大麻素受体CB1三维结构的同源模建及其对接研究

大麻素CB1受体属于G蛋白偶联受体. 以牛视紫红质的晶体结构为模板, 利用同源模建法对CB1受体的三维结构进行了模拟, 并采用分子动力学方法对模型进行了修正和优化. 在此基础上, 分析了活性位点的组成和结构, 研究了拮抗剂利莫那班与CB1受体的对接, 明确了CB1受体与利莫那班结合时起重要作用的氨基酸残基. 发现利莫那班与CB1受体残基Lys192形成氢键相互作用是CB1受体拮抗剂的重要分子作用基础.     

The Role of Lipids in Allosteric Modulation of Dopamine D2 Receptor—In Silico Study

The dopamine D₂ receptor, belonging to the class A G protein-coupled receptors (GPCRs), is an important drug target for several diseases, including schizophrenia and Parkinson’s disease. The D₂ receptor can be activated by the natural neurotransmitter dopamine or by synthetic ligands, which in both cases leads to the receptor coupling with a G protein. In addition to receptor modulation by orthosteric or allosteric ligands, it has been shown that lipids may affect the behaviour of membrane proteins. We constructed a model of a D₂ receptor with a long intracellular loop (ICL3) coupled with G_iα1 or G_iα2 proteins, embedded in a complex asymmetric membrane, and simulated it in complex with positive, negative or neutral allosteric ligands. In this study, we focused on the influence of ligand binding and G protein coupling on the membrane–receptor interactions. We show that there is a noticeable interplay between the cell membrane, G proteins, D₂ receptor and its modulators.     

Discovery of Homobivalent Bitopic Ligands of the Cannabinoid CB2 Receptor**

Single chemical entities with potential to simultaneously interact with two binding sites are emerging strategies in medicinal chemistry. We have designed, synthesized and functionally characterized the first bitopic ligands for the CB₂ receptor. These compounds selectively target CB₂ versus CB₁ receptors. Their binding mode was studied by molecular dynamic simulations and site-directed mutagenesis.     

CB1 Cannabinoid Receptor Signaling and Biased Signaling

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling—the preferential activation of a signaling transducer in detriment of another—have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.     

Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects—including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism—without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors–ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.     

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.     

Synthesis and In Vitro Characterization of Selective Cannabinoid CB2 Receptor Agonists: Biological Evaluation against Neuroblastoma Cancer Cells

1,8-naphthyridine-3-carboxamide structures were previously identified as a promising scaffold from which to obtain CB2R agonists with anticancer and anti-inflammatory activity. This work describes the synthesis and functional characterization of new 1,8-naphthyridin-2(1H)-one-3-carboxamides with high affinity and selectivity for CB2R. The new compounds were able to pharmacologically modulate the cAMP response without modulating CB2R-dependent β-arrestin2 recruitment. These structures were also evaluated for their anti-cancer activity against SH-SY5Y and SK-N-BE cells. They were able to reduce the cell viability of both neuroblastoma cancer cell lines with micromolar potency (IC₅₀ of FG158a = 11.8 μM and FG160a = 13.2 μM in SH-SY5Y cells) by a CB2R-mediated mechanism. Finally, in SH-SY5Y cells one of the newly synthesized compounds, FG158a, was able to modulate ERK1/2 expression by a CB2R-mediated effect, thus suggesting that this signaling pathway might be involved in its potential anti-cancer effect.     

A Critical Evaluation of Terpenoid Signaling at Cannabinoid CB1 Receptors in a Neuronal Model

In addition to phytocannabinoids, cannabis contains terpenoids that are claimed to have a myriad of effects on the body. We tested a panel of five common cannabis terpenoids, myrcene, linalool, limonene, α-pinene and nerolidol, in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons express a form of cannabinoid CB1 receptor-dependent retrograde plasticity while DRGs express a variety of transient receptor potential (TRP) channels. Most terpenoids had little or no effect on neuronal cannabinoid signaling. The exception was nerolidol, which inhibited endocannabinoid signaling. Notably, this is not via inhibition of CB1 receptors but by inhibiting some aspect of 2-arachidonoylglycerol (2-AG) production/delivery; the mechanism does not involve reducing the activity of the 2-AG-synthesizing diacylglycerol lipases (DAGLs). Nerolidol was also the only terpenoid that activated a sustained calcium response in a small (7%) subpopulation of DRGs. In summary, we found that only one of five terpenoids tested had notable effects on cannabinoid signaling in two neuronal models. Our results suggest that a few terpenoids may indeed interact with some components of the cannabinoid signaling system and may therefore offer interesting insights upon further study.     

Cannabinoid Receptor Modulation of Neurogenesis: ST14A Striatal Neural Progenitor Cells as a Simplified In Vitro Model

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.     

Insights into the Allosteric Effect of SENP1 Q597A Mutation on the Hydrolytic Reaction of SUMO1 via an Integrated Computational Study

Small ubiquitin-related modifier (SUMO)-specific protease 1 (SENP1) is a cysteine protease that catalyzes the cleavage of the C-terminus of SUMO1 for the processing of SUMO precursors and deSUMOylation of target proteins. SENP1 is considered to be a promising target for the treatment of hepatocellular carcinoma (HCC) and prostate cancer. SENP1 Gln597 is located at the unstructured loop connecting the helices α4 to α5. The Q597A mutation of SENP1 allosterically disrupts the hydrolytic reaction of SUMO1 through an unknown mechanism. Here, extensive multiple replicates of microsecond molecular dynamics (MD) simulations, coupled with principal component analysis, dynamic cross-correlation analysis, community network analysis, and binding free energy calculations, were performed to elucidate the detailed mechanism. Our MD simulations showed that the Q597A mutation induced marked dynamic conformational changes in SENP1, especially in the unstructured loop connecting the helices α4 to α5 which the mutation site occupies. Moreover, the Q597A mutation caused conformational changes to catalytic Cys603 and His533 at the active site, which might impair the catalytic activity of SENP1 in processing SUMO1. Moreover, binding free energy calculations revealed that the Q597A mutation had a minor effect on the binding affinity of SUMO1 to SENP1. Together, these results may broaden our understanding of the allosteric modulation of the SENP1−SUMO1 complex.     

Multiple Binding Sites Contribute to the Mechanism of Mixed Agonistic and Positive Allosteric Modulators of the Cannabinoid CB1 Receptor

The cannabinoid CB1 receptor (CB1R) is an abundant metabotropic G‐protein‐coupled receptor that has been difficult to address therapeutically because of CNS side effects exerted by orthosteric drug candidates. Recent efforts have focused on developing allosteric modulators that target CB1R. Compounds from the recently discovered class of mixed agonistic and positive allosteric modulators (Ago‐PAMs) based on 2‐phenylindoles have shown promising functional and binding properties as CB1R ligands. Here, we identify binding modes of both the CP 55,940 agonist and GAT228, a 2‐phenylindole allosteric modulator, by using our metadynamics simulation protocol, and quantify their affinity and cooperativity by atomistic simulations. We demonstrate the involvement of multiple adjunct binding sites in the Ago‐PAM characteristics of the 2‐phenylindole modulators and explain their ability to compete with orthosteric agonists at higher concentrations. We validate these results experimentally by showing the contribution of multiple sites on the allosteric binding of ZCZ011, another homologous member of the class, together with the orthosteric agonist.     

Subsynaptic Distribution,Lipid Raft Targeting and G Protein-Dependent Signalling of the Type 1 Cannabinoid Receptor in Synaptosomes from the Mouse Hippocampus and Frontal Cortex

Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level.     

Cannabinoid CB1 receptor recognition of endocannabinoids via the lipid bilayer: molecular dynamics simulations of CB1 transmembrane helix 6 and anandamide in a phospholipid bilayer

The phospholipid bilayer plays a central role in the lifecycle of the endogenous cannabinoid, N-arachidonoylethanolamine (anandamide, AEA). Therefore, the orientation and location of AEA in the phospholipid bilayer with respect to key membrane associated proteins, is a central issue in understanding the mechanism of endocannabinoid signaling. In this paper, we report a test of the hypothesis that a βXXβ motif (formed by beta branching amino acids, V6.43 and I6.46) on the lipid face of the cannabinoid CB1 receptor in its inactive state may serve as an initial CB1 interaction site for AEA. Eight 6 ns NAMD2 molecular dynamics simulations of AEA were conducted in a model system composed of CB1 transmembrane helix 6 (TMH6) in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer. In addition, eight 6 ns NAMD2 molecular dynamics simulations of a low CB1 affinity (20:2, n−6) analog of AEA were conducted in the same model system. AEA was found to exhibit a higher incidence of V6.43/I6.46 groove insertion than did the (20:2, n−6) analog. In certain cases, AEA established a high energy of interaction with TMH6 by first associating with the V6.43/I6.46 groove and then molding itself to the lipid face of TMH6 to establish a hydrogen bonding interaction with the exposed backbone carbonyl of P6.50. Based upon these results, we propose that the formation of this hydrogen bonded AEA/TMH6 complex may be the initial step in CB1 recognition of AEA in the lipid bilayer.     

DCN1-UBE2M相互作用抑制剂的3D-QSAR、分子对接和分子动力学研究

类泛素化是一种蛋白质翻译后修饰,其异常会导致神经退行性疾病和多种肿瘤的发生,因此它被视为有希望的抗肿瘤靶标.研究表明,抑制DCN1-UBE2M相互作用可选择性阻遏类泛素化.本文基于哌啶基脲类DCN1-UBE2M相互作用抑制剂进行3D-QSAR、分子对接和分子动力学模拟研究.利用3D-QSAR中的CoMFA和CoMSIA...     

A Computational Approach Applied to the Study of Potential Allosteric Inhibitors Protease NS2B/NS3 from Dengue Virus

Dengue virus (DENV) is a danger to more than 400 million people in the world, and there is no specific treatment. Thus, there is an urgent need to develop an effective method to combat this pathology. NS2B/NS3 protease is an important biological target due it being necessary for viral replication and the fact that it promotes the spread of the infection. Thus, this study aimed to design DENV NS2B/NS3pro allosteric inhibitors from a matrix compound. The search was conducted using the Swiss Similarity tool. The compounds were subjected to molecular docking calculations, molecular dynamics simulations (MD) and free energy calculations. The molecular docking results showed that two compounds, ZINC000001680989 and ZINC000001679427, were promising and performed important hydrogen interactions with the Asn152, Leu149 and Ala164 residues, showing the same interactions obtained in the literature. In the MD, the results indicated that five residues, Lys74, Leu76, Asn152, Leu149 and Ala166, contribute to the stability of the ligand at the allosteric site for all of the simulated systems. Hydrophobic, electrostatic and van der Waals interactions had significant effects on binding affinity. Physicochemical properties, lipophilicity, water solubility, pharmacokinetics, druglikeness and medicinal chemistry were evaluated for four compounds that were more promising, showed negative indices for the potential penetration of the Blood Brain Barrier and expressed high human intestinal absorption, indicating a low risk of central nervous system depression or drowsiness as the the side effects. The compound ZINC000006694490 exhibited an alert with a plausible level of toxicity for the purine base chemical moiety, indicating hepatotoxicity and chromosome damage in vivo in mouse, rat and human organisms. All of the compounds selected in this study showed a synthetic accessibility (SA) score lower than 4, suggesting the ease of new syntheses. The results corroborate with other studies in the literature, and the computational approach used here can contribute to the discovery of new and potent anti-dengue agents.     

分子动力学研究2-乙酸苯并噻吩在HIV-1蛋白酶与抑制剂结合中的作用

为了研究别构小分子2-乙酸苯并噻吩(2FX)在HIV-1蛋白酶与抑制剂结合中的作用, 利用分子动力学方法分别对未结合和结合2FX的HIV-1蛋白酶抑制剂体系进行了100 ns的模拟, 模拟计算中对每种体系均采用两种新的分子力场ff99SBildn和ff12SB. 研究了2FX对体系构象的影响和两体系在不同力场下的动力学行为, 分析了两体系的均方根偏差和残基的B因子, 比较了计算结构和晶体结构, 最后采用MM-PB/GBSA两种方法计算了两体系的结合自由能. 研究表明, 两种力场计算的结果虽有差异, 但都说明2FX的结合导致蛋白酶构象的变化, 使得体系更加稳定, 尤其是flap的柔性减弱, 使得蛋白酶和抑制剂的结合更牢固; 另外, 还发现ff12SB力场动力学过程更稳定. 研究结果有助于为设计新的别构抑制剂提供理论依据.     

Activation of β2-Adrenergic Receptor Induced by Three Catecholamine Agonists: a Docking and Molecular Dynamics Study

We studied the activation of β₂-adrenergic receptor(β₂AR) by norepinephrine, epinephrine and isoproterenol using docking and molecular dynamics(MD) simulation. The simulation was done on the assumption that β₂AR was surrounded with explicit water and infinite lipid bilayer membrane at body temperature. So the result should be close to that under the physiological conditions. We calculated the structure of binding sites in β₂AR for the three activators. We also simulated the change of the conformation of β₂AR in the transmembrane regions(TMs), in the molecular switches, and in the conserved DRY(Aspartic acid, Arginine and Tyrosine) motif. This study provides detailed information concerning the structure of β₂AR during activation process.     

Studying the Binding Modes of Novel 2-Aminopyridine Derivatives as Effective and Selective c-Met Kinase Type 1 Inhibitors Using Molecular Modeling Approaches

The mesenchymal epithelial cell transforming factor c-Met, encoded by c-Met proto-oncogene and known as a high-affinity receptor for Hepatocyte Growth Factor (HGF), is one of the receptor tyrosine kinases (RTKs) members. The HGF/c-Met signaling pathway has close correlation with tumor growth, invasion and metastasis. Thus, c-Met kinase has emerged as a prominent therapeutic target for cancer drug discovery. Recently a series of novel 2-aminopyridine derivatives targeting c-Met kinase with high biological activity were reported. In this study, 3D quantitative structure-activity relationship (QSAR), molecular docking and molecular dynamics simulations (MD) were employed to research the binding modes of these inhibitors.The results show that both the atom-based and docking-based CoMFA (Q² = 0.596, R² = 0.950 in atom-based model and Q² = 0.563, R² = 0.985 in docking-based model) and CoMSIA (Q² = 0.646, R² = 0.931 in atom-based model and Q² = 0.568, R² = 0.983 in docking-based model) models own satisfactory performance with good reliabilities and powerful external predictabilities. Molecular docking study suggests that Tyr1230 and Arg1208 might be the key residues, and electrostatic and hydrogen bond interactions were shown to be vital to the activity, concordance with QSAR analysis. Then MD simulation was performed to further explore the binding mode of the most potent inhibitor. The obtained results provide important references for further rational design of c-Met Kinase type I inhibitors.     

Allostery Inhibition of BACE1 by Psychotic and Meroterpenoid Drugs in Alzheimer’s Disease Therapy

In over a century since its discovery, Alzheimer’s disease (AD) has continued to be a global health concern due to its incurable nature and overwhelming increase among older people. In this paper, we give an overview of the efforts of researchers towards identifying potent BACE1 exosite-binding antibodies and allosteric inhibitors. Herein, we apply computer-aided drug design (CADD) methods to unravel the interactions of some proposed psychotic and meroterpenoid BACE1 allosteric site inhibitors. This study is aimed at validating the allosteric potentials of these selected compounds targeted at BACE1 inhibition. Molecular docking, molecular dynamic (MD) simulations, and post-MD analyses are carried out on these selected compounds, which have been experimentally proven to exhibit allosteric inhibition on BACE1. The SwissDock software enabled us to identify more than five druggable pockets on the BACE1 structural surface using docking. Besides the active site region, a melatonin derivative (compound 1) previously proposed as a BACE1 allostery inhibitor showed appreciable stability at eight different subsites on BACE1. Refinement with molecular dynamic (MD) simulations shows that the identified non-catalytic sites are potential allostery sites for compound 1. The allostery and binding mechanism of the selected potent inhibitors show that the smaller the molecule, the easier the attachment to several enzyme regions. This finding hereby establishes that most of these selected compounds failed to exhibit strong allosteric binding with BACE1 except for compound 1. We hereby suggest that further studies and additional identification/validation of other BACE1 allosteric compounds be done. Furthermore, this additional allosteric site investigation will help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric BACE1 inhibitors.