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Results for “target validation”

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PubMed2005

The development of molecularly targeted anticancer therapies: an Eli Lilly and Company perspective.

The ability to identify activated pathways that drive the growth and progression of cancer and to develop specific and potent inhibitors of key proteins in these pathways promises to dramatically change the treatment of cancer: A patient's cancer could be characterized at the molecular level and the information used to select the best treatment options. The development of successful therapies not only requires extensive target validation, but also new approaches to evaluating drug efficacy in animal models and in the clinic compared to the development of traditional cytotoxic agents. This article highlights Eli Lilly and Company's approach to developing targeted therapies, from target identification and validation through evaluation in the clinic. A selection of drugs in the Lilly Oncology pipeline is also discussed.

Animals↗

PubMed2007

The proteasome as a potential target for novel anticancer drugs and chemosensitizers.

A major challenge in cancer therapy is tumor drug resistance. To overcome it, it is essential to understand the mechanisms and identify the molecules involved, so that they can be specifically targeted in combination therapies. The proteasome is such a validated target: it plays a key role in cancer cell proliferation, inhibition of chemotherapy-induced apoptosis and drug resistance development. Bortezomib (Velcade, PS-341) was the first proteasome inhibitor to receive regulatory approval from the US Food and Drug Administration for the treatment of multiple myeloma. Clinical combination trials have demonstrated a chemo-sensitizing effect of bortezomib on conventional agents in hematological malignancies and some solid tumors such as androgen-independent prostate and ovarian cancer. Although generally well-tolerated, bortezomib still generates toxicity which underscores the need for less toxic proteasome inhibitors. Several naturally occurring products, such as green tea polyphenols and the antibiotic lactacystin, have been shown to be potent proteasome inhibitors. Significantly, green tea polyphenols, as well as several flavonoids such as genistein, curcumin and resveratrol, have also been shown to have chemo-sensitizing properties in prostate, breast, hepatic, and lung tumors. Further studies on natural proteasome inhibitors as chemo-sensitizers could lead to identification of more potent and less toxic compounds that could be used in combination therapies for drug-resistant tumors.

Antineoplastic Agents↗

PubMed2006

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study.

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.

Animals↗

PubMed1999

Scopolamine slows the orienting of attention in primates to cued visual targets.

The cholinergic agonist nicotine facilitates visuospatial attention shifting, but the role of muscarinic cholinergic drugs in this behavior is unclear. In order to establish the generality of cholinergic action in attention shifting, we administered the muscarinic antagonist scopolamine to two rhesus monkeys trained to perform a cued target detection (Posner) task. In this task, monkeys signaled the detection of a peripheral visual target by releasing a switch and their reaction times were measured. The location of the target's appearance was preceded by a cue that was either valid (target and cue in the same spatial location), invalid (target and cue to opposite hemifields), spatially uninformative (cues in both hemifields, target to one hemifield), or omitted altogether. Scopolamine produced a dose-dependent increase in all reaction times and a decrease in accuracy. The slowing was most prominent for valid cues in either visual field. However, slowing did not occur in trials whose cues lacked spatial information, or in tasks in which attention was directed to events at the fixation point, whether or not peripheral distractors were present. These results provide additional support for the hypothesis that acetylcholine plays a key role in reflexive attention shifting to peripheral visual targets.

Analysis of Variance↗

PubMed2006

Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs.

MicroRNAs (miRNAs) comprise 1 to 3% of all vertebrate genes, but their in vivo functions and mechanisms of action remain largely unknown. Zebrafish miR-430 is expressed at the onset of zygotic transcription and regulates morphogenesis during early development. By using a microarray approach and in vivo target validation, we find that miR-430 directly regulates several hundred target messenger RNA molecules (mRNAs). Most targets are maternally expressed mRNAs that accumulate in the absence of miR-430. We also show that miR-430 accelerates the deadenylation of target mRNAs. These results suggest that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early embryogenesis.

3' Untranslated Regions↗

PubMed2004

Multiplex GPCR assay in reverse transfection cell microarrays.

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.

Dose-Response Relationship, Drug↗

PubMed2006

Using genetically engineered mouse models of cancer to aid drug development: an industry perspective.

Recent developments in the generation and characterization of genetically engineered mouse models of human cancer have resulted in notable improvements in these models as platforms for preclinical target validation and experimental therapeutics. In this review, we enumerate the criteria used to assess the accuracy of various models with respect to human disease and provide some examples of their prognostic and therapeutic utility, focusing on models for cancers that affect the largest populations. Technological advancements that allow greater exploitation of genetically engineered mouse models, such as RNA interference in vivo, are described in the context of target and drug validation. Finally, this review discusses stratagems for, and obstacles to, the application of these models in the drug development process.

Algorithms↗

PubMed2006

Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development in Drosophila.

Suppression of wing fate and specification of haltere fate in Drosophila by the homeotic gene Ultrabithorax is a classical example of Hox regulation of serial homology (Lewis, E.B. 1978. Nature 276, 565-570) and has served as a paradigm for understanding homeotic gene function. We have used DNA microarray analyses to identify potential targets of Ultrabithorax function during haltere specification. Expression patterns of 18 validated target genes and functional analyses of a subset of these genes suggest that down-regulation of both anterior-posterior and dorso-ventral signaling is critical for haltere fate specification. This is further confirmed by the observation that combined over-expression of Decapentaplegic and Vestigial is sufficient to override the effect of Ubx and cause dramatic haltere-to-wing transformations. Our results also demonstrate that analysis of the differential development of wing and haltere is a good assay system to identify novel regulators of key signaling pathways.

Animals↗

PubMed2007

Control of spasticity in a multiple sclerosis model is mediated by CB1, not CB2, cannabinoid receptors.

BACKGROUND AND PURPOSE: There is increasing evidence to suggest that cannabis can ameliorate muscle-spasticity in multiple sclerosis, as was objectively shown in experimental autoimmune encephalomyelitis models. The purpose of this study was to investigate further the involvement of CB1 and CB2)cannabinoid receptors in the control of experimental spasticity. EXPERIMENTAL APPROACH: Spasticity was induced in wildtype and CB1-deficient mice following the development of relapsing, experimental autoimmune encephalomyelitis. Spastic-hindlimb stiffness was measured by the resistance to flexion against a strain gauge following the administration of CB1 and CB2 agonists. KEY RESULTS: As previously suggested, some CB2-selective agonists (RWJ400065) could inhibit spasticity. Importantly, however, the anti-spastic activity of RWJ400065 and the therapeutic effect of non-selective CB1/CB2 agonists (R(+)WIN55,212-2 and CP55, 940) was lost in spastic, CB1-deficit mice. CONCLUSIONS AND IMPLICATIONS: The CB1 receptor controls spasticity and cross-reactivity to this receptor appears to account for the therapeutic action of some CB2 agonists. As cannabinoid-induced psychoactivity is also mediated by the CB1 receptor, it will be difficult to truly dissociate the therapeutic effects from the well-known, adverse effects of cannabinoids when using cannabis as a medicine. The lack of knowledge on the true diversity of the cannabinoid system coupled with the lack of total specificity of current cannabinoid reagents makes interpretation of in vivo results difficult, if using a purely pharmacological approach. Gene knockout technology provides an important tool in target validation and indicates that the CB1 receptor is the main cannabinoid target for an anti-spastic effect.

Animals↗

PubMedDate not supplied

Identification of rat pancreatic carcinoma genes associated with lymphogenous metastasis.

In order to identify metastasis-associated and promoting genes of pancreatic carcinoma we investigated the transcriptional profile of rat pancreatic carcinoma cell lines BSp73-AS (non-metastatic) and BSp73-ASML (highly metastatic) with Affymetrix GeneChip Array technology. We analyzed the expression profile of 7000 genes. Two hundred and ten genes (3%) were up-regulated and 247 genes (3.5%) were down-regulated in the metastatic cell line based on a fold change of expression of at least 3 and a change factor quality of > or = 2. In order to classify the de-regulated genes we defined the following categories: proteases and protease-related genes, cytokines, receptor tyrosine kinases, other transmembrane proteins/receptors, transcription, cell cycle/apoptosis, signaling, adhesion/extracellular matrix, metabolism, detoxification, protein modification, trafficking, immune response and other genes. We identified de-regulated AP1, FRA-1 and c-myc-mediated transcription in cell line BSp73-ASML. Up-regulation of transmembrane tyrosine kinase receptors c-met, IGFR1, IGFR2 and EGFR family-related ligands such as HB-EGF, TGFa, amphiregulin and neuregulin as well as c-met ligand HGF point to a possible role of this system in metastasis. We identified 56 non-tyrosine kinase transmembrane receptors as new target candidates for inhibition of metastasis, four of them representing already validated targets. In addition, we identified MMP9, uPA, uPAR, cyclin D1 and S100A4 (mts1) as possible contributors of the metastatic phenotype.

Animals↗

PubMed2005

A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.

Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.

Base Sequence↗

PubMed2004

A structure-based strategy to identify new molecular scaffolds targeting the bacterial ribosomal A-site.

The need for novel antibiotics is widely recognized. A well validated target of antibiotics is the bacterial ribosome. Recent X-ray structures of the ribosome bound to antibiotics have shed new light on the binding sites of these antibiotics, providing fresh impetus for structure-based strategies aiming at identifying new ribosomal ligands. In that respect, the ribosomal decoding region of the aminoacyl-tRNA acceptor site (A-site) is of particular interest because oligonucleotide model systems of this site are available for crystallography, NMR and compound binding assays. This work presents how these different resources can be combined in a hierarchical screening strategy which has led to the identification of new A-site ligands. The approach exploits an X-ray structure of the A-site against which large and diverse libraries of compounds were computationally docked. The complementarity of the compounds to the A-site was assessed using a scoring function specifically calibrated for RNA targets. Starting from approximately 1 million compounds, the computational selection of candidate ligands allowed us to focus the experimental work on 129 compounds, 34 of which showed affinity for the A-site in a FRET-based binding assay. NMR experiments confirmed binding to the A-site for some compounds. For the most potent compound in the FRET assay, a tentative binding mode is suggested, which is compatible with the NMR data and the limited SAR in this series. Overall, the results validate the screening strategy.

Anti-Bacterial Agents↗

PubMed2006

From array to array: confirmation of genomic gains and losses discovered by array-based comparative genomic hybridization utilizing fluorescence in situ hybridization on tissue microarrays.

The combination of array-based comparative genomic hybridization (CGH) with fluorescence in situ hybridization utilizing custom-designed bacterial artificial chromosome (BAC) probes applied to tissue microarrays represents a powerful compendium of techniques-greatly enhancing the throughput of genomic analysis and subsequent target validation. Such approach can be automated at various levels and allows managing large volume of targets and samples in a few experiments. As such, this approach facilitates discovery, validation and implementation of findings in the process of identification of new diagnostic, prognostic and potentially therapeutic molecular markers.

DNA↗

PubMed2006

Drosophila in the study of neurodegenerative disease.

As populations benefit from increasing lifespans, neurodegenerative diseases have emerged as a critical health concern. How can the fruit fly, Drosophila melanogaster, contribute to curing human diseases of the nervous system? A growing number of neurodegenerative diseases, as well as other human diseases, are being modeled in Drosophila and used as a platform to identify and validate cellular pathways that contribute to neurodegeneration and to identify promising therapeutic targets by using a variety of approaches from screens to target validation. The unique properties and tools available in the Drosophila system, coupled with the fact that testing in vivo has proven highly productive, have accelerated the progress of testing therapeutic strategies in mice and, ultimately, humans. This review highlights selected recent applications to illustrate the use of Drosophila in studying neurodegenerative diseases.

Animals↗

PubMed2000

Genomics: from novel genes to new therapeutics in parasitology.

The advent of rapid DNA sequencing technologies is generating vast quantities of raw genomic information ranging from in-depth analysis of the expressed genes to complete sequencing of genomes at an increasing rate (bioinformatics). However, it is the functional characterisation of a specific gene product that is the key limiting factor for validation as targets for high throughput assay development. The challenge is to obtain the raw genomic information from parasites of economic importance and to effectively integrate broad technologies such as gene disruption and over-expression, DNA arrays, proteomics, antisense RNAs, with bioinformatics in a timely fashion to identify relevant biological targets. Screening of validated targets in a strategy that includes large numbers of chemistries with high diversity and predictive in vitro and in vivo assays should permit the successful identification of novel chemical entities with high specificity to the target parasite. It is proposed that this rational approach will permit the identification of new antiparasitic therapies able to surpass the current toxicological, environmental, and economic challenges of the marketplace.

Animals↗

PubMed2005

The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo.

Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multipotent C blastomere lineage in the C. elegans embryo. To identify PAL-1 regulatory target genes, we used microarrays to compare transcript abundance in wild-type embryos with mutant embryos lacking a C blastomere and to mutant embryos with extra C blastomeres. pal-1-dependent C-lineage expression was verified for select candidate target genes by reporter gene analysis, though many of the target genes are expressed in additional lineages as well. The set of validated target genes includes 12 transcription factors, an uncharacterized wingless ligand and five uncharacterized genes. Phenotypic analysis demonstrates that the identified PAL-1 target genes affect specification, differentiation and morphogenesis of C-lineage cells. In particular, we show that cell fate-specific genes (or tissue identity genes) and a posterior HOX gene are activated in lineage-specific fashion. Transcription of targets is initiated in four temporal phases, which together with their spatial expression patterns leads to a model of the regulatory network specified by PAL-1.

Analysis of Variance↗

PubMed2005

The tissue micro-array as a translational research tool for biomarker profiling and validation.

High-throughput molecular technologies have provided a wealth of putative biomarkers representing potential diagnostic, prognostic and therapeutic targets. Target validation commonly requires abundant and well-characterized tissue resources, as well as linked clinicopathologic data-sets. The tissue micro-array (TMA) is a validation and discovery platform of increasing popularity and necessity. TMAs provide a rapid means of examining in situ gene expression in a wide spectrum and large number of tissue samples. This paper describes the uses of TMAs as a translational research tool in biomarker profiling. The laboratory has performed numerous biomarker studies on several types of epithelial cancers. As an example, Epithelial Protein Cell Adhesion Molecule (EpCAM), a panepithelial antigen used to target tumours with immunotherapy, was examined in patients with renal cell carcinoma (RCC). EpCAM was an independent prognosticator for improved disease-specific survival in 318 clear cell RCCs examined, with a Cox proportional hazards multivariate hazard ratio of 0.63 (p=0.017; 95% confidence interval 0.43-0.92). Interestingly, despite a typically widespread epithelial expression, EpCAM is instead infrequently expressed in clear cell RCC, the most common type of renal cancer, making it a poor target for immunotherapy. In this capacity the TMA provided strong support for halting lengthy and costly clinical trials for this application.

Animals↗

PubMed2024

N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

Animals↗