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(A) The examples of hydrophobic ligand-binding G protein-coupled receptors (GPCRs). The N-terminal domain (purple) prevents the ligand access from the extracellular side. Instead, the possible entrance to the ligand binding site is located between helices TM1 and TM7 (red dashed ovals). (B) Modeling of a few residues in the N-terminal part of CB1 containing important disulfide bridge. For comparison, on the right, one of the probable conformations of the whole N-terminus of CB1 is shown.
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Most G protein-coupled receptors that bind the hydrophobic ligands (lipid receptors and steroid receptors) belong to the most populated class A (rhodopsin-like) of these receptors. Typical examples of lipid receptors are: rhodopsin, cannabinoid (CB), sphingosine-1-phosphate (S1P) and lysophosphatidic (LPA) receptors. The hydrophobic ligands access...
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... the fact that lipid receptors are closely related with other GPCRs from class A, they display one major structural difference compared to other members of that class: they lack the opening at the extracellular side. A large N-terminal domain forms a plug preventing access to the orthosteric binding site from the extracellular milieu ( Figure 1). Instead, lipid receptors exhibit a vast crevice between transmembrane helices TM1 and TM7 making the binding site accessible from the membrane. ...
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... means that this direction not only requires the smallest work but also there are the smallest forces on the way between the ligand binding site and the outside of the receptor. For the CB1 receptor, we have not used the whole modeled N-terminus ( Figure 1A) but only its fragment (residues 96-99) that is present but not visible in the crystal ( Figure 1B). This fragment contains the important disulfide bond (C98-C107) and the residues from this fragment (particularly F102 and M103) participate in the ligand binding in the orthosteric site. ...
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... means that this direction not only requires the smallest work but also there are the smallest forces on the way between the ligand binding site and the outside of the receptor. For the CB1 receptor, we have not used the whole modeled N-terminus ( Figure 1A) but only its fragment (residues 96-99) that is present but not visible in the crystal ( Figure 1B). This fragment contains the important disulfide bond (C98-C107) and the residues from this fragment (particularly F102 and M103) participate in the ligand binding in the orthosteric site. ...
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... pose, which we believe is a correct AEA binding pose, was reached after 181 ns of SuMD simulation. In this pose, there are π-π interactions formed between double bonds of AEA and residues F170 2.57 , F200 3.36 and W356 6.48 (Figure 7, 181 ns). 2.2.2. ...
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... lack of full entrance might have been caused by the strong ionic interactions of the phosphate group of those ligands formed with the residues K41 NT and R292 7.34 located near the entrance to the channel leading to the orthosteric ligand binding site ( Figure 8A). Nevertheless, the long hydrophobic tail of S1P is reaching the transmission switch (W269 6.48 ), so probably after the action of the switch and making more room in the receptor interior the deeper entrance of the agonist will be possible ( Figure 8A, 310 ns). The (S)-FTY720-P agonist remained half outside of the receptor bound to residues K41 NT and S44 1.31 . ...
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... (S)-FTY720-P agonist remained half outside of the receptor bound to residues K41 NT and S44 1.31 . This ligand is bulkier than S1P so it has to overcome higher steric barriers and this is why the 175 ns probably was not enough time to simulate the ligand entrance ( Figure 8B, 175 ns), compared with the time required for S1P for its partial entrance ( Figure 8A, 310 ns). ...
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... (S)-FTY720-P agonist remained half outside of the receptor bound to residues K41 NT and S44 1.31 . This ligand is bulkier than S1P so it has to overcome higher steric barriers and this is why the 175 ns probably was not enough time to simulate the ligand entrance ( Figure 8B, 175 ns), compared with the time required for S1P for its partial entrance ( Figure 8A, 310 ns). ...
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... also performed calculations of energy profiles during all SuMD simulations (Figure 10). For hydrophobic ligands THC and AEA ( Figure 10A,B) the electrostatic contribution of ligand-receptor interaction energy is close to zero, while the van der Waals energy goes down to about -40 kcal/mol and -56 kcal/mol, respectively, making such binding strong. ...
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... also performed calculations of energy profiles during all SuMD simulations (Figure 10). For hydrophobic ligands THC and AEA ( Figure 10A,B) the electrostatic contribution of ligand-receptor interaction energy is close to zero, while the van der Waals energy goes down to about -40 kcal/mol and -56 kcal/mol, respectively, making such binding strong. The entropy contribution is not regarded but since water is removed from the hydrophobic interior of the receptor the ligand binding is even stronger. ...
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... entropy contribution is not regarded but since water is removed from the hydrophobic interior of the receptor the ligand binding is even stronger. For S1P and LPA ligands entering their receptors ( Figure 10C,D), the van der Waals energy also contributes to the ligand binding: -25 kcal/mol for S1P and -36 kcal/mol for LPA. Both ligands have long hydrophobic tails but also positively and negatively charged groups which contribute to a large decrease in interaction energy resulting in strong binding to the receptor. ...
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... ligands have long hydrophobic tails but also positively and negatively charged groups which contribute to a large decrease in interaction energy resulting in strong binding to the receptor. Interestingly, in the case of ligand LPA, the electrostatic energy was positive during the initial stages of the ligand entering the receptor so the charged part of the ligand was repelled ( Figure 10D) and only van der Waals forces were responsible for attracting LPA into the receptor since the ligand was entering the receptor with its hydrophobic tail first (Figure 9). The (S)-FTY720-P only partially entered the S1P1 receptor but the obtained van der Walls energy was nearly the same as for the LPA ligand (-25 kcal/mol) indicating good binding of the hydrophobic part of this ligand. ...
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We recently showed that radiation-induced DNA damage in breast adipose tissue increases autotaxin secretion, production of lysophosphatidate (LPA) and expression of LPA1/2 receptors. We also established that dexamethasone decreases autotaxin production and LPA signaling in non-irradiated adipose tissue. In the present study, we showed that dexameth...
Citations
... Figure 2 shows a representative example of the comparison between AM11542 (a) and Raloxifene (b), with all poses into the channel, and a discarded compound (mupirocin) (c), with several scattered predicted poses. These channel analyses may provide an explanation for the different potency and selectivity of the CB1R ligands [49,50]. With this filter, 10 drugs were selected: Aminopterin (APGA), Avanafil, Ceftriaxone, Methotrexate, Miltefosine, PGE-1, Raloxifene, Raltegravir, Riociguat and Valsartan, as shown in Table 1. ...
... Interestingly, among these compounds, Methotrexate and APGA show similar structures, also shown in Table 1 for comparison. These channel analyses may provide an explanation for the different potency and selectivity of the CB1R ligands [49,50]. With this filter, 10 drugs were selected: Aminopterin (APGA), Avanafil, Ceftriaxone, Methotrexate, Miltefosine, PGE-1, Raloxifene, Raltegravir, Riociguat and Valsartan, as shown in Table 1. ...
... Light blue dots are poses with medium affinity (from −8.0 to −7.0 kcal/mol). These channel analyses may provide an explanation for the different potency and selectivity of the CB1R ligands [49,50]. With this filter, 10 drugs were selected: Aminopterin (APGA), Avanafil, Ceftriaxone, Methotrexate, Miltefosine, PGE-1, Raloxifene, Raltegravir, Riociguat and Valsartan, as shown in Table 1. ...
The cannabinoid receptor 1 (CB1R) plays a pivotal role in regulating various physiopathological processes, thus positioning itself as a promising and sought-after therapeutic target. However, the search for specific and effective CB1R ligands has been challenging, prompting the exploration of drug repurposing (DR) strategies. In this study, we present an innovative DR approach that combines computational screening and experimental validation to identify potential Food and Drug Administration (FDA)-approved compounds that can interact with the CB1R. Initially, a large-scale virtual screening was conducted using molecular docking simulations, where a library of FDA-approved drugs was screened against the CB1R’s three-dimensional structures. This in silico analysis allowed us to prioritize compounds based on their binding affinity through two different filters. Subsequently, the shortlisted compounds were subjected to in vitro assays using cellular and biochemical models to validate their interaction with the CB1R and determine their functional impact. Our results reveal FDA-approved compounds that exhibit promising interactions with the CB1R. These findings open up exciting opportunities for DR in various disorders where CB1R signaling is implicated. In conclusion, our integrated computational and experimental approach demonstrates the feasibility of DR for discovering CB1R modulators from existing FDA-approved compounds. By leveraging the wealth of existing pharmacological data, this strategy accelerates the identification of potential therapeutics while reducing development costs and timelines. The findings from this study hold the potential to advance novel treatments for a range of CB1R -associated diseases, presenting a significant step forward in drug discovery research.
... Interestingly, the ECL2 of S1P1, S1P3, LPA1, and CB1 interact with the N-terminus and form a cap over the binding site so that highly hydrophobic ligands enter into the binding site from the lipid bilayer. 47,97,98 Numbers of contacts between ECL2 and EC domains were used as an additional measure to describe ECL2 conformations. Extending this analysis to MD frames allowed us to investigate how the flexibility of ECL2 influences its interaction with other EC domains ( Figure 5). ...
The extracellular loop 2 (ECL2) is the longest and the most diverse loop among class A G protein-coupled receptors (GPCRs). It connects the transmembrane (TM) helices 4 and 5 and contains a highly conserved cysteine through which it is bridged with TM3. In this paper, experimental ECL2 structures were analyzed based on their sequences, shapes, and intra-molecular contacts. To take into account the flexibility, we incorporated into our analyses information from the molecular dynamics trajectories available on the GPCRmd website. Despite the high sequence variability, shapes of the analyzed structures, defined by the backbone volume overlaps, can be clustered into seven main groups. Conformational differences within the clusters can be then identified by intramolecular interactions with other GPCR structural domains. Overall, our work provides a reorganization of the structural information of the ECL2 of class A GPCR subfamilies, highlighting differences and similarities on sequence and conformation levels.
... MD simulations are often reported concurrently as the structural determination, as seen in the cases of 5-HT 3A [116], CXCR 2 [117], CRTH 2 /DP 2 [32], and CB 2 [56], where the simulations provide further conformational and binding insights. Steered MD simulations were used for investigating ligand entry and exit in CB 1 , S1P 1 , and LPA 1 [118]. MD simulations can also be used to probe and map orthosteric and allosteric binding sites [83,91,92,[119][120][121]. Advanced statistical mechanics tools such as Markov state models [122] have been used to investigate PAC1, VPAC1, and VPAC2 receptors [123], the adenosine A 2A receptor [124], and dopamine D 2 and D 3 receptors [125]. ...
Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.
... We first elucidated the structure of MPR of CB1R ( Figure 6)-see Section 3.1 of Methods for details. The structure of CB1R with MPR was used in our previous work [36] for investigating the entry pathway into the orthosteric binding site of the CB1 cannabinoid receptor. Next, the remaining residues 1-95 of the N-terminal domain were added according to the sequence of a wild-type human CB1R and the conformational space of the newly added part was extensively explored using the Replica Exchange Molecular Dynamics with Solute Tempering [37] simulations. ...
... We first elucidated the structure of MPR of CB 1 R ( Figure 6)-see Section 3.1 of Methods for details. The structure of CB 1 R with MPR was used in our previous work [36] for investigating the entry pathway into the orthosteric binding site of the CB 1 cannabinoid receptor. Next, the remaining residues 1-95 of the N-terminal domain were added according to the sequence of a wild-type human CB 1 R and the conformational space of the newly added part was extensively explored using the Replica Exchange Molecular Dynamics with Solute Tempering [37] simulations. ...
... However, in all simulations of ∆-88 (deletion of 1-88 residues) and ∆-98 (deletion of 1-98 residues) CB 1 R mutants (two simulations repeats for each mutant), the CBD binding mode was significantly distorted and the final pose was different for each of the four simulations ( Figure 11). In two out of four cases (one simulation for ∆-88 and one simulation for ∆-98) CBD exited the allosteric site completely and within less than 500 ns it moved toward the orthosteric ligand binding channel between helices I and VII (this gate was described by us in our previous paper [36]) distorting the conformation of MPR significantly. In one of the two simulations for the ∆-8 (deletion of 1-8 residues) CB 1 R, CBD dissociated from the receptor completely after 350 ns and remained dissolved in the membrane for the rest of the simulation time. ...
CB1 cannabinoid receptor (CB1R) contains one of the longest N termini among class A G protein-coupled receptors. Mutagenesis studies suggest that the allosteric binding site of cannabidiol (CBD) involves residues from the N terminal domain. In order to study the allosteric binding of CBD to CB1R we modeled the whole N-terminus of this receptor using the replica exchange molecular dynamics with solute tempering (REST2) approach. Then, the obtained structures of CB1R with the N terminus were used for ligand docking. A natural cannabinoid receptor agonist, Δ9-THC, was docked to the orthosteric site and a negative allosteric modulator, CBD, to the allosteric site positioned between extracellular ends of helices TM1 and TM2. The molecular dynamics simulations were then performed for CB1R with ligands: i) CBD together with THC, and ii) THC-only (1 µs each, 5 repeats for each case). Analyses of the differences in the residue-residue interaction patterns between those two cases allowed us to elucidate the allosteric network responsible for the modulation of the CB1R by CBD. In addition, we identified the changes in the orthosteric binding mode of Δ9-THC, as well as the changes in its binding energy, caused by the CBD allosteric binding. We have also found that the presence of a complete N-terminal domain is essential for a stable binding of CBD in the allosteric site of CB1R as well as for the allosteric-orthosteric coupling mechanism.
The G protein-coupled receptor GPR183 is a chemotactic receptor with an important function in the immune system and association with a variety of diseases. It recognizes ligands with diverse physicochemical properties as both the endogenous oxysterol ligand 7α,25-OHC and synthetic molecules can activate the G protein pathway of the receptor. To better understand the ligand promiscuity of GPR183, we utilized both molecular dynamics simulations and cell-based validation experiments. Our work reveals that the receptor possesses two ligand entry channels: one lateral between transmembrane helices 4 and 5 facing the membrane, and one facing the extracellular environment. Using enhanced sampling, we provide a detailed structural model of 7α,25-OHC entry through the lateral membrane channel. Importantly, the first ligand recognition point at the receptor surface has been captured in diverse experimentally solved structures of different GPCRs. The proposed ligand binding pathway is supported by in vitro data employing GPR183 mutants with a sterically blocked lateral entrance, which display diminished binding and signaling. In addition, computer simulations and experimental validation confirm the existence of a polar water channel which might serve as an alternative entrance gate for less lipophilic ligands from the extracellular milieu. Our study reveals knowledge to understand GPR183 functionality and ligand recognition with implications for the development of drugs for this receptor. Beyond, our work provides insights into a general mechanism GPCRs may use to respond to chemically diverse ligands.
Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well-characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re-evaluated. We evaluate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides in Callinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms increased almost two-fold, from 18 to 36. Product ion-triggered electron-transfer/higher-energy collision dissociation enabled the site-specific detection of 55 intact N- and O-linked glycoforms, while the faster stepped collision energy higher-energy collisional dissociation resulted in detection of 25. Applying this workflow to five neuronal tissues enabled the characterization of 36 glycoforms of known neuropeptides and 11 glycoforms of 9 putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro- and micro-heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides. This article is protected by copyright. All rights reserved.
X-ray crystallography and cryogenic electronic microscopy have provided significant advancement in the knowledge of GPCR structure and have allowed the rational design of GPCR ligands. The class A GPCRs cannabinoid receptor type 1 and type 2 are implicated in many pathophysiological processes and thus rational design of drug and tool compounds is of great interest. Recent structural insight into cannabinoid receptors has already led to a greater understanding of ligand binding sites and receptor residues that likely contribute to ligand selectivity. Herein, classes of heterocyclic covalent cannabinoid receptor ligands are reviewed in light of the recent advances in structural knowledge of cannabinoid receptors, with particular discussion regarding covalent ligand selectivity and rationale design.
