Negative allosteric modulators of cannabinoid receptor 2: protein modeling,binding site identification and molecular dynamics simulations in the presence of an orthosteric agonist

Abstract

Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of negative CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor’s allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, molecular dynamics (MD) simulations and binding free energy calculations to predict, characterize and validate allosteric sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans-β-caryophyllene (TBC) (note that TBC also shows agonist activity), at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid bilayer and subjected to a 200 ns MD simulation. The presence of an AM affected the CB2–CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π–π interaction between Phe117^3.36 and Trp258^6.48. Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 AMs.     

Target RNA activates the protease activity of Craspase to confer antiviral defense

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基于弹性网络模型的蛋白质变构路径与关键残基识别研究

目的 变构效应在蛋白质生物学功能执行过程中发挥着重要的调控作用，如何基于蛋白质空间结构，有效识别变构信号的传播路径和关键的残基位点是蛋白质结构-功能关系研究领域的热点科学问题。方法 本研究利用基于弹性网络模型（elastic network model，ENM）的力分布计算方法，通过分析蛋白质对外力的响应过程，来识别体系的变构路径以及变构过程中的关键残基。在该方法中，对蛋白质的关键变构位点施加外力，通过对体系形变以及内力分布情况的分析，有效识别与外力承载区域形变相耦合的关键残基，从而得到力信号在蛋白质结构内的传播路径。结果 利用该方法研究了人类磷酸甘油酸激酶（human phosphoglycerate kinase，hPGK）和蛋白质酪氨酸磷酸酶（protein tyrosine phosphatase，PTP）PDZ2结构域的变构调控路径和关键残基。对于hPGK，识别出从底物结合位点到铰链区的两条变构信号传导路径。对于PTP PDZ2，也成功识别出从配体结合位点传递到蛋白质远端的两条长程变构调控路径。计算结果与实验和分子动力学（molecular dynamics，MD）模拟得到的结果一致。结论 本研究为蛋白质体系关键残基识别及变构路径研究提供了有效的分析方法。     

Regulation and structure of aspartokinase in the genusBacillus

The aspartate pathway of amino acid biosynthesis in bacteria serves as paradigm for the evolution of patterns of enzyme regulation in response to specific physiological requirements. InBacillus species, the first step in the pathway is catalyzed by multiple forms of aspartokinase, which differ in their structure and feedback regulation. One form of aspartokinase (V-type) functions primarily during cell growth, another form (S-type) during sporulation. The V-type aspartokinase fromBacillus subtilis andBacillus polymyxa is discussed in some detail on account of its complex pattern of regulation by the pathway endproducts lysine and threonine and its unusual subunit structure. The enzyme is composed of two dissimilar subunits, the smaller of which corresponds to the carboxyl-terminal domain of the larger subunit. The coding sequence for the subunits ofBacillus subtilis aspartokinase has recently been cloned inEscherichia coli. The study of its structure and mode of expression has revealed that the two aspartokinase subunits are encoded by in-phase overlapping genes. These unusual features of aspartokinase suggest that important aspects of the regulation of the aspartate pathway are yet to be discovered.     

Explaining and Predicting Allostery with Allosteric Database and Modern Analytical Techniques

Allostery is a phenomenon that the protein activity is regulated when a non-functional site on it is bounded. This phenomenon is important in life process and disease therapy. However, it is difficult to study allostery due to the lack of knowledge. Facing this demand, we have created Allosteric Database (ASD) 10 years before to collect numerous kinds of allosteric data. In this review, we will introduce the 4 categories of data in ASD. For each category, we further reviewed how researchers applied ASD data to conduct studies. We focused on their research topics, analytical methods and conclusions. Several discoveries of new drug targets and allosteric modulators driven by ASD are also summarized. We hope this review could inspire researchers with new utilities of ASD data.     

Positive and negative allosteric modulation of GluK2 kainate receptors by BPAM344 and antiepileptic perampanel

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Therapeutic Targeting the Allosteric Cysteinome of RAS and Kinase Families

Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRAS^G12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy.