expression within brain that is distinct from the long PDE4A5 and short PDE4A1 isoforms.

In situ hybridisation methods were used to map the distribution of the novel long PDE4A10 isoform in the brain. PDE4A10 distribution was compared to that of the long PDE4A5 isoform and the short PDE4A1 isoform using probes specific for unique sequences within each of these isoforms. Coronal sections of the brain, taken at the level of the olfactory bulb, prefrontal cortex, striatum, thalamus, hippocampus and cerebellum, were analysed. Strongest expression of PDE4A isoforms was found in the olfactory bulb granular layer with high signals also in the piriform cortex, the dentate gyrus and the CA1 and CA2 pyramidal cells. For the two long forms, level general staining was noted throughout the striatum, thalamus and hippocampus but no signal was evident in the cerebellum. The long PDE4A10 and PDE4A5 isoforms localised to essentially the same regions throughout the brain, although PDE4A10 was uniquely expressed in the major island of Calleja. A signal for the short PDE4A1 isoform was found in regions in which the two long isoforms were both expressed, with the exception of the medial nucleus of the amygdala where weak signals for PDE4A5 and PDE4A10 were detected but PDE4A1 was absent. Uniquely, strong signals for PDE4A1 were detected in the glomerular layer of the olfactory bulb, the CA3 pyramidal cell region and the cerebellum; areas where signals for the two long forms were not evident. PDE4A transcripts for both PDE4A5 and PDE4A10 were not apparent in the brain stem and those for PDE4A1 were low. PDE4A isoforms are present in several key areas of the brain and therefore present valid targets for therapeutic interventions. Whilst the two long PDE4A isoforms show a remarkably similar distribution, in at least three regions there is clear segregation between their pattern of expression and that of the PDE4A1 short form. This identifies differential regulation of the expression of PDE4A long and short isoforms. We suggest that specific PDE4A isoforms may have distinct functional roles in the brain, indicating that PDE4A isoform-selective inhibitors may have specific therapeutic and pharmacologic properties.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Genetics and genomics in neuropsychopharmacology: the impact on drug discovery and development.

Genomics, the complete tabulation of all the genes in an organism, has made a major impact on the organisation of fully-integrated pharmaceutical companies. Drug discovery begins with bioinformatic elucidation of a human sequence encoding a potential drug target, followed by cloning and expression of the gene in a format for high throughput screening. Target validation is aided by reference to homologous genes in subhuman species as well as production of transgenic animals. In contrast, the impact of genetics on neuropsychopharmacology has been modest. It is interesting to compare the experience of genetics in the two major clinical disciplines dealing with disorders of the nervous system. Neurology has been at the forefront of human genetics with over 600 disorders mapped, of which causative mutations have been assigned to about 200 Mendelian disorders, each individually rare. Psychiatric genetics has been based on two log fewer diagnoses use of which has only yielded complex segregation patterns, a plethora of weak associations and no gene assignments. In neither case has genetics resulted in the development of a novel therapeutic agent. However, by refinements in diagnosis and genetic technology the promise for the future is great, not only for drug discovery, but also for subsequent preclinical and clinical development.

Animals↗

PubMed1998

Expression of ribozymes in gene transfer systems to modulate target RNA levels.

The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.

Animals↗

PubMed2003

RdlDv, a novel GABA-gated chloride channel gene from the American dog tick Dermacentor variabilis.

The American dog tick Dermacentor variabilis is a major transmitter of bacterial and viral pathogens in human and animal populations, and compounds active against this species would benefit both human and animal health. Invertebrate GABA-gated chloride channels are validated targets of commonly used insecticides and acaricides. We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from Dermacentor variabilis, RdlDv. The closest homologue of the predicted gene product of RdlDv is the RDL protein encoded by the GABA-gated chloride channel gene Drosophila Rdl (Resistance to Dieldrin), with which it shares 64% amino acid identity. When expressed in Xenopus oocytes, RdlDv produces GABA-activated currents blocked by the known insecticides and RDL antagonists fipronil and picrotoxinin. These results suggest that RdlDv encodes a GABA-gated chloride channel subunit, making it a potential target for compounds active against the tick D. variabilis.

Amino Acid Sequence↗

PubMed2003

Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei.

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.

Animals↗

PubMed2003

Recent progress in discovery of small-molecule CCR5 chemokine receptor ligands as HIV-1 inhibitors.

This review addresses key pharmacology and virology issues relevant in discovery and development of CCR5 antagonists as anti-HIV drugs, such as target validation, receptor internalization, allosterism, viral resistance and tropism. Recent progress in the discovery and development of CCR5 antagonists, SAR and clinical status are reviewed. Finally, modeling-based structure of CCR5 is discussed in the context of a small-molecule antagonism of the CCR5 receptor.

Animals↗

PubMed2002

Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase.

The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.

Amide Synthases↗

PubMed1996

Nature of expertise in searching mammograms for breast masses.

RATIONALE AND OBJECTIVES: The authors investigated how training and experience affect the performance of observers searching mammograms for breast masses. METHODS: Eye positions of mammographers, mammography technologists, mammography residents, and laypersons were compared to scan paths generated by a simulated scanner as each searched nine two-view digital mammogram pairs for breast masses. RESULTS: Analysis of time-to-hit data revealed that mammographers and mammography technologists with the most extensive training and experience had the fastest search times in the detection and confirmation of a breast mass on two views. Scanning patterns of less-experienced mammography residents were less efficient due to wider dispersion of visual attention between potential breast masses and perturbations in breast parenchyma. Because laypersons lacked both training and experience in mammography, bright blobs in the breast image were considered to be intuitively valid target candidates and these features distracted the search by capturing visual attention. CONCLUSION: Experience reading normal and abnormal mammograms plays a critical role in training radiologists. Experience combined with training provides the basis for generating efficient visual search strategies and developing distinctive conceptual criteria for perceptual differentiation and interpretation of true breast masses from image artifacts and structured noise that mimics breast abnormalities.

Artifacts↗

PubMed2003

Conditional protein alleles using knockin mice and a chemical inducer of dimerization.

We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.

Alleles↗

PubMed2001

Carbohydrate sulfotransferases: novel therapeutic targets for inflammation, viral infection and cancer.

Effective direct inhibition of adhesion receptors by small molecules has been hampered by extended receptor-ligand interfaces as well as the entropic penalties often associated with inhibition of cell adhesion. Therefore, alternative strategies have targeted enzymes that are centrally involved in the biosynthesis of recognition epitopes, which are crucial for productive adhesion. Two classes of enzymes shown to play a pivotal role in cell-cell and cell-matrix adhesions are the protein-tyrosine and carbohydrate sulfotransferases, which impart crucial sulfate moieties onto glycoproteins. The carbohydrate sulfotransferases will be discussed in terms of target validation and small-molecule inhibitor discovery.

Journal Article↗

PubMed2002

Engineering cellular microenvironments to improve cell-based drug testing.

Recent progress in the biology of cell adhesion is enabling cell culture models to better reproduce in vivo functions. Cues from adhesion to extracellular matrix and neighboring cells are important regulators of cell behaviors. The recent adaptation of semiconductor tools to spatially organize cells and their adhesions has enhanced our ability to engineer cell functions ex vivo. By using these tools to create more in vivo-like cultures, cell-based drug discovery and target validation could be improved. This review explores the biological advances made by these microfabrication tools and discusses how they could enable high-throughput cell-based assays.

Animals↗

PubMed2002

Probes for chemical genomics by design.

Chemical genomics represents a convergence of biology and chemistry in the era of global approaches to target identification and intervention. The success of genomics has led to a bottleneck in target validation that could be overcome by using small diverse organic compounds to interfere with biological processes. Because of the limitations of existing compound collections, this diversity can only fully be exploited using in silico design techniques to guide the selection of molecules with optimal binding properties. Structure-based design is used to create structures de novo that can be synthesized for use as chemical probes and drug leads.

Amino Acid Sequence↗