FS36作为新型人源Hsp90抑制剂的发现（英文）

热休克蛋白90(Hsp90)通过对几百种蛋白质底物(客户蛋白质)进行合理的折叠、成熟其构象并且激活,在肿瘤细胞的生长和繁殖中发挥重要作用.因此,Hsp90成为非常有吸引力、有前途的抗肿瘤药物靶点,并且超过20种抑制剂已经进入临床实验阶段.我们在这里设计并合成了一个小分子抑制剂:FS36.收集了Hsp90~N-FS36复合物晶体结构的X射线衍射实验数据.高分辨率X射线晶体结构表明,FS36在ATP结合位点上与Hsp90~N相互作用,并且FS36可能替代核苷酸与Hsp90~N结合.FS36和Hsp90~N的复合物晶体结构和相互作用为后期设计和优化新型抗肿瘤药物奠定基础.     

Hsp90抑制剂的研究进展

热激蛋白90(heat shock protein90,Hsp90)作为分子伴侣在调节细胞生长、分化、凋亡等方面发挥着重要的作用。Hsp90抑制剂能与Hsp90结合,使其功能丧失,造成细胞的多种生理活动缺陷,在Hsp90功能研究和癌症治疗方面具有潜在的价值。综述了不同来源的Hsp90抑制剂及其作用机制,同时对新型Hsp90抑制剂的来源进行了探讨。     

真菌Hsp90研究进展

近年来,随着广谱抗生素、化疗以及器官移植技术的广泛应用,真菌感染日益严重,从分子水平揭示病原真菌的致病机制对真菌感染的防控、治疗至关重要。微生物适应宿主微环境压力的能力在其共生与感染过程中发挥着关键作用,heat shock protein 90（Hsp90）是真核生物参与压力应答响应的分子伴侣,它不仅参与胞内蛋白质的折叠,还与许多底物蛋白相互作用共同调节病原真菌的形态发育、生物被膜形成、有性生殖、毒力以及耐药性。本文从真菌Hsp90的活性调节、底物蛋白,以及Hsp90与病原真菌形态发生、有性生殖、耐药性调控等方面综述了近年来真菌Hsp90信号通路的研究进展。     

Hsp90抑制剂对未折叠蛋白反应作用的研究进展

肿瘤细胞因癌基因突变、缺氧及营养受限而高度依赖未折叠蛋白反应(unfolded protein response,UPR)。细胞通过内质网(endoplasmic reticulum,ER)膜上3个跨膜蛋白感知未折叠蛋白信号,引起未折叠蛋白反应,动态调控内质网折叠能力,一方面通过暂时减缓翻译和加快蛋白质流出减少ER蛋白质折叠负担,另一方面通过转录因子提高伴侣分子合成,增加ER折叠能力。未折叠的蛋白质长时间积聚在ER会对细胞产生毒性,引起不能缓解的ER应激状态,启动细胞凋亡程序。热休克蛋白90(heat shock protein 90,Hsp90)是一种进化保守的伴侣分子,参与了300多种新生蛋白质的折叠与成熟,其中包括UPR重要信号IRE1α(inositol-requiring enzyme 1α)。Hsp90抑制剂导致细胞产生大量未折叠蛋白质,同时直接诱导IRE1α的降解,从而破坏UPR恢复蛋白质平衡的能力,诱导UPR相关凋亡。目前,Hsp90抑制剂可有效诱导分泌型肿瘤细胞如骨髓瘤以及RAS突变肿瘤UPR途径的凋亡。     

Ubiquitin B在HSP90抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用

目的 研究Ubiquitin B（Ubb）在热休克蛋白90（HSP90）抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用及机制.方法 不同浓度17-AAG处理HeLa细胞后,流式细胞术检测细胞周期分布,荧光分光光度法检测细胞蛋白酶体活性变化;Ubb siRNA 转染HeLa细胞后,Real Time PCR法检测Ubb干扰效应,Western 印迹检测细胞周期相关蛋白的表达改变.结果 17-AAG可以诱导HeLa细胞阻滞于G2/M期,同时显著增强细胞内糜蛋白酶样蛋白酶体活性,并且两者的变化均呈现剂量依赖性;干扰HeLa细胞内Ubb后,可以逆转17-AAG引起的G2/M期阻滞;17-AAG可明显下调HeLa细胞周期相关蛋白Cdk1和Hec1的表达,并且这一变化也是Ubb依赖的.结论 Ubb在17-AAG诱导的HeLa细胞周期阻滞中发挥重要作用,Ubb和HSP90抑制剂17-AAG在功能上相互关联,可能成为宫颈癌治疗的新靶点.     

热激蛋白90抑制剂

分子伴侣热激蛋白90(heat-shock protein 90,Hsp90)在生物体内具有重要的生理功能,它在许多肿瘤细胞中表达增加。临床研究发现Hsp90抑制剂单一用药或者联合用药都具有较好的抗肿瘤效果,因此目前Hsp90被认为是癌症治疗一个非常有潜力的靶标。本文总结了Hsp90的结构功能、Hsp90抑制剂的作用机理以及Hsp90抑制剂的临床应用前景,希望为设计和开发新的Hsp90抑制剂提供一定的参考。     

热休克蛋白90 抑制剂的活性初步评价

目的:初步评价经计算机模拟筛选的Fs系列化合物对A549细胞的增殖抑制作用与对热休克蛋白90活性的抑制。方法:首先采用SRB(磺基罗丹明B)法观察26个化合物对A549肿瘤细胞增殖抑制活性,再进一步应用Western Blot方法对此类化合物在蛋白水平上对热休克蛋白90活性的抑制进行评价。以热休克蛋白70的表达增加作为热休克蛋白90活性是否被抑制的参考指标。结果:在Fs系列化合物中Fs-1、Fs-8、Fs-24对A549细胞增殖有明显抑制,且能明显上调Hsp70蛋白的表达,但同时Hsp90蛋白的表达量并不受影响。其余化合物在两种方法中显示均无明显抑制作用。结论:在经计算机模拟筛选出的26种Fs系列化合物中Fs-1、Fs-8、Fs-24可抑制A549细胞的增殖,可能是通过对Hsp90活性的抑制而发挥作用,经筛选其余Fs系列化合物抑制A549细胞增殖与抑制Hsp90活性作用均不显著。Fs-1、Fs-8、Fs-24类化合物作用的初步探索将为研制Hsp90靶向抑制剂类药物开辟新途径。     

热休克蛋白90对血小板源衍生因子诱导的大鼠主动脉平滑肌细胞增殖的影响

目的:研究热休克蛋白90(HSP90)对血小板衍生因子(Platelet derived growth factor,PDGF)诱导的大鼠主动脉平滑肌细胞增殖的影响。方法:采用胶原酶消化法原代培养大鼠胸主动脉平滑肌细胞,应用脂质体细胞转染siRNA的方法抑制HSP90的表达,定量PCR和western blot的方法检测抑制效率。利用PDGF-bb诱导刺激平滑肌细胞增殖,CCK8法检测细胞增殖能力的变化,流式细胞术检测细胞生长周期的改变。结果:平滑肌细胞中转染HSP90的siRNA后,HSP90的mRNA和蛋白水平明显降低,分别为对照组的65.3%和57.6%(P0.05);PDGF-bb刺激明显促进平滑肌细胞生长,而降低HSP90水平显著影响PDGF-bb诱导的细胞增殖(P0.05);流式细胞术检测发现降低HSP90水平引起平滑肌细胞生长停滞,分布在细胞周期G1期的细胞比例明显增多(P0.05)。结论:HSP90通过调控平滑肌细胞的生长周期参与调节细胞增殖过程。     

热休克蛋白90抑制NIH3T3成纤维细胞凋亡

目的实验应用Hsp90过表达系统观察Hsp90抑制TNFα诱导的细胞凋亡,探讨其可能的作用机制。方法采用电穿孔技术建立稳定过表达Hsp90的细胞克隆,应用激光共聚焦显微镜和流式细胞仪观察TNFα和放线菌酮诱导的细胞凋亡,应用比色分析和Western blotting方法检测caspase的变化。结果1)在稳定过表达Hsp90的NIH3T3细胞中,Hsp90能够抑制TNFα诱导的细胞凋亡。2)在凋亡信号转导通路中,Hsp90作用于caspase-8下游、caspase-3上游。结论1)Hsp90能够在细胞凋亡信号转导通路中发挥负性调节因子的作用。2)Hsp90抑制剂作为抗肿瘤药物的机制之一,可能是通过促进caspase-3活化而促进肿瘤细胞凋亡。     

热休克蛋白90在缺氧心肌细胞中表达的初步研究

观察热休克蛋白90(HSP90)在缺氧心肌细胞中的表达变化规律,并初步阐明HSP90在早期心肌缺氧损害中的作用。方法:原代培养SD大鼠乳鼠心肌细胞,随机分为正常对照组、缺氧组和加HSP90阻断剂格尔霉素(geldanamycin,GA)预处理后再缺氧组(GA 缺氧组),蛋白免疫印迹法(western blot)及间接免疫荧光法检测缺氧1、3、6、12、24h后心肌细胞中HSP90蛋白分布表达情况,并检测细胞上清中肌酸激酶同工酶(CK-MB)及乳酸脱氢酶(LDH)的含量变化。结果:心肌细胞缺氧3h后HSP90蛋白在胞浆内表达量升高,持续到12h达到峰值。与单纯缺氧组相比,GA 缺氧组中LDH,CK-MB含量在不同时相点均有显著升高。结论:缺氧早期即可引起心肌细胞胞浆中HSP90蛋白表达增强,HSP90可能在早期心肌缺氧损害中发挥其内源性抗损伤机制。     

Hsp90 chaperones have an energetic hot‐spot for binding inhibitors

Although Hsp90‐family chaperones have been extensively targeted with ATP‐competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP‐binding pocket. A large‐scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen‐bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle‐specific Hsp90 paralogs. For inhibitors in the resorcinol family, the D93:inhibitor hydrogen‐bond is pH‐dependent because the associated inhibitor hydroxyl group is titratable, rationalizing a linked‐protonation event previously observed by the Matulis group. The inhibitor hydroxyl group pK_a associated with the D93 hydrogen‐bond is therefore critical for optimizing the affinity of resorcinol derivatives, and we demonstrate that spectrophotometric measurements can determine this pK_a value. Quantifying the energetic contribution of the D93 hotspot is best achieved with the mitochondrial Hsp90 paralog, yielding 3–6 kcal/mol of stabilization (35–60% of the total binding energy) for a diverse set of inhibitors. The Hsp90 Asp93?Asn substitution has long been known to abolish nucleotide binding, yet puzzlingly, native sequences of structurally similar ATPases, such as Topoisomerasese II, have an asparagine at this same crucial site. While aspartate and asparagine sidechains can both act as hydrogen bond acceptors, we show that a steric clash prevents the Hsp90 Asp93?Asn sidechain from adopting the necessary rotamer, whereas this steric restriction is absent in Topoisomerasese II.     

Extended conformational states dominate the Hsp90 chaperone dynamics

The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a β-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.     

SNX-2112, a Novel Hsp90 Inhibitor,Induces G2/M Cell Cycle Arrest and Apoptosis in MCF-7 Cells

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.     

Independent ATPase activity of Hsp90 subunits creates a flexible assembly platform

The ATPase activity of the molecular chaperone Hsp90 is essential for its function in the assembly of client proteins. To understand the mechanism of human Hsp90, we have carried out a detailed kinetic analysis of ATP binding, hydrolysis and product release. ATP binds rapidly in a two-step process involving the formation of a diffusion-collision complex followed by a conformational change. The rate-determining step was shown to be ATP hydrolysis and not subsequent ADP dissociation. There was no evidence from any of the biophysical measurements for cooperativity in either nucleotide binding or hydrolysis for the dimeric protein. A monomeric fragment, lacking the C-terminal dimerisation domain, showed no dependence on protein concentration and, therefore, subunit association for activity. The thermodynamic linkage between client protein binding and nucleotide affinity revealed ATP bound Hsp90 has a higher affinity for client proteins than the ADP bound form. The kinetics are consistent with independent Michaelis-Menten catalysis in each subunit of the Hsp90 dimer. We propose that Hsp90 functions in an open-ring configuration for client protein activation.     

热休克蛋白90三磷酸腺苷酶的调控机制研究

热休克蛋白90（heat shock protein90,Hsp90）是一类在生物进化中高度保守的蛋白,与细胞凋亡密切相关,作为抗癌新靶标已经得到了广泛的关注。相关研究表明,Hsp90可以通过多种方式调控ATPase的活性,如自身构象改变、与辅伴侣分子形成复合物以及转录后修饰等。在Hsp90基本构象改变的基础上,综述了不同因素对ATPase的调控作用,着重阐述近几年的研究进展,为进一步研究Hsp90调控ATPase的机制提供一定的参考。     

Discovery of novel heat shock protein (Hsp90) inhibitors based on luminespib with potent antitumor activity

A series of isosteric surrogates of the 4-phenyl group in luminespib were investigated as new scaffolds of the Hsp90 inhibitor for the discovery of novel antitumor agents. Among the synthesized surrogates of isoxazole and pyrazole, compounds 4a, 5e and 12b exhibited potent Hsp90 inhibition in ATPase activity and Her2 degradation assays and significant antitumor activity in A2780 and HCT116 cell lines. Animal studies indicated that compared to luminespib, their activities were superior in A2780 or NCI-H1975 tumor xenograft models. A molecular modeling study demonstrated that compound 4a could fit nicely into the N-terminal ATP binding pocket.     

Decomposition analysis of free energy profile for Hsp90-ADP association

The functional cycle of heat shock protein 90 (Hsp90) is driven and inhibited by the association/dissociation of ligand molecules. In order to understand the molecular mechanism of the association of N-terminal domain of Hsp90 (N-Hsp90) and its ligand molecule, it is necessary to investigate which part in the target system promotes or inhibits the association of N-Hsp90 and its ligand molecule. We apply the decomposition analysis for the association free energy of N-Hsp90 and ADP. The mean force calculated by thermodynamic integration method combined with molecular dynamic simulations is divided into the contributions from molecules in the target system. Van der Waals interaction of the solvent water molecules strongly stabilises the association. Three lysine residues on the surface of the N-Hsp90 pull ADP toward the binding pocket of N-Hsp90. This study elucidates the association process of ADP from the bulk region to the binding pocket of the N-terminal domain Hsp90. This approach is applicable to elucidate the association process of biomolecules.