二甲双胍是一种直接 sirt1激活化合物: 计算建模和实验验证

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Metformin Is a Direct SIRT1-Activating Compound: Computational Modeling and Experimental Validation

Metformin has been proposed to operate as an agonist of SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that mimics most of the metabolic responses to calorie restriction. Herein, we present an in silico analysis focusing on the molecular docking and dynamic simulation of the putative interactions between metformin and SIRT1. Using eight different crystal structures of human SIRT1 protein, our computational approach was able to delineate the putative binding modes of metformin to several pockets inside and outside the central deacetylase catalytic domain. First, metformin was predicted to interact with the very same allosteric site occupied by resveratrol and other sirtuin-activating compounds (STATCs) at the amino-terminal activation domain of SIRT1. Second, metformin was predicted to interact with the NAD+ binding site in a manner slightly different to that of SIRT1 inhibitors containing an indole ring. Third, metformin was predicted to interact with the C-terminal regulatory segment of SIRT1 bound to the NAD+ hydrolysis product ADP-ribose, a “C-pocket”-related mechanism that appears to be essential for mechanism-based activation of SIRT1. Enzymatic assays confirmed that the net biochemical effect of metformin and other biguanides such as a phenformin was to improve the catalytic efficiency of SIRT1 operating in conditions of low NAD+ in vitro. Forthcoming studies should confirm the mechanistic relevance of our computational insights into how the putative binding modes of metformin to SIRT1 could explain its ability to operate as a direct SIRT1-activating compound. These findings might have important implications for understanding how metformin might confer health benefits via maintenance of SIRT1 activity during the aging process when NAD+ levels decline.

二甲双胍被认为是 SIRT1的激动剂,SIRT1是一种依赖于烟酰胺腺嘌呤二核苷酸的去乙酰化酶,模拟了大多数对卡路里限制的代谢反应。在这里,我们提出了一个在计算机分析的重点是分子对接和动态模拟的假定相互作用二甲双胍和 SIRT1。利用人类 SIRT1蛋白8种不同的晶体结构,我们的计算方法能够描述二甲双胍与中心脱乙酰酶催化域内外的几个囊袋的假定结合模式。首先,二甲双胍被预测与白藜芦醇和其他去乙酰化酶激活化合物(STATCs)在 SIRT1的氨基末端激活区所占据的同样的变构位点相互作用。其次,二甲双胍与 NAD + 结合位点的相互作用方式与含有吲哚环的 SIRT1抑制剂略有不同。第三,二甲双胍被预测与 SIRT1的 c 末端调节片段相互作用,该片段与 NAD + 水解产物 ADP-ribose 结合,这是一个与“ c 口袋”相关的机制,似乎对 SIRT1的机制激活至关重要。酶法测定证实了二甲双胍和苯乙双胍等双胍的净生化效应是为了提高 SIRT1在低 NAD + 条件下的体外催化效率。即将到来的研究应该证实我们的计算机洞察力的机制相关性如何假定的结合模式二甲双胍 SIRT1可以解释其作为一个直接的 SIRT1激活化合物的能力。这些发现可能对理解二甲双胍如何在 NAD + 水平下降的衰老过程中通过保持 SIRT1活性而带来健康益处有重要的意义。

Introduction

引言

A small molecule capable of targeting aging and delaying the onset of aging-related multimorbidity has the potential to radically amend the way we understand (and practice) modern medicine (1). One such molecule is the biguanide metformin, which, 60 years after its introduction in Europe as a first-line therapeutic for type 2 diabetes (2), could have the potential to prevent multiple aging-related disorders (35). Against this background, the TAME (Targeting Aging with Metformin) clinical trial has been designed to evaluate the healthspan-promoting effects of metformin by enrolling patients aged 65–79 years diagnosed with one single age-associated condition, and then assessing the global impact of metformin on a composite outcome including cardiovascular events, cancer, dementia, mortality, and other functional and geriatric endpoints (6). Although the current consensus is that metformin has the ability to target multiple pathways of aging, it is still unclear whether such a capacity reflects downstream consequences of a primary action on a single mechanism or whether it involves direct effects on aging regulators (6).

一个小分子能够针对衰老和延缓衰老相关的多发病的发生,有可能从根本上改变我们理解(和实践)现代医学的方式(1)。其中一种分子是双胍二甲双胍,它作为第一线治疗2型糖尿病(2)的药物被引入欧洲60年后,有可能预防多种与衰老有关的疾病(3-5)。在此背景下,TAME (使用二甲双胍针对衰老)临床试验被设计用于评估二甲双胍促进健康跨度的效果,方法是招募年龄在65-79岁、被诊断患有单一年龄相关疾病的患者,然后评估二甲双胍对心血管事件、癌症、痴呆、死亡率以及其他功能性和老年人终点等综合结果的全球影响。虽然目前的共识是二甲双胍具有针对多种衰老途径的能力,但仍不清楚这种能力是否反映了单一机制的一个主要作用的下游结果,或者是否涉及对衰老调节器的直接影响(6)。

Metformin has been proposed to exert indirect pleiotropy on core metabolic hallmarks of aging such as the insulin/IGF-1 and AMPK/mTOR signaling pathways (4) downstream of its primary inhibitory action on mitochondrial respiratory complex I. Alternatively, but not mutually exclusive, its capacity to operate as a poly-therapeutic anti-aging agent might involve the direct targeting of the biologic machinery of aging per se. A systematic chemoinformatics approach established to computationally predict metformin targets recently revealed that the salutary effects of metformin on human cellular aging might involve its direct binding to core chromatin modifiers of the aging epigenome (78), such as the H3K27me3 demethylase KDM6A/UTX (911). The ability of metformin to directly interact with TGF-β1, thereby blocking its binding to TβRII and resulting in impaired downstream signaling (12), is another example of how metformin might exert pleiotropic effects on numerous (TGF-β1 hyperfunction-associated) aging diseases such as organ fibrosis and cancer, without necessarily involving changes in cellular bioenergetics.

二甲双胍被认为对衰老的核心代谢标志如胰岛素/igf-1和 AMPK/mTOR 信号通路产生间接多效性(4) ,是其对线粒体呼吸复合物的主要抑制作用的下游通路 i. 二甲双胍作为多治疗抗衰老药物的能力可能涉及直接针对衰老的生物机制本身。一个系统的化学信息学方法建立了计算机预测二甲双胍靶标最近揭示了二甲双胍对人类细胞衰老的有益影响可能涉及其直接结合的核心染色质修饰的老化表观基因组(7,8) ,如 H3K27me3去甲基化酶 KDM6A/UTX (9-11)。二甲双胍能够直接与 TGF-β1相互作用,从而阻断其与 TβRII 的结合,导致下游信号传导受损(12) ,这是二甲双胍可能对多种(TGF-β1高功能相关)衰老性疾病(如器官纤维化和癌症)产生多效性影响的另一个例子,而不一定涉及细胞生物能的改变。

SIRT1 is a member of the class III (NAD+-dependent) histone deacetylases (HDACs) that mimics most of the metabolic responses to calorie restriction and contributes to enhanced healthy aging, including a reduced incidence of cardiovascular and metabolic diseases, cancer, and neurodegeneration (1317). The regulation of SIRT1 by metformin is an archetypal example of its ability to indirectly and directly impact the aging process. Because of its enzymatic requirement for NAD+, SIRT1 is commonly viewed as a unique energy sensor that couples its function to the NAD+/NADH ratio of the cell or organism (1820). Accordingly, metformin-induced metabolic stress has been shown to induce SIRT1 expression and activity as a downstream consequence of AMPK activation-induced augmentation of cellular NAD+ levels (2124). Although the striking similarity between the pleiotropic effects of metformin and the physiological consequences of SIRT1 activation might merely represent the overlapping metabolic effects of SIRT1 and AMPK activators (2526), we are beginning to uncover evidence on the occurrence of energy crisis (i.e., AMPK/mTOR)-independent agonist effects of metformin on SIRT1 activity (2731). Nonetheless, both the putative molecular interactions on the atomic scale between metformin and SIRT1 and the mechanism of action of metformin as a direct modulator of SIRT1 activity remain elusive.

SIRT1是 III 类(NAD + 依赖性)组蛋白去乙酰化酶(HDACs)的一员,它模拟了大多数对卡路里限制的代谢反应,有助于促进健康衰老,包括降低心血管和代谢性疾病、癌症和神经退行性疾病的发病率(13-17)。二甲双胍对 SIRT1的调节是其间接和直接影响老化过程的一个典型例子。由于其对 NAD + 的酶促需求,SIRT1通常被认为是一种独特的能量传感器,它将其功能与细胞或生物体的 NAD +/NADH 比值(18-20)联系起来。因此,二甲双胍诱导的代谢应激可诱导 SIRT1的表达和活性,作为 AMPK 激活诱导的细胞 NAD + 水平(21-24)增加的下游结果。虽然二甲双胍的多效性效应和 SIRT1激活的生理后果之间惊人的相似性可能仅仅代表了 SIRT1和 AMPK 激活因子(25,26)的重叠代谢效应,但我们开始发现二甲双胍对 SIRT1活性(27-31)产生与能量危机(即 AMPK/mtor)无关的激动剂效应的证据。尽管如此,二甲双胍和 SIRT1之间原子尺度上的假定分子相互作用和二甲双胍作为 SIRT1活性的直接调节剂的作用机制仍然不清楚。

Here, we performed an in silico docking and molecular dynamics (MD) simulation study of the SIRT1-metformin complex coupled to laboratory-based experimental validation, aiming to interrogate the ability of metformin to directly enhance NAD+-dependent SIRT1 activity. Our findings present a first-in-class structural basis to understand the behavior of metformin as a direct SIRT1-activating compound.

在这里,我们进行了 SIRT1-二甲双胍复合物的计算机对接和分子动力学模拟研究,旨在审问二甲双胍直接增强 NAD + 依赖的 SIRT1活性的能力。我们的发现为理解二甲双胍作为直接 sirt1激活化合物的行为提供了一个一流的结构基础。

Materials and Methods

物料及方法

Computational Modeling of the Human SIRT1 Protein

人 SIRT1蛋白质的计算机模拟

To provide in silico insights into the binding pattern of metformin with SIRT1, we employed eight different crystal structures of the human SIRT1 protein, namely 4KXQ, 4IF6, 4ZZJ, 4ZZI, 4ZZH, 4I5I, 5BTR, and 4IG9. 4KXQ, and 4IF6 represent the heterodimeric (chains A and B), closed conformation of SIRT1 bound to adenosine-5-diphosphoribose (APR) (32). 4ZZJ represents the heterodimeric (chains A –SIRT1 and B –p53), open conformation of SIRT1 bound to small molecule sirtuin-activating compounds (STATCs) such as the non-hydrolyzable NAD+ analog carbaNAD (carba nicotinamide adenine dinucleotide) or to the carboxamide SIRT1 inhibitor 4TQ (33). 4ZZI represents the monomeric (chain A), open conformation of SIRT1 bound to the carboxamide SIRT1 inhibitors 4TQ and 1NS, whereas 4ZZH represents the monomeric (chain A), open conformation of SIRT1 bound to the carboxamide SIRT1 inhibitor 4TO (33). 4I5I represents the dimeric (chains A and B) conformation of SIRT1 bound to NAD or, alternatively, to the carboxamide SIRT1 inhibitor 4I5 (34). 5BTR represents the heterotrimeric (chains A, B, and C –SIRT1 and D, E, and F –p53), closed conformation of SIRT1 bound to resveratrol (35). Finally, 4IG9 represents a quaternary complex of SIRT1 with no bound ligand (32).

为了深入研究二甲双胍与 SIRT1的结合模式,我们采用了人 SIRT1蛋白的8种不同晶体结构,即4KXQ、4IF6、4ZZJ、4ZZI、4ZZH、4I5I、5BTR 和4IG9。4KXQ 和4IF6分别代表 SIRT1的异二聚体(a 链和 b 链) ,它们的结合位点是闭合构象。4ZZJ 代表异二聚体(链 a-SIRT1和 b-p53) ,SIRT1与小分子 sirtuin 激活化合物(STATCs)开放构象,如非水解 NAD + 类似物 carbaNAD (carba 烟酰胺腺嘌呤二核苷酸)或羧酰胺 SIRT1抑制剂4TQ (33)。4ZZH 表示 SIRT1与羧酰胺 SIRT1抑制剂4TQ 和1NS 的单体(链 a)开放构象,而4ZZH 表示 SIRT1与羧酰胺 SIRT1抑制剂4TO (33)的单体(链 a)开放构象。4I5I 表示 SIRT1与 NAD 或与羧酰胺 SIRT1抑制剂4I5(34)结合的二聚体(链 a 和链 b)构象。5BTR 代表异三聚体(a,b,c-SIRT1和 d,e,f-p53) ,是 SIRT1与白藜芦醇(35)结合的封闭构象。最后,4IG9表示无结合配体(32)的 SIRT1的四元配合物。

Docking Calculations

对接计算

All docking calculations were performed using Itzamna and Kin (www.mindthebyte.com), classical docking and blind-docking software tools. The above mentioned protein structures from RCSB Protein Data Bank (https://www.rcsb.org) were directly employed for docking calculations using the cavities defined by crystallographic ligands where available. Two runs were carried out for each calculation to avoid false positives.

所有的对接计算都是使用 Itzamna 和 Kin ( www.mindthebyte.com ) ,经典的对接和盲对接软件工具进行的。上面提到的来自 RCSB 蛋白质数据库的蛋白质结构被直接用于对接计算,使用由晶体配体定义的空腔。为了避免误报,每次计算都进行了两次运行。

Molecular Dynamics Simulations

分子动力学空间站模拟

Docking post-processing allowing conformational selections/induced fit events to optimize the interactions were performed via short (1 ns) MD simulations using NAMD version 2.10 over the best-docked complexes, which were selected based on the interaction energy. The Ambers99SB-ILDN and the GAFF forcefield set of parameters were employed for SIRT1 and metformin, respectively. The GAFF parameters were obtained using Acpype software, whereas the SIRT1 structures were modeled using the leap module of Amber Tools. Simulations were carried out in explicit solvent using the TIP3P water model with the imposition of periodic boundary conditions via a cubic box. Electrostatic interactions were calculated by the particle-mesh Ewald method using constant pressure and temperature conditions. Each complex was solvated with a minimum distance of 10 Å from the surface of the complex to the edge of the simulation box; Na+ or Cl ions were also added to the simulation to neutralize the overall charge of the systems. The temperature was maintained at 300 K using a Langevin thermostat, and the pressure was maintained at 1 atm using a Langevin Piston barostat. The time step employed was 2 fs. Bond lengths to hydrogens were constrained with the SHAKE algorithm. Before production runs, the structure was energy minimized followed by a slow heating-up phase using harmonic position restraints on the heavy atoms of the protein. Subsequently, the system was energy minimized until volume equilibration, followed by the production run without any position restraints.

对接后处理允许构象选择/诱导拟合事件来优化相互作用,通过短(1ns) MD 模拟使用 NAMD 版本2.10在最佳对接复合物上进行,这些最佳对接复合物是根据相互作用能选择的。分别采用 Ambers99SB-ILDN 和 GAFF 力场参数集对 SIRT1和二甲双胍进行测定。GAFF 参数采用 Acpype 软件获得,SIRT1结构采用 Amber Tools 的飞跃模块建模。利用 TIP3P 水模型,通过立方盒子施加周期性边界条件,在显式溶剂中进行了模拟。采用粒子-网格 Ewald 方法,在恒压和恒温条件下计算了静电相互作用。每个配合物被溶解,从配合物表面到模拟盒边缘的最小距离为1000nm; 模拟中还添加了 Na + 或 Cl-离子以中和体系的总电荷。使用 Langevin 恒温器,温度保持在300k,使用 Langevin Piston 气压调节器,压力保持在1个大气压。使用的时间步长为2 fs。键长到氢被限制与 SHAKE 算法。在生产运行之前,结构的能量最小,随后缓慢升温阶段使用谐波位置约束的重原子的蛋白质。随后,系统能量最小化,直到体积达到平衡,随后生产运行没有任何位置限制。

Binding Free Energy Analysis

结合自由能分析

Molecular Mechanics/Generalized Borne Surface Area (MM/GBSA) calculations were performed to calculate the alchemical binding free energy (ΔGbind) of metformin against SIRT1. MM/GBSA rescoring was performed using the MMPBSA.py algorithm within AmberTools. The snapshots generated in the 1 ns MD simulation were imputed into the post-simulation MM/GBSA calculations of binding free energy. Graphical representations were prepared using PyMOL program and PLIP version 1.3.0.

采用分子力学/广义表面积(MM/GBSA)计算方法,计算了二甲双胍对 SIRT1的醛化结合自由能(δgbind)。使用 AmberTools 中的 MMPBSA.py 算法进行 MM/GBSA 重新取样。将1ns 分子动力学模拟中产生的快照输入到结合自由能的后模拟计算中。使用 PyMOL 程序和 PLIP 1.3.0版制作了图形表示。

Interaction Analysis

互动分析

The predicted binding site residues of metformin to SIRT1 were defined using evidence-based interaction analyses of known SIRT1 activators/ inhibitors with well-defined binding residues.

通过对已知具有明确结合残基的 SIRT1激活剂/抑制剂的循证相互作用分析,确定了二甲双胍与 SIRT1的预测结合位点残基。

SIRT1 Enzymatic Assay

SIRT1酶法测定

The effects of metformin on SIRT1 activity were assessed using the SIRTainty™ Class III HDAC Assay (Cat. #17-10090, Millipore) and the Epigenase™ Universal SIRT1 Activity/Inhibition Assay Kit (Cat. # P-4027, Epigentek), as per the manufacturers’ instructions. In the former assay, purified SIRT1 enzyme, β-NAD, acetylated peptide substrate, metformin, and nicotinamidase enzyme were combined and incubated for 30 min. During this time the acetylated peptide substrate is deacetylated by SIRT1 and produces nicotinamide. In a secondary reaction, the nicotinamidase enzyme converts nicotinamide into nicotinic acid and free ammonia (NH+33+). To generate a signal for readout, a proprietary developer reagent is added and the signal is read (420ex/460em nm) using a fluorescent plate reader. In the latter assay, an acetylated histone SIRT1 substrate is stably coated onto microplate wells; active SIRT1 binds to the substrate and removes acetyl groups from the substrate and the amount of SIRT1-deacetylated products, which is proportional to the enzyme activity, can be measured using a specific antibody. The ratio or amount of deacetylated product, which is proportional to the enzyme activity, is fluorometrically measured by reading the fluorescence at 530ex/590em nm. Metformin, phenformin, and buformin (Sigma-Aldrich Ltd.) were added from aqueous stock solutions, and proguanil (Sigma-Aldrich Ltd.) from stock solutions in DMSO.

目的: 研究二甲双胍对小鼠 SIRT1活性的影响,探讨二甲双胍对 SIRT1活性的影响。# 17-10090,Millipore)和表观 asetm 通用 SIRT1活性/抑制试剂盒(Cat。# P-4027,Epigentek) ,按照制造商的说明。前者将纯化的 SIRT1酶、 β-nad 酶、乙酰化肽底物、二甲双胍酶和烟酰胺酶组合在一起,培养30min。在此期间,乙酰化多肽底物被 SIRT1脱乙酰基,生成烟酰胺。在二次反应中,烟酰胺酶将烟酰胺转化为烟酸和游离氨(nh3 + 3 +) 。为了产生读出信号,加入专有显影剂,并使用荧光板读出器读出信号(420ex/460em nm)。在后一种方法中,乙酰化的组蛋白 SIRT1底物被稳定地涂覆在微板上,活性的 SIRT1与底物结合并从底物中去除乙酰基,而且 SIRT1脱乙酰化产物的量与酶的活性成正比,可以用特异性抗体来测定。在530ex/590em nm 处读取荧光,荧光法测定脱乙酰产物的比例或数量与酶的活性成正比。二甲双胍、苯乙双胍和丁甲双胍(Sigma-Aldrich ltd.)分别从溶液中加入,丙胍(Sigma-Aldrich ltd.)从溶液中加入。

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