Fragment analysis in small molecule discovery.

Cheminformatics is playing an ever-increasing role in small molecule drug discovery. The widespread use of high-throughput screening (HTS) and combinatorial chemistry techniques has led to the generation of large amounts of pharmacological data which, in turn, has catalyzed the development of computational methods designed to reduce the time and cost in identifying molecules suitable for pharmaceutical development. This review focuses on recent advances in the field of substructure analysis, an increasingly popular data mining technique with applications at many levels of the discovery process, including HTS, compound library design, virtual screening and the prediction of biological activity.

Reduction of amyloid load and cerebral damage in a transgenic mouse model of Alzheimer's disease by treatment with a beta-sheet breaker peptide.

Genetic, neuropathological, and biochemical studies have provided strong evidence for a central role of amyloid in the pathogenesis of Alzheimer's disease (AD). We have proposed previously that peptides designed as beta-sheet breakers may be useful in preventing the formation of amyloid plaques. In this study, we describe a modified beta-sheet breaker peptide with improved pharmacological properties, a high rate of penetration across the blood-brain barrier, and the ability to induce a dramatic reduction in amyloid deposition in two different transgenic AD models.

Converting a peptide into a drug: strategies to improve stability and bioavailability.

The discovery of peptide hormones, growth factors and neuropeptides implicated in vital biological functions of our organism has increased interest in therapeutic use of short peptides. However, the development of peptides as clinically useful drugs is greatly limited by their poor metabolic stability and low bioavailability, which is due in part to their inability to readily cross membrane barriers such as the intestinal and blood-brain barriers. The aim of peptide medicinal chemistry is, therefore, to develop strategies to overcome these problems.

MIG--differential gene expression in mouse brain endothelial cells.

Different diseases of the CNS are associated with blood-brain barrier (BBB) damage and mononuclear cell infiltration. In order to study genes that may play a role in endothelial cell regulation in inflammatory CNS diseases, we performed differential gene expression (DGE) analysis using a mouse brain endothelial cell line. We found that interferon-gamma (IFNgamma)-induced monokine (MIG), a chemokine that plays a role in T lymphocyte and monocyte chemoattraction, is highly expressed in the presence of inflammatory cytokines.

Chemokine receptors--the next therapeutic target for HIV?

To date, the available therapies for the treatment of HIV infection are targeted against proteins encoded by the virus itself. Thus, combination drug therapies for HIV with reverse transciptase and protease inhibitors have resulted in spectacular reductions of viraemia, often leading to a remarkable improvement in symptoms and recovery from disease in infected people. There is still however, a great need for improved therapies since many patients are unable to take these drugs, either for reasons of intolerance, strain resistance, complexity of regimen or prohibitive cost.

Chemokine receptors: multifaceted therapeutic targets.

Chemokines and their receptors are involved in the pathogenesis of diseases ranging from asthma to AIDS. Chemokine receptors are G-protein-coupled serpentine receptors that present attractive tractable targets for the pharmaceutical industry. It is only ten years since the first chemokine receptor was discovered, and the rapidly expanding number of antagonists holds promise for new medicines to combat diseases that are currently incurable.

IL-18-binding protein expression by endothelial cells and macrophages is up-regulated during active Crohn's disease.

The pathogenesis of Crohn's disease (CD) remains under intense investigation. Increasing evidence suggests a role for mature IL-18 in the induction of proinflammatory cytokines and Th1 polarization in CD lesions. The aim of this study was to investigate the contribution of the IL-18-neutralizing (a and c) and non-neutralizing (b and d) isoforms of IL-18-binding protein (IL-18BP) during active CD.

Biochemical and structural studies of the prion protein polymorphism.

A hallmark event in transmissible spongiform encephalopathies is the conversion of the physiological prion protein into the disease-associated isoform. A natural polymorphism at codon 129 of the human prion gene, resulting in either methionine or valine, has profound influence on susceptibility and phenotypic expression of the disease in humans. In this study, we investigated the local propensity of synthetic peptides, corresponding to the region of the polymorphism and containing either methionine or valine, to adopt a beta-sheet-rich structure similar to the pathological protein.

Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease.

Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion.

Two major intracellular apoptosis signaling cascades have been characterized, the mitochondrial pathway and the death receptor pathway. The mitochondrial pathway is regulated by members of the Bcl-2 protein family. The members of this family can be subdivided into anti- and pro-apoptotic proteins.

The chemokine system: novel broad-spectrum therapeutic targets.

Chemokines are cytokines that specifically direct the trafficking of immune cells in the body. They offer a novel point of therapeutic intervention, as inhibiting specific chemokines and receptors could prevent the excessive recruitment of leukocytes to sites of inflammation. This approach could be considered to act upstream of the therapies used today which, for the most part, act on the cells already at the site of inflammation.

Cellular assays of chemokine receptor activation.

This unit describes procedures for measuring the activation of chemokine receptors by their ligands. Chemokines are chemoattractant proteins and two assays are described for analyzing the chemoattractant properties (chemotaxis) of these proteins. Chemokine receptors are coupled to G proteins, and activation of the receptors results in the mobilization of intracellular Ca²⁺ stores.

Characterization of chemokine receptors.

This unit describes the procedures for measuring binding of a radiolabeled chemokine to chemokine receptors in cells or cell membranes. The whole-cell binding assay can be used for both purified leukocytes as well as transfected cell lines expressing chemokine receptors. Two basic protocols are described.

Detecting the impact of sequencing errors on SAGE data.

Unlabelled: SAGE data are obtained by sequencing short DNA tags. Due to the mistakes in DNA sequencing, SAGE data contain errors. We propose a new approach to identify tags whose abundance is biased by sequencing errors.

Pulling strings below the surface: hormone receptor signaling through inhibition of protein tyrosine phosphatases.

Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptor's tyrosine residues. At any given moment, the receptor's phosphorylation state depends on the balance of kinase and phosphatase activities.

Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.

Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1).

Protein misfolding and disease; protein refolding and therapy.

Diverse human disorders, including several neurodegenerative diseases and systemic amyloidosis, are thought to arise from the misfolding and aggregation of an underlying protein. Recent findings strongly support this hypothesis and have increased our understanding of the molecular mechanism of protein conformational disorders. Many questions are still pending, but the data overall suggest that correction of protein misfolding constitutes a viable therapeutic strategy for conformational diseases.

Prions: disease propagation and disease therapy by conformational transmission.

Transmissible spongiform encephalopathies - also known as prion-related diseases - are a group of fatal neurodegenerative disorders associated with the misfolding of prion protein. Several unprecedented scientific findings, which have directly confronted popular dogmas in biology, have put prion research in the spotlight. The experimental evidence supports an entirely novel disease mechanism, involving disease transmission by replication of protein conformation.

Inhibiting the conversion of soluble amyloid-beta peptide into abnormally folded amyloidogenic intermediates: relevance for Alzheimer's disease therapy.

Alzheimer's disease is a degenerative disorder of the brain for which there is no cure or effective treatment. Recent studies suggest that cerebral amyloid plaques are central to the disease process. However, it is not clear which of the species going from the normal soluble amyloid-beta peptide to the mature amyloid plaque is the toxic agent in the brain.

Synthesis and characterization of fluorescent and photoactivatable MIP-1alpha ligands and interactions with chemokine receptors CCR1 and CCR5.

Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) were designed, synthesized, characterized, and applied to probe MIP-1alpha interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1alpha ligand, BP-MIP-1alpha, and the fluorescent ligand, Flu-MIP-1alpha were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1alpha. Both ligands BP-MIP-1alpha and Flu-MIP-1alpha retained high binding affinity and agonist potency at CCR1 and CCR5.

Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells.

Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane.

The strategy of blocking the chemokine system to combat disease.

One of the key characteristics of inflammation is the recruitment of leukocytes to the site of inflammation. Most anti-inflammatory strategies act intracellularly on the target cells, but after the cells have migrated to the site. We therefore propose that the prevention of cellular recruitment by blockade of the relevant chemokine receptor/ligand pair would present a novel therapy in that it would act upstream of the therapies currently in use.

The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets.

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors.