Sandrine Sagan
CNRS - Université Pierre et Marie Curie, ENS - Department of Chemistry, Department Member
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... 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,...
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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... 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.
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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... 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.
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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,... 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.
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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... 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.
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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)... 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.
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ABSTRACT
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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... 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.
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Trojan peptides or cell-penetrating peptides (CPP) are natural or designed peptides identified as cellular membrane-crossing molecules, in particular through their potency to vehiculate various kinds of compounds to the cytoplasm and... more
Trojan peptides or cell-penetrating peptides (CPP) are natural or designed peptides identified as cellular membrane-crossing molecules, in particular through their potency to vehiculate various kinds of compounds to the cytoplasm and nucleus of living cells. The indirect methods used so far to detect these peptides in cells led to controversial hypotheses on the mechanism of their cell entry. Therefore, we have developed a MALDI-TOF mass spectrometry-based quantification method to track these peptides inside cells. This new method is presented in this review.
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Antimicrobial and cell-penetrating peptides have inspired developments of abiotic membrane-active polymers that can coat, penetrate, or break lipid bilayers in model systems. Application to cell cultures is more recent, but remarkable... more
Antimicrobial and cell-penetrating peptides have inspired developments of abiotic membrane-active polymers that can coat, penetrate, or break lipid bilayers in model systems. Application to cell cultures is more recent, but remarkable bioactivities are already reported. Synthetic polymer chains were tailored to achieve (i) high biocide efficiencies, and selectivity for bacteria (Gram-positive/Gram-negative or bacterial/mammalian membranes), (ii) stable and mild encapsulation of viable isolated cells to escape immune systems, (iii) pH-, temperature-, or light-triggered interaction with cells. This review illustrates these recent achievements highlighting the use of abiotic polymers, and compares the major structural determinants that control efficiency of polymers and peptides. Charge density, sp. of cationic and guanidinium side groups, and hydrophobicity (including polarity of stimuli-responsive moieties) guide the design of new copolymers for the handling of cell membranes. While ...
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The PH-20 protein present on the membrane of guinea pig sperm was characterized using a monoclonal antibody [(1991) J. Cell Biol. 111, 2939-2949]. We have isolated the cDNA encoding the human PH-20 protein from a testis library. This cDNA... more
The PH-20 protein present on the membrane of guinea pig sperm was characterized using a monoclonal antibody [(1991) J. Cell Biol. 111, 2939-2949]. We have isolated the cDNA encoding the human PH-20 protein from a testis library. This cDNA was expressed in RK 13 cells using a vaccinia virus expression system. Cells expressing the human PH-20 protein possess hyaluronidase activity. Treatment with PI-PLC releases the hyaluronidase into the the medium with a concomitant large increase in enzymatic activity. These results demonstrate that the human PH-20 protein has hyaluronidase activity.
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The human NK-1 receptor transfected in Chinese hamster ovary (CHO) cells was studied with use of different tachykinin analogs: Substance P, [Pro9]SP, [Sar9, Met(O2)11]SP, [Gly9 psi (CH2CH2) Leu10]SP, Ac-Arg-septide, septide, [Gly9 psi... more
The human NK-1 receptor transfected in Chinese hamster ovary (CHO) cells was studied with use of different tachykinin analogs: Substance P, [Pro9]SP, [Sar9, Met(O2)11]SP, [Gly9 psi (CH2CH2) Leu10]SP, Ac-Arg-septide, septide, [Gly9 psi (CH2CH2) Gly10]SP, NKA, [pGlu6]SP(6-11) and [Lys5]NKA(4-10). Binding experiments with [3H][Pro9]SP discriminated two classes of peptides with either high affinity (K iota in the nanomolar range) for the human NK-1 receptor or with low affinity (K iota in the micromolar range); this second group of peptides included NKA and [pGlu6]SP(6-11). In spite of these differences, both peptide families evoked potent stimulation of phosphatidylinositol hydrolysis (EC50 in the nanomolar range). In contrast, only NK-1 agonists, with high affinity, stimulated with great potency cyclic AMP formation (EC50 from 8 to 50 nM), whereas the second family of peptides were only weak agonists (EC50 in the micromolar range). RP 67580, CP 96345 and GR 94800, a NK-2 antagonist, w...
Research Interests: Animals and Substance P
Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the... more
Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.
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Research Interests: Physiology, Animals, Male, Peptides, Rat Brain, and 4 moreRats, Radioligand Assay, Substance P, and Binding Site
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The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific mu-opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a... more
The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific mu-opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide (2.4 micrograms/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro-dermorphin related peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, was established by co-chromatography with synthetic peptides on reverse-phase HPLC, immunological analysis, gas-phase sequencing, mass spectrometry and by pharmacological assays. Opioid-binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the delta-opioid receptors. Because of its origin and its delta-opioid (enkephalin) activity and specificity, this novel D-amino acid containing peptide is named dermenkephalin.
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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... 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.
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Cell penetrating peptides (CPPs) belong to the large family of membrane active peptides that comprises antimicrobial and viral fusion peptides with whom they share many properties. CPPs have been increasingly used to transport a wide... more
Cell penetrating peptides (CPPs) belong to the large family of membrane active peptides that comprises antimicrobial and viral fusion peptides with whom they share many properties. CPPs have been increasingly used to transport a wide range of molecules and nanoparticles inside cells. Despite their recognized potential transporting properties, their mode of action is far from being understood and has been a matter of debate. Penetratin, a widely used CPP is one of the first discovered CPPs, yet its mechanism of action remains obscure. Herein an overview on studies regarding cellular and liposomal uptake and the interaction with lipid model systems of CPPs and more particularly penetratin is provided. Special emphasis will be given to biophysical approaches to investigate penetratin/lipid interaction and subsequent lipid reorganization using lipid model systems.
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ABSTRACT Numerous backbone constraints can be used to develop pseudopeptides or pseudomimetics of biologically active peptides. Among those, N- and Calpha-methyl amino acids that can be incorporated by solid-phase peptide synthesis in a... more
ABSTRACT Numerous backbone constraints can be used to develop pseudopeptides or pseudomimetics of biologically active peptides. Among those, N- and Calpha-methyl amino acids that can be incorporated by solid-phase peptide synthesis in a bioactive sequence represent important tools to restrict phi and psi angles of peptide backbone. This review will focus on the chemical syntheses of N- and Calpha-methyl amino acids, their effects on peptide conformation and structure, and their role on the peptide stability towards enzymatic degradation and on the biological activities of the resulting analogues.
Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Propsi(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Propsi((E)-CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP,... more
Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Propsi(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Propsi((E)-CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C-terminal tripeptide, Gly-Leu-Met-NH2, of SP. Propsi((Z)-CH=C(CH2CH(CH3)2)-CONH)Met-NH2, with an isobutyl substituent to mimic the Leu side-chain, was also incorporated in place of the C-terminal tripeptide. The substituted-SP analogs were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK-1 receptor. The most potent SP analogs [Pro9psi((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9psi ((E)CH=C(CH3)CONH)Leu10]SP, are about 100-fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)- or (E)-CH=C(CH3)- or (Z)-CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C-terminal tripeptide of SP within both the NK-1M- and NK-1m-specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.
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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)... 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.
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... References 1. Hruby, VJ; Toth, G.; Gehrig, CA; Kao, LF; Knapp, R.; Lui, GK; Yamamura, HI; Kramer, HT; Davies, P.; Burks, TFJ Med. Chem. ... 1992, 35, 4498. 3. Toth, G.; Russell, KC; Landis, G.; Kramer, TH; Fang, L.; Knapp, R.; Davies,... more
... References 1. Hruby, VJ; Toth, G.; Gehrig, CA; Kao, LF; Knapp, R.; Lui, GK; Yamamura, HI; Kramer, HT; Davies, P.; Burks, TFJ Med. Chem. ... 1992, 35, 4498. 3. Toth, G.; Russell, KC; Landis, G.; Kramer, TH; Fang, L.; Knapp, R.; Davies, P.; Burks, TF; Yamamura, HI; Hruby, VJJ Med. ...
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Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to... more
Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.