The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the e... more The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol-induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212-2, CP 55940 and anandamide also inhibited the isoproterenol-evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212-2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G-protein-coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]-SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G-protein-coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.
Histone lysine methylation is associated with essential biological functions like transcription a... more Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol,...
Angewandte Chemie (International ed. in English), Jan 9, 2017
Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocat... more Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles depending on their phospholipid content. To evaluate the phospholipid preference of Penetratin, as the first step of translocation, we have exploited the benzophenone triplet kinetics of hydrogen abstraction, slower for secondary than for allylic hydrogens. Using multilamellar vesicles (MLVs) of various phospholipid content, we have identified and characterized the crosslinked products by MALDI-TOF mass spectrometry. Penetratin shows a preference for negatively charged (vs zwitterionic) polar heads, for unsaturated (vs saturated) and short (vs long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.
The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the developmen... more The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.10(8) for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.
The effect of (R)-methanandamide, a chiral analog of the endogenous cannabimimetic anandamide, wa... more The effect of (R)-methanandamide, a chiral analog of the endogenous cannabimimetic anandamide, was investigated on calcium signalling in cultured rat astrocytes loaded with Indo-1. Pretreatment of astrocytes with (R)-methanandamide resulted in the inhibition of calcium responses induced by endothelin-1 or glutamate. Test of the filling level of internal calcium stores and biochemical assays of phospholipase C activity suggested that this inhibition resulted from the depletion of internal pools. This effect occurred in a reversible time-and dose-dependent manner, and was prevented by treating the cells with pertussis toxin (PTX) but was not reproduced by similar concentrations of arachidonic acid. Altogether, these observations demonstrate that this stable analog of anandamide controls calcium signalling in astrocytes through a PTX-sensitive mechanism which leads to the depletion of fast mobilizable internal stores.
Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokin... more Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Cα methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Cα methylated analogues of the undecapeptide SP (from position 5–11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Cα methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Cα methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [αMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Cα methylation of SP are sequence dependent. Moreover, a single Cα methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an α-aminoisobutyric acid. Furthermore, Cα methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4–8 domain of other potent SP analogues.
CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious ... more CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.
Conformationally and configurationally restricted rotameric probes of phenylalanine have been inc... more Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2-for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S. 3R)- and (2S, 3S)-indanylglycines, E- and Z-alpha, beta-dehydrophenylalanines, and 2(S)-alpha, beta-cyclopropylphenylalanines [delta E Phe. delta Z Phe. inverted delta E2 (S) Phe, and inverted delta Z 2 (S) Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (delta E Phe, delta Z Phe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (-) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is small since the other rotamer can be detected in [(2S, 3R)Ing7]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe7 substituted analogues of SP must come from steric hindrance in the S7 binding site, which drastically restricts the space filling around the C alpha-C beta bond of residue 7.
Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokin... more Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.
The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the e... more The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol-induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212-2, CP 55940 and anandamide also inhibited the isoproterenol-evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212-2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G-protein-coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]-SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G-protein-coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.
Histone lysine methylation is associated with essential biological functions like transcription a... more Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol,...
Angewandte Chemie (International ed. in English), Jan 9, 2017
Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocat... more Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles depending on their phospholipid content. To evaluate the phospholipid preference of Penetratin, as the first step of translocation, we have exploited the benzophenone triplet kinetics of hydrogen abstraction, slower for secondary than for allylic hydrogens. Using multilamellar vesicles (MLVs) of various phospholipid content, we have identified and characterized the crosslinked products by MALDI-TOF mass spectrometry. Penetratin shows a preference for negatively charged (vs zwitterionic) polar heads, for unsaturated (vs saturated) and short (vs long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.
The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the developmen... more The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.10(8) for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.
The effect of (R)-methanandamide, a chiral analog of the endogenous cannabimimetic anandamide, wa... more The effect of (R)-methanandamide, a chiral analog of the endogenous cannabimimetic anandamide, was investigated on calcium signalling in cultured rat astrocytes loaded with Indo-1. Pretreatment of astrocytes with (R)-methanandamide resulted in the inhibition of calcium responses induced by endothelin-1 or glutamate. Test of the filling level of internal calcium stores and biochemical assays of phospholipase C activity suggested that this inhibition resulted from the depletion of internal pools. This effect occurred in a reversible time-and dose-dependent manner, and was prevented by treating the cells with pertussis toxin (PTX) but was not reproduced by similar concentrations of arachidonic acid. Altogether, these observations demonstrate that this stable analog of anandamide controls calcium signalling in astrocytes through a PTX-sensitive mechanism which leads to the depletion of fast mobilizable internal stores.
Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokin... more Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Cα methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Cα methylated analogues of the undecapeptide SP (from position 5–11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Cα methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Cα methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [αMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Cα methylation of SP are sequence dependent. Moreover, a single Cα methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an α-aminoisobutyric acid. Furthermore, Cα methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4–8 domain of other potent SP analogues.
CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious ... more CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.
Conformationally and configurationally restricted rotameric probes of phenylalanine have been inc... more Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2-for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S. 3R)- and (2S, 3S)-indanylglycines, E- and Z-alpha, beta-dehydrophenylalanines, and 2(S)-alpha, beta-cyclopropylphenylalanines [delta E Phe. delta Z Phe. inverted delta E2 (S) Phe, and inverted delta Z 2 (S) Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (delta E Phe, delta Z Phe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (-) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is small since the other rotamer can be detected in [(2S, 3R)Ing7]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe7 substituted analogues of SP must come from steric hindrance in the S7 binding site, which drastically restricts the space filling around the C alpha-C beta bond of residue 7.
Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokin... more Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.
Uploads
Papers by Sandrine Sagan