Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening.

The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells.

Lung MRI for experimental drug research.

Current techniques to evaluate the efficacy of potential treatments for airways diseases in preclinical models are generally invasive and terminal. In the past few years, the flexibility of magnetic resonance imaging (MRI) to obtain anatomical and functional information of the lung has been explored with the scope of developing a non-invasive approach for the routine testing of drugs in models of airways diseases in small rodents. With MRI, the disease progression can be followed in the same animal.

Trends in drug discovery technologies - meeting highlights from MipTec 2007.

The 10th anniversary of MipTec, which took place in Basel, Switzerland on 7 - 10 May 2007, demonstrated that this meeting has truly evolved into the leading European event for drug discovery technologies. The rich programme included sessions on drug discovery processes, structure-based drug design, pharmacodynamics and biomarkers, maximizing compound value, drug discovery technologies or emerging absorption, distribution, metabolism and excretion/toxicity technologies together with keynote presentations providing the larger picture, an excellent poster session and a well-attended vendor exhibition. In summary, MipTec more than ever provides a much needed hub for scientific exchange and networking within the European drug discovery community.

Structural modification of the P2' position of 2,7-dialkyl-substituted 5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamides: the discovery of aliskiren, a potent nonpeptide human renin inhibitor active after once daily dosing in marmosets.

Due to its function in the rate limiting initial step of the renin-angiotensin system, renin is a particularly promising target for drugs designed to control hypertension, a growing risk to health worldwide. Despite vast efforts over more than two decades, no orally efficacious renin inhibitor had reached the market. As a result of a structure-based topological design approach, we have identified a novel class of small-molecule inhibitors with good oral blood-pressure lowering effects in primates.

Novel 2,7-dialkyl-substituted 5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamide transition state peptidomimetics are potent and orally active inhibitors of human renin.

The action of renin is the rate-limiting step of the renin-angiotensin system (RAS), a key regulator of blood pressure. Effective renin inhibitors directly block the RAS entirely at source and, thus, may provide a vital weapon for hypertension therapy. Our efforts toward identifying novel small-molecule peptidomimetic renin inhibitors have resulted in the design of transition-state isosteres such as 1 bearing an all-carbon 8-phenyl-octanecarboxamide framework.

Allosteric modulators for mGlu receptors.

The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions.

Insight into the structural determinants for selective inhibition of matrix metalloproteinases.

The matrix metalloproteinase (MMP) family has been a pharmaceutical target for over 20 years. Despite massive research and development efforts, only one MMP inhibitor (Periostat) has been approved by the FDA for the treatment of periodontal disease. Possible reasons for the low success rate of MMP inhibitors in the clinic include unwanted side effects caused by their lack of selectivity, poor oral bioavailability and decreased potency in vivo.

Synergistic effect of formoterol and mometasone in a mouse model of allergic lung inflammation.

Background And Purpose: Controversy still exists as to whether or not inhaled beta (2)-adrenoceptor agonists and corticosteroids act synergistically in vivo. In this study, we have used a murine model of lung inflammation to study the synergistic effect of an inhaled beta (2)-adrenoceptor agonist (formoterol) and an inhaled corticosteroid (mometasone).

Experimental Approach: Actively sensitized mice were challenged with aerosolized ovalbumin, once a day, for three consecutive days.

siRNAs in drug discovery: target validation and beyond.

'Omics' technologies, combined with knowledge management, have changed the way pharmaceutical companies approach drug discovery activities. The rapid generation of more reliable putative drug targets from gain- or loss-of-function genome scale screens means that there is a strong need to filter and prioritize targets that will be considered in the drug discovery process. Short interfering RNAs (siRNAs) can be used to validate genomic drug targets.

Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is critically involved in the embryonic development of the cardiovascular and central nervous systems. In the adult, S1P can produce cytoskeletal re-arrangements in many cell types to regulate immune cell trafficking, vascular homeostasis and cell communication in the central nervous system. S1P is contained in body fluids and tissues at different concentrations, and excessive production of the pleiotropic mediator at inflammatory sites may participate in various pathological conditions.

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism.

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis).

Classical PKC isoforms in cancer.

Protein kinases C (PKCs) are a family of serine/threonine kinases which are involved in tumor formation and progression. Although PKC over-expression has been shown to contribute to cell transformation, PKCs are not considered to be classical oncogenes which are activated by mutations, but considered as tumor promoters that enhance multiple cellular signaling pathways. Over the last two decades a lot of evidence has emerged demonstrating a role for various PKC isoforms in cancer.

Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells.

A detailed protocol is described allowing the generation of essentially pure populations of glutamatergic neurons from mouse embryonic stem (ES) cells. It is based on the culture of ES cells that are kept undifferentiated by repeated splitting and subsequently amplified as non-adherent cell aggregates. Treatment with retinoic acid causes these ES cells to essentially become neural progenitors with the characteristics of Pax6-positive radial glial cells.

SAR of the arylpiperazine moiety of obeline somatostatin sst1 receptor antagonists.

The SAR of over 50 derivatives of octahydrobenzo[g]quinoline (obeline)-type somatostatin sst(1) receptor antagonist 1 is presented, focusing on the modification of its arylpiperazine moiety. Sst(1) affinities in this series cover a range of five orders of magnitude with the best derivatives displaying subnanomolar sst(1) affinities and >10,000-fold selectivities over the sst(2) receptor subtype as well as promising pharmacokinetic properties.

Identification and SAR of potent and selective non-peptide obeline somatostatin sst1 receptor antagonists.

A novel class of non-peptide somatostatin receptor ligands bearing the octahydrobenzo[g]quinoline (obeline) structural element has been identified. SAR studies have been performed that led to the discovery of derivatives with high affinity (pK(d) r sst(1) > or = 9) and selectivity (> or = 150-fold for h sst(1) over h sst(2)-h sst(5)) for somatostatin receptor subtype sst(1). In a functional assay, the compounds act as antagonists at human recombinant sst(1) receptors.

Specific roles of GABA(B(1)) receptor isoforms in cognition.

The GABA(B) receptor is a heterodimer of GABA(B(1)) and GABA(B(2)) subunits. There are two isoforms of the GABA(B(1)) subunit: GABA(B(1a)) and GABA(B(1b)). Recent studies with mutant mice suggest a differential role for the two GABA(B(1)) isoforms in behavioural processes.

In vivo mouse imaging and spectroscopy in drug discovery.

Imaging modalities such as micro-computed tomography (micro-CT), micro-positron emission tomography (micro-PET), high-resolution MRI, optical imaging, and high-resolution ultrasound have become invaluable tools in preclinical pharmaceutical research. They can be used to non-invasively investigate, in vivo, rodent biology and metabolism, disease models, and pharmacokinetics and pharmacodynamics of drugs. The advantages and limitations of each approach usually determine its application, and therefore a small-rodent imaging laboratory in a pharmaceutical environment should ideally provide access to several techniques.

RNA interference for studying the molecular basis of neuropsychiatric disorders.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by sequence-specific mRNA degradation, which is triggered by short double-stranded RNA. This mechanism can be efficiently induced both in vitro and in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, or by the application of short interfering RNA (siRNA). The design of highly selective and efficacious siRNA and shRNA has become commonplace, owing to continuous progress in modeling, bioinformatics and chemistry.

Phosphorylated FTY720 stimulates ERK phosphorylation in astrocytes via S1P receptors.

Sphingosine-1-phosphate receptors (S1P1-5) are activated by the endogenous agonist S1P and are expressed in the central nervous system. In astrocytes, activation of S1P receptors leads to phosphorylation of extracellular-signal regulated kinase (ERK), a signaling cascade which plays intimate roles in cell proliferation. Fingolimod (FTY720) is in phase III clinical trials for the treatment of multiple sclerosis and its phosphorylated version (FTY720P) activates S1P receptors.

An automated microseed matrix-screening method for protein crystallization.

A microseed-matrix procedure has been established with the aim of influencing the nucleation event in standard crystallization screens. The method is based on the original description of matrix seeding described by Ireton & Stoddard (2004, Acta Cryst. D60, 601-605).

IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and toll-like receptor 7-mediated signaling and gene expression.

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD).

Computer-assisted reading in drug discovery.

We are witnessing an exponential increase in the available publications, patents and textual documents that can no longer be assimilated by individual scientists. The complexity of the information landscape is further enhanced by the constantly growing number and diversity of databases and web-based information sources. Therefore, much scientific information might go unnoticed or untapped by a large portion of the scientific community.

Aspartic proteases in drug discovery.

Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin.

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices.

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods.

Optimization of protein expression systems for modern drug discovery.

The expression of high levels of stable and functional proteins remains a bottleneck in many scientific endeavors, including the determination of structures in a high-throughput fashion or the screening for novel active compounds in modern drug discovery. Recently, numerous developments have been made to improve the production of soluble and active proteins in heterologous expression systems. These include modifications to the expression constructs, the introduction of new and/or improved pro- and eukaryotic expression systems, and the development of improved cell-free protein synthesis systems.