Application de la modélisation moléculaire à de nouvelles approches thérapeutiques du cancer [Application of molecular modeling to new therapeutic cancer approaches].

Recent progress in the experimental determination of protein structures allow to understand, at a very detailed level, the molecular recognition mechanisms that are at the basis of the living matter. This level of understanding makes it possible to design rational therapeutic approaches, in which effectors molecules are adapted or created de novo to perform a given function. An example of such an approach is drug design, were small inhibitory molecules are designed using in silico simulations and tested in vitro. In this article, we present a similar approach to rationally optimize the sequence of killer T lymphocytes receptors to make them more efficient against melanoma cells. The architecture of this translational research project is presented together with its implications both at the level of basic research as well as in the clinics.

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Fifteen years SIB Swiss Institute of Bioinformatics: life science databases, tools and support.

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) was created in 1998 as an institution to foster excellence in bioinformatics. It is renowned worldwide for its databases and software tools, such as UniProtKB/Swiss-Prot, PROSITE, SWISS-MODEL, STRING, etc, that are all accessible on ExPASy.org, SIB's Bioinformatics Resource Portal. This article provides an overview of the scientific and training resources SIB has consistently been offering to the life science community for more than 15 years.

Peroxisome proliferator-activated receptor-beta as a target for wound healing drugs.

Healing of cutaneous wounds, which is crucial for survival after an injury, proceeds via a well-tuned pattern of events including inflammation, re-epithelialisation, and matrix and tissue remodelling. These events are regulated spatio-temporally by a variety of growth factors and cytokines. The inflammation that immediately follows injury increases the expression of peroxisome proliferator-activated receptor (PPAR)-beta in the wound edge keratinocytes and triggers the production of endogenous PPARbeta ligands that activate the newly produced receptor. This elevated PPARbeta activity results in increased resistance of the keratinocytes to the apoptotic signals released during wounding, allowing faster re-epithelialisation. The authors speculate that, in parallel, ligand activation of PPARbeta in infiltrated macrophages attenuates the inflammatory response, which also promotes repair. Thus, current understanding of the roles of PPARbeta in different cell types implicated in tissue repair has revealed an intriguing intercellular cross-talk that coordinates, spatially and temporally, inflammation, keratinocyte survival, proliferation and migration, which are all essential for efficient wound repair. These novel insights into the orchestrating roles of PPARbeta during wound healing may be helpful in the development of drugs for acute and chronic wound disorders.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

To Monty Kier, a friendly tribute.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

EV02: a Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone.

The aim of this randomised controlled trial was to see if the addition of 4 mg/ml DNA-C priming given by the intramuscular route at weeks 0 and 4 to NYVAC-C at weeks 20 and 24, safely increased the proportion of participants with HIV-specific T-cell responses measured by the interferon (IFN)-gamma ELISpot assay at weeks 26 and/or 28 compared to NYVAC-C alone. Although 2 individuals discontinued after the first DNA-C due to adverse events (1 vaso-vagal; 1 transient, asymptomatic elevation in alanine transaminase), the vaccines were well tolerated. Three others failed to complete the regimen (1 changed her mind; 2 lost to follow-up). Of the 35 that completed the regimen 90% (18/20) in the DNA-C group had ELISpot responses compared to 33% (5/15) that received NYVAC-C alone (p=0.001). Responses were to envelope in the majority (21/23). Of the 9 individuals with responses to envelope and other peptides, 8 were in the DNA-C group. These promising results suggest that DNA-C was an effective priming agent, that merits further investigation.

Pharmacovigilance update [Pharmacovigilance update]

Main pharmacovigilance signals and alerts issued in 2009 are reviewed. Efalizumab was withdrawn from the market due to increased risks, including progressive multifocal leukoencephalopathy (PML) and questionable efficacy. New cases of PML are still being reported with rituximab and natalizumab. Rare cases of pure red cell aplasia have been observed with mycophenate. Gastrointestinal perforation, severe skin rashes and various ocular disorders have been reported during erlotinib use. Severe skin rashes have been related to etravirine. Acute renal failure and pancreatitis can occur with exenatide. A link between sitagliptin and pancreatitis is suspected. Raised concerns of causality between insuline glargine and malignant tumors are not supported by strong evidence. Proton pump inhibitors seem to blunt clopidogrel benefit. Aliskiren can cause angioedema.

Tests pharmacogénétiques: bientôt avant chaque prescription [Pharmacogenetic testing: soon before every prescription?]

Genetic polymorphisms have currently been described in more than 200 systems affecting pharmacological responses (cytochromes P450, conjugation enzymes, transporters, receptors, effectors of response, protection mechanisms, determinants of immunity). Pharmacogenetic testing, i.e. the profiling of individual patients for such variations, is about to become largely available. Recent progress in the pharmacogenetics of tamoxifen, oral anticoagulants and anti-HIV agents is reviewed to discuss critically their potential impact on prescription and contribution/limits for improving rational and safe use of pharmaceuticals. Prospective controlled trials are required to evaluate large-scale pharmacogenetic testing in therapeutics. Ethical, social and psychological issues deserve particular attention.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Molecular basis of selective PPARgamma modulation for the treatment of Type 2 diabetes.

Peroxisome proliferator-activated receptors (PPARs) (alpha, beta/delta and gamma) are lipid sensors capable of adapting gene expression to integrate various lipid signals. As such, PPARs are also very important pharmaceutical targets, and specific synthetic ligands exist for the different isotypes and are either currently used or hold promises in the treatment of major metabolic disorders. In particular, compounds of the class of the thiazolinediones (TZDs) are PPARgamma agonists and potent insulin-sensitizers. The specific but still broad expression patterns of PPARgamma, as well as its implication in numerous pathways, constitutes also a disadvantage regarding drug administration, since this potentially increases the chance to generate side-effects through the activation of the receptor in tissues or cells not affected by the disease. Actually, numerous side effects associated with the administration of TZDs have been reported. Today, a new generation of PPARgamma modulators is being actively developed to activate the receptor more specifically, in a cell and time-dependent manner, in order to induce a specific subset of target genes only and modulate a restricted number of metabolic pathways. We will discuss here why and how the development of such selective PPARgamma modulators is possible, and summarize the results obtained with the published molecules.

Carence en fer sans anémie: où en est-on en 2012 [Iron deficiency without anemia: where are we in 2012?].

Should we treat iron deficiency without anemia? The simple fact that the question can be formulated already leads to controversies. During the past years, the development of a new formulation of intravenous iron has helped fuel the controversy. What is the situation in 2012? This article gives a practical point of view on the actual situation and provides indications on the use of new intravenous medications.

Chemodiversity and molecular plasticity: recognition processes as explored by property spaces.

In the last few years, a need to account for molecular flexibility in drug-design methodologies has emerged, even if the dynamic behavior of molecular properties is seldom made explicit. For a flexible molecule, it is indeed possible to compute different values for a given conformation-dependent property and the ensemble of such values defines a property space that can be used to describe its molecular variability; a most representative case is the lipophilicity space. In this review, a number of applications of lipophilicity space and other property spaces are presented, showing that this concept can be fruitfully exploited: to investigate the constraints exerted by media of different levels of structural organization, to examine processes of molecular recognition and binding at an atomic level, to derive informative descriptors to be included in quantitative structure--activity relationships and to analyze protein simulations extracting the relevant information. Much molecular information is neglected in the descriptors used by medicinal chemists, while the concept of property space can fill this gap by accounting for the often-disregarded dynamic behavior of both small ligands and biomacromolecules. Property space also introduces some innovative concepts such as molecular sensitivity and plasticity, which appear best suited to explore the ability of a molecule to adapt itself to the environment variously modulating its property and conformational profiles. Globally, such concepts can enhance our understanding of biological phenomena providing fruitful descriptors in drug-design and pharmaceutical sciences.

Averting inflammation by targeting the cytokine environment.

Cytokines are key instigators and regulators of immune responses and therefore hold great potential as targets for new therapeutic strategies. However, the selection of which cytokines to target, and in particular the identification of which cytokines regulate the rate-limiting steps of disease pathways, is crucial to the success of such strategies. Moreover, balancing the need for ablating pathological inflammatory responses and simultaneously maintaining the ability to control infectious agents is a key consideration. Recent advances in our understanding of cytokine networks, as well as technical progress in blocking cytokines in vivo, are likely to be a source for new drugs that can control chronic inflammatory diseases.