Báo cáo khoa học: Investigation of the interaction between the atypical agonist c[YpwFG] and MOR

Investigation of the interaction between the atypical agonist c[YpwFG] and MOR.

Tác giả: Luca Gentilucci, Federico Squassabia, Rossella Demarco, Roberto Artali, Giuliana Cardillo, Alessandra Tolomelli, Santi Spampinato, Andrea Bedini

Lĩnh vực: Dược lý học, Hóa học

Nội dung tài liệu: Nghiên cứu này khám phá sự tương tác giữa một chất chủ vận atypical µ-opioid receptor (MOR) là c[YpwFG] và thụ thể MOR. Các tác giả đã tổng hợp và thử nghiệm một thư viện các peptide vòng được biến đổi, đồng thời sử dụng phân tích cấu trúc bằng NMR và động lực học phân tử. Nghiên cứu cũng sử dụng phương pháp mô phỏng đặt mục tiêu phân tử (molecular docking) và cơ học lượng tử/cơ học phân tử (QM/MM) để hiểu rõ hơn về cơ chế tương tác và các định hướng liên kết tiềm năng của các peptide vòng này trong vị trí gắn kết của thụ thể MOR. Kết quả cho thấy các peptide vòng này có thể tương tác với thụ thể thông qua một cơ chế khác biệt so với các chất chủ vận điển hình, nhưng vẫn duy trì khả năng kích hoạt thụ thể.

Mục lục chi tiết:

• Keywords
• atypical agonist; cyclopentapeptide; induced fit; molecular docking; opioid receptor
• Correspondence
• (Received 8 January 2008, revised 22 February 2008, accepted 6 March 2008)
• doi:10.1111/j.1742-4658.2008.06386.x
• Endogenous and exogenous opiates are currently considered the drugs of choice for treating different kinds of pain. However, their prolonged use produces several adverse symptoms, and in addition, many forms of pain are resistant to any kind of therapy. Therefore, the discovery of compounds active towards µ-opioid receptors (MORs) by alternative pharmacological mechanisms could be of value for developing novel classes of analgesics.
• There is evidence that some unusual molecules can bind opioid receptors, albeit lacking some of the typical opioid pharmacophoric features. In particular, the recent discovery of a few compounds that showed agonist behavior even in the absence of the primary pharmacophore, namely a protonable amine, led to a rediscussion of the importance of ionic interactions in stabilizing the ligand-receptor complex and in activating signal transduction.
• Very recently, we synthesized a library of cyclic analogs of the endogenous, MOR-selective agonist endomorphin-1 (YPWF-NH2), containing a Gly5 bridge between Tyrl and Phe4. The cyclopeptide c[YpwFG] showed good affinity and agonist behavior. This atypical MOR agonist does not have the protonable Tyr amine.
• In order to gain more information about plausible mechanisms of interaction between c[YpwFG] and the opioid receptor, we synthesized a selected set of derivatives containing different bridges between Tyrl and Phe4, and tested their affinities towards µ-opioid receptors.
• We performed conformational analysis of the cyclopeptides by NMR spectroscopy and molecular dynamics, and investigated plausible, unprecedented modes of interaction with the MOR by molecular docking.
• The successive quantum mechanics/molecular mechanics investigation of the complexes obtained by the molecular docking procedure furnished a more detailed description of the binding mode and the electronic properties of the ligands.
• The comparison with the binding mode of the potent agonist JOM-6 seems to indicate that the cyclic endomorphin-1 analogs interact with the receptor by way of an alternative mechanism, still maintaining the ability to activate the receptor.
• In recent years, various research groups have described opioid receptor (OR)-active molecules lacking some crucial pharmacological requisites. In particular, several papers have stressed the role of Tyrl in the interaction of native or synthetic opioid peptides with µ-opioid receptors (MORs). In certain cases, the…
• Abbreviations
• Aib, a-aminoisobutyric acid; CPP, cyclopentapeptide; DAMGO, H-Tyr-D-Ala-Gly-N-MePhe-glyol; DOR, 8-opioid receptor; DPPA, diphenylphosphorylazide; EL, extracellular loop; EM-1, endomorphin-1; KOR, к-opioid receptor; MD, molecular dynamics; MM, molecular mechanics; MOR, µ-opioid receptor; QM, quantum mechanics; TMH, transmembrane helix; VT, variable temperature.
• FEBS Journal 275 (2008) 2315-2337© 2008 The Authors Journal compilation 2008 FEBS
• 2315
• The atypical opioid agonist c[YpwFG]
• L. Gentilucci et al.
• modification of the phenolic OH group had no consequences for the ability to bind the receptor. Indeed, the transposition [1,2], removal [3,4], duplication [5] or substitution with a surrogate [6] of Tyrl gave analogs that showed comparable binding affinities and potencies to those of the parent peptides.
• A more relevant modification is the removal or derivatization of the positively charged N-terminal amino group. In general, these modifications are responsible for transforming agonists into antagonists, confirming the fundamental role of the amino group in receptor activation.
• Noteworthy examples are the somewhat 8-opioid receptor (DOR)-selective casomorphin derivatives, in which the terminal amino group is eliminated or formylated [7], the carbamate-peptide PhCH2OC(O)-Pro-Trp-PheNH2, which showed nanomolar affinity for MORs [8], the potent enkephalin-derived DOR antagonist containing a deaminated Tyr [9], the enkephalin and к-opioid receptor (KOR)-selective DynA analogs obtained by replacement of Tyrl with 3-(2′,6′-dimethyl-4′-hydroxyphenyl)propanoic acid and (2S)-2-methyl-3-(2′,6′-dimethyl-4′-hydroxyphenyl)-propionic acid (Mdp) [10], and finally the cyclic DynA analog lacking the basic N-terminus, which showed good KOR affinity [11].
• In contrast, a few compounds lacking the amino group have demonstrated an agonist nature: the MOR-selective bicyclic compound 1, designed to mimic enkephalin or endomorphin ẞ-turn models, the K-selective neoclerodane diterpene salvinorin A (compound 2), and the cyclic endomorphin-1 (EM-1) analog active towards MOR c[YpwFG] (compound 3) (Fig. 1).
• The highly constrained 6,6-bicyclic compound 1, which has no N-terminal amino group, showed an initial level of analgesic activity similar to that of morphine, but with a shorter in vivo half-life [12]. On the basis of 2D-NMR analysis and molecular mechanics (MM) computations, the authors noticed a certain superimposition of the structure of compound 1 with a…
• trans-EM-1 type III ẞ-turn-like structure. According to this partial superimposition and the MOR selectivity profile, they implicitly suggested that the interaction of compound 1 with the receptor could mimic that of EM-1 or enkephalins, even in the absence of a ionic interaction.
• Salvinorin A (compound 2), a naturally occurring hallucinogen isolated from Salvia divinorum [13], is a unique, non-nitrogen-containing selective KOR agonist. An earlier docking analysis, based in turn on models originally developed for non-opioid KOR agonists such as U69593 [14], led to a preliminary model. However, by using an improved model of the receptor, and screening of salvinorin derivatives [15], the same authors substantially modified the original model [16]. More recently, acquired structure-function data of salvinorin analogs [17,18] led to the proposal of a third different model [19].
• The cyclopeptide compound 3, c[YpwFG], showed good MOR affinity (Table 1), and agonist behavior (forskolin-stimulated cAMP production inhibition test) [20]. Cyclic peptides have been widely used as conformationally restricted frameworks [21], useful for arranging the pharmacophores in different reciprocal orientations, and in particular, cyclic pentapeptides containing one or two D-amino acids have been successfully utilized as ẞ-turn or y-turn models [22-27].
• The hypothesis that EM-1 derivatives could adopt at the receptor a folded structure stabilized by some kind of y-turn or ẞ-turn has been stressed in recent papers [8,28,29].
• For the atypical structure and the highly lipophilic character, we planned further studies to provide insights into how c[YpwFG] might interact with the receptor. We synthesized and tested a selected mini-library of new cyclopeptides derived from compound 3, and we performed a computational investigation intended to investigate the possible orientations of the biologically active cyclopeptides when docked into the binding site defined by the MOR model. We first…
• HO
• 1
• N.
• 2
• H
• HO
• N
• H
• 3
• Fig. 1. Examples of opioid agonists lacking a protonable amino group.
• 2316
• FEBS Journal 275 (2008) 2315-2337© 2008 The Authors Journal compilation 2008 FEBS
• L. Gentilucci et al.
• The atypical opioid agonist c[YpwFG]
• modification of the phenolic OH group had no consequences for the ability to bind the receptor. Indeed, the transposition [1,2], removal [3,4], duplication [5] or substitution with a surrogate [6] of Tyrl gave analogs that showed comparable binding affinities and potencies to those of the parent peptides.
• A more relevant modification is the removal or derivatization of the positively charged N-terminal amino group. In general, these modifications are responsible for transforming agonists into antagonists, confirming the fundamental role of the amino group in receptor activation.
• Noteworthy examples are the somewhat 8-opioid receptor (DOR)-selective casomorphin derivatives, in which the terminal amino group is eliminated or formylated [7], the carbamate-peptide PhCH2OC(O)-Pro-Trp-PheNH2, which showed nanomolar affinity for MORs [8], the potent enkephalin-derived DOR antagonist containing a deaminated Tyr [9], the enkephalin and к-opioid receptor (KOR)-selective DynA analogs obtained by replacement of Tyrl with 3-(2′,6′-dimethyl-4′-hydroxyphenyl)propanoic acid and (2S)-2-methyl-3-(2′,6′-dimethyl-4′-hydroxyphenyl)-propionic acid (Mdp) [10], and finally the cyclic DynA analog lacking the basic N-terminus, which showed good KOR affinity [11].
• In contrast, a few compounds lacking the amino group have demonstrated an agonist nature: the MOR-selective bicyclic compound 1, designed to mimic enkephalin or endomorphin ẞ-turn models, the K-selective neoclerodane diterpene salvinorin A (compound 2), and the cyclic endomorphin-1 (EM-1) analog active towards MOR c[YpwFG] (compound 3) (Fig. 1).
• The highly constrained 6,6-bicyclic compound 1, which has no N-terminal amino group, showed an initial level of analgesic activity similar to that of morphine, but with a shorter in vivo half-life [12]. On the basis of 2D-NMR analysis and molecular mechanics (MM) computations, the authors noticed a certain superimposition of the structure of compound 1 with a…
• trans-EM-1 type III ẞ-turn-like structure. According to this partial superimposition and the MOR selectivity profile, they implicitly suggested that the interaction of compound 1 with the receptor could mimic that of EM-1 or enkephalins, even in the absence of a ionic interaction.
• Salvinorin A (compound 2), a naturally occurring hallucinogen isolated from Salvia divinorum [13], is a unique, non-nitrogen-containing selective KOR agonist. An earlier docking analysis, based in turn on models originally developed for non-opioid KOR agonists such as U69593 [14], led to a preliminary model. However, by using an improved model of the receptor, and screening of salvinorin derivatives [15], the same authors substantially modified the original model [16]. More recently, acquired structure-function data of salvinorin analogs [17,18] led to the proposal of a third different model [19].
• The cyclopeptide compound 3, c[YpwFG], showed good MOR affinity (Table 1), and agonist behavior (forskolin-stimulated cAMP production inhibition test) [20]. Cyclic peptides have been widely used as conformationally restricted frameworks [21], useful for arranging the pharmacophores in different reciprocal orientations, and in particular, cyclic pentapeptides containing one or two D-amino acids have been successfully utilized as ẞ-turn or y-turn models [22-27].
• The hypothesis that EM-1 derivatives could adopt at the receptor a folded structure stabilized by some kind of y-turn or ẞ-turn has been stressed in recent papers [8,28,29].
• For the atypical structure and the highly lipophilic character, we planned further studies to provide insights into how c[YpwFG] might interact with the receptor. We synthesized and tested a selected mini-library of new cyclopeptides derived from compound 3, and we performed a computational investigation intended to investigate the possible orientations of the biologically active cyclopeptides when docked into the binding site defined by the MOR model. We first…
• HO
• 1
• N.
• 2
• H
• HO
• N
• H
• 3
• Fig. 1. Examples of opioid agonists lacking a protonable amino group.
• 2316
• FEBS Journal 275 (2008) 2315-2337© 2008 The Authors Journal compilation 2008 FEBS
• Table 1. Synthesis, analytical characterization and receptor affinities (means ± SE of three experiments) of DAMGO and compounds 3-8.
• Compound
• Sequence
• Yield (%)ª
• Purity (%)
• MS/calculated [M + 1]
• K (м)
• IC50 (M)
• DAMGO
• YaG-NMeF-Glyol
• 1.6 ± 0.3 × 10-9
• 9.9 ± 0.6 x 10-9
• 3
• c[YpwFG]
• 62
• 96
• 651.2/651.1
• 3.4 ± 0.7 x 10-8
• 4.4 ± 0.6 × 10-8
• 4
• c[YpwF-ẞAla]
• 58
• 93
• 665.3/665.3
• 6.1 ± 0.5 × 10-6
• 1.6 ± 0.2 x 10-5
• 5
• c[YpwF-GABA]
• 64
• 93
• 679.5/679.3
• 3.2 ± 0.4 x 10-6
• 8.4 ± 0.8 x 10-6
• 6
• c[YpwF-Aib]
• 55
• 95
• 679.2/679.3
• 2.9 ± 0.3 × 10-6
• 7.6 ± 0.7 x 10-6
• 7
• c[YpwFp]
• 53
• 96
• 691.6/691.3
• 3.2 ± 0.2 x 10-5
• 8.3 ± 0.9 × 10-5
• 8
• c[YpwFP]
• 59
• 95
• 691.5/691.3
• 7.2 ± 0.5 x 10-7
• 9.0 ± 0.4 x 10-7
• a Yield of the cyclization step after purification.
• explored the possible binding positions and binding modes of the ligand within the rigid receptor environment, and the solutions obtained from this docking study were subsequently optimized by means of the combined quantum mechanics QM/MM approach, using a flexible receptor environment that allows for simulation of the receptor adaptation upon ligand binding (induced fit).
• The conformations adopted in dimethylsulfoxide were used as starting structures for docking the ligands into the entire channel pore with AUTODOCK [30], without prior specification of the binding site, by using the so-called ‘blind docking’ approach, a technique introduced for the detection of possible binding sites and modes of binding of peptide ligands by searching the entire surface of protein targets [31,32].
• The main potential orientations have been evaluated using the QM/MM optimization of the complexes [33,34], providing a more detailed description of the binding mode and the electronic and steric properties of the c[Y