药学学报

Disease	Type	Sample	Conclusion	Reference
Cancer	Liver cancer	Hep3B cells	Chamaejasmenin E induces apoptosis in hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo	[44]
HepG2 cells	Hydroxethonine hydrochloride induces cellular senescence by regulating the LIF/AMPK pathway, thereby inhibiting the development and progression of hepatocellular carcinoma	[45]
Non-small cell carcinoma	A549 and H1299 cells	Evodiamine affects the Notch3 signaling pathway through inhibition of γ-secretase, thereby inhibiting non-small cell lung carcinogenesis	[46]
A549, H1299, and HLF cells	Cucurbitacin B exerts anti-non-small cell lung cancer effects by initiating cell pyroptosis	[47]
Colorectal cancer	HCT116, HT29, and SW480 cells	The interaction between Zuojin capsule and seven intracellular targets verified the mechanism of inhibition of colorectal cancer	[48]
Prostate cancer	293T, LNCaP, 22Rv1, C4-2, PC3, and DU145 cells	Found an inhibitor of the orphan receptor COUP-TFII to treat prostate cancer by inhibiting receptor activity	[49]
Mammary cancer	MCF-7 cells	Asiatic acid achieves anticancer effects against doxorubicin-resistant breast cancer cells through an AMPK dependent pathway	[50]
Tumour	Myelomatosis	H929 and U266 cells	Adenin specifically binds IKKβ thereby inhibiting NF-κB activation and inhibiting multiple myeloma cell proliferation	[51]
Osteosarcoma	HOS-MNNG, KHOS, MG63, U2OS, SJSA-1, G292, Cal72, and 143B cells	The MTH1 inhibitor TH1579 binds to MTH1, indicating the therapeutic effect of TH1579 on human osteosarcoma cells	[52]
Glioblastoma	U251 and U87 cells	Discovery of a novel KHS101 analog as a TACC3 inhibitor for the treatment of glioblastoma	[53]
Nervous system diseases	Neurodegenerative disease	BV-2 and HEK293T cells	Eupalinolide B can target the noncatalytic domain of ubiquitin-specific protease 7 to inhibit neuroinflammation and treat neurodegenerative diseases	[54]
Depression	Whole brain of DBA/2J and DBA/2 Ola mice	Found a novel binding target of the selective serotonin reuptake inhibitor paroxetine, the phosphofructokinase protein, to investigate its antidepressant mechanisms	[55]
Neuralgia	BV-2 cells	The TLR4 antagonist lovastatin inhibits TLR4 signaling by binding to myeloid differentiation protein 2, a coreceptor of TLR4	[56]
Organ disease	Heart	RAW264.7 cells	Iminostilbene targeting pyruvate kinase isoenzyme M2 reduces macrophage inflammation, thereby significantly attenuates myocardial ischemia/reperfusion injury	[57]
Liver	LO2 cells	Potential targets of heterotexin for acute liver injury are PTEN, PI3K, and BiP	[58]
LX-2 cells and mHSCs	18β-Glycyrrhetinic acid ameliorates liver fibrosis by inducing ROS- mediated apoptosis by targeting PRDX1/2 in cells	[59]
Kidney	mTECs	The TGF-β type I receptor inhibitor AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway	[60]
Vascellum	Dami cell	Proanthocyanidin A1 promotes platelet production by activating the JAK2/STAT3 pathway to improve chemotherapy-induced thrombocytopenia	[61]

Disease

Type

Sample

Conclusion

Reference

Cancer

Liver cancer

Hep3B cells

Chamaejasmenin E induces apoptosis in hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo

[44]

HepG2 cells

Hydroxethonine hydrochloride induces cellular senescence by regulating the LIF/AMPK pathway, thereby inhibiting the development and progression of hepatocellular carcinoma

[45]

Non-small cell carcinoma

A549 and H1299 cells

Evodiamine affects the Notch3 signaling pathway through inhibition of γ-secretase, thereby inhibiting non-small cell lung carcinogenesis

[46]

A549, H1299, and HLF cells

Cucurbitacin B exerts anti-non-small cell lung cancer effects by initiating cell pyroptosis

[47]

Colorectal cancer

HCT116, HT29, and SW480 cells

The interaction between Zuojin capsule and seven intracellular targets verified the mechanism of inhibition of colorectal cancer

[48]

Prostate cancer

293T, LNCaP, 22Rv1, C4-2, PC3, and DU145 cells

Found an inhibitor of the orphan receptor COUP-TFII to treat prostate cancer by inhibiting receptor activity

[49]

Mammary cancer

MCF-7 cells

Asiatic acid achieves anticancer effects against doxorubicin-resistant breast cancer cells through an AMPK dependent pathway

[50]

Tumour

Myelomatosis

H929 and U266 cells

Adenin specifically binds IKKβ thereby inhibiting NF-κB activation and inhibiting multiple myeloma cell proliferation

[51]

Osteosarcoma

HOS-MNNG, KHOS, MG63, U2OS, SJSA-1, G292, Cal72, and 143B cells

The MTH1 inhibitor TH1579 binds to MTH1, indicating the therapeutic effect of TH1579 on human osteosarcoma cells

[52]

Glioblastoma

U251 and U87 cells

Discovery of a novel KHS101 analog as a TACC3 inhibitor for the treatment of glioblastoma

[53]

Nervous system diseases

Neurodegenerative disease

BV-2 and HEK293T cells

Eupalinolide B can target the noncatalytic domain of ubiquitin-specific protease 7 to inhibit neuroinflammation and treat neurodegenerative diseases

[54]

Depression

Whole brain of DBA/2J and DBA/2 Ola mice

Found a novel binding target of the selective serotonin reuptake inhibitor paroxetine, the phosphofructokinase protein, to investigate its antidepressant mechanisms

[55]

Neuralgia

BV-2 cells

The TLR4 antagonist lovastatin inhibits TLR4 signaling by binding to myeloid differentiation protein 2, a coreceptor of TLR4

[56]

Organ disease

Heart

RAW264.7 cells

Iminostilbene targeting pyruvate kinase isoenzyme M2 reduces macrophage inflammation, thereby significantly attenuates myocardial ischemia/reperfusion injury

[57]

Liver

LO2 cells

Potential targets of heterotexin for acute liver injury are PTEN, PI3K, and BiP

[58]

LX-2 cells and mHSCs

18β-Glycyrrhetinic acid ameliorates liver fibrosis by inducing ROS- mediated apoptosis by targeting PRDX1/2 in cells

[59]

Kidney

mTECs

The TGF-β type I receptor inhibitor AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway

[60]

Vascellum

Dami cell

Proanthocyanidin A1 promotes platelet production by activating the JAK2/STAT3 pathway to improve chemotherapy-induced thrombocytopenia

[61]

Disease	Type	Sample	Conclusion	Reference
Cancer	Liver cancer	Hep3B cells	Chamaejasmenin E induces apoptosis in hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo	[44]
HepG2 cells	Hydroxethonine hydrochloride induces cellular senescence by regulating the LIF/AMPK pathway, thereby inhibiting the development and progression of hepatocellular carcinoma	[45]
Non-small cell carcinoma	A549 and H1299 cells	Evodiamine affects the Notch3 signaling pathway through inhibition of γ-secretase, thereby inhibiting non-small cell lung carcinogenesis	[46]
A549, H1299, and HLF cells	Cucurbitacin B exerts anti-non-small cell lung cancer effects by initiating cell pyroptosis	[47]
Colorectal cancer	HCT116, HT29, and SW480 cells	The interaction between Zuojin capsule and seven intracellular targets verified the mechanism of inhibition of colorectal cancer	[48]
Prostate cancer	293T, LNCaP, 22Rv1, C4-2, PC3, and DU145 cells	Found an inhibitor of the orphan receptor COUP-TFII to treat prostate cancer by inhibiting receptor activity	[49]
Mammary cancer	MCF-7 cells	Asiatic acid achieves anticancer effects against doxorubicin-resistant breast cancer cells through an AMPK dependent pathway	[50]
Tumour	Myelomatosis	H929 and U266 cells	Adenin specifically binds IKKβ thereby inhibiting NF-κB activation and inhibiting multiple myeloma cell proliferation	[51]
Osteosarcoma	HOS-MNNG, KHOS, MG63, U2OS, SJSA-1, G292, Cal72, and 143B cells	The MTH1 inhibitor TH1579 binds to MTH1, indicating the therapeutic effect of TH1579 on human osteosarcoma cells	[52]
Glioblastoma	U251 and U87 cells	Discovery of a novel KHS101 analog as a TACC3 inhibitor for the treatment of glioblastoma	[53]
Nervous system diseases	Neurodegenerative disease	BV-2 and HEK293T cells	Eupalinolide B can target the noncatalytic domain of ubiquitin-specific protease 7 to inhibit neuroinflammation and treat neurodegenerative diseases	[54]
Depression	Whole brain of DBA/2J and DBA/2 Ola mice	Found a novel binding target of the selective serotonin reuptake inhibitor paroxetine, the phosphofructokinase protein, to investigate its antidepressant mechanisms	[55]
Neuralgia	BV-2 cells	The TLR4 antagonist lovastatin inhibits TLR4 signaling by binding to myeloid differentiation protein 2, a coreceptor of TLR4	[56]
Organ disease	Heart	RAW264.7 cells	Iminostilbene targeting pyruvate kinase isoenzyme M2 reduces macrophage inflammation, thereby significantly attenuates myocardial ischemia/reperfusion injury	[57]
Liver	LO2 cells	Potential targets of heterotexin for acute liver injury are PTEN, PI3K, and BiP	[58]
LX-2 cells and mHSCs	18β-Glycyrrhetinic acid ameliorates liver fibrosis by inducing ROS- mediated apoptosis by targeting PRDX1/2 in cells	[59]
Kidney	mTECs	The TGF-β type I receptor inhibitor AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway	[60]
Vascellum	Dami cell	Proanthocyanidin A1 promotes platelet production by activating the JAK2/STAT3 pathway to improve chemotherapy-induced thrombocytopenia	[61]

Disease

Type

Sample

Conclusion

Reference

Cancer

Liver cancer

Hep3B cells

Chamaejasmenin E induces apoptosis in hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo

[44]

HepG2 cells

Hydroxethonine hydrochloride induces cellular senescence by regulating the LIF/AMPK pathway, thereby inhibiting the development and progression of hepatocellular carcinoma

[45]

Non-small cell carcinoma

A549 and H1299 cells

Evodiamine affects the Notch3 signaling pathway through inhibition of γ-secretase, thereby inhibiting non-small cell lung carcinogenesis

[46]

A549, H1299, and HLF cells

Cucurbitacin B exerts anti-non-small cell lung cancer effects by initiating cell pyroptosis

[47]

Colorectal cancer

HCT116, HT29, and SW480 cells

The interaction between Zuojin capsule and seven intracellular targets verified the mechanism of inhibition of colorectal cancer

[48]

Prostate cancer

293T, LNCaP, 22Rv1, C4-2, PC3, and DU145 cells

Found an inhibitor of the orphan receptor COUP-TFII to treat prostate cancer by inhibiting receptor activity

[49]

Mammary cancer

MCF-7 cells

Asiatic acid achieves anticancer effects against doxorubicin-resistant breast cancer cells through an AMPK dependent pathway

[50]

Tumour

Myelomatosis

H929 and U266 cells

Adenin specifically binds IKKβ thereby inhibiting NF-κB activation and inhibiting multiple myeloma cell proliferation

[51]

Osteosarcoma

HOS-MNNG, KHOS, MG63, U2OS, SJSA-1, G292, Cal72, and 143B cells

The MTH1 inhibitor TH1579 binds to MTH1, indicating the therapeutic effect of TH1579 on human osteosarcoma cells

[52]

Glioblastoma

U251 and U87 cells

Discovery of a novel KHS101 analog as a TACC3 inhibitor for the treatment of glioblastoma

[53]

Nervous system diseases

Neurodegenerative disease

BV-2 and HEK293T cells

Eupalinolide B can target the noncatalytic domain of ubiquitin-specific protease 7 to inhibit neuroinflammation and treat neurodegenerative diseases

[54]

Depression

Whole brain of DBA/2J and DBA/2 Ola mice

Found a novel binding target of the selective serotonin reuptake inhibitor paroxetine, the phosphofructokinase protein, to investigate its antidepressant mechanisms

[55]

Neuralgia

BV-2 cells

The TLR4 antagonist lovastatin inhibits TLR4 signaling by binding to myeloid differentiation protein 2, a coreceptor of TLR4

[56]

Organ disease

Heart

RAW264.7 cells

Iminostilbene targeting pyruvate kinase isoenzyme M2 reduces macrophage inflammation, thereby significantly attenuates myocardial ischemia/reperfusion injury

[57]

Liver

LO2 cells

Potential targets of heterotexin for acute liver injury are PTEN, PI3K, and BiP

[58]

LX-2 cells and mHSCs

18β-Glycyrrhetinic acid ameliorates liver fibrosis by inducing ROS- mediated apoptosis by targeting PRDX1/2 in cells

[59]

Kidney

mTECs

The TGF-β type I receptor inhibitor AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway

[60]

Vascellum

Dami cell

Proanthocyanidin A1 promotes platelet production by activating the JAK2/STAT3 pathway to improve chemotherapy-induced thrombocytopenia

[61]

Technology combination	Advantage	Disadvantage	Application	Reference
MS-CETSA	High throughput, high sensitivity, and good stability	There are limitations in detecting low-abundance proteins and proteins in complex mixtures, potentially leading to false negatives and incomplete proteome coverage	Proteome analysis Analysis of protein structural changes induced by drugs or other small molecules Development of antimicrobial drugs, drug mechanism of action, new efficacy, toxicity biomarkers or therapeutic drug targets	[1, 11, 13, 14]
ITDR-CETSA	Simple operation, a wide range of application	There are limitations in identifying targets with altered post-translational modifications or low-affinity interactions	Determine the optimal drug dose and monitor drug resistance	[11, 34]
2D-TPP	Compared with TPP, its analysis speed, sensitivity, specificity and accuracy are greatly improved The false positive rate was low	The generation of the spectrum map requires a high sample concentration, which is not convenient for large-scale screening The profiles of the multicomponent complexes are very complex and difficult to resolve	High-efficiency screening and optimization of drug candidates Acquired drug resistance study	[3, 13, 14, 23, 24]
PISA	Compared to TPP, the flux and specificity are further increased, which can effectively reduce sample consumption, shorten the running time of MS instruments, while retaining most of the information from 2D format	Loss of information on the non-CETSA abundance effect It would be difficult to detect small ΔAUC shifts and proteins with non-S type melting patterns	High-throughput, rapid analysis of compound binding to the target site For target studies of rare or precious samples	[17, 25, 26]
Alpha-CETSA	High sensitivity, small background interference, and a wide range of application than the BRET technology Less interfering and more sensitive for both serum and plasma samples	Specific donor-acceptor pairs are required	Detection of both protein-protein and protein-small molecule interactions High-throughput compound screening in biological samples	[29, 33, 34]
SplitLuc-CETSA	High sensitivity, small background interference, and a wide range of application	Specific donor-acceptor pairs are required It is not as applicable as Alpha	Detection of both protein-protein and protein-small molecule interactions High-throughput compound screening in biological samples	[29, 36, 37]
DARTS-CETSA	Improve the data analysis ability and reduce the number of proteolysis conditions Facilitate the analysis of the possible protein-protein interactions	The high abundance requirement of target proteins, some very sensitive proteins or proteins highly resistant to proteases may lead to the loss of target information	It is beneficial to predict the efficacy of small molecules and improve the dosage Drug screening and target identification	[5, 39]
Keap1-glow CETSA	Fast analysis speed, good specificity	The operation is more complex	Study of specific protein targets Providing strategies for disease prevention and treatment	[10]

Technology combination

Advantage

Disadvantage

Application

Reference

MS-CETSA

High throughput, high sensitivity, and good stability

There are limitations in detecting low-abundance proteins and proteins in complex mixtures, potentially leading to false negatives and incomplete proteome coverage

Proteome analysis
Analysis of protein structural changes induced by drugs or other small molecules
Development of antimicrobial drugs, drug mechanism of action, new efficacy, toxicity biomarkers or therapeutic drug targets

[1, 11, 13, 14]

ITDR-CETSA

Simple operation, a wide range of application

There are limitations in identifying targets with altered post-translational modifications or low-affinity interactions

Determine the optimal drug dose and monitor drug resistance

[11, 34]

2D-TPP

Compared with TPP, its analysis speed, sensitivity, specificity and accuracy are greatly improved
The false positive rate was low

The generation of the spectrum map requires a high sample concentration, which is not convenient for large-scale screening
The profiles of the multicomponent complexes are very complex and difficult to resolve

High-efficiency screening and optimization of drug candidates
Acquired drug resistance study

[3, 13, 14, 23, 24]

PISA

Compared to TPP, the flux and specificity are further increased, which can effectively reduce sample consumption, shorten the running time of MS instruments, while retaining most of the information from 2D format

Loss of information on the non-CETSA abundance effect
It would be difficult to detect small ΔAUC shifts and proteins with non-S type melting patterns

High-throughput, rapid analysis of compound binding to the target site
For target studies of rare or precious samples

[17, 25, 26]

Alpha-CETSA

High sensitivity, small background interference, and a wide range of application than the BRET technology
Less interfering and more sensitive for both serum and plasma samples

Specific donor-acceptor pairs are required

Detection of both protein-protein and protein-small molecule interactions
High-throughput compound screening in biological samples

[29, 33, 34]

SplitLuc-CETSA

High sensitivity, small background interference, and a wide range of application

Specific donor-acceptor pairs are required
It is not as applicable as Alpha

Detection of both protein-protein and protein-small molecule interactions
High-throughput compound screening in biological samples

[29, 36, 37]

DARTS-CETSA

Improve the data analysis ability and reduce the number of proteolysis conditions
Facilitate the analysis of the possible protein-protein interactions

The high abundance requirement of target proteins, some very sensitive proteins or proteins highly resistant to proteases may lead to the loss of target information

It is beneficial to predict the efficacy of small molecules and improve the dosage
Drug screening and target identification

[5, 39]

Keap1-glow CETSA

Fast analysis speed, good specificity

The operation is more complex

Study of specific protein targets
Providing strategies for disease prevention and treatment

[10]

Technology combination	Advantage	Disadvantage	Application	Reference
MS-CETSA	High throughput, high sensitivity, and good stability	There are limitations in detecting low-abundance proteins and proteins in complex mixtures, potentially leading to false negatives and incomplete proteome coverage	Proteome analysis Analysis of protein structural changes induced by drugs or other small molecules Development of antimicrobial drugs, drug mechanism of action, new efficacy, toxicity biomarkers or therapeutic drug targets	[1, 11, 13, 14]
ITDR-CETSA	Simple operation, a wide range of application	There are limitations in identifying targets with altered post-translational modifications or low-affinity interactions	Determine the optimal drug dose and monitor drug resistance	[11, 34]
2D-TPP	Compared with TPP, its analysis speed, sensitivity, specificity and accuracy are greatly improved The false positive rate was low	The generation of the spectrum map requires a high sample concentration, which is not convenient for large-scale screening The profiles of the multicomponent complexes are very complex and difficult to resolve	High-efficiency screening and optimization of drug candidates Acquired drug resistance study	[3, 13, 14, 23, 24]
PISA	Compared to TPP, the flux and specificity are further increased, which can effectively reduce sample consumption, shorten the running time of MS instruments, while retaining most of the information from 2D format	Loss of information on the non-CETSA abundance effect It would be difficult to detect small ΔAUC shifts and proteins with non-S type melting patterns	High-throughput, rapid analysis of compound binding to the target site For target studies of rare or precious samples	[17, 25, 26]
Alpha-CETSA	High sensitivity, small background interference, and a wide range of application than the BRET technology Less interfering and more sensitive for both serum and plasma samples	Specific donor-acceptor pairs are required	Detection of both protein-protein and protein-small molecule interactions High-throughput compound screening in biological samples	[29, 33, 34]
SplitLuc-CETSA	High sensitivity, small background interference, and a wide range of application	Specific donor-acceptor pairs are required It is not as applicable as Alpha	Detection of both protein-protein and protein-small molecule interactions High-throughput compound screening in biological samples	[29, 36, 37]
DARTS-CETSA	Improve the data analysis ability and reduce the number of proteolysis conditions Facilitate the analysis of the possible protein-protein interactions	The high abundance requirement of target proteins, some very sensitive proteins or proteins highly resistant to proteases may lead to the loss of target information	It is beneficial to predict the efficacy of small molecules and improve the dosage Drug screening and target identification	[5, 39]
Keap1-glow CETSA	Fast analysis speed, good specificity	The operation is more complex	Study of specific protein targets Providing strategies for disease prevention and treatment	[10]

Technology combination

Advantage

Disadvantage

Application

Reference

MS-CETSA

High throughput, high sensitivity, and good stability

There are limitations in detecting low-abundance proteins and proteins in complex mixtures, potentially leading to false negatives and incomplete proteome coverage

[1, 11, 13, 14]

ITDR-CETSA

Simple operation, a wide range of application

There are limitations in identifying targets with altered post-translational modifications or low-affinity interactions

Determine the optimal drug dose and monitor drug resistance

[11, 34]

2D-TPP

Compared with TPP, its analysis speed, sensitivity, specificity and accuracy are greatly improved
The false positive rate was low

High-efficiency screening and optimization of drug candidates
Acquired drug resistance study

[3, 13, 14, 23, 24]

PISA

Loss of information on the non-CETSA abundance effect
It would be difficult to detect small ΔAUC shifts and proteins with non-S type melting patterns

High-throughput, rapid analysis of compound binding to the target site
For target studies of rare or precious samples

[17, 25, 26]

Alpha-CETSA

High sensitivity, small background interference, and a wide range of application than the BRET technology
Less interfering and more sensitive for both serum and plasma samples

Specific donor-acceptor pairs are required

Detection of both protein-protein and protein-small molecule interactions
High-throughput compound screening in biological samples

[29, 33, 34]

SplitLuc-CETSA

High sensitivity, small background interference, and a wide range of application

Specific donor-acceptor pairs are required
It is not as applicable as Alpha

Detection of both protein-protein and protein-small molecule interactions
High-throughput compound screening in biological samples

[29, 36, 37]

DARTS-CETSA

Improve the data analysis ability and reduce the number of proteolysis conditions
Facilitate the analysis of the possible protein-protein interactions

The high abundance requirement of target proteins, some very sensitive proteins or proteins highly resistant to proteases may lead to the loss of target information

It is beneficial to predict the efficacy of small molecules and improve the dosage
Drug screening and target identification

[5, 39]

Keap1-glow CETSA

Fast analysis speed, good specificity

The operation is more complex

Study of specific protein targets
Providing strategies for disease prevention and treatment

[10]

非修饰靶点发现细胞热位移分析技术的变革与应用进展

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刘广苑 , 李亚慧 , 张炜 ^* , 孔德志 ^*

药学学报 | 综述 2024,59(1): 25-34

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药学学报 | 综述 2024, 59(1): 25-34

非修饰靶点发现细胞热位移分析技术的变革与应用进展

全屏

刘广苑, 李亚慧, 张炜^*, 孔德志^*

作者信息

河北医科大学中西医结合研究所, 河北石家庄 050017

通讯作者:

^*张炜, E-mail: weizhang@hebmu.edu.cn;

孔德志, E-mail: kongdezhi@hebmu.edu.cn

The evolution and application progress of non-modified drug target discovery CETSA technology

Guang-yuan LIU, Ya-hui LI, Wei ZHANG^*, De-zhi KONG^*

Affiliations

Institute of Integrated Traditional Chinese and Western Medicine, Hebei Medical University, Shijiazhuang 050017, China

出版时间: 2024-01-12 doi: 10.16438/j.0513-4870.2023-0415

文章导航

摘要

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了解药物的蛋白靶点研究方法对于新药研发、药物的临床应用、药物的作用机制和疾病的致病机制都具有十分重要的意义。细胞热位移分析(cellular thermal shift assay, CETSA) 作为无修饰分析的靶点研究方法自开发以来就被广泛应用。经过不断地演化, 现在已经出现了多种基于CETSA的技术结合, 如基于质谱的细胞热位移分析(mass spectrometry-based cellular thermal shift assay, MS-CETSA)、结合等温剂量反应实验的细胞热位移分析(isothermal dose response-cellular thermal shift assay, ITDR-CETSA)、结合均相光激化学发光免疫分析的细胞热位移分析(amplified luminescent primity homogeneous assay-cellular thermal shift assay, Alpha-CETSA) 等。这些技术融合了各自的优势, 进一步扩展了CETSA的应用范围, 适用于从药物筛选到监测药物在患者体内的靶点结合和脱靶毒性研究的整个药物开发链。本文结合作者的研究经验, 综述了近年来CETSA和相关结合技术的原理及其在靶点发现上的应用、数据处理分析的进展, 旨在为CETSA的进一步应用提供参考和借鉴。

关键词

细胞热位移分析 / 热蛋白质组分析 / 靶点发现 / 技术结合

Abstract

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Understanding the research methods for drug protein targets is crucial for the development of new drugs, clinical applications of drugs, drug mechanisms, and the pathogenesis of diseases. Cellular thermal shift assay (CETSA), a target research method without modification, has been widely used since its development. Now, there are various CETSA-based technology combinations, such as mass spectrometry-based cellular thermal shift assay (MS-CETSA), isothermal dose response-cellular thermal shift assay (ITDR-CETSA), amplified luminescent proximity homogeneous assay-cellular thermal shift assay (Alpha-CETSA), etc., which combine their respective advantages and further expand the application scope of CETSA. These technologies are suitable for the entire drug development chain, from drug screening to monitoring the target binding and off-target toxicity of drugs in patients. Based on the author's research experience, this paper reviews the principles of CETSA and related binding technologies, their application in target discovery, and the progress of data processing and analysis in recent years, aiming to provide reference and reference for the further application of CETSA.

Key words

cellular thermal shift assay / thermal proteome profiling / target discovery / technology combination

引用本文

刘广苑, 李亚慧, 张炜, 孔德志. 非修饰靶点发现细胞热位移分析技术的变革与应用进展. 药学学报, 2024 , 59 (1) : 25 -34 . DOI: 10.16438/j.0513-4870.2023-0415

Guang-yuan LIU, Ya-hui LI, Wei ZHANG, De-zhi KONG. The evolution and application progress of non-modified drug target discovery CETSA technology[J]. Acta Pharmaceutica Sinica, 2024 , 59 (1) : 25 -34 . DOI: 10.16438/j.0513-4870.2023-0415

正文

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热位移分析(thermal shift assay, TSA) 是目前研究靶点结合最广泛使用的无修饰方法之一, 它通过测量不同条件下蛋白质熔点温度的变化来探究靶点蛋白是否发生了相互作用^[1]。由于TSA只适用于纯化蛋白, Pär Nordlund团队^[2]于2013年开发了细胞热位移分析(cellular thermal shift assay, CETSA) 以研究各种细胞或组织中的药物-蛋白相互作用。该技术的原理与TSA类似: 当化合物与目标蛋白发生结合时蛋白质的构象、分子内疏水性等可能会发生改变从而影响蛋白的热稳定性^[3], 根据蛋白质热熔曲线的变化即可确定是否发生了靶点结合。蛋白质的热稳定性一般用半数蛋白质发生解折叠时对应的温度即热变性中点温度(melting temperature, T_m) 来表示。当蛋白与配体结合后, 其稳定性一般比未发生配体结合的蛋白更好, T_m值更高。因此通过比较化合物处理后蛋白的T_m值和热熔曲线是否存在差异即可确定化合物的靶点蛋白^[3], 通过测定化合物处理后不同温度下的细胞裂解液中蛋白质含量的多少来研究靶点结合程度。

CETSA技术具有操作简单^[4]、适用范围广^[5]、稳定性好、兼容性强等优势, 能与其他技术结合从而增强其检测能力和应用范围^[6]。在CETSA技术中蛋白质的鉴定最初采用特异性强的Western blotting法, 但该法无法预测未知靶蛋白, 此外在通量和多路复用方面也受到限制。近年来各种基于CETSA的技术层出不穷: 随着质谱技术^[7]和基于发光的检测^[8]等高通量技术的发展, CETSA技术可鉴定的蛋白质范围大大提升; 邻位延伸分析技术(proximity extension assay, PEA)^[9]能将蛋白浓度信号转换成核酸信号从而实现低丰度蛋白的检测, 结合PEA后的CETSA将允许在小样本中对大量特定的蛋白质进行平行量化, 灵敏地测量药物靶点结合。CETSA技术与其他技术的结合使其在药物靶点发现等领域发挥了重要作用, 现将CETSA的技术变革及其在药物靶点发现中的应用进行阐述, 该方法的流程及已报道的各种与CETSA结合的技术见图 1。

1 质谱技术与CETSA结合

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为了解决Western blotting在鉴定靶蛋白时存在的问题^[10], Savitski等^[11]将CETSA与质谱技术相结合即基于质谱的细胞热位移分析(mass spectrometry-based cellular thermal shift assay, MS-CETSA) 技术。该技术的原理在本质上与CETSA方法完全一致^[12], 不同之处在于MS-CETSA能通过质谱技术同时鉴定多个蛋白, 计算出蛋白质的热熔曲线、T_m值、pEC₅₀值以检测全蛋白质组中每种蛋白质在不同温度下的热变性状态。因此MS-CETSA也可称为热蛋白质组分析(thermal proteome profiling, TPP)。该技术灵敏度高、稳定性好, 是一种完整的蛋白质组分析方法, 可作为一种独立或补充的策略来表征完整细胞中的蛋白质^[13]。

TPP为定量蛋白质组学评估细胞中的蛋白质水平提供了正交信息^[14], 这有助于检测蛋白质的生理变化状态(蛋白质-蛋白质相互作用、蛋白质-代谢物相互作用)^[13], 从而开发新的治疗监测策略。位于完整细胞和细胞环境的TPP分析可以用于区分配体结合引起的热位移和下游修饰引起的热位移^[1]以发现药物的作用机制, 从而揭示药物新疗效、毒性生物标志物或治疗性药物靶点^[14]。此外, TPP在蛋白质-配体相互作用的大规模分析中可用于识别药物分子的靶点和脱靶效应^{[1, 11]}, 有助于去孤儿化和解释药物的不良反应。目前该方法还具有无需修饰、通量高(能够同时评估不同药物与人类细胞中7 000多个蛋白质的结合) 等优点^[4], 已被广泛地应用于药物或其他小分子诱导的蛋白质结构变化的分析^[1]。Azimi等^[15]首次实现了基于磷酸化修饰蛋白组学的MS-CETSA, 阐明了用BRAF抑制剂和/或Hsp90抑制剂单一和联合治疗黑色素瘤的机制。Ma等^[16]发现TPP还可应用于植物化学遗传学, 在完整的拟南芥细胞中进行靶点识别。另外, 该方法已成功应用于病原体治疗, 为抗菌药物的开发和作用机制研究提供了价值^[14]。

为了在蛋白质组范围内确定蛋白质的热稳定性, 并具有区域级分辨率, Liu等^[17]开发了一种结构蛋白质组方法即有限蛋白水解质谱技术(limited proteolysis-mass spectrometry, LiP-MS), 该技术中的蛋白提取物经过了双蛋白酶消化步骤。首先用广谱性的蛋白酶对天然构象条件下的蛋白进行酶切。当蛋白与小分子结合后, 蛋白质的构象发生变化从而暴露出不同的广谱性蛋白酶酶切位点, 产生不同的蛋白酶切片段; 然后再将蛋白样品变性, 用序列特异性蛋白酶完全消化蛋白; 最后通过质谱分析处理后的多肽样本, 比较不同处理条件下样品中全酶切及半酶切肽段的信号强度变化可以发现结构发生变化的特征多肽片段, 进而确定药物作用位点。将LiP-MS技术与细胞热位移分析的药物靶点识别方法相结合, 可以增加蛋白质组的覆盖率^[18]、扩大应用范围。

据文献^[3]报道配体稳定化靶点识别法(target identification by ligand stabilization, TILS)、微粒辅助沉淀筛选法(microparticle-assisted precipitation screening, MAPS) 和沉淀组分支持的热蛋白质组学分析(precipitate-supported thermal proteome profiling, PSTPP)^[19]与TPP方法相近, 它们通过对不溶性沉淀物进行热分析, 可作为TPP在药物靶点发现中的一种补充^[1]。此外, Fang等^[20]开发了一种名为氢-氘交换质谱集成蛋白质热损耗(hydrogen-deuterium exchange mass spectrometry with integrated protein thermal depletion, PTD-HDX-MS) 的策略用于蛋白质靶点鉴定。该方法通过加热去除非靶蛋白降低了样品的复杂性, 可作为TPP在药物靶点发现中的一种补充以交叉验证其他方法所识别的靶点^[20]。

2 等温剂量反应实验与CETSA结合

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为了研究不同浓度范围的配体对蛋白结合的影响, 研究人员开发了结合等温剂量反应实验的细胞热位移分析(isothermal dose response-cellular thermal shift assay, ITDR-CETSA) 技术。该实验先利用系列浓度的配体处理细胞然后在同一温度下对样本加热, 随后对蛋白进行鉴定和定量得到蛋白质的剂量反应曲线。靶蛋白的剂量反应曲线与配体药物呈剂量依赖型, 然而, 非靶蛋白的剂量反应曲线不会随着配体浓度发生改变。需要注意的是, ITDR实验所用的固定温度一般由热熔曲线的数据来确定^[21], 且该温度一般稍高于纯化蛋白的解链温度^[12]。ITDR-CETSA通过测定配体浓度与靶蛋白结合的关系及结合亲和力的大小, 有助于确定最佳的药物剂量、监测耐药性以发挥最大药效。Dziekan等^[22]进行了ITDR-CETSA分析以确定奎宁和甲氟喹的蛋白靶点, 对暴露于奎宁(0~10 μmol·L^-1) 或甲氟喹(0~100 μmol·L^-1) 的样品应用ITDR (51 ℃) 方案, 发现嘌呤核苷磷酸化酶是唯一可信稳定的靶蛋白。

3 ITDR-CETSA与TPP结合

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利用多路液相色谱串联质谱技术, Becher等^[23]设计了二维热蛋白质组分析(two dimensional-thermal proteome profiling, 2D-TPP) 策略, 该方法的实质是将ITDR-CETSA与基于温度范围的TPP方法相结合, 蛋白样本与系列浓度化合物反应的同时在不同温度下进行热处理, 从而使靶点识别的速度、灵敏度、特异性和准确性都大大提升^{[3, 13]}。2D-TPP能在同一质谱中对包括是否进行药物处理在内的所有变化的条件进行比较, 从而得到更精确的量化指标^{[3, 13]}。此外, 在2D-TPP方法中不同化合物浓度下蛋白质的稳定性考察增加了对数据额外的质控, 可排除一定的假阳性结果^{[23, 24]}。

Becher等^[23]利用2D-TPP来研究组蛋白去乙酰化酶的小分子抑制剂帕比司他对靶蛋白的剂量依赖性效应, 该实验还发现了帕比司他的脱靶点苯丙氨酸羟化酶, 提示该分子可能用于治疗酪氨酸血症^[1]。此外该研究还探究了不同细胞状态的生物样品间蛋白质稳定性变化和蛋白质水平变化, 文章中用不同的细胞周期状态取代药物浓度, 测量了10个温度下蛋白质的稳定性, 每种蛋白质的多维测量为细胞状态之间的转变提供了一个特征分析, 实现了一种新的2D-TPP策略。研究者在K562细胞中进行多维蛋白质相互作用状态的综合调节分析, 在细胞周期进程中测量了蛋白质相互作用状态的变化。MS-CETSA测得的水平信息与细胞内蛋白质形态水平变化的垂直信息基本正交, 可以为处于不同细胞周期阶段的细胞提供获得性耐药^[14]等有价值的信息。

2018年, Becher等^[25]报道了多维2D-TPP策略, 又称为蛋白质组整体溶解度改变(proteome integral solubility alteration, PISA)^[26]。该策略将每个热反应步骤的样本集中到一个样本中分析, 能够有效减少样本消耗、缩短MS仪器运行时间、同时保留来自2D格式的大部分信息^{[17, 26]}。PISA相较于TPP, 其通量和特异性进一步增加。在PISA中, 熔体曲线数据不是以2D格式的热图表示结果而是被压缩为ΔAUC, 即每个浓度下阴性对照和测试化合物之间的曲线下面积, 适用于用曲线拟合来探测蛋白质稳定性或溶解度的分析方法^[26]。样本的汇集往往也会带来一定的局限性: 一方面将导致非CETSA丰度效应信息的丢失, 例如蛋白表达水平或降解的变化, 这可以通过制备相同处理的未加热样品和对照样品来克服^[26]。另一方面将很难检测到微小的ΔAUC位移和具有非S型熔化模式的蛋白质, 为了克服这一问题, Li等^[27]通过模拟和实验表明只选择三种温度下的样本进行集中可以提高分辨率, 并有助于找到独特的曲线位移。

4 高通量技术与CETSA结合

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CETSA鉴定分子与靶点相互作用的效用促进了高通量CETSA技术(high-throughput cellular thermal shift assays, HT-CETSA) 的出现^[28]。多种HT-CETSA格式的发展而产生的数据集为研究化合物的生物化学活性和感兴趣靶点的细胞机制提供了依据^[28]。

Shaw等^[6]采用了HT-CETSA一次性对多个样本进行测定来提高检测通量。最近的研究表明^[28], 使用纳米荧光素酶报告基因和AlphaLISA测定法等技术可以在标准的96孔、384孔和1536孔微量滴定板格式中进行HT-CETSA。目前HT-CETSA检测已成功应用于30多个靶点, 包括定位于细胞核、线粒体、细胞质和质膜的靶点^[29]。

Owens等^[30]开发了一种基于声学反相蛋白阵列的HT-CETSA策略(acoustic reversed-phase protein arrays-based high-throughput cellular thermal shift assays, HT-CETSA-aRPPA) 来测量96孔或384孔板中未修饰的蛋白质。该方法利用声滴喷射将纳升体积的裂解物直接转移到硝酸纤维素膜上, 消除了SDS-PAGE分离蛋白质的需求, 可以在单个膜上检测多达1 536个裂解物样品, 大大节省了时间和资源。HT-CETSA-aRPPA提供了一个平台, 可用于分析多个类别的靶点和大量样本, 能够通过筛选激酶抑制剂的剂量-反应图谱来检测MEK1靶点的结合^[13]。

HT-CETSA可以用于从高通量筛选中选择和表征化合物, 并通过促进剂量-反应实验来优化先导化合物的优先级。Feng等^[13]使用这种方法从一组896个激酶抑制剂中鉴定出13个化合物, 其中8个是B-Raf抑制剂。这表明B-Raf的HT-CETSA实验可以用于筛选和鉴定B-Raf缀合物。该法还被用于在内源性表达雄激素受体(androgen receptor, AR) 的前列腺癌细胞系中筛选AR的直接结合物^[6], 实验确定了已知的AR拮抗剂和激动剂并提供了明显的细胞内抑制常数。此外HT-CETSA还可以验证一个给定的假设靶点从而显著减少早期的时间和资源消耗、提高药物开发速率、增强对于疾病生物学的理解。除了筛选化合物文库外, HT-CETSA还可以用于结构-活性关系(structure-activity relationship, SAR) 研究^[21]。在进行SAR研究时, 首先要运行一个大型SAR库来对未发生结合和低结合的化合物进行排序, 然后用PISA来确定剩余测试化合物的共同靶点和脱靶蛋白。然而, 医药化学家是否可以利用CETSA的效价直接决定受SAR影响的配体并对感兴趣的化合物进行排序还有待研究^[28]。

5 基于发光的技术和CETSA结合

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基于发光的检测策略具有高灵敏度、背景干扰小和适用范围较广等优点, 其在早期药物发现中起着至关重要的作用^[29]。这类技术的发展前沿包括均相光激化学发光免疫分析(amplified luminescent proximity homogeneous assay, Alpha) 和纳米生物发光共振能量转移(nano bioluminescence resonance energy transfer, NanoBRET) 技术。这两种技术的原理基本相同, 都涉及邻近状态下的发光能量转移: 激发态能量从供体转移到受体且能量转移的效率取决于供受体间的距离。不同的是二者使用不同的供体和受体对: NanoBRET使用荧光素酶和荧光团对, 而Alpha技术使用供体和受体微珠分别产生和检测单线态氧。此外二者的检测能力也不同: Alpha技术能测定最大距离为200 nm的相互作用而BRET技术的最大测定距离约为10 nm^[31], 即Alpha技术比BRET技术测定范围更广, 两者在检测范围方面具有一定的互补作用。这两种方法都可以应用于检测蛋白质-蛋白质和蛋白质-小分子相互作用, 也可用于生物样品中的高通量化合物筛选, 这为分析细胞裂解物等环境中复合物的分子生物物理特性提供了许多可能性。

5.1 均相光激化学发光免疫分析和细胞热位移分析结合

Alpha技术有两种形式: AlphaScreen和AlphaLISA。二者使用相同的供体微珠, 但所用的受体微珠的发射光谱不同。AlphaScreen微珠具有较宽的发射光波长范围, 其区间在520~620 nm; 而AlphaLISA微珠的发射光波长仅为615 nm, 受500~600 nm吸收光干扰的可能性更低, 因此AlphaLISA对血清和血浆样品的干扰较小且灵敏度更高, 可作为复杂的酶联免疫吸附测定或细胞内蛋白质免疫测定的良好替代方法。

结合了Alpha技术的CETSA即Alpha-CETSA (amplified luminescent proximity homogeneous assay-cellular thermal shift assay) 使用基于双抗体的接近系统将靶点结合转换为可读的信号以检测未发生沉淀而保持稳定的蛋白质。该方法先用感兴趣的化合物与蛋白样品共同孵育, 随后对体系进行加热, 加热完成后向体系加入Alpha-CETSA检测试剂盒以使蛋白质和化合物分别与Alpha受体珠或供体珠标记的抗体连接。在发生靶点结合的情况下供体微珠和受体微珠会相互靠近, 产生均匀的Alpha信号且信号强度与样品中可溶性蛋白的量成正比。相反, 未与化合物结合的蛋白经加热后将发生变性沉淀而不产生Alpha信号。美国PerkinElmer公司提供了一种包含抗小鼠免疫球蛋白G供体和抗兔免疫球蛋白G受体珠的Alpha-CETSA TE工具箱检测试剂盒, 该试剂盒能够在无需洗涤和分离的步骤下快速、灵敏、定量地检测目标蛋白。Chen等^[32]利用AlphaLISA-CETSA技术在体外和微量小鼠异种移植肿瘤中测定了CB-839与结肠癌细胞系中的谷氨酰胺酶特异性结合, 此外还评估了CB-839在肿瘤活检中的药效学。

由于基于微珠的化学检测相对容易实现自动化, 因此可被用于高通量CETSA实验^{[33, 34]}。Alpha-HT-CETSA能够使用均质分析平台以可靠和高通量的模式检测并筛选感兴趣的靶点。微量滴定板形式的Alpha-HT-CETSA首先在HL60活细胞中使用人蛋白激酶p38α进行了测定^[34]。为了验证测量结果的可靠性, 将Alpha-HT-CETSA与传统Western blotting方法的结果数据进行比较发现两种检测方法的T_m和EC₅₀值具有较好的相关性^[33]。该法已实际应用于人类胸苷酸合成酶的复合筛选^[35], 能在一天内对10 000多种化合物进行筛选并最终确定了65种稳定的化合物。

5.2 纳米生物发光共振能量转移技术和CETSA结合

NanoBRET能够验证并实时定量地研究靶点与活细胞中小分子之间的相互作用, 是临床前药物发现的重要途径。NanoBRET与Alpha法最大的不同在于它需要表达荧光素酶标记的靶蛋白细胞系和荧光素酶的底物^[21]。其原理是配体与目标蛋白结合后蛋白质的构象改变, 荧光素酶活性降低^[36]从而改变了荧光信号强度, 根据荧光信号强度的变化可确定是否发生了靶点结合。目前NanoBRET-CETSA技术包含HiBiT热位移分析(BiTSA) 和分裂纳米荧光素酶热位移分析(split nano luciferase cellular thermal shift assay, SplitLuc CETSA)。

Mortison等^[37]结合CETSA和部分NanoBRET开发了一种称为BiTSA的无抗体高通量检测系统。BiTSA使用CRISPR/Cas9基因编辑技术将11-氨基酸肽标签HiBiT连接到靶蛋白和加热后添加到实验体系的互补伙伴LgBiT之间。HiBiT和LgBiT连接形成的复合物具有荧光素酶活性, 能产生生物发光, 且荧光素酶信号随与目标蛋白结合的配体数量增多而呈线性衰减^{[36, 37]}; 当靶点蛋白变性时LgBiT不能与HiBiT结合, 因此不会产生荧光信号。

Martinez等^[38]提出了一个同质、标准化、靶点独立和高通量的CETSA平台: SplitLuc CETSA。文章中具体描述了该法的开发和验证过程, 同时使用SplitLuc CETSA试验初步筛选了乳酸脱氢酶A和细胞周期依赖蛋白激酶9的抑制剂。该方法广泛适用于多种靶点、能为中通量和高通量应用提供靶点结合研究的依据、为表型或其他基于细胞的检测方法不容易获得的靶点提供快速检测开发和筛选的平台^[21]。

6 药物亲和反应靶稳定性和CETSA结合

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药物亲和反应靶稳定性(drug affinity response target stability, DARTS) 的原理是小分子药物与其靶点蛋白结合后, 靶蛋白对蛋白酶的敏感性下降从而保护了靶蛋白的活性和功能。由于无需药物保护性修饰且无药物活性依赖性, DARTS可广泛应用于药物筛选与靶点鉴定。近年来随着DARTS技术的发展, 对该方法的优化研究不断涌现。DARTS结合CETSA的方法可根据药物处理后不同蛋白在酶作用下的降解差异来分析靶点, 该法在提高了数据分析能力的同时还减少了蛋白水解条件的测试次数^[39]。此外, 该方法便于分析可能的蛋白质相互作用, 更有利于预测小分子的疗效和改进剂量。当然这种方法也有一定的局限性, 如对靶蛋白的丰度要求较高、一些非常敏感的蛋白或对蛋白酶有高抗性的蛋白可能会导致靶信息丢失。

7 特定蛋白靶点的鉴定技术和CETSA结合

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对于特定蛋白靶点的研究有助于深刻了解疾病的机制, 然而目前对于蛋白-蛋白相互作用(protein-protein interactions, PPI) 的鉴定技术仍然有限。已知Keap1和Nrf2的PPI与细胞氧化应激反应相关, 是许多自身免疫疾病、神经退行性疾病以及癌症的重要治疗靶点。Dayalan Naidu等^[10]为了促进细胞环境中作用于Keap1-Nrf2的PPI界面的小分子抑制剂的识别和验证, 详细介绍了两种细胞热位移实验方案。其中一种是基于免疫印迹的方法Keap1-CETSA, 用于检测内源性Keap1。另外一种是他们开发的基于中通量微量滴定板的筛选法Keap1-glow CETSA, 该方法为识别靶向Keap1的小分子提供了平台, 且在较宽的温度范围内不需要执行费力耗时的半定量Western blotting。研究中使用基于荧光的微量滴定板法检测过表达的荧光标记Keap1, 药物抑制Keap1-Nrf2复合物的PPI界面导致Nrf2的激活和细胞保护基因的表达, 这为疾病的预防和治疗提供了一种有前途的策略。Keap1-glow CETSA有潜力区分Keap1抑制剂的作用模式但可能不适用于其他Keap1抑制剂, 如Keap1中可逆结合半胱氨酸传感器的亲电试剂^[10]。

8 CETSA技术数据处理的进展

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CETSA实验数据分析常通过比较化合物处理前后热熔曲线中T_m的差异(ΔT_m) 来确定靶蛋白, 但是这种方法可能会导致假阴性^[3]。Childs等^[40]提出用响应曲线的非参数分析(nonparametric analysis of response curves, NPARC) 来替代传统的数据分析方法, 它不依赖于ΔT_m而是通过比较整个熔解曲线来确定靶蛋白, 因此数据分析的敏感性和特异性都大大提升。类似的还有PISA^[26], 该法利用蛋白质的丰度差异即曲线下面积的差异ΔS_m寻找化合物靶点。一般而言蛋白的ΔT_m或ΔS_m越大, 越可能是化合物的靶点。Gaetani等^[26]还利用1D PISA和2D PISA中构建的Ft函数和Fc函数综合对所有的靶蛋白进行优先等级排序以选择最接近真实的靶点。Fang等^[41]建立了贝叶斯半参数模型, 可对偏离预测的S形熔解曲线的蛋白质进行全面分析。Kurzawa等^[24]也开发了一种针对结果数据进行错误发生率(false discovery rate, FDR) 控制分析的统计方法, 利用该方法在先前发表的2D-TPP数据集中发现了帕比司他的新靶点。目前常用的CETSA数据处理R软件包^[7]有: mine CETSA^[42]、TPP^[43]、mstherm (https://CRAN.R-project.org/pa-ckage=mstherm) 等。

9 CETSA技术的不足与展望

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CETSA技术作为一种非修饰化学蛋白组学方法, 利用配体与靶蛋白结合后蛋白的热稳定性会发生改变的原理来鉴定化合物的靶点。其操作简便、适用范围广、稳定性好、兼容性强, 能与其他技术结合应用于整个药物开发链。此外, CETSA能在保持实验条件恒定的情况下对中药未知药效成分进行靶点研究^[12]、帮助确定合适的药物剂量、检测获得性耐药性等^[1]。当前, CETSA技术已用于多种疾病药物的开发和治疗研究, 详见表 1^[44-61]。

但是该技术也存在一定的局限性, 主要表现在以下几个方面:

① 存在假阳性^[62]。这种结果可由热邻近共聚合(thermal proximity coaggregation, TPCA) 现象和加热后细胞的膜通透性改变造成。TPCA增加了假阳性的发生率, 已知多维MS-CETSA方法有助于发现热邻近共聚合现象从而提高蛋白质靶点检测的灵敏度和准确性。加热会改变细胞膜的通透性, 原本不能进入细胞的药物可能会进入细胞结合胞浆内蛋白, 导致后续结合蛋白鉴定时出现假阳性结果。目前可使用较温和的NP-40裂解液代替蛋白裂解液从而保持膜蛋白的完整。

② 非特异性蛋白质的干扰。一些低丰度的靶向蛋白可能会被高丰度的非靶向蛋白所淹没, 即使利用“鸟枪法”定量蛋白组学也很难检测出低丰度的靶向蛋白^[11]。

③ 某些蛋白质与化合物的结合不影响热稳定性, 这可以归因于配体和蛋白质相互作用的特殊性质^[62]。例如, 当蛋白质的展开是由蛋白质结构域决定的, 而蛋白质结构域不受化合物结合的影响; 或者当蛋白质靶点与另一蛋白质发生相互作用后更稳定而这种相互作用又不受有机分子结合的影响时, 熔解曲线的偏移就不太可能发生。通过设计尺寸更小的报告结构域融合结构可解决此类问题。据报道, CRISPR/Cas9可以方便地将标签插入到与疾病相关的细胞系甚至是用于HT-CETSA的原代细胞, 这为HT-CETSA提供了另一种尚未实现的途径。

④ 大多数蛋白质在标准温度范围(40~75 ℃) 内熔化, 而有些蛋白质需要更高的温度, 如白蛋白和超氧化物歧化酶1^{[62, 63]}。某些蛋白质需要极端的温度来检测, 这可能会显著影响蛋白质^[64]的其他部分。

⑤ 一些膜蛋白和一些可溶性蛋白在CETSA过程中可能不发生反应。例如整体跨膜蛋白天生就很难转移, 在化合物结合时只产生很小的热稳定性变化。这通常可以通过适当优化这些个别靶点的分析条件(主要是选择洗涤剂) 来克服。

此外, 关于CETSA和相关技术联用的优缺点与应用范围的总结见表 2^{[1, 3, 5, 10, 11, 13, 14, 17, 23-26, 29, 33, 34, 36, 37, 39]}。

10 总结

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对于细胞内研究, 区分药物的直接相互作用和下游作用仍然存在挑战, CETSA的未来发展趋势是紧密联系MS技术, 采用更先进的多路复用技术、着重于减少样本消耗量、增大样本适用性以实现在珍贵和相关的材料中进行分析, 提高分析效率。同时, 一种方法获得的信息是有限的, 使用多学科的方法来综合分析是化合物靶点鉴定的未来发展趋势^[62]。有研究^[64]整合了细胞热位移分析、定量蛋白质组学、代谢组学、计算机辅助对接和靶点验证方法以演示交互组学工作流的开发, 简化了多组学数据中的冗余信息, 并确定了一种环境毒物代谢产物: 邻苯二甲酸单乙基己酯的新蛋白靶点。除交互组学工作流外, 通过改变影响蛋白质稳定性的因素对于研究靶点结合也具有重要的意义。蛋白质的稳定性不但与自身结构有关, 也与存储环境(pH、温度、盐离子浓度、表面活性剂等) 相关。当前已有许多研究通过改变蛋白所处的环境辅助鉴定靶点, 而对于通过改变蛋白质结构帮助鉴定靶点的研究较少。如果将能影响氢键、二硫键、范德华力等相互作用力的技术与CETSA结合, 可能有利于提升靶点鉴定技术的应用范围, 扩宽CETSA的应用领域。

作者贡献: 孔德志和张炜提供文章思路、指导文章撰写及修改; 刘广苑参与文章框架的构思, 并负责文章的撰写; 李亚慧对论文进行了检查和部分修改。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然科学基金资助项目(82174004)
国家重点研发计划“中医药现代化”重点专项(2022YFC3500501)

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2024年第59卷第1期

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doi: 10.16438/j.0513-4870.2023-0415

接收时间：2023-04-06
首发时间：2025-11-28
出版时间：2024-01-12

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出版历史

收稿日期：2023-04-06
修回日期：2023-07-17

基金

国家自然科学基金资助项目(82174004)

国家重点研发计划“中医药现代化”重点专项(2022YFC3500501)

作者信息

河北医科大学中西医结合研究所, 河北石家庄 050017

通讯作者:

^*张炜, E-mail: weizhang@hebmu.edu.cn;

孔德志, E-mail: kongdezhi@hebmu.edu.cn

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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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Disease	Type	Sample	Conclusion	Reference
Cancer	Liver cancer	Hep3B cells	Chamaejasmenin E induces apoptosis in hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo	[44]
HepG2 cells	Hydroxethonine hydrochloride induces cellular senescence by regulating the LIF/AMPK pathway, thereby inhibiting the development and progression of hepatocellular carcinoma	[45]
Non-small cell carcinoma	A549 and H1299 cells	Evodiamine affects the Notch3 signaling pathway through inhibition of γ-secretase, thereby inhibiting non-small cell lung carcinogenesis	[46]
A549, H1299, and HLF cells	Cucurbitacin B exerts anti-non-small cell lung cancer effects by initiating cell pyroptosis	[47]
Colorectal cancer	HCT116, HT29, and SW480 cells	The interaction between Zuojin capsule and seven intracellular targets verified the mechanism of inhibition of colorectal cancer	[48]
Prostate cancer	293T, LNCaP, 22Rv1, C4-2, PC3, and DU145 cells	Found an inhibitor of the orphan receptor COUP-TFII to treat prostate cancer by inhibiting receptor activity	[49]
Mammary cancer	MCF-7 cells	Asiatic acid achieves anticancer effects against doxorubicin-resistant breast cancer cells through an AMPK dependent pathway	[50]
Tumour	Myelomatosis	H929 and U266 cells	Adenin specifically binds IKKβ thereby inhibiting NF-κB activation and inhibiting multiple myeloma cell proliferation	[51]
Osteosarcoma	HOS-MNNG, KHOS, MG63, U2OS, SJSA-1, G292, Cal72, and 143B cells	The MTH1 inhibitor TH1579 binds to MTH1, indicating the therapeutic effect of TH1579 on human osteosarcoma cells	[52]
Glioblastoma	U251 and U87 cells	Discovery of a novel KHS101 analog as a TACC3 inhibitor for the treatment of glioblastoma	[53]
Nervous system diseases	Neurodegenerative disease	BV-2 and HEK293T cells	Eupalinolide B can target the noncatalytic domain of ubiquitin-specific protease 7 to inhibit neuroinflammation and treat neurodegenerative diseases	[54]
Depression	Whole brain of DBA/2J and DBA/2 Ola mice	Found a novel binding target of the selective serotonin reuptake inhibitor paroxetine, the phosphofructokinase protein, to investigate its antidepressant mechanisms	[55]
Neuralgia	BV-2 cells	The TLR4 antagonist lovastatin inhibits TLR4 signaling by binding to myeloid differentiation protein 2, a coreceptor of TLR4	[56]
Organ disease	Heart	RAW264.7 cells	Iminostilbene targeting pyruvate kinase isoenzyme M2 reduces macrophage inflammation, thereby significantly attenuates myocardial ischemia/reperfusion injury	[57]
Liver	LO2 cells	Potential targets of heterotexin for acute liver injury are PTEN, PI3K, and BiP	[58]
LX-2 cells and mHSCs	18β-Glycyrrhetinic acid ameliorates liver fibrosis by inducing ROS- mediated apoptosis by targeting PRDX1/2 in cells	[59]
Kidney	mTECs	The TGF-β type I receptor inhibitor AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway	[60]
Vascellum	Dami cell	Proanthocyanidin A1 promotes platelet production by activating the JAK2/STAT3 pathway to improve chemotherapy-induced thrombocytopenia	[61]

Disease

Type

Sample

Conclusion

Reference

Cancer

Liver cancer

Hep3B cells

Chamaejasmenin E induces apoptosis in hepatocellular carcinoma cells by targeting c-Met in vitro and in vivo

[44]

HepG2 cells

Hydroxethonine hydrochloride induces cellular senescence by regulating the LIF/AMPK pathway, thereby inhibiting the development and progression of hepatocellular carcinoma

[45]

Non-small cell carcinoma

A549 and H1299 cells

Evodiamine affects the Notch3 signaling pathway through inhibition of γ-secretase, thereby inhibiting non-small cell lung carcinogenesis

[46]

A549, H1299, and HLF cells

Cucurbitacin B exerts anti-non-small cell lung cancer effects by initiating cell pyroptosis

[47]

Colorectal cancer

HCT116, HT29, and SW480 cells

The interaction between Zuojin capsule and seven intracellular targets verified the mechanism of inhibition of colorectal cancer

[48]

Prostate cancer

293T, LNCaP, 22Rv1, C4-2, PC3, and DU145 cells

Found an inhibitor of the orphan receptor COUP-TFII to treat prostate cancer by inhibiting receptor activity

[49]

Mammary cancer

MCF-7 cells

Asiatic acid achieves anticancer effects against doxorubicin-resistant breast cancer cells through an AMPK dependent pathway

[50]

Tumour

Myelomatosis

H929 and U266 cells

Adenin specifically binds IKKβ thereby inhibiting NF-κB activation and inhibiting multiple myeloma cell proliferation

[51]

Osteosarcoma

HOS-MNNG, KHOS, MG63, U2OS, SJSA-1, G292, Cal72, and 143B cells

The MTH1 inhibitor TH1579 binds to MTH1, indicating the therapeutic effect of TH1579 on human osteosarcoma cells

[52]

Glioblastoma

U251 and U87 cells

Discovery of a novel KHS101 analog as a TACC3 inhibitor for the treatment of glioblastoma

[53]

Nervous system diseases

Neurodegenerative disease

BV-2 and HEK293T cells

Eupalinolide B can target the noncatalytic domain of ubiquitin-specific protease 7 to inhibit neuroinflammation and treat neurodegenerative diseases

[54]

Depression

Whole brain of DBA/2J and DBA/2 Ola mice

Found a novel binding target of the selective serotonin reuptake inhibitor paroxetine, the phosphofructokinase protein, to investigate its antidepressant mechanisms

[55]

Neuralgia

BV-2 cells

The TLR4 antagonist lovastatin inhibits TLR4 signaling by binding to myeloid differentiation protein 2, a coreceptor of TLR4

[56]

Organ disease

Heart

RAW264.7 cells

Iminostilbene targeting pyruvate kinase isoenzyme M2 reduces macrophage inflammation, thereby significantly attenuates myocardial ischemia/reperfusion injury

[57]

Liver

LO2 cells

Potential targets of heterotexin for acute liver injury are PTEN, PI3K, and BiP

[58]

LX-2 cells and mHSCs

18β-Glycyrrhetinic acid ameliorates liver fibrosis by inducing ROS- mediated apoptosis by targeting PRDX1/2 in cells

[59]

Kidney

mTECs

The TGF-β type I receptor inhibitor AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway

[60]

Vascellum

Dami cell

Proanthocyanidin A1 promotes platelet production by activating the JAK2/STAT3 pathway to improve chemotherapy-induced thrombocytopenia

[61]

Technology combination	Advantage	Disadvantage	Application	Reference
MS-CETSA	High throughput, high sensitivity, and good stability	There are limitations in detecting low-abundance proteins and proteins in complex mixtures, potentially leading to false negatives and incomplete proteome coverage	Proteome analysis Analysis of protein structural changes induced by drugs or other small molecules Development of antimicrobial drugs, drug mechanism of action, new efficacy, toxicity biomarkers or therapeutic drug targets	[1, 11, 13, 14]
ITDR-CETSA	Simple operation, a wide range of application	There are limitations in identifying targets with altered post-translational modifications or low-affinity interactions	Determine the optimal drug dose and monitor drug resistance	[11, 34]
2D-TPP	Compared with TPP, its analysis speed, sensitivity, specificity and accuracy are greatly improved The false positive rate was low	The generation of the spectrum map requires a high sample concentration, which is not convenient for large-scale screening The profiles of the multicomponent complexes are very complex and difficult to resolve	High-efficiency screening and optimization of drug candidates Acquired drug resistance study	[3, 13, 14, 23, 24]
PISA	Compared to TPP, the flux and specificity are further increased, which can effectively reduce sample consumption, shorten the running time of MS instruments, while retaining most of the information from 2D format	Loss of information on the non-CETSA abundance effect It would be difficult to detect small ΔAUC shifts and proteins with non-S type melting patterns	High-throughput, rapid analysis of compound binding to the target site For target studies of rare or precious samples	[17, 25, 26]
Alpha-CETSA	High sensitivity, small background interference, and a wide range of application than the BRET technology Less interfering and more sensitive for both serum and plasma samples	Specific donor-acceptor pairs are required	Detection of both protein-protein and protein-small molecule interactions High-throughput compound screening in biological samples	[29, 33, 34]
SplitLuc-CETSA	High sensitivity, small background interference, and a wide range of application	Specific donor-acceptor pairs are required It is not as applicable as Alpha	Detection of both protein-protein and protein-small molecule interactions High-throughput compound screening in biological samples	[29, 36, 37]
DARTS-CETSA	Improve the data analysis ability and reduce the number of proteolysis conditions Facilitate the analysis of the possible protein-protein interactions	The high abundance requirement of target proteins, some very sensitive proteins or proteins highly resistant to proteases may lead to the loss of target information	It is beneficial to predict the efficacy of small molecules and improve the dosage Drug screening and target identification	[5, 39]
Keap1-glow CETSA	Fast analysis speed, good specificity	The operation is more complex	Study of specific protein targets Providing strategies for disease prevention and treatment	[10]

Technology combination

Advantage

Disadvantage

Application

Reference

MS-CETSA

High throughput, high sensitivity, and good stability

There are limitations in detecting low-abundance proteins and proteins in complex mixtures, potentially leading to false negatives and incomplete proteome coverage

[1, 11, 13, 14]

ITDR-CETSA

Simple operation, a wide range of application

There are limitations in identifying targets with altered post-translational modifications or low-affinity interactions

Determine the optimal drug dose and monitor drug resistance

[11, 34]

2D-TPP

Compared with TPP, its analysis speed, sensitivity, specificity and accuracy are greatly improved
The false positive rate was low

High-efficiency screening and optimization of drug candidates
Acquired drug resistance study

[3, 13, 14, 23, 24]

PISA

Loss of information on the non-CETSA abundance effect
It would be difficult to detect small ΔAUC shifts and proteins with non-S type melting patterns

High-throughput, rapid analysis of compound binding to the target site
For target studies of rare or precious samples

[17, 25, 26]

Alpha-CETSA

High sensitivity, small background interference, and a wide range of application than the BRET technology
Less interfering and more sensitive for both serum and plasma samples

Specific donor-acceptor pairs are required

Detection of both protein-protein and protein-small molecule interactions
High-throughput compound screening in biological samples

[29, 33, 34]

SplitLuc-CETSA

High sensitivity, small background interference, and a wide range of application

Specific donor-acceptor pairs are required
It is not as applicable as Alpha

Detection of both protein-protein and protein-small molecule interactions
High-throughput compound screening in biological samples

[29, 36, 37]

DARTS-CETSA

Improve the data analysis ability and reduce the number of proteolysis conditions
Facilitate the analysis of the possible protein-protein interactions

The high abundance requirement of target proteins, some very sensitive proteins or proteins highly resistant to proteases may lead to the loss of target information

It is beneficial to predict the efficacy of small molecules and improve the dosage
Drug screening and target identification

[5, 39]

Keap1-glow CETSA

Fast analysis speed, good specificity

The operation is more complex

Study of specific protein targets
Providing strategies for disease prevention and treatment

[10]