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Results for “target validation”

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At least 253 records · Page 14Linked to original sources

PubMed2002

Molecular interactions of the CcdB poison with its bacterial target, the DNA gyrase.

The ccd poison/antidote system of the F plasmid encodes CcdB, a toxin targeting the essential DNA gyrase of E. coli, and CcdA, the unstable antidote that interacts with CcdB to neutralise its toxicity. Gyrase belongs to the topoisomerase II class of enzymes and is a well-validated target for efficient therapeutic drugs, i. e. the quinolones. CcdB acts on gyrase in a similar way as quinolones do, both compounds induce double-strand breaks in DNA. Interestingly, the CcdB-binding domain of gyrase is different than that of quinolones. Therefore, novel classes of therapeutic drugs could be derived from the analysis of the interaction between CcdB and gyrase.

Bacterial Proteins↗

PubMed2000

Hammerhead ribozymes: biochemical and chemical considerations.

Ribozymes can be used to cleave specific mRNAs so as to prevent their translation. This presents an alternative to the use of antisense oligonucleotides for drug target validation or therapeutic purposes. The present review covers only one class of ribozymes, the hammerheads, which are small enough for chemical synthesis. Following an account of their structure and ability to catalyze the cleavage of target RNA is a discussion of some problems associated with their use as exogenous therapeutics. Chemical modifications that are used to address these issues are described.

Animals↗

PubMed2006

Progress towards validating the NMDA receptor hypofunction hypothesis of schizophrenia.

This article describes recent progress towards validation of the N-methyl-D-aspartate (NMDA) receptor hypofunction hypothesis of schizophrenia in preclinical models. Schizophrenia, a complex disease characterized by positive, negative and cognitive symptoms, affects 1% of the world population and requires lifelong, daily maintenance therapy. For the last several decades, thinking in this field has been dominated by the hypothesis that hyperfunction of dopamine pathways played a key role in schizophrenia. However, the therapeutic agents developed from this hypothesis have a slow onset of action and tend to improve only the positive symptoms of the disease. The NMDA receptor antagonist PCP has been shown to induce the positive, negative and cognitive symptoms of schizophrenia in healthy patients and cause a resurgence of symptoms in stable patients. These observations led to the NMDA receptor hypofunction hypothesis as an alternative theory for the underlying cause of schizophrenia. According to this hypothesis, any agent that can potentiate NMDA receptor currents has the potential to ameliorate the symptoms of schizophrenia. To date, NMDA receptor currents can be modulated by either direct action on modulatory sites on the NMDA receptor (i.e., the glycine co-agonist binding site) or indirectly by activation of G-protein coupled receptors (GPCRs) known to potentiate NMDA receptor function (i.e., mGluR5). This review will discuss the NMDA receptor hypofunction hypothesis, the NMDA receptor as an emerging target for the development of novel antipsychotic agents and progress towards in vivo target validation with GlyT1 inhibitors and mGluR5 positive allosteric modulators. Other potential targets for modulating NMDA receptor currents (polyamine sites, muscarinic receptors, etc...) will also be addressed briefly.

Allosteric Regulation↗

PubMed2006

EGFR as a target: rationale for therapy.

OBJECTIVES: To review the biology of the EGFR, its structure, and the associated signal transduction pathways. To provide an overview of the role of EGFR in normal physiology and the pathophysiology of malignancy. Current anti-EGFR treatments are also discussed. DATA SOURCES: Research articles. CONCLUSION: EGFR is a valid target in the treatment of solid tumors. EGFR abnormalities and dysfunction are involved in various aspects of carcinogenesis and tumor progression, and EGFR is overexpressed in several tumor types. The development of anti-EGFR therapies represents an important advance in cancer therapy. IMPLICATIONS FOR NURSING PRACTICE: Anti-EGFR therapy is currently available in the clinical setting. Nurses involved in the care of patients with cancer can benefit from an increased understanding of the normal and abnormal function of EGFR in the body and the mechanisms by which anti-EGFR therapies act.

Antibodies, Monoclonal↗

PubMed2005

On the delivery of small interfering RNAs into mammalian cells.

RNA interference is becoming the technique of choice for analysing gene function and drug target validation. In this process, sequence-specific gene inhibition is initiated by small RNA duplexes, known as small interfering RNAs (siRNAs). The possibility that exogenously delivered siRNAs or endogenously expressed hairpin siRNAs can cause the destruction of specific target mRNA in vitro and in animal models has been demonstrated. However, the key challenges for the development of siRNAs as human therapeutics is largely dependent on the development of suitable delivery agents and improved siRNA specificity. This review highlights recent advances in siRNA delivery, as well as challenging problems related to immune stimulation.

Animals↗

PubMed2005

Targeting ligand cleavage to inhibit the ErbB pathway in cancer.

ADAMs (a disintegrin and metalloproteases) are zinc-dependent transmembrane metalloproteases that shed the extracellular domains of membrane-bound growth factors, cytokines, and receptors. Recently, ADAMs have emerged in ErbB signaling pathways as sheddases or multiple ErbB ligands. As the ErbB pathway is a validated target for anticancer drugs, upstream activators of ErbB ligands, their sheddases, become new drug targets in the ErbB pathway. We have identified selective small molecule inhibitors of ADAM proteases that block shedding and activation of multiple ErbB ligands, and we are planning to test the compounds in the clinic.

ADAM Proteins↗

PubMed2001

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

Bacterial Proteins↗

PubMed2002

Specific and potent RNA interference in terminally differentiated myotubes.

Double-stranded RNA (dsRNA) interference is a potent mechanism for sequence-specific silencing of gene expression and represents an invaluable approach for investigating gene function in normal and diseased states as well as for drug target validation. Here, we report that skeletal muscle myoblasts and terminally differentiated myotubes are susceptible to RNA interference. We employed an approach in which dsRNA is generated by cellular transcription from plasmids containing long (1 kilobase) inverted DNA repeats of the target gene rather than using dsRNA synthesized in vitro. We show that gene silencing by this method is effective for endogenously expressed genes as well as for exogenous reporter genes. An analysis of the expression of several endogenous genes and exogenous reporters demonstrates that the silencing effect is specific for the target gene containing sequences within the inverted repeat. Our method eliminates the need to chemically synthesize dsRNA and is not accompanied by global repression of gene expression. Furthermore, we show for the first time that sequence-specific dsRNA-mediated gene silencing is possible in differentiated, multinucleated skeletal muscle myotubes. These findings provide an important molecular tool for the examination of protein function in terminally differentiated muscle cells and provide alternative approaches for generating disease models.

Animals↗

PubMed2003

Fulfilling the promise: drug discovery in the post-genomic era.

The genomic era has brought with it a basic change in experimentation, enabling researchers to look more comprehensively at biological systems. The sequencing of the human genome coupled with advances in automation and parallelization technologies have afforded a fundamental transformation in the drug target discovery paradigm, towards systematic whole genome and proteome analyses. In conjunction with novel proteomic techniques, genome-wide annotation of function in cellular models is possible. Overlaying data derived from whole genome sequence, expression and functional analysis will facilitate the identification of causal genes in disease and significantly streamline the target validation process. Moreover, several parallel technological advances in small molecule screening have resulted in the development of expeditious and powerful platforms for elucidating inhibitors of protein or pathway function. Conversely, high-throughput and automated systems are currently being used to identify targets of orphan small molecules. The consolidation of these emerging functional genomics and drug discovery technologies promises to reap the fruits of the genomic revolution.

Animals↗

PubMed2005

Targeting malaria with specific CDK inhibitors.

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum. Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC50 values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor-ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.

Adenosine Triphosphate↗

PubMed2001

Novel compounds in the therapy of breast cancer: opportunities for integration with docetaxel.

Increasingly, novel agents are being developed specifically at inhibition of growth factor receptors and events within the signal transduction pathway. These agents include the epidermal growth factor tyrosine kinase inhibitors, the farnesyl transferase inhibitors, and bcl-2 antisense oligonucleotides. Along with these new approaches to molecular targeting, it will be necessary to develop new study designs for drug evaluation. Target validation in both normal surrogate tissues and tumor tissue becomes increasingly relevant in early clinical trials. Furthermore, antitumor efficacy may no longer correlate with normal hematological or nonhematological toxicity, and it may be more appropriate in phase I trials to identify the maximum target inhibition dose rather than the maximum tolerated dose. Moreover, measures of cytoreduction, such as complete and partial response, may be less relevant than disease stabilization for some of these novel agents which have limited cytotoxic effects and would be considered cytostatic agents. Assessment of single-agent activity and the future role in conjunction with cytostatic agents represents the single most important challenge facing the clinical development of these molecular targeted therapies.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Catalytic DNA: a novel tool for gene suppression.

RNA, as an intermediate in the production of every gene encoded protein and the genetic material of many pathogenic viruses, presents an attractive target for both biological and therapeutic manipulation. Despite its extensive involvement in living systems, its chemical diversity based on four units is relatively low compared with protein. This provides the opportunity for a generic approach to targeting with specificity based on primary structure rather than complex higher order structures. This form of recognition occurs naturally in complementary nucleic acids, due to an ability to bind their single stranded target through Watson-Crick interactions. The most established nucleic acid based approach to gene suppression at the RNA level is through antisense oligodeoxynucleotides (ODNs). These compounds form heteroduplex with target RNA which are thought to either block its function or mediate its destruction by activation of RNase H. Alternatively, RNA can be targeted by catalytic RNA such as the hammerhead ribozyme. Ribozymes have the advantage of being equipped with their own RNA cleavage apparatus and are therefore independent of host nuclear protein activity. At present, the utility of ribozyme oligonucleotides is restricted by the relative difficulty synthesising active molecules with sufficient resistance to nuclease degradation. Recently the power of in vitro selection has been used to evolve catalytic DNA sequences with RNA cleavage specificity and activity rivalling the very best ribozymes, while maintaining the more robust chemistry of an ODN. These deoxyribozymes or DNAzymes have tremendous potential as gene suppression agents for both target validation and therapeutic applications. A number of studies evaluating the biological activity of these compounds have shown promising results. However, as with other oligonucleotide based strategies, future exploitation of this approach may depend on accessory technology to assist with the accessibility of a target which is folded by its own secondary structure and hidden within the intracellular compartment.

Animals↗

PubMed1995

The Rac and Rho pathways as a source of drug targets for Ras-mediated malignancies.

The small GTP-binding proteins Rac and Rho are key control elements in the reorganization of the actin cytoskeleton induced by growth factors or oncogenic Ras. It has been established recently that Rac and Rho also play a crucial role in Ras transformation. This suggests that the elements in the pathways regulated by Rac and Rho are valid targets for cancer therapy. Several important components of those signaling pathways have now have identified.

Animals↗

PubMed2003

Targeting NS5B RNA-dependent RNA polymerase for anti-HCV chemotherapy.

The global prevalence of persistent hepatitis C virus (HCV) infection and the lack of a highly effective and well-tolerated antiviral therapy have spurred intensive efforts to discover and develop novel anti-HCV therapy in the pharmaceutical industry. HCV NS5B RNA-dependent RNA polymerase (RdRp), the centerpiece for viral replication, constitutes a valid target for drug discovery. Compared to the host RNA and DNA polymerases, NS5B RdRp has distinct subcellular localization at the interface of the endoplasmic reticulum (ER) membrane and cytoplasm, a novel catalytic mechanism and many unique structural features, all of which make it an attractive target for developing effective anti-HCV therapeutics. High genetic variation among the major HCV genotypes commands that any efficacious NS5B inhibitors have to be broadly active against NS5Bs from various genotypes. Rapid viral replication and its inherent genetic diversity will certainly culminate drug resistance to any NS5B inhibitors. Therefore, iterative drug design and combination therapies of drugs that intervene with different steps in the HCV replicative cycle are needed to combat the viral infection. Many classes of nucleoside and non-nucleoside inhibitors of NS5B RdRp have been identified and appeared in literatures and patent applications. These progresses hold a considerable promise to the development of novel, specific and highly effective therapeutics to achieve sustained response and ultimately the eradication of HCV infection.

Amino Acid Sequence↗

PubMed2005

Identification of a drug target motif: an anti-tumor drug NK109 interacts with a PNxxxxP.

The synthetic compound NK109 shows anti-tumor effects against a number of human cancer cell lines. The mechanism of action is thought to involve the inhibition of DNA topoisomerase II. However, NK109 also exhibits potent anti-tumor activities against doxorubicin-, cisplatin- and etoposide-resistant human cell lines. This paper describes target validation of NK109 using biotinylated NK109 and a T7 phage library screening procedure. Phage particles displaying an affinity for NK109 were isolated and the DNA sequence determined. The amino acid sequences of selected peptides, and the results of mutation experiments by alanine scanning, confirmed that the binding target motif of NK109 is PNxxxxP. In silico analysis of the interaction between NK109 and the peptide, by docking simulation and molecular dynamics, supported this conclusion. The PNxxxxP motif exists in the C2 domain of protein kinase Calpha. NK109 was confirmed to bind the C2 domain from surface plasmon resonance analysis. Furthermore, NK109 moderately inhibited protein kinase C activity in vitro. Our results show that the anti-tumor activity of NK109 stems from interactions with multiple protein targets.

Amino Acid Motifs↗

PubMed2002

Transgenic systems in drug discovery: from target identification to humanized mice.

Pharmaceutical companies are faced with the challenge that only approximately 10% of compounds tested in costly clinical trials eventually become a new drug. Investment in early discovery research can decrease this attrition in late-stage R&D and focus resources on the best targets. Transgenic technology influences decision-making in target identification, target validation, and can also provide better models for human diseases, as well as models designed to alert researchers early about potential issues with drug metabolism and toxicity. Here we review how transgenic technology can reduce the late-stage attrition by increasing the quality of both the target and the compound.

Animals↗

PubMed2002

Proteomics-based anticancer drug discovery and development.

Proteins are important targets for drug discovery and this applied to cancer as well because there is a defect in the protein machinery of the cell in malignancy. Proteomic technologies are now being integrated with genomic approaches for cancer drug discovery and target validation. Among the large number of proteomic technologies available for this purpose, the most important ones are 3-D protein structure determination, protein microarrays, laser capture microdissection and study of protein-protein and protein-drug interactions. Cancer biomarkers and several cell pathways are important drug targets. Several companies are involved in using proteomic technologies for drug discovery. Finally, proteomic approaches will play an important role in the discovery and development of personalized medicines.

Antineoplastic Agents↗

PubMed2002

Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis.

BACKGROUND: Despite the high prevalence of benign prostatic hyperplasia (BPH) in the aging male, little is known regarding the etiology of this disease. A better understanding of the molecular etiology of BPH would be facilitated by a comprehensive analysis of gene expression patterns that are characteristic of benign growth in the prostate gland. Since genes differentially expressed between BPH and normal prostate tissues are likely to reflect underlying pathogenic mechanisms involved in the development of BPH, we performed comparative gene expression analysis using cDNA microarray technology to identify candidate genes associated with BPH. METHODS: Total RNA was extracted from a set of 9 BPH specimens from men with extensive hyperplasia and a set of 12 histologically normal prostate tissues excised from radical prostatectomy specimens. Each of these 21 RNA samples was labeled with Cy3 in a reverse transcription reaction and cohybridized with a Cy5 labeled common reference sample to a cDNA microarray containing 6,500 human genes. Normalized fluorescent intensity ratios from each hybridization experiment were extracted to represent the relative mRNA abundance for each gene in each sample. Weighted gene and random permutation analyses were performed to generate a subset of genes with statistically significant differences in expression between BPH and normal prostate tissues. Semi-quantitative PCR analysis was performed to validate differential expression. RESULTS: A subset of 76 genes involved in a wide range of cellular functions was identified to be differentially expressed between BPH and normal prostate tissues. Semi-quantitative PCR was performed on 10 genes and 8 were validated. Genes consistently upregulated in BPH when compared to normal prostate tissues included: a restricted set of growth factors and their binding proteins (e.g. IGF-1 and -2, TGF-beta3, BMP5, latent TGF-beta binding protein 1 and -2); hydrolases, proteases, and protease inhibitors (e.g. neuropathy target esterase, MMP2, alpha-2-macroglobulin); stress response enzymes (e.g. COX2, GSTM5); and extracellular matrix molecules (e.g. laminin alpha 4 and beta 1, chondroitin sulfate proteoglycan 2, lumican). Genes consistently expressing less mRNA in BPH than in normal prostate tissues were less commonly observed and included the transcription factor KLF4, thrombospondin 4, nitric oxide synthase 2A, transglutaminase 3, and gastrin releasing peptide. CONCLUSIONS: We identified a diverse set of genes that are potentially related to benign prostatic hyperplasia, including genes both previously implicated in BPH pathogenesis as well as others not previously linked to this disease. Further targeted validation and investigations of these genes at the DNA, mRNA, and protein levels are warranted to determine the clinical relevance and possible therapeutic utility of these genes.

Adult↗