药学学报

Gene	Protein	Heat inducible	Subcellular localization	Homology (to HSPA1A)/%
HSPA1A	Hsp70-1A, Hsp72	Yes	Cytoplasm/nucleus	100
HSPA1L	Hsp70-1L	No	Cytoplasm/nucleus	89
HSPA1B	Hsp70-1B	Yes	Cytoplasm/nucleus	99
HSPA2	Hsp70-2	No	Cytoplasm/nucleus	85
HSPA5	Bip, Grp78	Yes	Endoplasmic reticulum	64
HSPA6	Hsp70-6	Yes	Cytoplasm/nucleus	83
HSPA8	Hsc70, Hsp70-8	No	Cytoplasm/nucleus	87
HSPA9	mtHsp70, Grp75, mortalin	No	Mitochondria	52
HSPA7	Hsp70-7	N/A	Cytoplasm/nucleus	33
HSPA12A	Hsp70-12A	N/A	Cytoplasm/nucleus	29
HSPA12B	Hsp70-12B	N/A	Cytoplasm/nucleus	25
HSPA13	Hsp70-13	N/A	Cytoplasm/nucleus	40
HSPA14	Hsp70-14	N/A	Cytoplasm/nucleus	35

Gene

Protein

Heat inducible

Subcellular localization

Homology (to HSPA1A)/%

HSPA1A

Hsp70-1A, Hsp72

Yes

Cytoplasm/nucleus

100

HSPA1L

Hsp70-1L

Cytoplasm/nucleus

HSPA1B

Hsp70-1B

Yes

Cytoplasm/nucleus

HSPA2

Hsp70-2

Cytoplasm/nucleus

HSPA5

Bip, Grp78

Yes

Endoplasmic reticulum

HSPA6

Hsp70-6

Yes

Cytoplasm/nucleus

HSPA8

Hsc70, Hsp70-8

Cytoplasm/nucleus

HSPA9

mtHsp70, Grp75, mortalin

Mitochondria

HSPA7

Hsp70-7

N/A

Cytoplasm/nucleus

HSPA12A

Hsp70-12A

N/A

Cytoplasm/nucleus

HSPA12B

Hsp70-12B

N/A

Cytoplasm/nucleus

HSPA13

Hsp70-13

N/A

Cytoplasm/nucleus

HSPA14

Hsp70-14

N/A

Cytoplasm/nucleus

Gene	Protein	Heat inducible	Subcellular localization	Homology (to HSPA1A)/%
HSPA1A	Hsp70-1A, Hsp72	Yes	Cytoplasm/nucleus	100
HSPA1L	Hsp70-1L	No	Cytoplasm/nucleus	89
HSPA1B	Hsp70-1B	Yes	Cytoplasm/nucleus	99
HSPA2	Hsp70-2	No	Cytoplasm/nucleus	85
HSPA5	Bip, Grp78	Yes	Endoplasmic reticulum	64
HSPA6	Hsp70-6	Yes	Cytoplasm/nucleus	83
HSPA8	Hsc70, Hsp70-8	No	Cytoplasm/nucleus	87
HSPA9	mtHsp70, Grp75, mortalin	No	Mitochondria	52
HSPA7	Hsp70-7	N/A	Cytoplasm/nucleus	33
HSPA12A	Hsp70-12A	N/A	Cytoplasm/nucleus	29
HSPA12B	Hsp70-12B	N/A	Cytoplasm/nucleus	25
HSPA13	Hsp70-13	N/A	Cytoplasm/nucleus	40
HSPA14	Hsp70-14	N/A	Cytoplasm/nucleus	35

Gene

Protein

Heat inducible

Subcellular localization

Homology (to HSPA1A)/%

HSPA1A

Hsp70-1A, Hsp72

Yes

Cytoplasm/nucleus

100

HSPA1L

Hsp70-1L

Cytoplasm/nucleus

HSPA1B

Hsp70-1B

Yes

Cytoplasm/nucleus

HSPA2

Hsp70-2

Cytoplasm/nucleus

HSPA5

Bip, Grp78

Yes

Endoplasmic reticulum

HSPA6

Hsp70-6

Yes

Cytoplasm/nucleus

HSPA8

Hsc70, Hsp70-8

Cytoplasm/nucleus

HSPA9

mtHsp70, Grp75, mortalin

Mitochondria

HSPA7

Hsp70-7

N/A

Cytoplasm/nucleus

HSPA12A

Hsp70-12A

N/A

Cytoplasm/nucleus

HSPA12B

Hsp70-12B

N/A

Cytoplasm/nucleus

HSPA13

Hsp70-13

N/A

Cytoplasm/nucleus

HSPA14

Hsp70-14

N/A

Cytoplasm/nucleus

Hsp70 inhibitors	Targeted Hsp70s^*	Mode of actions	Refs
VER-155008, compounds 2 and 8	Hsp72, Hsp70, Bip	ATP competitive	[33-36]
Apoptozole, Az-TPP-O3	Hsp72, Hsc70	ATP competitive	[37, 38]
Hsp70-36	Hsp72	ATP competitive	[39]
MKT-077 and JG series	mtHsp70, Hsc70	Allosteric	[40-42]
YK5	Hsp72, Hsc70, Hsp70-6	Allosteric	[43-45]
HS-72	Hsp72	Allosteric	[46]
Oridonin	Hsp72	Allosteric	[47]
MAL3-101 and 115-7c	Hsp72, Hsc70	Hsp70-JDPs PPIs	[18, 48]
S1g-2, S1g-6 and S1g-10	Hsp72, Hsc70, mtHsp70	Hsp70-Bim PPIs	[49, 50]
HA-15	Bip	Unknown	[51]
Azure C, methylene blue and myricetin	Hsc70	Unknown	[52]
PES, PES-Cl, PET-16 and AP-4-139B	Hsp72	Allosteric	[53]
Novolactone	Hsp72, Hsc70, Bip	Allosteric	[54]
Ritterostatin Gn1N	Bip	Unknown	[55]
Hexachlorophene	Bip	SBD-peptide Interactions	[56]
Compound 8	Bip	SBD-peptide Interactions	[57]
15-Deoxyspergualin	Hsc70	EEVD	[58]
AEAC	Unknown	Unknown	[59]

Hsp70 inhibitors

Targeted Hsp70s^*

Mode of actions

Refs

VER-155008, compounds 2 and 8

Hsp72, Hsp70, Bip

ATP competitive

[33-36]

Apoptozole, Az-TPP-O3

Hsp72, Hsc70

ATP competitive

[37, 38]

Hsp70-36

Hsp72

ATP competitive

[39]

MKT-077 and JG series

mtHsp70, Hsc70

Allosteric

[40-42]

YK5

Hsp72, Hsc70, Hsp70-6

Allosteric

[43-45]

HS-72

Hsp72

Allosteric

[46]

Oridonin

Hsp72

Allosteric

[47]

MAL3-101 and 115-7c

Hsp72, Hsc70

Hsp70-JDPs PPIs

[18, 48]

S1g-2, S1g-6 and S1g-10

Hsp72, Hsc70, mtHsp70

Hsp70-Bim PPIs

[49, 50]

HA-15

Bip

Unknown

[51]

Azure C, methylene blue and myricetin

Hsc70

Unknown

[52]

PES, PES-Cl, PET-16 and AP-4-139B

Hsp72

Allosteric

[53]

Novolactone

Hsp72, Hsc70, Bip

Allosteric

[54]

Ritterostatin Gn1N

Bip

Unknown

[55]

Hexachlorophene

Bip

SBD-peptide Interactions

[56]

Compound 8

Bip

SBD-peptide Interactions

[57]

15-Deoxyspergualin

Hsc70

EEVD

[58]

AEAC

Unknown

[59]

Hsp70 inhibitors	Targeted Hsp70s^*	Mode of actions	Refs
VER-155008, compounds 2 and 8	Hsp72, Hsp70, Bip	ATP competitive	[33-36]
Apoptozole, Az-TPP-O3	Hsp72, Hsc70	ATP competitive	[37, 38]
Hsp70-36	Hsp72	ATP competitive	[39]
MKT-077 and JG series	mtHsp70, Hsc70	Allosteric	[40-42]
YK5	Hsp72, Hsc70, Hsp70-6	Allosteric	[43-45]
HS-72	Hsp72	Allosteric	[46]
Oridonin	Hsp72	Allosteric	[47]
MAL3-101 and 115-7c	Hsp72, Hsc70	Hsp70-JDPs PPIs	[18, 48]
S1g-2, S1g-6 and S1g-10	Hsp72, Hsc70, mtHsp70	Hsp70-Bim PPIs	[49, 50]
HA-15	Bip	Unknown	[51]
Azure C, methylene blue and myricetin	Hsc70	Unknown	[52]
PES, PES-Cl, PET-16 and AP-4-139B	Hsp72	Allosteric	[53]
Novolactone	Hsp72, Hsc70, Bip	Allosteric	[54]
Ritterostatin Gn1N	Bip	Unknown	[55]
Hexachlorophene	Bip	SBD-peptide Interactions	[56]
Compound 8	Bip	SBD-peptide Interactions	[57]
15-Deoxyspergualin	Hsc70	EEVD	[58]
AEAC	Unknown	Unknown	[59]

Hsp70 inhibitors

Targeted Hsp70s^*

Mode of actions

Refs

VER-155008, compounds 2 and 8

Hsp72, Hsp70, Bip

ATP competitive

[33-36]

Apoptozole, Az-TPP-O3

Hsp72, Hsc70

ATP competitive

[37, 38]

Hsp70-36

Hsp72

ATP competitive

[39]

MKT-077 and JG series

mtHsp70, Hsc70

Allosteric

[40-42]

YK5

Hsp72, Hsc70, Hsp70-6

Allosteric

[43-45]

HS-72

Hsp72

Allosteric

[46]

Oridonin

Hsp72

Allosteric

[47]

MAL3-101 and 115-7c

Hsp72, Hsc70

Hsp70-JDPs PPIs

[18, 48]

S1g-2, S1g-6 and S1g-10

Hsp72, Hsc70, mtHsp70

Hsp70-Bim PPIs

[49, 50]

HA-15

Bip

Unknown

[51]

Azure C, methylene blue and myricetin

Hsc70

Unknown

[52]

PES, PES-Cl, PET-16 and AP-4-139B

Hsp72

Allosteric

[53]

Novolactone

Hsp72, Hsc70, Bip

Allosteric

[54]

Ritterostatin Gn1N

Bip

Unknown

[55]

Hexachlorophene

Bip

SBD-peptide Interactions

[56]

Compound 8

Bip

SBD-peptide Interactions

[57]

15-Deoxyspergualin

Hsc70

EEVD

[58]

AEAC

Unknown

[59]

热休克蛋白70小分子抑制剂的研究进展

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朱金言 , 何明慧 , 吴凡 , 余荧蓝 , 罗雷 ^* , 邵豪 ^*

药学学报 | 专题报道: 蛋白成熟与翻译后修饰的化学干预 2024,59(11): 2962-2974

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药学学报 | 专题报道: 蛋白成熟与翻译后修饰的化学干预 2024, 59(11): 2962-2974

热休克蛋白70小分子抑制剂的研究进展

全屏

朱金言, 何明慧, 吴凡, 余荧蓝, 罗雷^*, 邵豪^*

作者信息

西南大学药学院·中医药学院, 重庆 400715

通讯作者:

^*罗雷, Tel: 86-23-68251225, E-mail: haoshao@swu.edu.cn;

邵豪, Tel: 86-23-68251225, E-mail: drluolei@swu.edu.cn

Current status and perspectives of small molecule inhibitors of heat shock protein 70

Jin-yan ZHU, Ming-hui HE, Fan WU, Ying-lan YU, Lei LUO^*, Hao SHAO^*

Affiliations

College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China

出版时间: 2024-11-12 doi: 10.16438/j.0513-4870.2024-0462

文章导航

摘要

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热休克蛋白70 (heat shock protein 70, Hsp70) 是一类细胞中广泛存在的分子伴侣, 具有维持细胞内蛋白质稳态的功能。Hsp70在多种疾病的发生发展中也发挥了重要的作用, 包括癌症、神经退行性疾病和传染性疾病等, 是治疗这些疾病的潜在靶点, 因此有必要开发小分子抑制剂对这一重要靶点进行验证。近年来针对Hsp70小分子抑制剂的研究取得了显著进展, 通过不同方式抑制Hsp70功能的小分子化合物都有报道。本文简要介绍了Hsp70的结构域、共伴侣蛋白及相关的疾病, 系统总结了其小分子抑制剂的发现过程及作用特点, 以期为Hsp70小分子抑制剂的进一步研发提供参考。

关键词

热休克蛋白70 / 分子伴侣 / 小分子抑制剂 / 变构抑制剂 / 蛋白-蛋白相互作用

Abstract

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Heat shock protein 70 (Hsp70) is a class of molecular chaperones essential for maintaining protein homeostasis in cells. Hsp70s also play important roles in the pathogenesis of a variety of diseases, including cancer, neurodegenerative diseases and infectious diseases, which makes them potential targets for the treatment of these diseases. It is necessary to develop small molecule inhibitors to validate this class of important therapeutic targets. In recent years, the discovery of small molecule inhibitors for Hsp70s has made remarkable progress, and Hsp70 inhibitors with different modalities have been reported. In this paper, Hsp70 and relevant diseases are briefly introduced, and the discovery of Hsp70 small molecule inhibitors with distinct modalities are summarized, providing reference for the further discovery and development of Hsp70 small molecule inhibitors.

Key words

heat shock protein 70 / molecular chaperone / small molecule inhibitor / allosteric inhibitor / protein-protein interaction

引用本文

朱金言, 何明慧, 吴凡, 余荧蓝, 罗雷, 邵豪. 热休克蛋白70小分子抑制剂的研究进展. 药学学报, 2024 , 59 (11) : 2962 -2974 . DOI: 10.16438/j.0513-4870.2024-0462

Jin-yan ZHU, Ming-hui HE, Fan WU, Ying-lan YU, Lei LUO, Hao SHAO. Current status and perspectives of small molecule inhibitors of heat shock protein 70[J]. Acta Pharmaceutica Sinica, 2024 , 59 (11) : 2962 -2974 . DOI: 10.16438/j.0513-4870.2024-0462

正文

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热休克蛋白(heat shock protein, Hsp) 70是一种分子伴侣蛋白, 是细胞内维持蛋白质稳态的核心成员^[1]。Hsp70家族蛋白有13个成员, 研究较多的主要有组成型表达的Hsc70 (heat shock cognate 71-kDa protein)、应激诱导表达的Hsp72、内质网中的免疫球蛋白结合蛋白(immunoglobulin binding protein, Bip) 和线粒体中的mtHsp70 (mitochondrial hsp70), 其中Hsc70和Hsp72都位于细胞浆中^[2]。Hsp70与共伴侣蛋白J结构域蛋白(J-domain proteins, JDPs)、核苷酸交换因子(nucleotide exchange factors, NEFs) 和四肽重复蛋白(tetratricopeptide repeat proteins, TPRs) 协作通过多种功能来维持细胞内蛋白质稳态, 包括折叠新合成的蛋白质、重新折叠错误折叠的蛋白、增加蛋白聚集体的溶解性、协助蛋白质的跨膜转运、调控多蛋白复合物的形成和解离、通过蛋白酶体和自噬途径降解有害蛋白质等^[3]。

除了在正常的生理活动中发挥着重要的作用, Hsp70与癌症、神经退行性疾病和感染性疾病等的发生发展也密切相关, 因此Hsp70抑制剂的研究受到了很大的关注。早期的Hsp70抑制剂大多是有生物活性的天然产物或者人工合成小分子, 通过靶点垂钓发现其结合Hsp70。近些年报道的部分抑制剂则是通过计算机辅助设计或高通量筛选得到的^{[4, 5]}。Hsp70与共伴侣蛋白协同通过水解ATP发挥其功能, 因此有多种方式抑制Hsp70蛋白复合物, 包括ATP竞争性抑制剂、变构抑制剂和蛋白-蛋白相互作用(protein-protein interactions, PPIs) 抑制剂等。Hsp70抑制剂的发展进程远远落后于分子伴侣Hsp90, 目前已经报道的Hsp70抑制剂均处于临床前研究, 而Hsp90抑制剂TAS-116已在日本获批上市用于治疗胃肠道间质瘤^[6]。

本文首先简要介绍Hsp70的结构域、共伴侣蛋白及相关的疾病, 然后基于Hsp70的核苷酸结合域(nucleotide binding domain, NBD) 和底物结合域(substrate binding domain, SBD) 将其抑制剂分为两大类, 并总结了其小分子抑制剂的发现过程、作用方式及特点, 最后对该领域的发展方向进行展望。

1 Hsp70的结构域及其共伴侣蛋白

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1.1 Hsp70的结构域

Hsp70家族的所有成员均具有一个N端核苷酸结合域(NBD) 和一个C端底物结合域(SBD), 两者由一个短肽连接(图 1)^[3]。NBD由两个亚结构域Ⅰ和Ⅱ组成, 进一步划分为A区和B区。SBD由具有底物结合位点的β夹层结构域(β-SBD) 和α-螺旋盖子(α-SBD) 组成。Hsp70的NBD可以水解ATP, 而SBD则结合来自未折叠蛋白的疏水性肽段。SBD对序列的选择性较低, 可以结合大多数由非极性氨基酸组成的肽段。除了水解ATP, Hsp70发挥分子伴侣功能需要共伴侣蛋白的协助作用。

1.2 Hsp70的共伴侣蛋白及分子伴侣循环

Hsp70发挥其功能需要与共伴侣蛋白协同作用, 以调节其活性并决定底物蛋白的命运。人的基因组中有大约13种Hsp70 (表 1)、45种JDPs、14种NEFs和48种TPRs^{[7, 8]}, 分布在细胞浆和不同的细胞器中。基因及分布的多样性使得Hsp70能够形成多种复合物, 从而实现其功能的多样化。这些共伴侣蛋白通过PPIs调控Hsp70的功能, 其中JDPs和NEFs能够加速Hsp70水解ATP的活性, TPRs可作为衔接蛋白通过连接Hsp70和其他蛋白与相关通路相互作用^{[9, 10]}。共伴侣蛋白与Hsp70的相互作用调节了Hsp70的活性和底物结合能力, 从而参与了多种细胞过程。Hsp70及其共分子伴侣的循环主要包括以下几个步骤(图 2): ① JDPs与未折叠蛋白质或错误折叠蛋白结合并将其传递给Hsp70, 此时NBD与ATP结合, α-SBD呈开放状态, Hsp70与底物亲和力弱; ② JDPs促进ATP水解为ADP, 导致α-SBD关闭, 底物与SBD亲和力较强; ③ NEFs促进ADP替换为ATP, Hsp70释放正确折叠的蛋白质。

2 与Hsp70相关的疾病

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Hsp70与多种疾病的发生发展相关, 如癌症、神经退行性疾病和感染性疾病等(图 3)^[4], 对Hsp70与疾病关系的研究主要集中在上面提到的几个常见的Hsp70s。本文仅简单列举几个与Hsp70相关的疾病, 未详细总结各亚型和疾病的相关性。

2.1 癌症

癌细胞的异常增殖需要分子伴侣帮助其折叠生长所需的必须蛋白, 包括Hsp70、Hsp90和Hsp60等, 这一现象被称为“非致癌基因成瘾”^[11]。Hsp70在多种癌症中表达水平升高并与患者预后呈负相关, 包括乳腺癌、子宫内膜癌、口腔癌、结肠直肠癌、前列腺癌和白血病等^[4]。Hsp70通过多种机制促进癌症的发生发展, 包括抑制多种细胞凋亡途径、促进肿瘤免疫逃逸、促进血管生成和肿瘤转移等。Hsp70的过表达还与一些抗癌药物的耐药性相关, 如伊马替尼和顺铂等^{[12, 13]}。大部分研究发现需要同时敲除2个不同的Hsp70亚型才能导致癌细胞凋亡, 因此用于治疗癌症的小分子可能需要同时抑制至少两个不同的Hsp70亚型^[14]。目前报道的Hsp70抑制剂在肿瘤模型中研究得最多。

2.2 神经退行性疾病

阿尔茨海默病、帕金森病和亨廷顿氏症等神经退行性疾病共同的病理特征是错误折叠的蛋白质形成寡聚体或淀粉样纤维, 包括Tau、α-突触核蛋白和多聚谷氨酰胺蛋白等^{[15, 16]}。Hsp70与共伴侣蛋白参与调控这些蛋白质的稳态, 包括重新折叠错误折叠的蛋白质、抑制错误折叠蛋白质的聚集及降解错误折叠的蛋白质。鉴于Hsp70在调控这些底物蛋白稳态中的双重作用, 目前有两种思路来治疗神经退行性疾病。一种是提高Hsp70的活性, 可以通过小分子激活Hsp70或者基因过表达Hsp70来提高其蛋白水平^{[17, 18]}; 另一种是使用小分子化合物抑制Hsp70, 将Hsp70稳定在底物结合状态, 延长Hsp70与底物的结合时间, 使得热休克同源蛋白70羧基端相互作用蛋白(carboxyl terminus of Hsc70-interacting protein, CHIP) 能够将底物泛素化进而降解^[19]。Abisambra等^[19]发现仅抑制Hsc70就可以使tau蛋白降解, 并不会导致细胞凋亡, 因此治疗神经退行性疾病需要高选择性的Hsp70亚型抑制剂。

2.3 传染病

细菌中Hsp70的同源蛋白DnaK对细菌的生长至关重要, 对金黄色葡萄球菌和大肠杆菌生物被膜的形成也起着关键作用^[20]。文献^[20-23]中已有报道Dnak的小分子或者多肽抑制剂具有杀菌效果, 或增强已有抗生素的疗效。

病毒利用宿主细胞的营养物质、能量及蛋白质机器完成复制, 分子伴侣在其入侵、复制、组装和转运过程中起着重要作用。通过靶向宿主细胞的分子伴侣蛋白是预防和治疗多种病毒感染的潜在方法。通过抑制Hsp70来治疗黄病毒感染的研究相对较多, 尤其是寨卡病毒(Zika virus)。Hsp70及其共伴侣蛋白参与了寨卡病毒的入侵、复制和颗粒产生, Hsp70变构抑制剂MKT-077衍生物在小鼠模型中可以减慢病毒复制^{[24, 25]}。Hsp70在其他几种病毒感染过程中也是必不可少的, 包括乙肝病毒、甜菜黑色焦枯病毒、多瘤病毒和呼吸道合胞体病毒等^[26-29]。

2.4 其他疾病

一些研究发现Hsp70还与Ⅱ型糖尿病、风湿性关节炎和肺纤维化等有关^[30-32], 大部分仍处于探索阶段, Hsp70能否作为治疗这些疾病的靶点仍有待于进一步研究。

3 Hsp70抑制剂

收起

根据Hsp70的结构域将其抑制剂分为结合于NBD和SBD的化合物(表 2)^{[18, 33-59]}, 表中同时列出了文献中报道其结合的Hsp70亚型。

3.1 结合于NBD区域的化合物

结合在NBD区域的Hsp70抑制剂根据其作用方式可以分为3类: ATP竞争性抑制剂、变构抑制剂和蛋白-蛋白相互作用抑制剂(图 4)。目前仅有VER-155008系列化合物与蛋白的复合物结构被解析(图 1B)。

3.1.1 ATP竞争性抑制剂VER-155008

由于Hsp70对ATP和ADP的结合能力很强(K_d~500 nmol·L^-1), Williamson等^[33]以腺苷为先导化合物基于结构优化得到VER-155008 (1, 图 4), 该化合物是第一个靶向Hsp70 ATP结合口袋的Hsp70抑制剂。表面等离子共振实验表明VER155008与Hsp72的K_d值为0.3 µmol·L^-1, 在使用FITC-ATP作为探针的荧光偏振试验中测得VER-155008抑制探针与Hsp72、Hsc70和Bip结合的IC₅₀值分别为0.5、2.6和2.6 µmol·L^-1[60]。Hsc70/Bag-1 (Bcl-2-associated athanogene 1) X-衍射共晶结果表明, 腺嘌呤的N-1和Ser275形成氢键, N-3与Ser345形成由水介导的氢键, 核糖上3位的羟基与Lys271形成氢键, 4位的羟基与Asp234形成由水介导的氢键, 喹啉环和氰基取代的苯环分别与Arg272和Tyr15形成π-π堆积作用。Hsp70对ATP的高亲和力使得VER-155008很难与细胞内高浓度的ATP (1~5 mmol·L^-1) 竞争, 是其低细胞毒活性的潜在原因。VER-155008可抑制多种癌细胞的生长, 包括结肠癌、乳腺癌、多发性骨髓瘤和急性髓系白血病等, 在20 µmol·L^-1可降低Her2和Raf-1的蛋白水平^[34]。

Pettinger等^[35]对VER155008优化得到了Hsp72的共价抑制剂化合物2 (2, 图 4)。该系列化合物最初设计是靶向半胱氨酸Cys17, 而在验证过程中发现其与赖氨酸Lys56共价结合。该团队对化合物2的共价弹头进一步优化得到了化合物18 (3, 图 4), 其共价抑制效率提高了100多倍^[36]。该策略有潜力克服Hsp70对ATP结合能力很强的问题, 从而提高细胞活性。但对这些共价抑制剂的研究仅停留在生物化学水平上, 其细胞活性和作用机制尚未报道。

3.1.2 ATP竞争性抑制剂apoptozole (Az)

Williams等^[37]筛选了216个咪唑衍生物, 发现apoptozole (4, 图 4) 可以诱导细胞凋亡, 然后用亲和探针发现其靶点为Hsc70和Hsp72 (K_d分别为0.14和0.21 μmol·L^-1)。Apoptozole在细胞中主要聚集在溶酶体, 可增加溶酶体膜通透性, 抑制自噬并导致溶酶体介导的细胞凋亡。通过引入三苯基磷基团得到Az-TPP-O3 (5, 图 4), 不同于apoptozole, Az-TPP-O3主要位于线粒体, 抑制mortalin与p53的相互作用从而增加线粒体外膜通透性并促进线粒体介导的细胞凋亡。这两个化合物由于在细胞中的分布不同而通过不同的机制诱导细胞凋亡 ^[38]。然而目前关于apoptozole的研究仍存在争议, Evans等^[61]通过多个实验证明apoptozole并不与Hsp72和Hsc70的NBD区域结合。

3.1.3 HSP70-36

Zeng等^[39]针对ATP结合口袋采用虚拟筛选发现一类新型哌啶衍生物, 通过结构优化获得代表性化合物HSP70-36 (6, 图 4)。表面等离子共振实验表明, HSP70-36与Hsp70的K_d值为2.46 μmol·L^-1。化合物HSP70-36能显著抑制人类乳腺癌细胞BT474和BT/LapR1.0的增殖, 其IC₅₀值分别为1.41和1.47 μmol·L^-1, 同时可以显著抑制对拉帕替尼产生耐药的乳腺癌细胞和其他肿瘤细胞的生长。

3.1.4 变构抑制剂MKT-077和JG系列

MKT-077 (7, 图 4) 是一种阳离子罗达氰素染料, Koya等^[62-64]发现其在多种癌细胞系上有较好的细胞毒活性, 包括膀胱癌、结肠癌、乳腺癌、黑色素瘤和胰腺癌等。他们使用生物素标记的MKT-077发现其与mtHsp70结合, 释放出抑癌蛋白p53从而杀死癌细胞^[40]。基于MKT-077在动物模型上的良好表现, MKT-077作为抗癌候选药物进入了Ⅰ期临床实验, 然而由于其肾毒性和血药浓度达不到有效浓度而导致临床试验终止^{[65, 66]}。Aikaterini等^[67]发现MKT-077结合在Hsc70 NBD的变构口袋, 并抑制与其共伴侣蛋白Bag3的相互作用。针对MKT-077的活性低和代谢稳定性差的问题, Li和Shao等^{[41, 42]}合成了一系列MKT-077衍生物获得了二代抑制剂JG-98 (8, 图 4) 和JG-231, 大大提高了其细胞活性和成药性, 在多种肿瘤动物模型中有好的治疗效果^[68-71]。Shao等^[72]同时发现第一代和第二代化合物在细胞中的分布及化学遗传相互作用不尽相同, 第一代抑制剂的抗登革热病毒活性更好, 第二代抑制剂的抗癌活性更好。该研究表明, 可通过调控化合物的空间分布达到抑制不同Hsp70亚型, 为靶向具有多个结构保守家族成员的药物设计提供了新的思路。

MKT-077及其衍生物被证明在多种模型上可降低tau蛋白水平, 有应用于治疗老年痴呆等tau蛋白疾病的潜力^[73]。但是由于MKT-077等化合物带正电使其难以通过血脑屏障, Miyata等^[74]合成了一系列中性化合物YM-08 (9, 图 4) 和JG-48 (10, 图 4), 其中YM-08可穿透血脑屏障。但这些化合物的活性比较低, 成药性也有待于进一步提高, 目前仅作为工具化合物使用。

3.1.5 变构抑制剂YK5

Rodina等^{[43, 44]}通过计算机同源建模构建了Hsp72模型发现3个潜在的结合口袋, 并设计了共价抑制剂YK5 (11, 图 4)。YK5结合在Hsp72 NBD的变构口袋, 通过与Cys267形成共价键来发挥作用。生物素标记的YK5探针表明YK5也可以结合Hsp70-6和Hsc70。体外生化实验表明, YK5可抑制Hsp70水解ATP和重新折叠失活荧光素酶的活性。在细胞水平上YK5通过干扰Hsp70-HOP-Hsp90复合物的形成从而释放致癌蛋白, 如Her2、Raf-1和Akt等, 并导致这些蛋白水平下降。此外, YK5在诱导癌细胞凋亡的同时并不会诱发热休克反应。在进一步的研究中, Taldone等^[45]对YK5进行结构修饰获得了一系列非共价抑制剂, 代表化合物27c (12, 图 4) 在同等浓度下与YK5有相同的生物活性和作用机制, 证明了共价结合不是这类化合物具有生物活性的必要条件。

3.1.6 变构抑制剂HS-72

HS-72 (13, 图 4) 是通过筛选3 379个嘌呤类似物获得的选择性Hsp72变构抑制剂, 不与Hsc70或Bip结合, 并且该变构口袋只有Hsp72与ATP结合时才能呈现^[46]。当Cys306突变后, HS-72不能与Hsp72结合表明HS-72可能直接与Cys306相互作用或者Cys306突变后影响了变构口袋的形成。HS-72对多个乳腺癌和前列腺癌细胞有很好的杀伤效果, 提高了自发性乳腺癌小鼠的存活率。另外, HS-72还具有抗病毒活性, 通过破坏Hsp72与登革热病毒受体复合物的结合, 阻止登革热病毒进入宿主细胞, 从而达到抵抗登革热病毒感染的目的^[75]。HS-72是通过筛选发现的苗头化合物, Howe^[46]等对其作用方式和生物活性进行了研究, 并未对其进一步优化以提高生物活性和成药性。

3.1.7 变构抑制剂冬凌草甲素(oridonin)

冬凌草甲素(oridonin) (14, 图 4) 是从唇形科香茶菜属碎米桠(Isodon rubescens) 中分离得到的一种对映贝壳杉烷型二萜类化合物, 具有抗肿瘤活性^[76]。作用机制研究表明, 冬凌草甲素可以诱导细胞周期停滞、细胞凋亡和自噬, 但其具体靶点未知。Dal Piaz等^[47]采用生物素标记的冬凌草甲素鉴定其靶点为Hsp72。并进一步通过质谱确定其α, β-不饱和酮部分与位于NBD的Cys267形成共价键发挥作用。等离子共振实验表明冬凌草甲素与Hsp72的K_d值为26 nmol·L^-1, 并且可以抑制Hsp72水解ATP和折叠失活柠檬酸合成酶的活性。此外, 还通过分子对接结合分子动力学模拟阐明了冬凌草甲素与Hsp72的结合模式。目前关于冬凌草甲素的结构优化尚无报道。

3.1.8 Hsp70-JDPs蛋白-蛋白相互作用调节剂MAL3-101和115-7c

Fewell等^{[48, 77]}筛选了一系列Hsp70激活剂15-DSG的衍生物, 部分化合物功能与15-DSG类似, 可激活Hsp70水解ATP。其中3个化合物抑制JDPs刺激Hsp70水解ATP的活性, 代表性化合物是MAL3-101 (15, 图 4)。MAL3-101是通过阻断JDPs与Hsp70s的PPIs来发挥作用, 并不抑制Hsp70自身的ATP水解活性。MAL3-101在多种癌细胞系上有活性, 包括梅克尔细胞癌^[78]和横纹肌肉瘤^[14]等, 通过激活未折叠蛋白反应导致细胞凋亡。基因敲除实验证明MAL3-101通过抑制Hsp72和Hsc70来激活未折叠蛋白反应导致细胞凋亡。Wisén等^[18]对MAL3-101进行结构改造获得了Hsp70激活剂115-7c (16, 图 4), 该化合物在Hsp70功能障碍的多聚谷氨酸疾病酵母模型中有效。Fewell^[48]和Wisén^[18]虽然对这一系列化合物进行了优化, 但其生物活性仍相对较弱。

3.1.9 Hsp70-Bim蛋白-蛋白相互作用抑制剂S1g-2、S1g-6和S1g-10

Hsp70与Bcl-2家族蛋白BH3的成员Bim共同维持致癌蛋白Akt和Raf1的稳定, 因此特异性地阻断Hsp70与Bim的PPIs被认为是治疗酪氨酸激酶抑制剂耐药的慢性粒细胞白血病(chronic myelogenous leukemia, CML) 和莫昔芬耐药的ER⁺乳腺癌的潜在策略^{[49, 79-82]}。Wang^[83]和Li^[84]等通过荧光偏振法筛选了一系列BH3类似物获得了Hsp70-Bim PPIs抑制剂S1g-2 (17, 图 4) 和S1g-6 (18, 图 4), S1g-6抑制Hsp70-Bim PPIs的IC₅₀和结合Hsp70的K_d值分别为0.45和1.01 µmol·L^-1。亲和探针证明, S1g-6与Hsp72、Hsc70和mortalin结合, S1g-6可降低Akt和Raf1的蛋白水平并导致CML细胞凋亡。该团队对S1g-2进行进一步优化得到了S1g-10 (19, 图 4), 其体外Hsp70/Bim抑制活性提高了10倍, 对CML细胞的活性提高了5~10倍, 在酪氨酸激酶抑制剂耐药的CML动物模型上有较好的效果 ^[50]。

3.1.10 HA-15

HA-15 (20, 图 4) 是通过对噻唑烷二酮类胰岛素增敏药物曲格列酮进行优化获得的, 通过生物素标记的HA-15探针发现其靶点为Bip, 不与Hsp70其他亚型结合^[85]。HA-15能抑制Bip水解ATP的活性, 等温滴定量热法表明其与Bip的NBD结合, 但具体的结合位点尚不明确^[51]。荧光显微镜实验显示HA-15主要分布在内质网中, 作用机制研究表明, HA-15能够激活内质网应激, 可诱导自噬并导致黑色素瘤细胞凋亡, 并在黑色素瘤动物模型上抑制肿瘤生长。HA-15对BRAF抑制剂耐受的黑色素瘤很有效, 在细胞和动物模型上都得到了验证。虽然HA-15在细胞和动物模型上都展现出疗效, 但其在酶和细胞水平上的活性仍有待提高。

3.1.11 二甲基蓝、亚甲基蓝和槲皮素

Jinwal等^[52]采用Hsc70-JDP-NEF复合物筛选了2 800个活性化合物, 发现二甲基蓝(21, 图 4)、亚甲基蓝(22, 图 4) 和槲皮素(23, 图 4) 这3个化合物可抑制复合物的ATP水解活性。这些化合物在tau蛋白过表达的细胞模型中可降低tau蛋白水平 ^[86]。NMR研究证明, 槲皮素与Hsp70 NBD中的IB和IIB结构域结合, 通过变构作用阻止了Hsp70与JDP的相互作用 ^[87]。NMR实验表明, 亚甲基蓝与Hsc70的NBD结合, 其去甲基化衍生物二甲基蓝可能也结合在同一区域, 但具体位置和作用机制目前并不清楚^[86]。这3个化合物是通过高通量筛选发现的苗头化合物, 对Hsc70-JDP-NEF复合物有较弱的抑制活性, 其活性和成药性均有待于进一步提高。

3.2 结合于SBD区域的化合物

结合在SBD的化合物包括变构抑制剂和底物竞争性抑制剂, 部分化合物仅知其与SBD结合, 但具体位点未知(图 5)。SBD底物结合位点呈长槽状、小且疏水, 目前仅有底物短肽NRLLLTG和变构抑制PET-16 (26, 图 5) 与Dnak SBD及novolactone (28, 图 5) 与Hsp72 SBD的复合物结构被解析(图 1C)。SBD抑制剂中仅有变构抑制剂2-苯基乙炔磺酰胺系列(代表性化合物为26, 图 5)、底物竞争性抑制剂六氯酚(30, 图 5) 和化合物8 (31, 图 5) 有初步的构效关系研究, 其他几个化合物尚未进行结构优化。

3.2.1 变构抑制剂2-苯基乙炔磺酰胺(2-phenylethynesulfonamide, PES) 及其衍生物PES-Cl、PET-16和AP-4-139B

2-苯基乙炔磺酰胺(24, 图 5) 是Strom等^[88]在Chembridge DIVERSet类药小分子库中筛选得到能够抑制p53介导的细胞凋亡的化合物。生物素标记的PES探针发现其靶点为Hsp72, 并且不与Hsc70、Grp78或Hsp90结合。PES与Hsp72 SBD结合, 并阻止底物与SBD的相互作用 ^{[53, 89]}。Yang等^[90]发现PES可与Hsp72 SBD的Cys574和Cys603发生共价结合。PES抑制自噬并改变溶酶体功能, 促进p62的寡聚和聚集, 诱导癌细胞凋亡。其衍生物PES-Cl (25, 图 5) 抗癌活性优于PES, 对5种黑色素瘤的半数抑制浓度IC₅₀值在2~5 µmol·L^-1之间 ^[91]。

Leu等^[89]解析了PES衍生物PET-16 (26, 图 5) 与DnaK-SBD (aa389-607) 的晶体结构, 阐明了该类化合物抑制Hsp70的结构生物学基础。PET-16与Hsp70 SBD的一个变构口袋结合, 相互作用的氨基酸主要有Leu392、Pro396、Leu399、Gly482、Ala503和Ser504, 该口袋只有在ADP与Hsp70结合时才能呈现。PET-16与Hsp72和Dnak SBD的K_d值分别为2.9和6.0 µmol·L^-1。进一步对PET-16优化获得了活性更好的AP-4-139B (27, 图 5), 该化合物靶向癌细胞的多个区室, 包括线粒体^[92]。AP-4-139B对结直肠癌细胞具有线粒体毒性和细胞毒性, 但对正常结肠上皮细胞没有毒性。AP-4-139B在原发性和转移性结直肠癌小鼠模型上都有效, 并且在同系结直肠癌小鼠肿瘤模型上增加了免疫细胞向肿瘤的募集。

3.2.2 变构抑制剂novolactone

诺华公司通过在酵母上进行化学基因组筛选发现天然产物novolactone (28, 图 5) 的靶点可能是Hsp70^{[54, 93]}。Novolactone抑制JDPs激活Hsp72水解ATP活性(IC₅₀ = 0.25 µmol·L^-1) 和折叠失活荧光素酶的活性。体外生物化学实验表明, novolactone中的内酯环与Hsp72 SBD的Glu444形成共价键, 两者的共结晶也证明了这一结果(PDBID: 4WV7)。此外, novolactone与Leu399形成水介导的氢键, 与Pro398、Leu456、Ile485和Asn505等多个氨基酸通过范德华力相互作用。由于Glu444在多个Hsp70亚型中都存在, novolactone可能没有亚型选择性。进一步实验证明, novolactone同时抑制细胞质和内质网中的Hsp70s, 用novolactone处理癌细胞后会诱导Hsp72的表达, 降低EGFR和MET的蛋白水平并诱导细胞凋亡。

3.2.3 Ritterostatin G_N1_N

Ritterostatin G_N1_N (29, 图 5) 是天然产物cephalostatin和ritterazine的合成杂化分子, 三者均为吡嗪双甾体螺酮类化合物, 具有较好的抗癌活性并且作用机制相同, 但具体的靶点未知^[94]。Ambrose等^[55]使用ritterostatin G_N1_N免疫亲和荧光探针发现其与多个Hsp70s结合, 但主要靶点为Bip。等温滴定量热法显示, ritterostatin G_N1_N与Bip、Hsp72和Hsc70的亲和力K_d值分别为0.19、0.625和1.63 µmol·L^-1。进一步测试发现其与Bip SBD的亲和力K_d值为3.12 µmol·L^-1, 而不与NDB结合。细胞成像实验显示, ritterostatin G_N1_N免疫亲和荧光探针在加入细胞30 min后主要聚集在内质网中, 并且可以激活内质网未折叠蛋白反应并导致细胞凋亡。综合ritterostatin G_N1_N细胞定位和作用机制, Ambrose等^[55]认为该化合物的主要靶点为Bip, 但其具体结合位点仍不清楚。

3.2.4 底物竞争性抑制剂六氯酚

Ambrose等^[56]采用荧光偏振法筛选了2 560个化合物发现了Bip SBD底物竞争性抑制剂六氯酚(30, hexachlorophene, 图 5), 该方法用的荧光探针是FITC-NRLLLTG。短肽NRLLLTG最初是采用噬菌体展示技术寻找与DnaK相互作用的多肽时发现的, 与Bip和mtHsp70 SBD结合的K_d值约为100 nmol·L^-1, 不与其他几个Hsp70s结合^[95]。六氯酚可竞争性地抑制荧光探针与SBD的结合(IC₅₀ = 9.1 µmol·L^-1), 但具体如何与SBD相互作用并不清楚。六氯酚在HCT-116细胞上可激活未折叠的蛋白质反应, 诱导自噬和细胞凋亡。

3.2.5 底物竞争性抑制剂化合物8

Ambrose等^[57]采用与六氯酚相同的方法筛选了70 000个化合物发现苗头化合物, 对其进行结构优化获得化合物8 (31, 图 5)。化合物8抑制荧光探针与Bip结合的IC₅₀约为500 nmol·L^-1, 对mtHsp70和Hsp70-2的选择性分别为7倍和23倍, 对Bip、Hsp72和Hsp70-6的选择性大于70倍。化合物8及其衍生物对2D培养的一组癌细胞在10 µmol·L^-1浓度下几乎没有细胞毒活性, 在胶质母细胞瘤细胞U251和肺癌细胞H520类器官模型上有较弱的活性。

3.2.6 15-Deoxyspergualin (15-DSG)

15-Deoxyspergualin (32, 15-DSG, 图 5) 最早于1982年由Iwasawa等^[58]在合成抗生素spergualin的衍生物时发现。直到1992年, Nadler等^[96]鉴定15-DSG的靶点为Hsc70。随后进一步发现15-DSG可以与Hsc70 SBD的EEVD序列结合, 该序列在Hsp90中也存在, 因此15-DSG也与Hsp90结合^[97]。15-DSG已被批准用于治疗异体移植排斥反应^[98], 但抗癌效果较差。Fewell等^[48]基于15-DSG寻找新的Hsp70抑制剂最终获得了MAL3-101等衍生物, 但其作用机制完全不同。

3.2.7 N-氨基乙基氨基秋水仙碱(N-amino-ethylamino derivative of colchicine, AEAC)

Lazarev等^[59]采用Hsp70与底物结合和Hsp70折叠失活底物两种方法筛选了InterBioScreen化合物库, 发现AEAC (33, 图 5) 抑制Hsp70与底物羧甲基化乳清蛋白结合和折叠失活荧光素的活性。微量热涌动实验显示AEAC对Hsp70的亲和力K_d值为149 nmol·L^-1。分子对接结果表明, AEAC可能结合在NBD的1个口袋或SBD的2个口袋, 其中与SDB结合的分子对接打分结果最好, 基于此, 本文将其放在结合SBD的化合物部分, 该结果仍有待于进一步验证。AEAC对C6神经胶质瘤和B16黑色素瘤细胞的半数抑制浓度分别为195和98 µmol·L^-1。虽然细胞毒活性较差, 但在动物模型上可显著提高多柔比星的抗癌效果, 明显抑制肿瘤生长并延长小鼠的生存期。

4 总结与展望

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尽管Hsp70抑制剂的研究已经取得了很大的进展, 但所有化合物仍处于临床前研究阶段, 可能由以下几个原因造成: 第一个原因是Hsp70系统生物学的复杂性, Hsp70家族成员和共伴侣蛋白数量众多, 针对某一种疾病应该靶向哪一个亚型或者哪一对PPIs仍有待于基础生物学和致病机制的进一步深入研究; 第二个原因是仍然缺乏高质量的Hsp70抑制剂, 目前没有任何一个化合物在所有Hsp70亚型上测试过其选择性, 另外还有一部分化合物是通过表型筛选得到的, 生化水平上结合Hsp70或抑制Hsp70活性较差, 这一类化合物不适合用来进行靶点验证; 第三个原因是Hsp70抑制剂的成药性仍有待提高, 目前已经报道的一部分化合物并没有进行构效关系研究和成药性优化, 只是对其靶点和作用机制进行了研究。

提高Hsp70抑制剂的选择性和成药性仍是该领域亟需解决的问题。由于Hsp70家族蛋白结构保守, 使得选择性的靶向某一亚型非常困难, 但仍有可能实现。Hsp70家族蛋白对ATP和短肽NRLLLTG的亲和力不同, 表明ATP和底物结合口袋氨基酸序列存在差异, 可以被用来设计亚型特异性抑制剂。目前在该领域已经有了初步的成果, 但是化合物的活性和选择性仍有待于进一步提高。另外一个潜在的方法是通过调控化合物的空间分布来提高其亚型选择性, 例如MKT-077系列化合物、HA-15和ritterostatin G_N1_N。这几个化合物空间分布是其自身的物理化学性质决定的, 如何通过结构修饰调控化合物的亚细胞空间分布还不清楚, 但仍是一个可以努力的方向。

作者贡献: 朱金言负责文献的收集及文章的撰写; 余荧蓝、何明慧和吴凡负责文章部分内容的撰写并提出修改建议; 邵豪、罗雷负责构思文章框架和指导文章撰写等工作。

利益冲突: 本文所有作者声明不存在利益冲突关系。

基金

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国家自然科学基金青年科学基金(82104001)
中央高校基本科研业务费专项资金项目(5330501000)
中央高校基本科研业务费专项资金项目(5330501059)

参考文献

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文献

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2024年第59卷第11期

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文章信息

doi: 10.16438/j.0513-4870.2024-0462

接收时间：2024-05-14
首发时间：2025-11-24
出版时间：2024-11-12

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收稿日期：2024-05-14
修回日期：2024-09-11

基金

国家自然科学基金青年科学基金(82104001)

中央高校基本科研业务费专项资金项目(5330501000)

中央高校基本科研业务费专项资金项目(5330501059)

作者信息

西南大学药学院·中医药学院, 重庆 400715

通讯作者:

^*罗雷, Tel: 86-23-68251225, E-mail: haoshao@swu.edu.cn;

邵豪, Tel: 86-23-68251225, E-mail: drluolei@swu.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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Gene	Protein	Heat inducible	Subcellular localization	Homology (to HSPA1A)/%
HSPA1A	Hsp70-1A, Hsp72	Yes	Cytoplasm/nucleus	100
HSPA1L	Hsp70-1L	No	Cytoplasm/nucleus	89
HSPA1B	Hsp70-1B	Yes	Cytoplasm/nucleus	99
HSPA2	Hsp70-2	No	Cytoplasm/nucleus	85
HSPA5	Bip, Grp78	Yes	Endoplasmic reticulum	64
HSPA6	Hsp70-6	Yes	Cytoplasm/nucleus	83
HSPA8	Hsc70, Hsp70-8	No	Cytoplasm/nucleus	87
HSPA9	mtHsp70, Grp75, mortalin	No	Mitochondria	52
HSPA7	Hsp70-7	N/A	Cytoplasm/nucleus	33
HSPA12A	Hsp70-12A	N/A	Cytoplasm/nucleus	29
HSPA12B	Hsp70-12B	N/A	Cytoplasm/nucleus	25
HSPA13	Hsp70-13	N/A	Cytoplasm/nucleus	40
HSPA14	Hsp70-14	N/A	Cytoplasm/nucleus	35

Gene

Protein

Heat inducible

Subcellular localization

Homology (to HSPA1A)/%

HSPA1A

Hsp70-1A, Hsp72

Yes

Cytoplasm/nucleus

100

HSPA1L

Hsp70-1L

Cytoplasm/nucleus

HSPA1B

Hsp70-1B

Yes

Cytoplasm/nucleus

HSPA2

Hsp70-2

Cytoplasm/nucleus

HSPA5

Bip, Grp78

Yes

Endoplasmic reticulum

HSPA6

Hsp70-6

Yes

Cytoplasm/nucleus

HSPA8

Hsc70, Hsp70-8

Cytoplasm/nucleus

HSPA9

mtHsp70, Grp75, mortalin

Mitochondria

HSPA7

Hsp70-7

N/A

Cytoplasm/nucleus

HSPA12A

Hsp70-12A

N/A

Cytoplasm/nucleus

HSPA12B

Hsp70-12B

N/A

Cytoplasm/nucleus

HSPA13

Hsp70-13

N/A

Cytoplasm/nucleus

HSPA14

Hsp70-14

N/A

Cytoplasm/nucleus

Hsp70 inhibitors	Targeted Hsp70s^*	Mode of actions	Refs
VER-155008, compounds 2 and 8	Hsp72, Hsp70, Bip	ATP competitive	[33-36]
Apoptozole, Az-TPP-O3	Hsp72, Hsc70	ATP competitive	[37, 38]
Hsp70-36	Hsp72	ATP competitive	[39]
MKT-077 and JG series	mtHsp70, Hsc70	Allosteric	[40-42]
YK5	Hsp72, Hsc70, Hsp70-6	Allosteric	[43-45]
HS-72	Hsp72	Allosteric	[46]
Oridonin	Hsp72	Allosteric	[47]
MAL3-101 and 115-7c	Hsp72, Hsc70	Hsp70-JDPs PPIs	[18, 48]
S1g-2, S1g-6 and S1g-10	Hsp72, Hsc70, mtHsp70	Hsp70-Bim PPIs	[49, 50]
HA-15	Bip	Unknown	[51]
Azure C, methylene blue and myricetin	Hsc70	Unknown	[52]
PES, PES-Cl, PET-16 and AP-4-139B	Hsp72	Allosteric	[53]
Novolactone	Hsp72, Hsc70, Bip	Allosteric	[54]
Ritterostatin Gn1N	Bip	Unknown	[55]
Hexachlorophene	Bip	SBD-peptide Interactions	[56]
Compound 8	Bip	SBD-peptide Interactions	[57]
15-Deoxyspergualin	Hsc70	EEVD	[58]
AEAC	Unknown	Unknown	[59]

Hsp70 inhibitors

Targeted Hsp70s^*

Mode of actions

Refs

VER-155008, compounds 2 and 8

Hsp72, Hsp70, Bip

ATP competitive

[33-36]

Apoptozole, Az-TPP-O3

Hsp72, Hsc70

ATP competitive

[37, 38]