Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket.

Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain dicarboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al., 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both dicarboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore-gating modulation. KEY POINTS: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved 'in series', a mechanism which may have relevance for eukaryote receptors. We show that short-chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand-gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs /PNa ) of 0.54 for GLIC ion pore.

Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State.

Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates.

Bioinspired Design and Oriented Synthesis of Immunogenic Site-Specifically Penicilloylated Peptides.

ß-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4+ T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naïve CD4+ T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naïve CD4+ T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naïve CD4+ T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.

Palladium-Catalyzed Oxidative Synthesis of Unsymmetrical Azophenols.

A straightforward palladium-catalyzed oxidative hydroxylation of azobenzenes is reported. The developed methodology tolerates various functional groups and allows the synthesis of diverse unsymmetrical azophenols under mild conditions in good to excellent yields. A complementary procedure was also investigated by in situ generation of PIFA. This study represents the first general method for the synthesis of o-hydroxyazobenzenes starting from simple azoarenes.

Asymmetric reduction of ß-ketoesters and chiral ß-iminoesters: impact of a α-quaternary stereocenter.

Diastereomeric reduction of nonactivated, hindered ß-keto and chiral ß-iminoesters are described. The influence of a α-stereocontrolled center on the efficiency and stereoselectivity of the reduction was studied. Reaction conditions were optimized to synthesize ß-hydroxy- and ß-aminoesters in good yields. In the case of chiral ß-iminoesters, influence of matched/mismatched diastereomeric pairs has been assessed.

A facile and stereocontrolled synthesis of γ-substituted γ-fluoroglutamates by conjugate addition: conflicting effect between fluorinated enaminoester and hinderered Michael acceptor.

Asymmetric Michael addition of chiral 2-fluoroenaminoesters derived from (S)-1-phenylethylamine to α-substituted methyl acrylate leads to diastereomeric γ-substituted γ-fluoroglutamate precursors. The tertiary center bearing the amino acid function in its natural configuration is generated with a high level of stereocontrol in contrast to the quaternary carbon center. Diastereomeric γ-substituted γ-fluoroglutamates were efficiently separated and isolated as thioketal derivatives harboring very good enantioselectivity. The Michael addition diastereoselectivity was studied for the asymmetric conjugate addition of fluorinated chiral ß-enaminoester to methyl α-acetamidoacrylate by (19)F and (1)H NMR experiments as well as ab initio computations. An interfering conjunction between hindrance of the electrophile and a destabilizing effect of the fluorine atom borne by the nucleophile is revealed.

Synthesis of new aza-analogs of staurosporine, K-252a and rebeccamycin by nucleophilic opening of C2-symmetric bis-aziridines.

Stable, water-soluble aminosugar staurosporine, K-252a and rebeccamycin analogs have been prepared by nucleophilic opening of C(2)-symmetric N-activated bis-aziridines by bis-indolylmaleimides. This divergent strategy allows the synthesis of unsymmetrical substituted derivatives and provides an easy access to the piperidine and pyrrolidine analogs.

Synthesis and antimalarial activity of carbamate and amide derivatives of 4-anilinoquinoline.

A series of 4-anilinoquinolines bearing an amino side chain linked to the aromatic ring with a carbamate or an amide bond were synthesized and evaluated for their antimalarial activity and their cytotoxicity upon MRC-5 cells. Among the 17 compounds, a majority was found to be active in the low nanomolar range against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro with relative low cytotoxicity. Two compounds were then tested on mice infected by Plasmodium berghei and were found to exhibit reasonable in vivo activity.

Synthesis and antimalarial activity of new analogues of amodiaquine.

In order to determine the real significance of the 4'-phenolic group in the antimalarial activity and/or cytotoxicity of amodiaquine (AQ), analogues for which this functionality was shifted or modified were synthesized. Good in vitro antimalarial activity was obtained for compounds unable to form intramolecular hydrogen bond. Among the compounds synthesized, new amino derivative 5 displayed the greatest selectivity index towards the most CQ-resistant strain tested and was active in mice infected by Plasmodium berghei.

Asymmetric Michael reaction involving chiral imines/secondary enamines: stereocontrolled synthesis of 2,2-disubstituted tetrahydrothiophen-3-ones.

[reaction: see text] The asymmetric Michael reaction involving a chiral imine derived from 2-methyltetrahydrothiophenone-3-one and enantiopure (R)-1-phenylethylamine with a variety of electrophilic alkenes furnished 2,2-disubstituted tetrahydrothiophenone-3-ones with good yields and excellent stereoselectivity.

Identification of cinnamic acid derivatives as novel antagonists of the prokaryotic proton-gated ion channel GLIC.

Pentameric ligand gated ion channels (pLGICs) mediate signal transduction. The binding of an extracellular ligand is coupled to the transmembrane channel opening. So far, all known agonists bind at the interface between subunits in a topologically conserved "orthosteric site" whose amino acid composition defines the pharmacological specificity of pLGIC subtypes. A striking exception is the bacterial proton-activated GLIC protein, exhibiting an uncommon orthosteric binding site in terms of sequence and local architecture. Among a library of Gloeobacter violaceus metabolites, we identified a series of cinnamic acid derivatives, which antagonize the GLIC proton-elicited response. Structure-activity analysis shows a key contribution of the carboxylate moiety to GLIC inhibition. Molecular docking coupled to site-directed mutagenesis support that the binding pocket is located below the classical orthosteric site. These antagonists provide new tools to modulate conformation of GLIC, currently used as a prototypic pLGIC, and opens new avenues to study the signal transduction mechanism.