Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks.

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Chemical Composition of the Unexplored Volatile Fraction of Betula glandulosa, a Prevalent Shrub in Nunavik, Québec.

The volatile fraction of the leaves of Betula glandulosa Michx. has been investigated for its secondary metabolite composition by GC/MS and GC/FID. The rapid expansion of this shrub species in subarctic landscapes, like the ones found in Nunavik (Northern Québec, Canada), highly impacts ecosystem dynamics. Yet, despite its abundance, few phytochemical investigations have yet been conducted on this species. In this study, we present the first phytochemical investigation of the volatile metabolites of B. glandulosa leaves. Although no essential oil was isolated, volatile compounds were extracted from the hydrosol by steam distillation. The main metabolites observed were linalool (14.6-19.0 %), C6 oxylipins (known as green leaf volatiles, GLV; total of 18.2-40.2 %), eugenol (1.6-8.6 %) and α-terpineol (3.3-4.8 %). Dwarf birch is an important food source for insects and herbivores, so knowledge of its metabolite composition could help understand parts of its functional role in subarctic ecosystems. The composition of the volatile fraction could serve as marker for differentiating B. glandulosa from other dwarf birch species like Betula nana L.

Development of sulfahydantoin derivatives as ß-lactamase inhibitors.

Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of ß-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as ß-lactamase inhibitors using two common Class A ß-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the ß-lactamases with IC50 values between 130 and 510 µM and inferred Ki values between 32 and 55 µM.

Preventing Candida albicans biofilm formation using aromatic-rich piperazines.

The global increase in microbial resistance is an imminent threat to public health. Effective treatment of infectious diseases now requires new antimicrobial therapies. We report herein the discovery of aromatic-rich piperazines that inhibit biofilm formation by C. albicans. 22 piperazines, including 16 novel ones, were prepared efficiently using a combination of solid- and solution phase synthesis. The most potent compound prevents morphological switching under several hypha-inducing conditions and reduces C. albicans' ability to adhere to epithelial cells. These processes are essential to the development of Candida biofilms, which are associated with its increased resistance to immune defenses and antifungal agents.

Total Synthesis and Antimalarial Activity of Dominicin, a Cyclic Octapeptide from a Marine Sponge.

Dominicin, a macrocyclic peptide isolated from the marine sponge Eurypon laughlini, has been synthesized for the first time by solid-phase peptide synthesis. The strategy uses oxime resin and takes advantage of the nucleophile susceptibility of the oxime ester bond. The synthesis relies on the preparation of a linear precursor followed by on-resin head-to-tail concomitant cyclization-cleavage. This is the first report of the use of a Boc/OtBu biorthogonal protection strategy on oxime resin to facilitate concomitant N-terminal and side-chain tert-butyl ether deprotection cyclization of unprotected peptides. Also, we report the first antimalarial investigation of dominicin. Interestingly, the natural macrocyclic peptide demonstrates effective low micromolar activity (1.8 µM) against the chloroquine-mefloquine-pyrimethamine-resistant Dd2 strain of Plasmodium falciparum.

Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against oral pathogens.

Microorganisms embedded in a biofilm are significantly more resistant to antimicrobial agents and the defences of the human immune system, than their planktonic counterpart. Consequently, compounds that can inhibit biofilm formation are of great interest for novel therapeutics. In this study, a screening approach was used to identify novel cyclic dipeptides that have anti-biofilm activity against oral pathogens. Five new active compounds were identified that prevent biofilm formation by the cariogenic bacterium Streptococcus mutans and the pathogenic fungus Candida albicans. These compounds also inhibit the adherence of microorganisms to a hydroxylapatite surface. Further investigations were conducted on these compounds to establish the structure-activity relationship, and it was deduced that the common cleft pattern is required for these molecules to act effectively against biofilms.

A novel route towards cycle-tail peptides using oxime resin: teaching an old dog a new trick.

Two anabaenopeptins, Schizopeptin 791 and anabaenopeptin NZ825, have similar structural features and have been synthesized via a novel acid-catalyzed head-to-side-chain concomitant cyclization/cleavage reaction on oxime resin. The methodology gave rapid access to the anabaenopeptin scaffold by taking advantage of a combined solid-phase/solution-phase synthetic strategy. Also, as side-products of the synthesis, large C2-symmetric 38-member cyclic peptides ring bearing two endocyclic lysine side-chains were isolated, constituting a novel cyclic peptide scaffold.

Lobaric acid and pseudodepsidones inhibit NF-κB signaling pathway by activation of PPAR-γ.

Herein we report the anti-inflammatory activity of lobaric acid and pseudodepsidones isolated from the nordic lichen Stereocaulon paschale. Lobaric acid (1) and three compounds (2, 7 and 9) were found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1ß and TNF-α) in LPS-stimulated macrophages. Inhibition and docking simulation experiments provided evidence that lobaric acid and pseudodepsidones bind to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists. These findings suggest that lobaric acid and pseudodepsidones reduce the expression of pro-inflammatory cytokines by blocking the NF-κB pathway via the activation of PPAR-γ.

Synthesis and anti-inflammatory activity of isoquebecol.

We report here the synthesis of isoquebecol, an unprecedented constitutional isomer of quebecol, a polyphenolic compound discovered in maple syrup. The methodology used to prepare isoquebecol involves, as key steps, the formation of a dibromoalkene from an α-ketoester precursor, followed by a double Suzuki-Miyaura reaction. The anti-inflammatory activity of isoquebecol was studied on macrophage cells by monitoring its ability to inhibit LPS-induced IL-6 secretion. Results show that this new compound has an improved bioactivity over that of its natural isomer. Precursors and derivatives of quebecol, isoquebecol and model analog 2,3,3-triphenylpropanol were also prepared and tested in this study. Comparison between the three series of compounds led to establishing new SARs concerning the aryl ring substitution pattern on the triarylpropanol scaffold and substructure functionalization.

Dibenzofurans and Pseudodepsidones from the Lichen Stereocaulon paschale Collected in Northern Quebec.

Chemical investigation of the methanol extract of the lichen Stereocaulon paschale collected in Nunavik, Canada, led to the isolation and identification of two new dibenzofurans (1 and 3) and 11 known lichen metabolites. The structures of the new compounds were established by analysis of 1D and 2D NMR spectroscopic and high-resolution mass spectrometric data. Herein, the first isolation of ascomatic acid dibenzofuran derivatives (1-3) from a whole lichen organism is reported. In addition, some of the isolated metabolites showed antibacterial activity against the oral pathogens Porphyromonas gingivalis and Streptococcus mutans.

Amphiphilicity Is a Key Determinant in the Membrane Interactions of Synthetic 14-mer Cationic Peptide Analogues.

Cationic antimicrobial peptides are a component of the innate immune system of several organisms and represent an interesting alternative to fight multiresistant bacteria. In this context, we have elaborated a synthetic peptide scaffold allowing the study of the impact of different molecular determinants on the membrane interactions. The aim of the present study was to elucidate the mechanism of action of two cationic peptides that derive from a neutral 14-mer template peptide and where the hydrophilic portion is composed of a crown ether. The R5R10 peptide is active in the presence of both negatively charged and zwitterionic membranes (nonselective) and adopts an α-helical conformation, whereas the R4R11 peptide is more active in the presence of negatively charged membranes (selective) and forms intermolecular ß-sheet structures. Both the membrane topology and the location of the peptides have been assessed using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopy. In addition, fluorescence experiments have been performed on different membrane mixtures to evaluate the ability of the peptides to induce a positive curvature to the membrane. Overall, for both the R5R10 and R4R11 peptides, the results are consistent with a mechanism of action similar to the sinking-raft model in which the peptides are mainly lying flat on the membrane surface and impose a bending stress to the membrane, thus leading to the formation of pores. Furthermore, the difference of membrane selectivity between R5R10 and R4R11 peptides is due to their differing amphipathic properties which modulate the membrane activity on zwitterionic model membranes.

Anti-inflammatory properties of quebecol and its derivatives.

Herein we report our results on the anti-inflammatory activity of quebecol, a polyphenolic compound discovered in maple syrup. Bioassays demonstrated that quebecol has an anti-inflammatory effect on LPS-induced NF-κB activation and inhibits the secretion of two pro-inflammatory cytokines, IL-6 and TNF-α. We also prepared and tested precursors of quebecol and its derivatives corresponding to its substructures of interest, with the aim to study the structure-activity relationships. Comparing the results obtained for all tested compounds allowed the identification of the main moiety responsible for the anti-inflammatory activity of quebecol.

Investigation of the mechanism of action of novel amphipathic peptides: insights from solid-state NMR studies of oriented lipid bilayers.

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by (31)P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular ß-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective ß-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Crown ether helical peptides are preferentially inserted in lipid bilayers as a transmembrane ion channels.

Oriented circular dichroism was used to study the alignment crown ether-modified peptides. The influence of different N- and C-functionalities was assessed using at variable peptide:lipid ratios from 1:20 to 1:200. Neither the functionalities nor the concentration had any major effect on the orientation. The alignment of the 21-mer peptides was also examined with lipid membranes of different bilayer thickness. The use of synchrotron radiation as light source allowed the study of peptide:lipid molar ratios from 1:20 to 1:1000. For all conditions studied, the peptides were found to be predominantly incorporated as a transmembrane helix into the membrane, especially at low peptide concentration, but started to aggregate on the membrane surface at higher peptide:lipid ratios. The structural information on the preferred trans-bilayer alignment of the crown ether functional groups explains their ion conductivity and is useful for the further development of membrane-active nanochemotherapeutics.

Exploiting peptide nanostructures to construct functional artificial ion channels.

Natural ion channel proteins possess remarkable properties that researchers could exploit to develop nanochemotherapeutics and diagnostic devices. Unfortunately, the poor stability, limited availability, and complexity of these structures have precluded their use in practical devices. One solution to these limitations is to develop simpler molecular systems through chemical synthesis that mimic the salient properties of artificial ion channels. Inspired by natural channel proteins, our group has developed a family of peptide nanostructures thatcreate channels for ions by aligning crown ethers on top of each other when they adopt an α-helical conformation. Advantages to this crown ether/peptide framework approach include the ease of synthesis, the predictability of their conformations, and the ability to fine-tune and engineer their properties. We have synthesized these structures using solid phase methods from artificial crown ether amino acids made from L-DOPA. Circular dichroism and FTIR spectroscopy studies in different media confirmed that the nanostructures adopt the predicted α-helical conformation. Fluorescence studies verified the crown ether stacking arrangement. We confirmed the channel activity by single-channel measurements using a modified patch-clamp technique, planar lipid bilayer (PLB) assays, and various vesicle experiments. From the results, we estimate that a 6 Å distance between two relays is ideal for sodium cation transport, but relatively efficient ion transport can still occur with an 11 Å distance between two crown ethers. Biophysical studies demonstrated that peptide channels operate as monomers in an equilibrium between adsorption at the surface and an active, transmembrane orientation. Toward practical applications of these systems, we have prepared channel analogs that bear a biotin moiety, and we have used them as nanotransducers successfully to detect avidin. Analogs of channel peptide nanostructures showed cytotoxicity against breast and leukemia cancer cells. Overall, we have prepared well-defined nanostructures with designed properties, demonstrated their transport abilities, and described their mechanism of action. We have also illustrated the advantages and the versatility of polypeptides for the construction of functional nanoscale artificial ion channels.

Squaramide Tethered Clindamycin, Chloroquine, and Mortiamide Hybrids: Design, Synthesis, and Antimalarial Activity.

Malaria remains one of the major health problems in the world. In this work, a series of squaramide tethered chloroquine, clindamycin, and mortiamide D hybrids have been synthesized to assess their in vitro antiplasmodial activity against 3D7 (chloroquine-sensitive) and Dd2 strains of Plasmodium falciparum. The most active compound, a simple chloroquine analogue, displayed low nanomolar IC50 value against both strains (3 nM for 3D7 strain and 18 nM for Dd2 strain). Moreover, all molecular hybrids incorporating the hydroxychloroquine scaffold showed the most potent activities, exemplified with a chloroquine dimer, IC50 = 31 nM and 81 nM against 3D7 and Dd2 strains, respectively. These results highlight the first time use of clindamycin and mortiamide D as antimalarial molecular hybrids and establish these valuable hits for future optimization.

Chemical Composition and Antiplasmodial Activity of the Essential Oil of Rhododendron subarcticum Leaves from Nunavik, Québec, Canada.

Dwarf Labrador tea, Rhododendron subarcticum Harmaja, is a popular medicinal plant in use by First Nations of Northern Canada, but its phytochemistry has remained largely unexplored. We have isolated and characterized the essential oil from a population of this species harvested near the treeline in Nunavik, Québec. Analyses by gas chromatography-mass spectrometry (GC-MS) and gas chromatography/flame-ionization detection (GC/FID) led to the identification of 53 compounds; the main secondary metabolites were ascaridole (64.7% of the total FID area) and p-cymene (21.1%). Such a composition resembles a chemotype observed for R. tomentosum, a close relative found mainly in Europe and Asia, but has never been attributed to R. subarcticum. Growth inhibition assays against different strains of Plasmodium falciparum (3D7, Dd2), the parasite responsible for the most severe form of malaria, were conducted with either the R. subarcticum's essential oil or the isolated ascaridole. Our results show that the essential oil's biological activity can be attributed to ascaridole as its IC50 is more than twice that of ascaridole [ascaridole's IC50 values are 147.3 nM (3D7) and 104.9 nM (Dd2)].

Determining the Predominant Conformations of Mortiamides A-D in Solution Using NMR Data and Molecular Modeling Tools.

Macrocyclic peptidomimetics have been seriously contributing to our arsenal of drugs to combat diseases. The search for nature's discoveries led us to mortiamides A-D (found in a novel fungus from Northern Canada), which is a family of cyclic peptides that clearly have demonstrated impressive pharmaceutical potential. This prompted us to learn more about their solution-state properties as these are central for binding to target molecules. Here, we secured and isolated mortiamide D, and then acquired high-resolution nuclear magnetic resonance (NMR) data to learn more about its structure and dynamics attributes. Sets of two-dimensional NMR experiments provided atomic-level (through-bond and through-space) data to confirm the primary structure, and NMR-driven molecular dynamics (MD) simulations suggested that more than one predominant three-dimensional (3D) structure exist in solution. Further steps of MD simulations are consistent with the finding that the backbones of mortiamides A-C also have at least two prominent macrocyclic shapes, but the side-chain structures and dynamics differed significantly. Knowledge of these solution properties can be exploited for drug design and discovery.

Determining the mode of action involved in the antimicrobial activity of synthetic peptides: a solid-state NMR and FTIR study.

We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular ß-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular ß-sheet peptides on both types of bilayers. (31)P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. (2)H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core.

Interfering with hepatitis C virus assembly in vitro using affinity peptides directed towards core protein.

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.