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PubMed1995

Evaluation of the efficacy and safety of isepamicin compared with amikacin in the treatment of nosocomial pneumonia and septicaemia.

Isepamicin is a new aminoglycoside antibiotic which possesses greater stability to aminoglycoside-inactivating enzymes compared with other available aminoglycosides. In this prospective, randomised, open trial, the safety and efficacy of intravenous administration of isepamicin was compared with that of intravenous amikacin in seriously ill adults with nosocomial pneumonia or septicaemia. Each study aminoglycoside was administered concurrently with ceftazidime or imipenem. Patients were randomised to receive isepamicin 15 mg/kg once daily, isepamicin 7.5 mg/kg twice daily or amikacin 7.5 mg/kg twice daily. For patients with nosocomial pneumonia, the proportions of patients in the intent-to-treat population (n = 130) who were clinically cured at the end of treatment were similar in each treatment group: 18/44 (41%) isepamicin once daily; 19/45 (42%) isepamicin twice daily; and 17/41 (42%) amikacin. Corresponding results for the efficacy population (n = 58) were: 12/20 (60%) isepamicin once daily; 14/21 (67%) isepamicin twice daily; 9/17 (53%) amikacin. In patients with septicaemia, clinical cure was achieved in 8/10 (80%) patients treated with isepamicin once daily, compared with 8/13 (62%) patients who received isepamicin twice daily, and 7/12 (58%) patients treated with amikacin. For both diagnoses, there were no statistically significant differences between the treatment groups in clinical cure rate. The most commonly isolated target pathogen was Pseudomonas aeruginosa. For both nosocomial pneumonia and septicaemia, the proportion of patients in the intent-to-treat population whose pretreatment valid target pathogens were eliminated was similar in each treatment group. In total, 51 patients (30%) died during study, mostly due to disease progression or complications, or concurrent illness. All three treatment regimens were well tolerated. The proportion of patients experiencing at least one adverse event was 11%, 25% and 9% for isepamicin once daily, isepamicin twice daily and amikacin, respectively. The incidence of ototoxicity and nephrotoxicity was relatively low in both treatment groups.

Amikacin↗

PubMed2004

Potassium channel subtypes as molecular targets for overactive bladder and other urological disorders.

Potassium channels have re-emerged as attractive targets for overactive bladder and other urological diseases in recent years, in part due to an enhanced understanding of their molecular heterogeneity, tissue distribution, functional roles and regulation in physiological and pathological states. Cloning and heterologous expression analysis, coupled with the advancement of improved high-throughput screening techniques, have enabled expeditious identification of selective small-molecule openers and blockers for ATP-sensitive K+ channels, Ca2+-activated K+ channels and voltage-dependent K+ channel-KQT-like subfamily (KCNQ) members, and has paved the way in the assessment of efficacy and adverse effects in preclinical models. This review focuses on the rationale for molecular targeting of K+ channels, the current status of target validation, including preclinical proof-of-concept studies, and provides perspectives on the limitations and hurdles to be overcome in realising the potential of these targets for diverse urological indications such as overactive bladder, erectile dysfunction and prostate diseases.

Aged↗

PubMed2003

Prospects for pharmacologic inhibition of hepatic glucose production.

Type 2 diabetes is a widespread disease where effective pharmacologic therapies can have a profound beneficial public health impact. Increased hepatic glucose production (HGP) is observed in diabetics and its moderation by currently available agents provides therapeutic benefits. This review describes the challenges associated with the discovery of small molecules that inhibit HGP. Gluconeogenesis, glycogenolysis, liver architecture, and hepatocyte composition are described to provide background information on hepatic function. Current methods of target validation for drug discovery, HGP measurement, diabetes animal models, as well as current drug therapies are covered. In the accompanying review article the new drug targets being probed to produce the next generation of therapies are described. Significant pharmaceutical and academic efforts to pharmacologically inhibit HGP has the opportunity to provide new therapeutics for type 2 diabetics.

Animals↗

PubMedDate not supplied

Targeting E3 ubiquitin ligases for cancer therapy.

E3 ubiquitin ligases are a large family of proteins that can be classified into three major structurally distinct types: N-end rule E3s, E3s containing the HECT (Homology to E6AP C-Terminus) domain, and E3s with the RING (Really Interesting New Gene) finger, including its derivatives, the U- Box and the PHD (Plant Homeo-Domain). E3 ubiquitin ligases exist as single polypeptide or multimeric complexes. Together with ubiquitin activating enzyme E1 and ubiquitin conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of protein substrates for targeted degradation via the 26S proteasome. E3 ubiqutin ligases, therefore, play an essential role in regulation of many biological processes. Furthermore, E3s are enzymes that determine the specificity of protein substrates; they represent a class of "drugable" targets for pharmaceutical intervention. In this review, I will mainly focus on E3 ubiquitin ligases as potential cancer targets and discuss three of the most promising E3s, Mdm2/Hdm2, IAPs, and SCF, for their target rationales, target validation, and critical issues associated with them. These E3 ligases or their components are overexpressed in many human cancers and their inhibition leads to growth suppression or apoptosis. In addition, I will evaluate two current methodologies available for the high throughput screening for small molecular weight chemical inhibitors of the E3 ubiquitin ligases. Although targeting E3 ubiquitin ligases is still in its infancy, speedy approval of the general proteasome inhibitor, Velcade (bortezomib) by the FDA for the treatment of relapsed and refractory multiple myeloma suggests the promise of specific E3 inhibitors in anti-cancer therapy. Emerging technologies, such as siRNA, will provide a better validation of many E3s. It is anticipated that E3 ubiquitin ligases will represent an important new target platform for future mechanism-driven drug discovery.

Animals↗

PubMed2003

Novel molecular targets in pain control.

PURPOSE OF REVIEW: The complexity of pain processing in clinical pain conditions and in animal models has revealed many time-related changes and an abundance of molecular drug targets. There continues to be insecurity, however, about new target validation in clinical pain and thus most analgesia development is of high risk for evolving new pain therapies. The present review highlights a number of molecular targets being pursued for pain control. RECENT FINDINGS: Many pain targets are critically dependent on the pain model/lesion type. Neural and glial plasticity, ranging from changes in molecular expression and receptor phosphorylation to profound morphological reorganization, has been described under these conditions. Pain modulation has been shown to involve all major families of regulatory proteins such as the G-protein coupled receptors, ion channels, regulatory enzymes, neurotrophins, and kinases, offering an abundance of targets and therapeutic opportunities for symptomatic pain relief. SUMMARY: Many molecular targets have been highlighted with some being the focus of current analgesia research. Some of these (e.g. vanilloid receptor 1, cannabinoid receptor 1, sodium channel NaV 1.8) have been evaluated in animal studies and in preliminary clinical studies, but others are highly novel and riskier analgesia pain targets (e.g. metabotropic glutamate receptors, sensory neurone specific receptors, kinase inhibitors).

Journal Article↗

PubMed2006

RNAi: a novel antisense technology and its therapeutic potential.

Antisense oligonucleotide agents induce the inhibition of target gene expression in a sequence-specific manner by exploiting the ability of oligonucleotides to bind to target RNAs via Watson-Crick hybridization. Once bound, the antisense agent either disables or induces the degradation of the target RNA. This technology may be used for therapeutic purposes, functional genomics, and target validation. There are three major categories of gene-silencing molecules: (1) antisense oligonucleotide derivatives that, depending on their type, recruit RNase H to cleave the target mRNA or inhibit translation by steric hindrance; (2) ribozymes and deoxyribozymes--catalytically active oligonucleotides that cause RNA cleavage; (3) small interfering double-stranded RNA molecules that induce RNA degradation through a natural gene-silencing pathway called RNA interference (RNAi). RNAi is the latest addition to the family of antisense technologies and has rapidly become the most widely used approach for gene knockdown because of its potency. In this mini-review, we introduce the RNAi effect, briefly compare it with existing antisense technologies, and discuss its therapeutic potential, focusing on recent animal studies and ongoing clinical trials. RNAi may provide new therapeutics for treating viral infections, neurodegenerative diseases, septic shock, macular degeneration, cancer, and other illnesses, although in vivo delivery of small interfering RNAs remains a significant obstacle.

Animals↗

PubMed2005

How many genomics targets can a portfolio afford?

The pharmaceutical industry can look back at a history of successful innovations. Although genomics technologies have provided drug discovery pipelines with a plethora of new potential drug targets, solid target validation is crucial to avoiding high attrition rates. Biomarkers for patient stratification and approaches for personalized medicine will further help to reduce the risk associated with new targets. To achieve an overall risk balance, portfolios have to be supplemented with precedented targets, me-too approaches and line extensions of existing drugs. However, capitalizing on genomics investments and working on unprecedented targets is essential for a continuous stream of innovative drugs.

Drug Industry↗

PubMed2003

CNIO cancer conference: targeted search for anticancer drugs.

The topics discussed at the conference covered many aspects of cancer research, from the genetic search for new targets, target validation and drug discovery, all the way to preclinical and clinical development of oncology drugs. Here the presentations on new metabolic, angiogenic, cell cycle and other molecular targets, as well as recent developments with experimental drugs with action on some of these targets, are summarised. Particular emphasis is placed on the emerging realisation that changes in the metabolic phenotype lie at the heart of cellular transformation. New insights into the biological links between cancer cell metabolism and the balance between survival and death signalling are likely to lead to the identification of a new category of anticancer targets.

Animals↗

PubMed2003

Chemogenomics knowledge-based strategies in drug discovery.

In the postgenomic age of drug discovery, targets can no longer be viewed as singular objects having no relationship to one another. All targets are now visible and the systematic exploration of selected target families appears to be a promising way to speed up and further industrialize target-based drug discovery. Chemogenomics refers to such systematic exploration of target families and aims to identify all possible ligands of all target families. Because biology works by applying prior knowledge to an unknown entity, chemogenomics approaches are expected to be especially effective within the previously well-explored target families, for which, in addition to the protein sequence and structure information, considerable knowledge of pharmacologically active structural classes and structure-activity relationships exists. For the new target families, chemical knowledge will have to be generated and beyond biological target validation, the emphasis is on chemistry to provide the molecules with which their novel biology and pharmacology can be studied. Using examples from the previously most successfully explored target families, the GPCR family in particular, we summarize herein our current chemogenomics knowledge-based strategies for drug discovery, which are founded on the high integration of chem and bioinformatics, thereby providing a molecular informatics frame for the exploration of the new target families.

Computational Biology↗

PubMed2002

Aptamers and aptazymes: accelerating small molecule drug discovery.

Synthetic nucleic acid ligands, known as aptamers, are versatile tools that can greatly enhance the efficiency of modern drug development. Exhibiting binding characteristics comparable to or even better than monoclonal antibodies, these ligands can be used as detection probes, highly efficient inhibitors of protein function or specific competitors in high-throughput screening (HTS) assays. Thus, aptamer technology can be exploited to address the growing demand for multi-parallel analysis of proteomes, functional prioritization of potential drug targets and accelerated small molecule lead identification. The unique advantages of this technology are the rapid automated generation of sophisticated ligands against almost any target molecule and the convenient structural or chemical modification of the nucleic acid probes. Depending on the strategy, an RNA aptamer can be expressed transgenically to investigate and inactivate an endogenous protein in an animal model, or it can be designed to function as a highly sensitive nucleic acid biosensor. More recently, the technology has been extended to directly link functional target validation with HTS, accelerating the process of drug discovery.

Animals↗

PubMed2005

Modulation of human dUTPase using small interfering RNA.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is responsible for maintaining low intracellular levels of dUTP, thus preventing the incorporation of dUTP into DNA. A 21 bp double-stranded RNA molecule (siRNAdUT3) targeted against motif 3 of human dUTPase resulted in a time- and dose-dependent decrease in dUTPase activity in transfected cells. dUTPase activity was reduced approximately 95+/-5% in all cell lines tested 48 h after transfection with 2 microg siRNAdUT3 and it was maintained at this decreased level for at least 72 h. Down-regulation of dUTPase resulted in a significant increase in intracellular dUTP and a decreased proliferation of the transfected cells. Therefore, we conclude that dUTPase activity/expression can be down-regulated using siRNA specifically targeted to dUTPase mRNA and that this approach can be used to elucidate the role of dUTPase in DNA metabolism, as well as, to determine whether dUTPase is a valid target for drug development.

Cell Line, Tumor↗

PubMed2003

RNA interference: new mechanisms for targeted treatment?

Nucleic acid-based sequence-specific therapeutic intervention offers the potential for treatment of particular cancers without side effects. RNA interference (RNAi) induced by small interfering RNA (siRNA) (19-21 bp) is a normal cellular mechanism leading to highly specific and extraordinarily efficient degradation of the corresponding mRNA. The mechanism of RNAi as well as strategies for the design and delivery of siRNA are described. The growing role of RNAi in target validation for cancer-specific genetic aberrations is discussed. We attempt an early assessment of the potential for using RNAi technologies to treat cancer directly, especially hematologic malignancies. Promising targets for specific gene silencing in hematologic oncology include oncogenic fusion proteins and oncogenes activated by point mutations. Potency and specificity of gene silencing are the major advantages of the new RNAi technology over other nucleic acid-based gene targeting approaches. Crucial questions for pharmaceutical interventions remain. Advances in the areas of delivery, systemic spreading and duration of the silencing effect are necessary before the methodology can enter clinical oncology.

Animals↗

PubMed2003

Advances in high content screening for drug discovery.

Cell-based target validation, secondary screening, lead optimization, and structure-activity relationships have been recast with the advent of HCS. Prior to HCS, a computational approach to the characterization of the functions of specific target proteins and other cellular constituents, along with whole-cell functions employing fluorescence cell-based assays and microscopy, required extensive interaction among the researcher, instrumentation, and software tools. Early HCS platforms were instrument-centric and addressed the need to interface fully automated fluorescence microscopy, plate-handling automation, and seamless image analysis. HCS has since evolved into an integrated solution for accelerated drug discovery by encompassing the workflow components of assay and reagent design, robust instrumentation for automated fixed-end-point and live cell kinetic analysis, generalized and specific BioApplication software (Cellomics, Pittsburgh, PA) modules that produce information on drug responses from cell image data, and informatics/bioinformatics solutions that build knowledge from this information while providing a means to globalize HCS throughout an entire organization. This review communicates how these recent advances are incorporated into the drug discovery workflow by presenting a real-world use case.

Drug Design↗

PubMed2005

Designing scaffolds of peptides for phage display libraries.

Phage display is a powerful method for the discovery of peptide ligands that are used for analytical tools, drug discovery, and target validations. Phage display technology can produce a huge number of peptides and generate novel peptide ligands. Recently, phage display technology has successfully managed to create peptide ligands that bind to pharmaceutically difficult targets such as the erythropoietin receptor. As a result of the structural analysis of their ligands, we found that the conformational design of peptides in library is important for selecting high-affinity ligands that bind to every target from a phage peptide library. Key issues concern constraints on the conformation of peptides on the phage and the development of chemically synthesized peptides derived from peptides on phage. This review discusses studies related to the conformation of peptides selected from phage display peptide libraries in addition to the conversion from peptides to non-peptides.

Amino Acid Sequence↗

PubMed2006

Rethinking target discovery in polygenic diseases.

Despite an extraordinary investment in R&D the yield of successful new drugs has been disproportionately low in recent years, suggesting that the whole process of drug development requires rethinking and reform. Most analyses on this issue focus on molecular target discovery considerations. Target identification is characterized by a surplus of potential targets, but there is a translational bottleneck primarily due to limitations of currently employed target validation platforms. Meanwhile, the clinical entities, to which treatments are directed, are also highly complex in terms of pathophysiologic mechanisms and manifestations. In the present study we discuss the limitations of current molecular target discovery approaches mainly in regard to selectivity and efficacy. We also describe the constraints imposed on drug development by the current diagnostic constructs and the tendency towards dissecting the complex clinical phenotypes to component intermediate phenotypes. Finally, we describe how the reconsideration of molecular and clinical targets in polygenic diseases may lead to new strategies of pharmacological intervention directed against component dysfunctions, rather than the whole complex phenotype. Such strategies involve the combination of single ligands that act selectively on multiple molecules involved in a particular disease, or the employment of "multi-targeted" drugs, i.e. single drug molecules that hit selectively multiple receptors sharing common binding sites.

Animals↗

PubMed2005

Complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for C5b-9.

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.

Animals↗

PubMed1994

The Zimbabwe External Quality Assessment Scheme (ZEQAS) in clinical chemistry: results of the pilot programme.

A pilot programme for assessing laboratory performance in clinical chemistry laboratories in Zimbabwe is described (ZEQAS). Twenty four laboratories providing patient care services participated. Eight lyphilised bovine sera were distributed over one year. Consensus values and the spread of interlaboratory agreement were calculated for each of 12 analytes and compared with results previously obtained in a large mature national EQA scheme in the UK (UK NEQAS). For all analytes except phosphate, the mean consensus value obtained in ZEQAS was between 94 and 108 pc of the UK target, although the spread of results in ZEQAS was generally two to threefold greater for individual analytes than in UK NEQAS. It is concluded the ZEQAS consensus values for the analytes surveyed provide a valid target against which individual laboratory performance can be assessed. The wide spread of results from individual laboratories suggests there is considerable scope for improving interlaboratory agreement. This is being addressed by the continuing programme, with increased interaction and production of local specimens.

Chemistry, Clinical↗

PubMed2005

Sequence-based discovery of the human and rodent peroxisomal proteome.

BACKGROUND: Peroxisomes are metabolic organelles present in virtually all eukaryotic cells. They contain enzymes involved in hydrogen peroxide-based respiration and lipid metabolism. At present, only a small number of peroxisomal enzymes that are associated with oxidative stress response and metabolic disorders have been characterised biochemically. Therefore, we devised a sequence-based, multistep knowledge discovery strategy to identify potential novel peroxisomal protein candidates in small rodent model organisms and human. METHODS: Screening of 130,629 putative translations of GenBank rodent and primate mRNA sequences was limited to the classical type-1 peroxisomal targeting signal [SA]-K-L. This motif is over-represented among peroxisomal proteins and has a high targeting efficiency. Subsequent steps of identifying co-occurring motifs, secondary structure properties, orthologues and variants, in combination with literature searching and visual inspection by domain experts, aimed at reduction of both false positive and negative validation targets. RESULTS: Our method yielded 117 known peroxisome-targeted proteins and 29 novel candidate proteins. Of special interest were the mouse C530046K17Rik and 1300019N10Rik protein sequences that contain domains associated with enzymatic functions. C530046K17Rik showed no similarity to any known sequence of the animal kingdom, but weak similarity to the possible Leishmania quinone oxidoreductase and a putative cyanobacterium nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidoreductase. 1300019N10Rik contains two protease-related domains, glutamyl endopeptidase I and trypsin-like serine and cysteine proteases, which may have unique specificities to achieve efficient breakdown of proteins in the peroxisomes. CONCLUSION: One mouse C57BL/6J strain-specific isocitrate dehydrogenase 1 isoform might be suitable to investigate potential phenotypes associated with the deficit of the intraperoxisomal reduced form of NADP (NADPH) and 2-oxoglutarate. Our biological knowledge discovery strategy enabled not only the identification of peroxisomal enzymes already described in the literature, but also the prediction of several novel proteins with possible roles in peroxisomal biochemistry and metabolism that are currently under experimental validation.

Amino Acid Sequence↗