药学学报

Name	Principle	Application	Advantage	Disadvantage
DARTS	Proteolytic stability	In vitro/cell lysate	Simple operation, no drug activity dependence	Unable to detect binding to target proteins within cells/tissues
CETSA	Protein thermal stability	Cell/tissue	Binding to target proteins in tissues can be detected	Low abundances of target proteins are difficult to detect
MST	Temperature affects the movement of protein molecules	Purified protein	Low protein usage, no need for protein fixation	Requires high purity protein, protein cannot be recycled
BLI	Light interference	Purified protein	Protein does not require labeling, protein samples can be recycled, and sensors can be renewable and reused	Strong non-specific binding to small molecules containing multiple -OH/NH groups
SPR	Light refraction	Purified protein	High sensitivity and high throughput	Sensitive to sample composition and temperature, non-specific adsorption
ITC	Exothermic reaction	Purified protein	Proteins do not require fixation and can provide both thermodynamic and kinetic information	High protein consumption and low heat production, low throughput
DSF	Protein thermal stability	Purified protein	Dyes are cheap and the instrument (PCR instrument) is easy to operate	Not suitable for heat insensitive proteins
ICD	Absorption difference of left and right circularly polarized light	Purified protein	Detectable membrane protein and ligand binding	Require a large amount of purified protein
MS	Ionization of protein molecules into ions	Purified protein/intracellular overexpressed proteins	No need to set ligand concentration gradient and can provide binding site information	Difficult to distinguish between covalent and non-covalent binding
NMR	Magnetization transfer	Purified protein	Identify the functional groups that bind to the target protein	Isotopic labeling is required for 2D NMR
X-ray single crystal diffraction	Co-crystallization of small molecules and proteins	Co-crystallization	Specific amino acid residue binding site information can be provided	Obtaining small molecule protein eutectic is difficult and time-consuming
Cryo-EM	Rapid freezing+transmission electron microscopy	Purified protein	Obtaining structural information of target protein-ligand complexes	Expensive price and low penetration rate
Computer simulation	Virtual docking		No need for protein purification, high throughput	Further experimental verification is required

Name	Principle	Application	Advantage	Disadvantage
DARTS	Proteolytic stability	In vitro/cell lysate	Simple operation, no drug activity dependence	Unable to detect binding to target proteins within cells/tissues
CETSA	Protein thermal stability	Cell/tissue	Binding to target proteins in tissues can be detected	Low abundances of target proteins are difficult to detect
MST	Temperature affects the movement of protein molecules	Purified protein	Low protein usage, no need for protein fixation	Requires high purity protein, protein cannot be recycled
BLI	Light interference	Purified protein	Protein does not require labeling, protein samples can be recycled, and sensors can be renewable and reused	Strong non-specific binding to small molecules containing multiple -OH/NH groups
SPR	Light refraction	Purified protein	High sensitivity and high throughput	Sensitive to sample composition and temperature, non-specific adsorption
ITC	Exothermic reaction	Purified protein	Proteins do not require fixation and can provide both thermodynamic and kinetic information	High protein consumption and low heat production, low throughput
DSF	Protein thermal stability	Purified protein	Dyes are cheap and the instrument (PCR instrument) is easy to operate	Not suitable for heat insensitive proteins
ICD	Absorption difference of left and right circularly polarized light	Purified protein	Detectable membrane protein and ligand binding	Require a large amount of purified protein
MS	Ionization of protein molecules into ions	Purified protein/intracellular overexpressed proteins	No need to set ligand concentration gradient and can provide binding site information	Difficult to distinguish between covalent and non-covalent binding
NMR	Magnetization transfer	Purified protein	Identify the functional groups that bind to the target protein	Isotopic labeling is required for 2D NMR
X-ray single crystal diffraction	Co-crystallization of small molecules and proteins	Co-crystallization	Specific amino acid residue binding site information can be provided	Obtaining small molecule protein eutectic is difficult and time-consuming
Cryo-EM	Rapid freezing+transmission electron microscopy	Purified protein	Obtaining structural information of target protein-ligand complexes	Expensive price and low penetration rate
Computer simulation	Virtual docking		No need for protein purification, high throughput	Further experimental verification is required

小分子配体-靶蛋白相互作用及高通量配体筛选技术

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董睿方 , 夏元铮 ^* , 孔令义 ^*

药学学报 | 综述 2023,58(11): 3242-3253

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药学学报 | 综述 2023, 58(11): 3242-3253

小分子配体-靶蛋白相互作用及高通量配体筛选技术

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董睿方, 夏元铮^*, 孔令义^*

作者信息

中国药科大学中药学院, 天然药物活性组分与药效国家重点实验室, 江苏省生物活性天然产物研究重点实验室, 江苏南京 210009

通讯作者:

*夏元铮, Tel: 13913024226, E-mail: xiayz@cpu.edu.cn;

孔令义, Tel: 86-25-83271405, E-mail: cpu_lykong@126.com

The technologies of small molecule ligand-target protein interaction and high throughput ligand screening

Rui-fang DONG, Yuan-zheng XIA^*, Ling-yi KONG^*

Affiliations

Jiangsu Key Laboratory of Bioactive Natural Product Research, State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China

出版时间: 2023-11-12 doi: 10.16438/j.0513-4870.2023-0649

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摘要

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药物-靶蛋白相互作用研究是新药研发的重要环节, 药物通过结合靶蛋白的特定氨基酸位点, 从而影响靶蛋白的药理作用, 这是药物发挥治疗效果的前提。目前, 有多种技术应用于检测药物配体与靶蛋白相互作用。如基于热力学的细胞热转移分析(cellular thermal shift assay, CETSA)、差示扫描荧光(differential scanning fluorimetry, DSF) 及质谱、核磁共振技术等。此外, 高通量配体筛选技术为寻找靶蛋白特异性配体提供了技术便利, 是高效鉴定药物配体与靶蛋白相互作用的利器。本文对药物小分子与靶点蛋白相互作用及亲和力技术进行了总结, 并探讨了高通量配体筛选技术在药物研究的应用。

关键词

靶蛋白 / 配体 / 亲和力 / 相互作用 / 高通量

Abstract

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The interaction of drug and target protein is a critical part of new drug discovery. It is the premise for drugs to exert therapeutic effects by targeting specific binding sites of target proteins and thereby affecting its pharmacological activity. Currently, a variety of techniques are exploited to detect the interaction between drug ligands and target proteins. For example, cellular thermal shift assay (CETSA) and differential scanning fluorimetry (DSF) based on thermodynamics, mass spectrometry and nuclear magnetic resonance technology, etc. In addition, high-throughput ligand screening technology provides technical convenience for the search of specific ligand, and is a powerful tool to efficiently identify the interaction between drug ligand and target protein. Here, we summarize the detection techniques of interaction between small molecules and target proteins, and discuss the application of high-throughput ligand screening technology in drug research.

Key words

target protein / ligand / affinity / interaction / high throughput

引用本文

董睿方, 夏元铮, 孔令义. 小分子配体-靶蛋白相互作用及高通量配体筛选技术. 药学学报, 2023 , 58 (11) : 3242 -3253 . DOI: 10.16438/j.0513-4870.2023-0649

Rui-fang DONG, Yuan-zheng XIA, Ling-yi KONG. The technologies of small molecule ligand-target protein interaction and high throughput ligand screening[J]. Acta Pharmaceutica Sinica, 2023 , 58 (11) : 3242 -3253 . DOI: 10.16438/j.0513-4870.2023-0649

正文

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药物与靶蛋白之间的相互作用为药物的发现提供了重要的信息, 是药物发现的关键步骤, 有助于发现现有药物的新靶点或确定新药的靶点以及药物脱靶、毒性及不良反应机制, 并有针对性地进行结构修饰。小分子药物发挥作用的实质是药物与靶蛋白之间的相互作用, 而其药效的高低则取决于两者的作用方式和亲和力的大小。细胞的增殖、分化、基因表达、代谢、免疫和信号转导都需要配体和靶点的参与。确定小分子配体与靶标蛋白之间的相互结合是一个巨大的挑战。因此, 对小分子药物-靶蛋白相互作用研究具有较高的科学价值。本文对检测药物小分子-靶蛋白相互作用的技术进行了总结, 以期为药物机制的探索提供便利。

1 靶点验证及结合亲和力检测技术

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1.1 药物亲和反应靶点稳定性(drug affinity responsive target stability, DARTS)

DARTS (图 1A)^[1]是一种识别小分子靶点的简单技术, 有助于表征小分子和其他方法确定的假定靶点之间的相互作用。当小分子与蛋白质相互作用时, 它可能稳定或破坏蛋白质的结构, 从而降低或增加对蛋白酶作用的敏感性^[2]。DARTS可以用作靶点验证, 当验证小分子与假定的靶蛋白相互作用时, 可以使用Western blot检测。DARTS被证明是一种快速和可靠的方法来确定小分子(或代谢物) 与靶蛋白的直接结合, 而不需要大量的纯化蛋白, 甚至可以使用全细胞裂解物。在DARTS过程中, 蛋白质提取物被蛋白水解酶处理, 只有与小分子结合的蛋白质才能免于或加速蛋白酶水解。该方法的优点是不需要对小分子化合物修饰, 因此不受化学的限制, 可以用来识别任何小分子的结合靶点, 具有检测小分子-靶点相互作用的潜力^[3]。

DARTS实验中蛋白浓度要求为4~6 μg·μL^-1, 蛋白浓度太低或太高均影响实验结果, 且蛋白浓度不精确不利于寻找差异蛋白条带。DARTS技术中使用的蛋白水解酶主要有嗜热菌蛋白酶和链霉蛋白酶(pronase), 其中优先推荐使用pronase。实验选择非变性的裂解液, 如NP-40裂解液, 以保持天然蛋白构象。实验过程中一定不要涡旋蛋白裂解物, 避免破坏一些蛋白质的天然构象, 改变或消除它们的配体结合活性^[4]。

1.2 细胞热转移实验(cellular thermal shift assay, CETSA)

CETSA (图 1B) 被用于评估细胞和组织样本中配体小分子与靶蛋白的结合。该方法是基于配体诱导的靶蛋白热力学稳定性。一个典型输出结果是对表观熔化曲线(或者更准确地说, 温度诱导的聚合曲线) 的比较。其中, 蛋白质在配体存在和不存在的情况下都要接受一系列温度孵育, 以便评估潜在的热稳定性。此外, 等温剂量反应指纹图谱(isothermal dose-response fingerprints, ITDRF) 也可用于评估配体浓度对靶蛋白稳定性的影响, 在固定的温度和加热时间内, 用不同的配体浓度处理细胞裂解液, 以评估药物浓度效应^{[5, 6]}。无论哪种形式, CETSA都可以用于药物靶点发现, 以解决候选药物是否在生物学相关环境中真正作用于预期的靶点, 以及它们在何种浓度下发挥作用等基本问题, 但最容易的是等温剂量反应曲线。在药物临床前和临床应用上, 可用于检测体外或组织中药物是否到达预期的部位并与靶蛋白结合。因此, CETSA在验证药物-靶蛋白相互作用中应用较为广泛。

CETSA实验依赖于两个关键步骤: 第一, 加热步骤, 靶蛋白变性和沉淀; 第二, 在加热结束后把保持稳定的蛋白与变性和沉淀的蛋白区别开来, 可以通过离心分离出可溶性蛋白。在检测细胞内靶蛋白结合时, 药物孵育时间尽量在30~60 min内, 如果孵育时间延长到数小时, 化合物也可以通过改变转录和翻译活动影响蛋白质水平^[7]。

1.3 微量热泳动技术(microscale thermophoresis, MST)

MST (图 2A) 是一种生物物理分析方法, 用于定量分子之间的相互作用, 如蛋白质和小分子^{[8, 9]}。近年来, MST常被用于检测蛋白质-蛋白质和蛋白质-药物相互作用。该方法通过定量荧光分子响应温度梯度的热泳动来检测分子之间的相互作用。在实际应用中, 将荧光分子(靶蛋白) 与不同浓度的非荧光配体混合, 然后将混合物装载到毛细管中。利用红外激光器照射毛细管中的样品, 使之产生温度梯度, 并通过激发荧光团实时监测荧光分子在毛细管中的运动。随着时间的推移收集发射的荧光, 将不同浓度的非荧光配体对荧光分子运动的影响进行量化, 以测试分子之间的相互作用^{[10, 11]}。如果荧光分子与配体相互作用, 分子的性质(如电荷、大小和水化膜) 将影响分子的运动。蛋白一般需要荧光染料共价标记, 对于含有芳香族残基的蛋白质, 不需要荧光标记, 可以利用蛋白固有的荧光(主要由芳香族氨基酸苯丙氨酸、酪氨酸和色氨酸引起) 检测蛋白质的运动^[12]。在这种情况下, 需要一个无标签的MST仪器来检测。

MST方法不需要固定, 在生物溶液(天然的环境中、生理学实验条件、血清、细胞裂解液) 中即可测定, 样品用量小; 10 min之内测量任何(生物) 分子间亲和力(K_d, 解离常数)。可用于研究各种不同大小的分子: 离子、化合物、核酸、多肽、蛋白、糖类、脂质体、纳米颗粒和病毒等。

1.4 生物膜层干涉(bio-layer interferometry, BLI)

BLI (图 2B)^[13]是一种测量生物分子相互作用的非标记技术, 它是一种光学分析技术。分析从生物传感器尖端的一层固定蛋白和一层内部参考层两个表面反射的白光产生的光干涉模式, 并将光干涉信号转化为实时的响应信号。固定在生物传感器尖端表面的配体与溶液中的分析物之间的结合使生物传感器尖端的光学厚度增加, 从而导致波长偏移, 这是直接测量生物层厚度变化的方法。通过对分子结合过程的实时监测, 系统会测定结合常数(k_a或k_on) 和解离常数(k_d或k_off), 以及起始结合速率, 并通过拟合计算分析得到亲和力(K_d) 和浓度信息^[13-15]。

小分子检测信号相对比较低, 需要将蛋白固化量提高, 使得小分子结合的量更多。所以建议使用SSA传感器(需要生物素化试剂和脱盐柱), 如果SSA传感器的固化量还是很低(< 2 nm) 并且蛋白上带有his-tag标签, 可以尝试使用NTA传感器。因为化合物测试的浓度比较大, 所以可能存在与单独传感器结合的背景信号, 需要用一系列不固化蛋白的传感器(或者用5 μg·μL^-1生物胞素封闭) 与固化蛋白的传感器运行同样的步骤。

目前, 主流产品是Fortebio分子相互作用仪。该方法无须标记, 蛋白样品可回收, 传感器可再生重复使用; 可对小分子化合物、DNA、多肽、蛋白质、寡糖或寡核苷酸、细胞、病毒或细菌等进行检测。小分子存在非特异性结合, 可在缓冲液中加入0.05%牛血清白蛋白或提高Tween-20含量, 增加NaCl浓度以降低非特异性结合。由于小分子类的分析物分子量比较小, 所以信号比较低, 要求蛋白固化的信号比较高, 至少5 nm。

1.5 表面等离子共振技术(surface plasmon resonance, SPR)

SPR (图 2C)^[16]技术被认为是检测生物分子相互作用的“金标准”, 因为它具有高精度、实时和无标记测量的显著特点, 可以同时测定动力学常数和解离平衡常数, 已成为药物研发中质量控制的必要技术, 并被2020版《中国药典》收录, 广泛应用于中药天然产物、化学合成药物小分子靶点的发现与相互作用研究及中药有效活性成分鉴定等领域^[17-21]。

SPR原理: 光在棱镜与金属膜表面发生全反射现象, 会形成消逝波进入光疏介质中, 而在介质中又存在一定的等离子波。当两波相遇时发生共振, 此时检测到的反射光强会大幅度减弱, 能量从光子转移到表面等离子。入射光的大部分能量被表面等离子波吸收, 使反射光的能量急剧减少。可以从反射光响应曲线看到一个最小的尖峰, 此时对应的入射波长为共振波长, 使反射光完全消失的入射角就是SPR角。SPR角随金膜表面折射率的变化而变化, 而折射率的变化又与金膜表面结合的分子质量成正比。因此, 可以通过对生物反应中SPR角的动态变化获取分子之间相互作用的特异信号^{[16, 22-26]}。

基于SPR-MS的垂钓技术已被用于“垂钓”天然产物靶点。将“靶点蛋白”固定在芯片表面, 当被检测样品流过芯片表面时, 未知的互作因子能够与“靶点蛋白”相结合, 实现对未知相互作用分子的俘获, 再结合质谱技术鉴定出未知因子结构^[27-30]。研究人员以肿瘤坏死因子α (tumor necrosis factor α, TNF-α) 受体为“捕获蛋白”, 采用SPR垂钓技术进行了5种中药的筛选和分离, 最终从大黄中分离回收得到了结合TNF-α的小分子配体^[31]。Chen等^[32]对32种中药进行SPR-MS分析, 从中筛选出了5个信号转导及转录激活蛋白3 (signal transducer and activator of transcription 3, STAT3) 的配体, 其中首次发现新黄芩素和白藜芦醇苷是潜在的STAT3配体, 能显著降低STAT3的磷酸化。随着SPR蛋白阵列的后续设计, 预计SPR-MS将进入高通量蛋白质相互作用发现及高通量靶向药物筛选。

此外, 局域等离子共振技术(localized surface plasmon resonance, LSPR) 是以纳米金颗粒为检测单位的新一代SPR技术。不同于传统SPR技术检测折射率改变引起的SPR角度的偏转, LSPR检测的是纳米金颗粒表面分子层厚度的变化产生的光吸收峰的位移^{[33, 34]}。LSPR中消逝波是由纳米颗粒的光散射产生的, 克服了SPR复杂的光学系统, 降低了设备成本。

1.6 等温滴定量热法(isothermal titration calorimetry, ITC)

ITC (图 2D)^[35]是一种直接测量两个分子反应过程中释放或消耗热量的物理技术。它是检测配体在恒温下与大分子相互作用的一种分析方法。它的工作原理是热力学的基本原理, 两个分子之间的接触产生热或吸收热, 取决于结合的类型, 即放热或吸热^{[36, 37]}。实验过程中, 参照池和样品池都保持在稳定的温度和压力下, 配体通过注射器打入样品池。大分子与配体的结合导致热释放或消耗, 从而导致样品池内温度的变化, 随着配体浓度的增加导致底物饱和, 从而减少了热量的释放或消耗。然而, 仪器将始终保持与参照池和样品池相同的温度不变。为了保持温度, 仪器根据相互作用提供相应的功率(更高或更低)。因此, 可连续、准确地监测和记录一个变化过程的量热曲线, 原位、在线和无损伤地同时提供热力学和动力学信息^[38]。通过直接测量配体与目标蛋白结合时的热变化, 可以计算或确定分子间相互作用的结合亲和力(k_a)、结合位点数(n) 以及焓(∆H) 和熵(∆S) 的变化、动力学参数如酶促反应的k_m和k_cat。

在ITC实验中, 所有的样品, 如蛋白质和测试化合物必须溶解在完全相同的缓冲成分。特别是pH值的差异不应超过0.05个pH单位。不稳定的还原剂如二硫苏糖醇(DTT) 应避免在结合缓冲液中, 如果反应需要还原剂, 建议使用磷酸三氯乙酯或β-巯基乙醇^[39]。与SPR不同的是, ITC不需要固定或修饰分子; ITC技术不受蛋白质或配体大小的限制, 不受样品任何潜在光学性质的干扰, 使其成为药物发现方面有价值的技术^[40]。然而, ITC存在一些严重的局限性, 如低产热量、低通量和低分辨率; 需要蛋白量较大, 浓度要在50 μmol·L^-1以上, 体积300 μL以上; 小分子的浓度约为蛋白浓度的10~20倍, 以确保滴定反应结束时反应达到饱和或接近饱和。因此, ITC更适合于二次筛选而不是一次筛选, 特别是对大型化合物库的筛选^{[41, 42]}。

此外, ITC还可用于检测酶动力学特征, Abis等^{[43, 44]}采用单次注射(single-injection method, SIM) 的方法来研究人可溶性环氧化物水解酶(hsEH) 的动力学, 这是一种通过环氧脂肪酸(EpFAs) 代谢参与心血管稳态、高血压、痛感和胰岛素敏感性的酶。在SIM方法中, 当底物耗尽时, 反应速率通过监测热功率来确定, 克服了合成底物的需要, 减少了后续处理。结果显示, ITC能够详细、快速和重复表征hsEH介导的几种天然EpFA底物的水解, 并且显示出在利用酶的生理底物快速筛选和发现新的hsEH抑制剂方面的能力。

随着技术的不断发展, 新的ITC技术, 如ITC的反向滴定和置换滴定法得以发展。反向滴定(reverse titration), 即互换高分子和配位体来测定二者的结合模式。通常, 小分子被注入注射器中, 目标蛋白质在样品池中。而反向滴定是用蛋白来滴定小分子^[45]。此外, 置换滴定法(displacement titration) 可确定皮摩尔范围的配体的完整结合热力学信息, 其基本原理是当另一个竞争性配体存在时, 结合反应的配体结合性质被改变^{[46, 47]}。

1.7 差示扫描荧光(differential scanning fluorimetry, DSF)

DSF (图 3A) 是一种快速和廉价的筛选小分子配体与靶蛋白结合的方法。蛋白质展开的温度是通过与蛋白质疏水部分亲和的染料的荧光增加来测量的; 当蛋白质展开时, 疏水部分会暴露出来, 荧光染料通过优先结合在展开时暴露的疏水蛋白表面而增强荧光信号。荧光对温度的依赖用一个S型函数来描述, 它可以拟合玻尔兹曼方程(Boltzmann)。该方程可以用来计算转变的拐点, 即蛋白质的熔化温度(T_m)。蛋白质展开的吉布斯(Gibbs) 自由能∆G与蛋白质的T_m呈正相关。配体结合通常会增加蛋白展开的∆G, 导致T_m的增加^[48-50]。通常与对照组相比会导致稳定的T_m正位移0.5~2.0 ℃^[51]。

DSF可使用常规的RT-PCR仪, 操作简单。荧光染料常用的是高信噪比的SYPRO orange。此外, SYPRO orange的激发波长相对较高, 接近500 nm, 减少了小分子化合物对荧光染料的影响。然而, 并不是所有的蛋白质都可以用SYPRO orange来分析。对于使用SYPRO orange没有观察到蛋白展开过渡的情况, 应测试其他染料^{[51, 52]}。如果不能检测到T_m移位, 并不一定意味着目标蛋白与小分子之间没有结合。配体的结合可能不影响蛋白质的热稳定性, 在这种情况下, 需要使用另一种方法来研究相互作用。

Nano Temper公司设计了一款更加高级的差示荧光扫描技术nanoDSF。该技术通过检测色氨酸自发荧光的微小变化来进行蛋白质稳定性的研究。此外, 小分子影响蛋白质对热变性的稳定性也可以用差示扫描量热法^[53] (differential L scanning caborimetry, DSC)、差示扫描光散射法^[54] (differential scanning light scattering, DSLS) 或基于荧光的热位移分析^{[50, 55]} (fluorescence thermal shift, FTS) 来测定。

1.8 诱导圆二色谱(induced circular dichroism, ICD)

ICD (图 3B) 是一种通过检测左、右圆偏振光的吸收差, 研究蛋白质构象行为的快速和相对简单的光谱技术; 对溶液中蛋白质、核酸等生物大分子二级及三级结构微小变化高度灵敏^[56]。ICD光谱的一个重要应用是测定配体和靶蛋白之间结合作用的平衡解离常数K_d, 可以通过非线性回归方法分析固定波长下CD数据与配体浓度的关系来计算^[57]。当小分子与蛋白质结合时, 它通过对其结构的手性扰动或电子重排获得ICD谱。这种ICD的波长是由配体自身的吸收光谱决定的, 而ICD光谱的强度是由它与蛋白质相互作用的强度和几何形状决定的^[58]。如果配体本身是无手性的, 那么它的CD谱就会从0变为某个值; 如果配体是手性的, 那么它的ICD是配体的CD光谱和蛋白质存在时的光谱之间的差值。此外, CD在小分子靶向结合DNA研究中应用较为广泛^{[59, 60]}。大量膜蛋白已被证明难以结晶或结构太复杂不能用于X射线晶体学结构测定和核磁共振结构研究。同步辐射CD (synchrotron radiation CD, SRCD) 可实现膜蛋白与配体结合的鉴定。Patching等^[61]首次利用SRCD光谱展示了膜蛋白-配体相互作用的定量数据。因此, ICD法可进行小分子与蛋白(膜蛋白) 结合的验证。

1.9 质谱(mass spectrometry, MS) 技术

在过去的30年中, 质谱及其相关技术已被应用于研究蛋白质-配体相互作用, 不仅用于共价复合物的研究, 而且还用于非共价复合物的研究。软电离质谱技术, 包括电喷雾质谱和基质辅助激光解吸电离质谱, 可以直接测定小分子与靶蛋白化学计量比、结合强度及结合氨基酸位点等信息^[62]。质谱法(图 3C) 鉴定蛋白质上的小分子结合位点已被应用于许多蛋白质与药物结合的研究。该方法不仅可以提供精确的质量和序列特异性信息, 还可以从滴定实验中提供蛋白质-配体复合体的解离常数(即K_d值)、结合时构象变化的信息(离子迁移率测量和碰撞截面计算)、结合位点的位置(表位映射方法)、结合化学计量学和结合特异性(竞争实验)^[63]。本课题组采用质谱法已经鉴定出了天然产物ginkgolide B^[64]、withangulatin A^{[65, 66]}、tubocapsenolide A^[67]、iso-seco-tanapartholide^[68]及polyphyllin D^[1]与靶蛋白的结合位点, 并阐明了其作用机制。Rogawski等^[69]开发了一种活性质谱(native mass spectrometry) 方法, 用于分析真核细胞中的药物摄取和靶点结合, 该方法基于直接从粗样品中使用活性质谱无标签分析细胞内过表达蛋白, 无须纯化。活性质谱可快速测定配体与靶蛋白的亲和力值(IC₅₀), 而不需要设置配体浓度梯度, 一次质谱进样即可完成。小分子蛋白质结合后会在蛋白的主峰附近再出一个小峰, 通过测定小峰和主峰的积分面积, 按特定公式可计算小分子与靶蛋白的K_d^[70]。

在配体筛选中, 亲和质谱技术(affinity selection mass spectrometry) 已广泛应用于受体、酶、膜蛋白等靶蛋白配体的筛选^[71]。分离纯化靶蛋白和配体复合物系统, 将配体从复合物解离下来进行液-质联用分析, 从而鉴定出其配体结构, 可实现高通量筛选。水雯箐课题组^[71]使用SCIEX TripleTOF^®6600系统开发了基于亲和质谱技术的高通量、无标记的配体筛选方法, 用于G-蛋白偶联受体小分子配体的筛选。为提高亲和质谱的通量, 水雯箐课题组对其进行了进一步改进, 建立了一种新型的迭代式亲和质谱方法。改进后的方法能够一次性从2万个化合物组成的混合物中高效富集结合特定G-蛋白偶联受体靶标的配体^[72]。由于膜蛋白的疏水性及脂质双分子层, 小分子与膜蛋白结合的检测仍具有挑战性。牛津大学Robinson课题组^[73]开发了一种高分辨率的、基于Orbitrap质谱的方法来分析完整的膜蛋白-配体复合物, 验证了各种不同质量的膜蛋白(从26~186 kDa, 二聚体、三聚体及五聚体) 结构特征和化学计量学。

1.10 核磁共振光谱(nuclear magnetic resonance spectroscopy, NMR) 技术

核磁共振光谱学是第一个被描述为筛选高亲和力的技术^[74]。无论是基于配体还是蛋白的NMR技术均已开发用于配体-蛋白结合的筛选。其中, 基于配体的NMR技术被广泛应用于小分子配体筛选, 因为它们不需要对蛋白质结构的精准认知, 也不限制蛋白质的大小^[75]。目前, 实验室常用的3种检测配体-靶蛋白相互作用的核磁共振技术包括: 饱和转移差谱(saturation transfer difference, STD) (图 3D)^{[76, 77]}、水配体记录梯度光谱(water-ligand observed by gradient spectroscopy, WaterLOGSY)^[78-80]和液体T2横向弛豫时间序列(carr-purcell-meiboom-gill, CPMG)^[81]。

STD涉及蛋白质特异性甲基质子共振的选择性扰动(0.5~1 ppm)^[76], 这种扰动迅速扩散到整个蛋白质, 通过180度脉冲选择性完成, 并通过核overhauser效应(NOE) 导致磁化从蛋白质转移到任何瞬时结合的配体。转移到配体的NOE的积累会导致相应的信号增强, 对应于STD光谱中配体的共振转移^[82]。在STD实验中, 影响信号强度的因素有很多, 包括蛋白质的大小、共振辐照的持续时间、辐照的频率、配体的解离常数、配体/蛋白质比。因此, 实验过程中改变配体和蛋白质浓度、照射频率和照射持续时间等参数是可行的。

在WaterLOGSY实验中, 磁化从自由态水转移到配体上, 主要是通过结合在配体结合位点上或靠近配体结合位点的水分子^[78]。NOE从水到配体的磁化转移发生在缓慢“颠倒”的状态下, NOE的信号将与观察到的磁化转移到溶液中游离的小分子(不与蛋白质结合) 的NOE信号相反; 如果配体被绑定, 负的NOE将主导信号^[80]。若添加蛋白质后, WaterLOGSY信号没有变化, 表明该配体未与蛋白结合。

CPMG是通过依赖配体核磁共振信号在束缚态和自由态之间弛豫速率的差异来检测配体结合的^[83]。在CPMG实验中, 采集前使用100~400 ms的时间延迟来过滤观察到的信号。小分子弛豫缓慢, 产生尖峰, 而蛋白质弛豫迅速, 往往显示出广泛的核磁共振信号。在短暂的配体与蛋白结合后, 由配体质子引起共振扩大^[84]。如果该配体与蛋白质结合, 则在蛋白质存在的情况下信号减弱。

基于蛋白观察核磁共振技术可识别配体的结合位点信息^[85]。二维nuclear overhauser effect spectroscopy (NOESY) 光谱^[86]、¹H-¹⁵N heteronuclear single quantum coherence (HSQC) 光谱^[87]和¹H-¹⁵N transverse relaxa-tion optimized spectroscopy (TROSY) 光谱是最常用的检测小分子配体结合位点的核磁共振技术^[88]。¹H-¹⁵N HSQC是对靶蛋白分子主链酰胺的¹H-¹⁵N信号进行化学位移归属, 用小分子滴定¹⁵N标记的靶蛋白, 记录一系列的¹H-¹⁵N HSQC谱, 检测每个氨基酸HSQC谱中峰化学位移变化, 从而确定配体与靶蛋白结合及具体结合的氨基酸残基位置^[89]。随着技术的改进, 通过二维转移-NOE光谱(2D TR-NOESY) (图 3E) 直接探测细胞系中各种配体-受体相互作用成为可能^{[86, 90]}。目前, 核磁共振技术在药物筛选及配体结合鉴定方面的应用越来越广泛。

1.11 X射线单晶衍射(小分子与靶蛋白共晶)

蛋白质的三维空间结构以及蛋白质和活性化合物分子的空间结合方式是揭示药物机制的关键环节。小分子与蛋白共晶是小分子与蛋白结合最直接的证据, 可以提供具体的氨基酸结合位点信息。但该方法获得小分子-蛋白共晶难度较大, 需耗费大量时间筛选培养条件, 甚至难以获得共晶。常用的共晶培养方法是蒸汽扩散法^{[91, 92]}, 包括坐滴法和悬滴法(图 3F), 将高浓度的蛋白(纯度97%以上) 与结晶缓冲液混匀形成小液滴, 液滴含有纯化蛋白、缓冲液和沉淀剂, 使其置于含有浓度较高的结晶缓冲液和沉淀剂的较大密闭存储池; 由于两者饱和蒸汽压不同, 水从小液滴蒸发并转移到存储池, 使小液滴中沉淀剂浓度增加, 蛋白浓度逐渐趋于饱和, 最终达到最适合结晶的水平, 开始形成晶核^{[91, 93]}。此时, 小液滴中的小分子配体会随着与蛋白的结合附着在蛋白晶体上。随着晶体的生长, 小液滴蛋白浓度降低, 最后晶体和液滴中蛋白达到平衡, 晶体停止生长。坐滴法常用于晶体生长条件初筛, 悬滴法常用于晶体生长条件的优化。

影响共晶形成的因素有^[91]: 蛋白质纯度、pH值、蛋白质浓度、温度和沉淀剂。为了保证足够的同质性, 蛋白质通常至少要有97%的纯度。pH条件也很重要, 因为不同的pH值会导致不同的结晶方向。缓冲剂, 如Tris-HCl, 通常是维持特定pH值所必需的。沉淀剂, 如硫酸铵或聚乙二醇, 是诱导蛋白质从溶液中沉淀出来。

1.12 冷冻电镜技术(cryo-EM)

冷冻电镜技术是将生物大分子溶液置于电镜载网上形成一层非常薄的水膜, 利用快速冷冻技术将其瞬间冷冻至液氮温度下, 使水膜形成一种玻璃态的冰, 生物大分子被固定在薄冰中, 在透射电子显微镜下观察, 从而获得生物大分子或生物大分子-配体复合物结构。由于冷冻电镜价格昂贵, 应用于靶蛋白-小分子结合检测尚未普及。科学家利用冷冻电镜解析了膜蛋白-瞬时感受器电位离子通道蛋白V1 (transient receptor potential vanilloid-1, TRPV1) 和辣椒素相互结合的结构信息, 辣椒素通过香草基和S4-S5连接子之间的“拉-接触”相互作用稳定TRPV1的开放状态^[94]。APN-1607是最近发现的用于阿尔茨海默症和其他tau蛋白病的正电子发射计算机断层扫描(positron emission tomography, PET) 成像示踪剂, Shi等^[95]利用电子冷冻显微镜确定APN-1607在阿尔茨海默症中的tau蛋白结合位点。西湖大学申怀宗课题组^[96]利用高分辨率冷冻电镜解析了人源钾电压阀门通道亚家族蛋白Q4 (potassium voltage-gated channel Q4, KCNQ4) 通道蛋白及其与药物小分子瑞替加滨和利诺吡啶结合的高分辨率结构。

1.13 基于计算机模拟靶蛋白-配体结合亲和力

分子对接是最常用的计算机模拟配体-靶蛋白相互作用技术, 按几何互补、能量互补的原则, 寻找小分子在靶蛋白活性位点处的合理取向和构象, 从整体考虑配体与靶蛋白结合效果^[97]。随着计算机技术的发展, 在线分子对接网站得以开发使用, SwissDock^[98]是瑞士生物信息研究所开发的一项免费在线分子对接平台, 用于小分子与靶蛋白对接(http://www.swissdock.ch/docking)。近期, 科研者之家推出了在线分子对接平台(https://www.dockeasy.cn/DockCompound), 均可实现在线的分子对接。

现有的高通量筛选实验用于确定药物和靶点之间的生物活性是一个昂贵费时的步骤。因此, 基于已经在临床试验中测量的相互作用, 使用统计学和机器模型来预测新的药物-靶点的相互作用的亲和力是一项重要的替代方案。随着计算机技术的发展, 越来越多的基于计算的方法被开发用于预测蛋白-配体相互作用的结合亲和力。除了常用的分子对接技术以外, 科学家相继开发了多种计算机模拟技术。目前, 用于预测蛋白-配体亲和性的深度学习模型主要有两种: ①基于3D-CNN (将蛋白-配体的结构分为一个个格点, 用格点周围原子对格点的占有率描述结构)^[99]; ②基于空间图的图神经网络(将蛋白-配体中的原子作为节点, 将原子间距离用邻近矩阵描述)^[99]。目前, 已开发20余种模拟技术, 如SimBoost^[100]、DStruBTarget^[101]、GraphDTA^[102]、DeepDTAF^[103]、MATT-DTI^[104]和ML-DTI^[105]等, 这里不再一一赘述。

基于以上描述, 本综述对以上方法的使用范围和优缺点进行了归纳总结, 详情见表 1。几种方法联合使用能更好地证明小分子配体与靶蛋白的结合。

2 高通量配体筛选

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药物发现的最初阶段越来越依赖于研究蛋白质-蛋白质和蛋白质-配体相互作用的分析方法的发展和改进。疾病相关靶标蛋白与小分子化合物的相互作用研究的高通量筛选已成为新药研发的热点领域。靶蛋白与药物配体结合亲和力及动力学参数已成为评估和预测其体内药效的重要指标。默沙东公司开发了一种高通量的筛选技术平台NanoSAR (nanoscale synthesis and affinity ranking), 对高通量偶联反应合成的小分子, 使用无标记的亲和选择-质谱法(affinity-selection mass spectrometry, ASMS) 直接测定反应产物对靶标蛋白的亲和性, 并进行排序^[106]。如前所述, 目前适用于高通量筛选技术有DSF、质谱、核磁等。质谱技术在高通量配体筛选上应用较为广泛, 包括亲和选择质谱(AS-MS)^[107]和矩阵辅助激光解吸/电离质谱(matrix-assisted laser desorption/ionization mass spectrometry, MALDI-MS)^[108]。此外, Sanchez等^[109]改进了CETSA方法, 采用分裂荧光素酶结合CETSA技术, 克服了CETSA筛选的低通量。将SPR-MS应用于配体筛选表现出较好的效果, Chen等^[32]对32种中药进行SPR-MS分析, 从中筛选出了5个STAT3的配体。基于荧光的荧光偏振(fluorescence polarization, FP) 及荧光共振能量转移(fluorescence resonance energy transfer, FRET) 的高通量筛选技术已应用于天然产物及小分子抑制剂的筛选。Camara等^[110]采用FP筛选出第一个腺苷转移酶(HYPE) 调节剂。FRET广泛应用于靶向DNA药物小分子筛选, 如靶向DNA G-quadruplex小分子筛选^[111]。DNA编码化学文库(DNA-encoded small molecule libraries, DELs)^[112]是一种将遗传学与化学相结合的配体高通量筛选技术, 并成功地用于药物筛选, 如筛选得到的化合物GSK2982772 (GSK特异性抑制剂) 已完成了银屑病的II期临床试验。然而, 该方法需要纯化蛋白, Petersen等^[113]改进了该方法, 使其可以在活细胞水平实现高通量筛选; Huang等^[114]对其进行改进, 使其能在活细胞水平筛选膜蛋白。

随着计算机及AI技术的发展, 虚拟筛选应运而生, 该方法基于配体-靶标的相互作用模式, 利用计算机筛选虚拟化合物库, 最终筛选出有苗头的化合物, 并进行验证。

3 总结与展望

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药物-靶蛋白相互作用系统在药物结构设计及新药研发中发挥着关键作用, 是优化减毒增效药物小分子的基础。对药物-靶蛋白亲和力的检测可指导用药以发挥最佳疗效, 减少毒副作用。大多数检测技术均需要纯化蛋白, 具有较大的限制。如何将药物-靶点相互作用技术推进到体内及临床环境, 以理解临床中药物-靶点相互作用及脱靶效应, 并确定新的治疗相关靶点, 仍面临巨大挑战。质谱技术表现出较强的临床应用价值, 可实现临床上检测药物与靶蛋白的结合, 对研究药物-靶点蛋白临床表现具有指导意义, 但也面临着临床检测灵敏度及分辨率低的问题。因此, 体内检测药物-靶蛋白相互作用仍需科学家继续探索。

作者贡献: 董睿方撰写正文及画图; 夏元铮和孔令义提供综述的全文思路和结构; 所有作者共同参与了文章的撰写及修改。

利益冲突: 本文作者声明没有利益冲突。

基金

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国家自然科学基金资助项目(81973524)

参考文献

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文献

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2023年第58卷第11期

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doi: 10.16438/j.0513-4870.2023-0649

接收时间：2023-05-18
首发时间：2025-11-21
出版时间：2023-11-12

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收稿日期：2023-05-18
修回日期：2023-07-10

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国家自然科学基金资助项目(81973524)

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中国药科大学中药学院, 天然药物活性组分与药效国家重点实验室, 江苏省生物活性天然产物研究重点实验室, 江苏南京 210009

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*夏元铮, Tel: 13913024226, E-mail: xiayz@cpu.edu.cn;

孔令义, Tel: 86-25-83271405, E-mail: cpu_lykong@126.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Name	Principle	Application	Advantage	Disadvantage
DARTS	Proteolytic stability	In vitro/cell lysate	Simple operation, no drug activity dependence	Unable to detect binding to target proteins within cells/tissues
CETSA	Protein thermal stability	Cell/tissue	Binding to target proteins in tissues can be detected	Low abundances of target proteins are difficult to detect
MST	Temperature affects the movement of protein molecules	Purified protein	Low protein usage, no need for protein fixation	Requires high purity protein, protein cannot be recycled
BLI	Light interference	Purified protein	Protein does not require labeling, protein samples can be recycled, and sensors can be renewable and reused	Strong non-specific binding to small molecules containing multiple -OH/NH groups
SPR	Light refraction	Purified protein	High sensitivity and high throughput	Sensitive to sample composition and temperature, non-specific adsorption
ITC	Exothermic reaction	Purified protein	Proteins do not require fixation and can provide both thermodynamic and kinetic information	High protein consumption and low heat production, low throughput
DSF	Protein thermal stability	Purified protein	Dyes are cheap and the instrument (PCR instrument) is easy to operate	Not suitable for heat insensitive proteins
ICD	Absorption difference of left and right circularly polarized light	Purified protein	Detectable membrane protein and ligand binding	Require a large amount of purified protein
MS	Ionization of protein molecules into ions	Purified protein/intracellular overexpressed proteins	No need to set ligand concentration gradient and can provide binding site information	Difficult to distinguish between covalent and non-covalent binding
NMR	Magnetization transfer	Purified protein	Identify the functional groups that bind to the target protein	Isotopic labeling is required for 2D NMR
X-ray single crystal diffraction	Co-crystallization of small molecules and proteins	Co-crystallization	Specific amino acid residue binding site information can be provided	Obtaining small molecule protein eutectic is difficult and time-consuming
Cryo-EM	Rapid freezing+transmission electron microscopy	Purified protein	Obtaining structural information of target protein-ligand complexes	Expensive price and low penetration rate
Computer simulation	Virtual docking		No need for protein purification, high throughput	Further experimental verification is required

Name

Principle

Application

Advantage

Disadvantage

DARTS

Proteolytic stability

In vitro/cell lysate

Simple operation, no drug activity dependence

Unable to detect binding to target proteins within cells/tissues

CETSA

Protein thermal stability

Cell/tissue

Binding to target proteins in tissues can be detected

Low abundances of target proteins are difficult to detect

MST

Temperature affects the movement of protein molecules

Purified protein

Low protein usage, no need for protein fixation

Requires high purity protein, protein cannot be recycled

BLI

Light interference

Purified protein

Protein does not require labeling, protein samples can be recycled, and sensors can be renewable and reused

Strong non-specific binding to small molecules containing multiple -OH/NH groups

SPR

Light refraction

Purified protein

High sensitivity and high throughput

Sensitive to sample composition and temperature, non-specific adsorption

ITC

Exothermic reaction

Purified protein

Proteins do not require fixation and can provide both thermodynamic and kinetic information

High protein consumption and low heat production, low throughput

DSF

Protein thermal stability

Purified protein

Dyes are cheap and the instrument (PCR instrument) is easy to operate

Not suitable for heat insensitive proteins

ICD

Absorption difference of left and right circularly polarized light

Purified protein

Detectable membrane protein and ligand binding

Require a large amount of purified protein

Ionization of protein molecules into ions

Purified protein/intracellular overexpressed proteins

No need to set ligand concentration gradient and can provide binding site information

Difficult to distinguish between covalent and non-covalent binding

NMR

Magnetization transfer

Purified protein

Identify the functional groups that bind to the target protein

Isotopic labeling is required for 2D NMR

X-ray single crystal diffraction

Co-crystallization of small molecules and proteins

Co-crystallization

Specific amino acid residue binding site information can be provided

Obtaining small molecule protein eutectic is difficult and time-consuming

Cryo-EM

Rapid freezing+transmission electron microscopy

Purified protein

Obtaining structural information of target protein-ligand complexes

Expensive price and low penetration rate

Computer simulation

Virtual docking

No need for protein purification, high throughput

Further experimental verification is required