Identification of Exosite-Targeting Inhibitors of Anthrax Lethal Factor by High-Throughput Screening

SummaryProtease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here, we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full-length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small-molecule library and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases

Unexpected Gating Behaviour of an Engineered Potassium Channel Kir

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point

Nouveaux inhibiteurs de la dimérisation de la protéase du VIH-1 (relations structure-activité, preuves mécanistiques et effets sur des protéases mutées)

Deux nouvelles séries d inhibiteurs de la dimérisation de la protéase du VIH-1 qui ciblent le feuillet bêta antiparallèle formé par l interdigitation des extrémités N- et C-terminales de chaque monomère ont été développées : les pinces moléculaires et les lipopeptides. Des variations structurales ont permis d'augmenter leur stabilité métabolique des molécules et leur solubilité. Elles ont conduit au meilleur inhibiteur de la dimérisation (Kid = 0,3 nM). Par ailleurs, l'ultracentrifugation analytique et la RMN ont permis d'apporter des preuves directes de l existence d un complexe monomère-inhibiteur. Un modèle de ce complexe a été proposé par modélisation moléculaire. Des molécules des deux séries inhibent des protéases mutées similaires à celles observées chez des patients infectés multi-résistants. Nos molécules actives au niveau nanomolaire peuvent donc être le point de départ de nouvelles molécules thérapeutiques susceptibles de prendre le relais des antiprotéases actuelles.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment

Here we study the intact stoichiometry and top-down fragmentation behavior of three integral membrane proteins which were natively reconstituted into detergent micelles: the mechano-sensitive ion channel of large conductance (MscL), the Kirbac potassium channel and the p7 viroporin from the hepatitis C virus. By releasing the proteins under nondenaturing conditions inside the mass spectrometer, we obtained their oligomeric sizes. Increasing the ion activation (collision energy) causes unfolding and subsequent ejection of a highly charged monomer from the membrane protein complexes. Further increase of the ion activation then causes collision-induced dissociation (CID) of the ejected monomers, with fragments observed which were predominantly found to stem from membrane-embedded regions. These experiments show how in a single experiment, we can probe the relation between higher-order structure and protein sequence, by combining the native MS data with fragmentation obtained from top-down MS

Structural and Functional highlights of Vacuolar Soluble Protein 1 from pathogen T. brucei. brucei

International audienceTrypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis or sleeping sickness. The causative parasite Trypanosoma encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. Latter are acidic membrane enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg 2+ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF-hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by small angle X-ray scattering (SAXS) and electron microscopy data. Solution scattering data suggests structural flexibility in EF-hand domains indicative of conformational plasticity within TbbVSP1

Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain

International audienceATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function

Peptidomimetic dimerization inhibitors of HIV-1 protease : further insights into structural and mechanism of action.

International audienceMutations that occur in response to the HIV-1 protease inhibitors (PIs) are responsible for the development of multi-drug cross-resistance to PIs in AIDS treatment. Virtually all PIs act through the same mechanism: they are transition-state analogs that target the active site of the homodimeric enzyme located at the junction of the two monomers. The emergence of resistance to one PI usually results in cross-resistance to other PIs. One alternative to inhibiting the active site of HIV-1 protease is to target the dimer interface of the enzyme at the antiparallel beta-sheet formed by the interdigitation of the C- and N-ends of each monomer. This region is highly conserved and is responsible for about 75% of the dimer stabilization energy. Here we describe new dimerization inhibitors in which new structural molecular variations have been introduced and the peptidic characteristics have been decreased by introducing peptidomimetic groups that have peptide-like hydrogen bonding properties. This led to an increase of the in vitro efficiency (subnanomolar level) against HIV-1 protease activity. Our dimerization inhibitors proved equally active in vitro against both wild-type and mutated proteases. The mechanism of inhibition was established using a combination of kinetic and biophysical methods. Using analytical ultracentrifugation and NMR, we obtained direct experimental evidence of non-covalent dissociative mode of interaction of the HIV-1 protease dimerization inhibitor

Global Analysis of Human Nonreceptor Tyrosine Kinase Specificity Using High-Density Peptide Microarrays

Protein kinases phosphorylate substrates in the context of specific phosphorylation site sequence motifs. The knowledge of the specific sequences that are recognized by kinases is useful for mapping sites of phosphorylation in protein substrates and facilitates the generation of model substrates to monitor kinase activity. Here, we have adapted a positional scanning peptide library method to a microarray format that is suitable for the rapid determination of phosphorylation site motifs for tyrosine kinases. Peptide mixtures were immobilized on glass slides through a layer of a tyrosine-free Y33F mutant avidin to facilitate the analysis of phosphorylation by radiolabel assay. A microarray analysis provided qualitatively similar results in comparison with the solution phase peptide library “macroarray” method. However, much smaller quantities of kinases were required to phosphorylate peptides on the microarrays, which thus enabled a proteome scale analysis of kinase specificity. We illustrated this capability by microarray profiling more than 80% of the human nonreceptor tyrosine kinases (NRTKs). Microarray results were used to generate a universal NRTK substrate set of 11 consensus peptides for in vitro kinase assays. Several substrates were highly specific for their cognate kinases, which should facilitate their incorporation into kinase-selective biosensors