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An inherited gain-of-function variant (E756del) in the mechanosensitive cationic channel PIEZO1 was shown to confer a significant protection against severe malaria. Here, we demonstrate in vitro that human red blood cell (RBC) infection by Plasmodium falciparum is prevented by the pharmacological activation of PIEZO1. Yoda1 causes an increase in intracellular calcium associated with rapid echinocytosis that inhibits RBC invasion, without affecting parasite intraerythrocytic growth, division or egress. Notably, Yoda1 treatment significantly decreases merozoite attachment and subsequent RBC deformation. Intracellular Na+/K+ imbalance is unrelated to the mechanism of protection, although delayed RBC dehydration observed in the standard parasite culture medium RPMI/albumax further enhances the resistance to malaria conferred by Yoda1. The chemically unrelated Jedi2 PIEZO1 activator similarly causes echinocytosis and RBC dehydration associated with resistance to malaria invasion. Spiky outward membrane projections are anticipated to reduce the effective surface area required for both merozoite attachment and internalization upon pharmacological activation of PIEZO1. Globally, our findings indicate that the loss of the typical biconcave discoid shape of RBCs, together with an altered optimal surface to volume ratio, induced by PIEZO1 pharmacological activation prevent efficient P. falciparum invasion.

The conversion of force into an electrical cellular signal is mediated by the opening of different types of mechanosensitive ion channels (MSCs), including TREK/TRAAK K2P channels, Piezo1/2, TMEM63/OSCA, and TMC1/2. Mechanoelectrical transduction plays a key role in hearing, balance, touch, and proprioception and is also implicated in the autonomic regulation of blood pressure and breathing. Thus, dysfunction of MSCs is associated with a variety of inherited and acquired disease states. Significant progress has recently been made in identifying these channels, solving their structure, and understanding the gating of both hyperpolarizing and depolarizing MSCs. Besides prototypical activation by membrane tension, additional gating mechanisms involving channel curvature and/or tethered elements are at play.

An inherited gain-of–function variant (E756 del) in the mechanosensitive cationic channel PIEZO1 was recently shown to confer a significant protection against severe malaria. Here, we demonstrate in vitro that human red blood cell (RBC) infection by Plasmodium falciparum is prevented by the pharmacological activation of PIEZO1. The PIEZO1 activator Yoda1 inhibits RBC invasion, without affecting parasite intraerythrocytic growth, division or egress. RBC dehydration, echinocytosis and intracellular Na+/K+ imbalance are unrelated to the mechanism of protection. Inhibition of invasion is maintained, even after a prolonged wash out of Yoda1. Similarly, the chemically unrelated activators Jedi1 and Jedi2 potently inhibit parasitemia, further indicating a PIEZO1-dependent mechanism. Notably, Yoda1 treatment significantly reduced RBC surface receptors of P. falciparum, and decreased merozoite attachment and subsequent RBC deformation. Altogether these data indicate that the pharmacological activation of Piezo1 in human RBCs inhibits malaria infection by impairing P. falciparum invasion.

sensaas is a tool developed for aligning and comparing molecular shapes and sub‐shapes. Alignment is obtained by registration of 3D point‐based representations of the van der Waals surface. The method uses local properties of the shape to identify the correspondence relationships between two point clouds containing up to several thousand colored (labeled) points. Our rigid‐body superimposition method follows a two‐stage approach. An initial alignment is obtained by matching pose‐invariant local 3D descriptors, called FPFH, of the input point clouds. This stage provides a global superimposition of the molecular surfaces, without any knowledge of their initial pose in 3D space. This alignment is then refined by optimizing the matching of colored points. In our study, each point is colored according to its closest atom, which itself belongs to a user defined physico‐chemical class. Finally, sensaas provides an alignment and evaluates the molecular similarity by using Tversky coefficients. To assess the efficiency of this approach, we tested its ability to reproduce the superimposition of X‐ray structures of the benchmarking AstraZeneca (AZ) data set and, compared its results with those generated by the two shape‐alignment approaches shaep and shafts. We also illustrated submatching properties of our method with respect to few substructures and bioisosteric fragments. The code is available upon request from the authors (demo version at https://chemoinfo.ipmc.cnrs.fr/SENSAAS).

International audience; Mechanotransduction plays a key role in vascular development, physiology and disease states. Piezo1 is a mechanosensitive non-selective cationic channel present in endothelial and vascular smooth muscle cells. It is activated by shear stress associated with increases in local blood flow, as well as by cell membrane stretch upon elevation of blood pressure. Here we briefly review the pharmacological modulators of Piezo and discuss the present state of knowledge on the role of Piezo1 in vascular mechanobiology and associated clinical disorders, such as atherosclerosis and hypertension. Ultimately, we believe that this recent research will help identify novel therapeutic strategies for the treatment of vascular diseases. Blood flow generates a frictional force acting on the endothelium (shear stress, parallel to the vessel wall), as well as wall distension (stretch; a force perpendicular to the vessel wall) in response to changes in transmural pressure [1-4]. Shear stress can arise due to either laminar (smooth flow with fluid layers sliding in parallel) or turbulent (rough) flow of blood through the vasculature. These mechanical forces have a significant impact on vascular development, physiology and are implicated in various disease states, including atherosclerosis and hypertension [1-4]. Multiple mechanosensors (see Glossary) detect these mechanical forces within vascular cells, including elements of the extracellular matrix (ECM), adhesion molecules, the

International audience; Acid-sensing ion channels (ASICs) are proton-gated cationic channels involved in pain and other processes, underscoring the potential therapeutic value of specific inhibitors such as the three-finger toxin mambalgin-1 (Mamb-1) from snake venom. A low-resolution structure of the human-ASIC1a/Mamb-1 complex obtained by cryo-electron microscopy has been recently reported, implementing the structure of the chicken-ASIC1/Mamb-1 complex previously published. Here we combine structure-activity relationship of both the rat ASIC1a channel and the Mamb-1 toxin with a molecular dynamics simulation to obtain a detailed picture at the level of side-chain interactions of the binding of Mamb-1 on rat ASIC1a channels and of its inhibition mechanism. Fingers I and II of Mamb-1 but not the core of the toxin are required for interaction with the thumb domain of ASIC1a, and Lys-8 of finger I potentially interacts with Tyr-358 in the thumb domain. Mamb-1 does not interfere directly with the pH sensor as previously suggested, but locks by several contacts a key hinge between α4 and α5 helices in the thumb domain of ASIC1a to prevent channel opening. Our results provide an improved model of inhibition of mammalian ASIC1a channels by Mamb-1 and clues for further development of optimized ASIC blockers.

Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å(3)and 977 Å(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.

Mutations in the polycystins PC1 or PC2 cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid-filled renal cysts that disrupt renal architecture and function, ultimately leading to kidney failure in the majority of patients. Although the genetic basis of ADPKD is now well established, the physiological function of polycystins remains obscure and a matter of intense debate. The structural determination of both the homomeric PC2 and heteromeric PC1-PC2 complexes, as well as the electrophysiological characterization of PC2 in the primary cilium of renal epithelial cells, provided new valuable insights into the mechanisms of ADPKD pathogenesis. Current findings indicate that PC2 can function independently of PC1 in the primary cilium of renal collecting duct epithelial cells to form a channel that is mainly permeant to monovalent cations and is activated by both membrane depolarization and an increase in intraciliary calcium. In addition, PC2 functions as a calcium-activated calcium release channel at the endoplasmic reticulum membrane. Structural studies indicate that the heteromeric PC1-PC2 complex comprises one PC1 and three PC2 channel subunits. Surprisingly, several positively charged residues from PC1 occlude the ionic pore of the PC1-PC2 complex, suggesting that pathogenic polycystin mutations might cause ADPKD independently of an effect on channel permeation. Emerging reports of novel structural and functional findings on polycystins will continue to elucidate the molecular basis of ADPKD.

International audience; Mambalgins are 57-amino acid peptides isolated from mamba venom. They produce potent analgesic effects in mice against inflammatory pain upon central intrathecal (i.t.), and peripheral local (i.pl.) injections, through inhibition of different ASICs subtypes and involvement of opioid-independent pathways. They produce fewer side effects than morphine and no apparent toxicity. We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical pain upon systemic intravenous (i.v.) administration and are effective against neuropathic pain by i.v., i.t. and i. pl. injections. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels in the i.v. effects of mambalgins against inflammatory pain. The potent analgesic effect on neuropathic pain involves two different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection, and an ASIC1a-dependent and partially naloxone-sensitive effect associated with i.t. injection. We have done in collaboration with the CEA iBiTecS Institute in Gif-sur-Yvette, the full stepwise solid-phase peptide synthesis of mambalgin-1, solved its 3D crystal structure, mapped the pharmacophore, and identified the binding site and the inhibitory mechanism on ASIC1a channels. These findings identify new roles for ASICs in pain pathways, and mambalgins as new potential analgesics against inflammatory and chronic neuropathic pain.

Invasion of the red blood cell by Plasmodium falciparum parasites requires formation of an electron dense circumferential ring called the Moving Junction (MJ). The MJ is anchored by a high affinity complex of two parasite proteins: Apical Membrane Antigen 1 (PfAMA1) displayed on the surface of the parasite and Rhoptry Neck Protein 2 that is discharged from the parasite and imbedded in the membrane of the host cell. Structural studies of PfAMA1 revealed a conserved hydrophobic groove local- ized to the apical surface that coordinates RON2 and invasion inhibitory peptides. In the present work, we em- ployed computational and biophysical methods to identify competitive P. falciparum AMA1-RON2 inhibitors with the goal of exploring the 'druggability' of this attractive antimalarial target. A virtual screen followed by molecular docking with the PfAMA1 crystal structure was performed using an eight million compound collection that included commercial molecules, the ChEMBL malaria library and approved drugs. The consensus approach resulted in the selection of inhibitor candidates. We also developed a fluorescence anisotropy assay using a modified inhibitory peptide to experimentally validate the ability of the se- lected compounds to inhibit the AMA1-RON2 interaction. Among those, we identified one compound that displayed significant inhibition. This study offers interesting clues to improve the throughput and reliability of screening for new drug leads.

[Image: see text] Presented here are several data sets that gather information collected from the labels of the FDA approved drugs: their molecular structures and those of the described active metabolites, their associated pharmacokinetics and pharmacodynamics data, and the history of their marketing authorization by the FDA. To date, 1852 chemical structures have been identified with a molecular weight less than 2000 of which 492 are or have active metabolites. To promote the sharing of data, the original web server was upgraded for browsing the database and downloading the data sets (http://chemoinfo.ipmc.cnrs.fr/edrug3d). It is believed that the multidimensional chemistry-oriented collections are an essential resource for a thorough analysis of the current drug chemical space. The data sets are envisioned as being used in a wide range of endeavors that include drug repurposing, drug design, privileged structures analyses, structure–activity relationship studies, and improving of absorption, distribution, metabolism, and elimination predictive models.

http://thcgerm.free.fr/spip.php?article123; National audience

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis–Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.

Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.

International audience; The AFF (AF4/FMR2) family of genes includes four members: AFF1/AF4, AFF2/FMR2, AFF3/LAF4 and AFF4/AF5q31. AFF2/FMR2 is silenced in FRAXE intellectual disability, while the other three members have been reported to form fusion genes as a consequence of chromosome translocations with the myeloid/lymphoid or mixed lineage leukemia (MLL) gene in acute lymphoblastic leukemias (ALLs). All AFF proteins are localized in the nucleus and their role as transcriptional activators with a positive action on RNA elongation was primarily studied. We have recently shown that AFF2/FMR2 localizes to nuclear speckles, subnuclear structures considered as storage/modification sites of pre-mRNA splicing factors, and modulates alternative splicing via the interaction with the G-quadruplex RNA-forming structure. We show here that similarly to AFF2/FMR2, AFF3/LAF4 and AFF4/AF5q31 localize to nuclear speckles and are able to bind RNA, having a high apparent affinity for the G-quadruplex structure. Interestingly, AFF3/LAF4 and AFF4/AF5q31, like AFF2/FMR2, modulate, in vivo, the splicing efficiency of a mini-gene containing a G-quadruplex structure in one alternatively spliced exon. Furthermore, we observed that the overexpression of AFF2/3/4 interferes with the organization and/or biogenesis of nuclear speckles. These findings fit well with our observation that enlarged nuclear speckles are present in FRAXE fibroblasts. Furthermore, our findings suggest functional redundancy among the AFF family members in the regulation of splicing and transcription. It is possible that other members of the AFF family compensate for the loss of AFF2/FMR2 activity and as such explain the relatively mild to borderline phenotype observed in FRAXE patients.

The yeast Kes1p/Osh4p protein functions as a sterol/PI(4)P exchanger between lipid membranes, which suggests the possibility of creating a sterol gradient via phosphoinositide metabolism., Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p–PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers.

International audience

Plasmodium falciparum is an obligate intracellular protozoan parasite that employs a highly sophisticated mechanism to access the protective environment of the host cells. Key to this mechanism is the formation of an electron dense ring at the parasite-host cell interface called the Moving Junction (MJ) through which the parasite invades. The MJ incorporates two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 have shown that a partially mobile loop, termed the DII loop, forms part of a deep groove in domain I and overlaps with the RON2 binding site. To investigate the mechanism by which the DII loop influences RON2 binding, we measured the kinetics of association and dissociation and binding equilibria of a PfRON2sp1 peptide with both PfAMA1 and an engineered form of PfAMA1 where the flexible region of the DII loop was replaced by a short Gly-Ser linker (ΔDII-PfAMA1). The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants and corresponding thermodynamic profiles. Our results indicate that both PfAMA1 constructs bound to the PfRON2sp1 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. Consistent with Isothermal Titration Calorimetry measurements, final complex formation was enthalpically driven and slightly entropically unfavorable. Importantly, our experimental data shows that the DII loop lengthened the complex half-life time by 18-fold (900 s and 48 s at 25°C for Pf and ΔDII-Pf complex, respectively). The longer half-life of the Pf complex appeared to be driven by a slower dissociation process. These data highlight a new influential role for the DII loop in kinetically locking the functional binary complex to enable host cell invasion.

Computational docking approaches are important as a source of protein-protein complexes structures and as a means to understand the principles of protein association. A key element in designing better docking approaches, including search procedures, potentials, and scoring functions is their validation on experimentally determined structures. Thus, the databases of such structures (benchmark sets) are important. The previous, first release of the DOCKGROUND resource (Douguet et al., Bioinformatics 2006; 22:2612-2618) implemented a comprehensive database of cocrystallized (bound) protein-protein complexes in a relational database of annotated structures. The current release adds important features to the set of bound structures, such as regularly updated downloadable datasets: automatically generated nonredundant set, built according to most common criteria, and a manually curated set that includes only biological nonobligate complexes along with a number of additional useful characteristics. The main focus of the current release is unbound (experimental and simulated) protein-protein complexes. Complexes from the bound dataset are used to identify crystallized unbound analogs. If such analogs do not exist, the unbound structures are simulated by rotamer library optimization. Thus, the database contains comprehensive sets of complexes suitable for large scale benchmarking of docking algorithms. Advanced methodologies for simulating unbound conformations are being explored for the next release. The future releases will include datasets of modeled protein-protein complexes, and systematic sets of docking decoys obtained by different docking algorithms. The growing DOCKGROUND resource is designed to become a comprehensive public environment for developing and validating new docking methodologies.

Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug–drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections., Cyclophilins play a key role in the life cycle of many viruses and represent important drug targets for broad-spectrum antiviral therapies. Here, the authors use fragment-based drug discovery to develop non-peptidic inhibitors of human cyclophilins with high activity against replication of a number of viral families.

Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.

Plasmodium falciparum and Toxoplama gondii are obligate intracellular protozoan parasites that invade and replicate within host cells. They both require the formation of a tight interaction with the host cell, called Moving Junctions (MJ), for successful infection. It has been shown that the MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 proteins have shown a versatile loop, called domain II loop that extends into domain I likely to hide the RON2 binding site from host immunity. In the present work, we have studied association, dissociation reactions and binding equilibria of PfAMA1 and TgAMA1 reacting with their respective RON2 short peptide ligand. Equally, we have studied a deltaDII-loop-PfAMA1 construct to elucidate the role of this loop upon RON2 peptide binding. The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants to calculate the thermodynamic profile and propose a reaction mechanism. Our results showed that PfAMA1 and TgAMA1 bind to their respective RON2 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. The reactions are both enthalpically and entropically favorable upon ligand binding thanks of the DII-loop induced fit folding down over the bound ligand forming a most stable final complex. The half life time of the complex at 25°C is 326s and 1077s for Pf and Tg complexes, respectively. By in vitro-in vivo extrapolation at 37°C, it is compatible with the time frame of erythrocyte invasion by Plasmodium falciparum merozoites. The elucidation of the binding mechanism brings new strategies for ligand discovery against these pharmacologically important targets.

We have developed a WWW server for the rapid structural analysis of protein sequence/structure alignments. Users can submit an amino acid sequence to perform structural predictions and fold recognition. Pairwise 1D/3D alignments are then visualized as colored alignments to highlight sequence similarities and the relative positions of secondary structure elements and insertions/deletions. A pseudo-energy score is computed for each block in the proposed structural alignment to help manual refinement of the threading if necessary. Modelling is then performed using MODELLER and directly validated using Verify3D. The Web interfaces, we have developed, allow rapid and automatic molecular modelling even in the case of low sequence identity level (20 –30%). Here, we report a structural analysis of the Thymidine Monophosphate Kinase (TMPK) from Campylobacter jejuni(hereafter, TMPKcj), a Gram-negative bacterium responsible for human diarrhea. Limited but significant sequence similarities could be detected with other nucleotide monophosphate kinases. The comparison of the TMPK with the crystal structure of other TMPK, strongly suggested that it might adopt a similar fold and revealed the conservation of the monomer-monomer interface. We propose a structural model of TMPKcj and analyze the slight differences at the levels of primary and 3D-structure suggesting putative variations in the binding of substrate analogs. © 2002 Elsevier Science Inc. All rights reserved.

Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the β-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and β-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.

Rational drug design involves finding solutions to large combinatorial problems for which an exhaustive search is impractical. Genetic algorithms provide a novel tool for the investigation of such problems. These are a class of algorithms that mimic some of the major characteristics of Darwinian evolution. LEA has been designed in order to conceive novel small organic molecules which satisfy quantitative structure-activity relationship based rules (fitness). The fitness consists of a sum of constraints that are range properties. The algorithm takes an initial set of fragments and iteratively improves them by means of crossover and mutation operators that are related to those involved in Darwinian evolution. The basis of the algorithm, its implementation and parameterization, are described together with an application in de novo molecular design of new retinoids. The results may be promising for chemical synthesis and show that this tool may find extensive applications in de novo drug design projects.

OBJECTIVE To assess the effects of various catecholaminergic agents on pulmonary venous tone. DESIGN Prospective, randomized, controlled, experimental study. SETTING Physiology laboratory of a university hospital. SUBJECTS Thirty anesthetized, mechanically ventilated adult sheep. INTERVENTIONS Four groups of six animals received 1-hr infusions of norepinephrine (0.5 microg/kg/min), epinephrine (0.5 microg/kg/ min), dopamine (10 microg/kg/min), or dobutamine (10 microg/kg/min). MEASUREMENTS AND MAIN RESULTS A 7-Fr pulmonary artery catheter was placed in a proximal location to measure cardiac output and pressure in a large pulmonary vein (Ppw) after balloon inflation. Another catheter wedged in a small pulmonary artery measured pressure in a small pulmonary vein (Pdw). A third catheter measured left atrial pressure (PLA ). This method was able to detect the pulmonary venoconstrictive effects of histamine in a separate group of six animals. Pdw-PLA increased from a mean of 2.0+/-1.7 to 3.0+/-1.5 (SD) cm H2O (p < .01), 2.3+/-1.6 to 4.4+/-1.3 cm H2O (p < .01), and 1.7+/-1.0 to 3.5+/-2.2 cm H2O (p < .05) with norepinephrine, epinephrine, and dopamine, respectively. All of these drugs increased Pdw-Ppw, but only norepinephrine and epinephrine increased Ppw-PLA . No change in either pressure difference was observed with dobutamine. Elevation of cardiac output alone could not account for these findings since the increase in cardiac output induced by fluid infusion did not change the pressure differences. CONCLUSION Norepinephrine, epinephrine, and dopamine at doses commonly used in humans increase pulmonary venous tone in sheep.

This study describes a non-invasive method for assessment of the lung transit time distribution of a tracer, using first-pass technetium-99m albumin angiocardiography and a model-free method of deconvolution. Ten patients received a first injection of 1 MBq kg-1 in the external jugular vein to position a gamma camera in the left anterior oblique position and two additional injections (5 MBq kg-1) to record first-pass angiocardiographic data. Right and left ventricular time-activity curves were derived from regions of interest every 0.5 s over a 1-min period. The left ventricular curve was deconvoluted by the right ventricular curve to obtain the lung transport function. The deconvolution procedure was based on a modified version of the Kalman filtering technique. The procedure was repeated at an interval of 30 min in eight patients. Two patients were re-examined up to 2 years later. Skewness, kurtosis and relative dispersion of the distributions did not change over time. We also found that the distribution, once normalized by its first moment, was independent of isolated changes in heart rate or cardiac output. Comparison of curve shapes at an interval of 30 min by point by point analysis demonstrated the reproducibility of the technique. We conclude that computation of the pulmonary transit time distribution of 99mTc-albumin from a standard angiocardiography procedure by model-free deconvolution is reliable and reproducible over time. We suggest that it may be a valuable tool for the non-invasive follow-up of the pulmonary circulation.

The tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K(+)-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.

TWIK1 belongs to the family of background K + channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K + selective at neutral pH, it becomes permeable to Na + at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1 −/− pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.

Motivation: In the drug discovery field, new uses for old drugs, selective optimization of side activities and fragment-based drug design (FBDD) have proved to be successful alternatives to high-throughput screening. e-Drug3D is a database of 3D chemical structures of drugs that provides several collections of ready-to-screen SD files of drugs and commercial drug fragments. They are natural inputs in studies dedicated to drug repurposing and FBDD. Availability: e-Drug3D collections are freely available at http://chemoinfo.ipmc.cnrs.fr/e-drug3d.html either for download or for direct in silico web-based screenings. Contact: douguet@ipmc.cnrs.fr Supplementary information: Supplementary data are available at Bioinformatics online.

International audience; Among mammalian secreted phospholipases A(2) (sPLA(2)s), group X sPLA(2) has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA(2) is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA(2) in HEK293 cells, which have been extensively used to analyze sPLA(2)-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA(2) inhibitors and protease inhibitors, we demonstrate that group X sPLA(2) is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA(2) inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA(2) maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.

Apicomplexan parasites such as Toxoplasma gondii and Plasmodium species actively invade host cells through a moving junction (MJ) complex assembled at the parasite–host cell interface. MJ assembly is initiated by injection of parasite rhoptry neck proteins (RONs) into the host cell, where RON2 spans the membrane and functions as a receptor for apical membrane antigen 1 (AMA1) on the parasite. We have determined the structure of Tg AMA1 complexed with a RON2 peptide at 1.95 angstrom resolution. A stepwise assembly mechanism results in an extensive buried surface area, enabling the MJ complex to resist the mechanical forces encountered during host cell invasion. Besides providing insights into host cell invasion by apicomplexan parasites, the structure offers a basis for designing therapeutics targeting these global pathogens.

Pulmonary artery occlusion pressure (Ppao) represents the pressure in a pulmonary vein (Ppv) and reflects left atrial pressure (Pla) provided alveolar pressure (Palv) does not exceed the pressure existing at the venous end of the collapsible alveolar vessels. Three major factors may influence the relationships between Palv and Ppv: mechanical ventilation with positive end-expiratory pressure (PEEP), vertical location of the pulmonary artery catheter (PAC) tip relative to the left atrium, and volemia. In an experimental and clinical study, we assessed a simple means to ensure meaningful measurements of Ppao during mechanical ventilation with PEEP, based on the following reasoning: if Ppao reflects Palv and not Ppv, it will increase during positive pressure inspiration by an amount (ΔPpao) similar to Palv; thus, ΔPpao will widely exceed the increase in pulmonary arterial pressure (ΔPpa), which approximates the increase in pleural pressure. Conversely, when Ppao actually reflects Ppv, ΔPpao will be close to ΔPpa. A preliminary experimental study in an animal with a left atrial catheter and PAC in place showed that Ppao widely exceeded Pla when ΔPpao/ΔPpa widely exceeded 1 during mechanical ventilation with PEEP. After fluid infusion, Pla and Ppao were almost identical and ΔPpao/ΔPpa was now close to 1. In 12 patients ventilated for adult respiratory distress syndrome and with a left ventricular catheter and PAC in place, we verified at four levels of PEEP (0, 10, 15, and 20 cm H 2 O) that a Ppao close to left ventricular end-diastolic pressure was always associated with a ΔPpao/ΔPpa ratio close to 1 (38 measurements). In 34 additional patients ventilated for noncardiogenic pulmonary edema, we also tested the ΔPpao/ΔPpa ratio during the same incremental PEEP protocol and found that ΔPpao/ΔPpa was close to 1 at each level of PEEP in 30 of the patients (108 measurements). In five measurements in four hypovolemic patients, ΔPpao/ΔPpa widely exceeded 1; only one of these four patients was found to have a PAC tip located above the left atrium level on a lateral chest x-ray film. We conclude that the bedside determination of the ΔPpao/ΔPpa ratio should identify the cases in which Ppao measurements are not valid more reliably than lateral chest x-ray film, thus avoiding misleading interpretations of Ppao during PEEP ventilation.

Mechanosensitive K + channels TREK1 and TREK2 form a subclass of two P-domain K + channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K + channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.

Summary: HELIQUEST calculates the physicochemical properties and amino acid composition of an α-helix and screens databank to identify protein segments possessing similar features. This server is also dedicated to mutating helices manually or automatically by genetic algorithm to design analogues of defined features. Availability: http://heliquest.ipmc.cnrs.fr Contact: gautier@ipmc.cnrs.fr

International audience; A series of N(1)-(4-substituted-benzyl)-pyrimidines were synthesized as potential inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). Key SAR parameters included the chain length substitution in para position of the benzyl ring, the functional group terminating the alkyl chain, and the substituent on the C-5 pyrimidine ring. Synthesized molecules were assayed against both recombinant enzyme and mycobacteria cultures. The most potent compounds have K(i) values in the micromolar range and an MIC(50) of 50microg/mL against Mycobacterium bovis. These results will guide the design of a new generation of lead compounds.

Tuberculosis (TB) ranks among the leading causes of death worldwide from a single infectious agent, particularly in developing countries. The emergence of multidrug resistant strains of Mycobacteri...