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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.

[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

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.

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.

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.

Without crystal structures to study the CYP26 family of drug metabolizing enzymes, homology models were used to characterize CYP26A1, CYP26B1 and CYP26C1 and to identify substrates and inhibitors of the enzymes. Computational models of each isoform based on structural homology to CYP120 were validated by docking all-trans retinoic acid, an endogenous ligand of CYP26. Docking of retinoic acid receptor agonists and antagonists suggested that tazarotenic acid (TA) and adapalene may be metabolic substrates for CYP26, data which was confirmed using in vitro metabolite identification assays. Phenotyping experiments determined that CYP26s played a major role in the metabolism of these compounds in vitro. The kinetics of TA sulfoxidation by CYP26A1 and CYP26B1 were characterized and the compound was proposed as an in vitro probe of CYP26 activity in single enzyme expression systems. Structural characterization efforts identified similarities between the CYP26 homology models and the known crystal structure of CYP2C8, in agreement with previously published reports. Using TA as a probe, the IC50’s of known CYP2C8 inhibitors was measured against CYP26A1 and CYP26B1, with a statistically significant correlation observed between CYP26A1 and CYP2C8. Additional in vitro and computational experiments were used to characterize the inhibition mechanism for the most potent inhibitors. The observed in vitro inhibition was then used to predict the likelihood of CYP26 inhibition being involved in clinically relevant drug interactions. As a whole, the results presented support the role of the CYP26s in the metabolism of xenobiotic compounds as well as in potential in vivo drug interactions., En l’absence de structures tridimensionnelles expérimentales des cytochromes P450 CYP26A1, CYP26B1 et CYP26C1, la caractérisation de leur substrats et ligands s’est basée sur l’analyse des modèles structuraux obtenus par modélisation par homologie avec la structure expérimentale du cytochrome P450 CYP120. La justesse des modèles a été validée par l’amarrage de l’acide rétinoïque all-trans dans des configurations compatibles avec les métabolites attendus. L’amarrage d’agonistes et d’antagonistes des récepteurs nucléaires RARs prédirent l’acide tazaroténique (TA) et l’adapalène comme des substrats potentiels. Les expériences in vitro confirmèrent la métabolisation de ces 2 médicaments par les CYP26s. L’analyse de la cinétique de sulfoxidation du TA par CYP26A1 and CYP26B1 a permis d’établir le TA comme la référence contrôle de l’activité de ces enzymes. Puis, la comparaison des modèles des CYP26s avec la structure cristalline de CYP2C8 a permis d’identifier des similarités structurales de leurs inhibiteurs. Une corrélation entre l’inhibition de CYP26A1 et de CYP2C8 par des inhibiteurs connus de CYP2C8 a été démontrée après détermination de leurs IC50 pour CYP26A1 et CYP26B1 en utilisant le TA comme substrat de référence. La mesure de l’inhibition in vitro fut ensuite utilisée pour évaluer la possibilité que les CYP26s soient impliquées dans des interactions médicamenteuses observées pour certaines molécules. Cette thèse caractérise et appuie le rôle encore mal connu des CYP26s dans la métabolisation in vivo de certains xénobiotiques ainsi que l’effet potentiel de leur inhibition qui favoriserait la survenue d'effets indésirables.