表观遗传相关药物对癌症的治疗

表观遗传变化是指核酸序列不发生变化的情况下发生的可遗传变化。表观遗传变化在癌症发生和恶性转移的过程中发挥着重要的作用。目前,已有数种表观药物通过FDA认证并用于癌症的治疗,主要为DNA甲基转移酶抑制剂和组蛋白去乙酰化酶抑制剂;还有许多的新型表观药物处于实验室或者临床试验阶段,包括组蛋白乙酰化酶、组蛋白甲基化酶、组蛋白去甲基化酶以及表观解读蛋白的小分子抑制剂,它们都表现出显著的抗肿瘤活性。该文对这些表观修饰小分子抑制剂的最新进展和将来的应用前景进行了整理、总结。     

HIV-1整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

FS36作为新型人源Hsp90抑制剂的发现

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．     

靶向Ras蛋白的抗癌药物

Ras蛋白是Ras信号通路调控的关键分子,与30%人类肿瘤的发生和进展密切相关,研发针对Ras蛋白的抗癌药物具有重要意义。经过数十年的努力,过去因技术限制和对Ras性质及结构认识不足导致的Ras靶向药物研发缓慢的局面如今已有新突破。目前已有多种抗Ras蛋白的单克隆抗体和单链抗体问世。Ras蛋白的这些抗体通过与Ras蛋白特异结合使其失去调控Ras信号通路的作用,目前正在解决这些抗体穿透肿瘤细胞膜进入细胞的难题。已合成多种Ras蛋白抑制剂,如化合物SML-10701、3144、DCAI、AMG 510、MRTX 849和Cyclorasin等,其中AMG 510已进行Ⅲ期临床试验,其他的抑制剂在细胞膜穿透能力和肿瘤抑制效果方面有较大改善。Ras翻译后修饰抑制剂GGTI-2418、NSC1011和Cysmethynil等通过影响Ras蛋白的细胞内定位而阻断Ras信号通路,提高这类抑制剂抑制效果的研究仍在继续。本文从抗Ras蛋白抗体、Ras蛋白抑制剂和Ras翻译后修饰抑制剂三个方面综述近些年Ras蛋白靶向药物的研究进展,为ras基因驱动肿瘤的治疗提供参考。     

HIV—1复制抑制剂的新靶标

现在市场上的多种治疗ＡＩＤＳ的药物都是病毒逆转录酶和蛋白水解酶的特异性抑制剂 ,但是发现有 2 0 %以上的病人不能在短期内承受这些药物的治疗 ,而且长期使用蛋白水解酶抑制剂对人体正常新陈代谢的影响也越来越突出 ,现已发现ＨＩＶ 1的耐药变种已经出现并正以很快的速度传播。另外 ,这些药物不能直接清除感染ＨＩＶ 1病人体内的病毒。所以现在需要发现新型的治疗ＡＩＤＳ病的药物 ,这些药物可以长期使用 ,能补充或者部分代替已有的药物 ,而这就需要先找到ＨＩＶ 1复制抑制剂的新靶标。ＨＩＶ 1病毒的生活周期是这样的 :病毒被膜和细胞表…     

流感病毒神经氨酸酶抑制剂的筛选

以神经氨酸酶为药物靶点筛选神经氨酸酶抑制剂, 是研究和开发抗流感病毒药物的重要途径. 应用虚拟药物筛选方法, 从化合物数据库中选出部分待测化合物, 然后应用已建立的 神经氨酸酶抑制剂的高通量筛选模型, 检测了这些化合物的抑制活性, 从中发现了3个活性较高、结构新颖的化合物, 其IC50在0.1~3 μmol/L之间, 这些活性化合物的结构特点, 对于新型神经氨酸酶抑制剂的设计与开发, 将提供重要的信息指导. 流感病毒神经氨酸酶抑制剂的筛选结果表明, 虚拟筛选技术与高通量筛选技术的合理结合, 将有利于促进药物筛选与药物发现.     

Autodock Vina与Discovery Studio在虚拟筛选耐药蛋白抑制剂中的比较

随着大量与细菌耐药相关的基因的发现和其表达蛋白结构的成功测定,从已有的化合物中通过计算机模拟方法筛选对耐药蛋白靶点有作用的候选化合物,成为了药物发现的一个标准途径。虚拟筛选在耐药基因抑制剂的发现中可以提高效率、降低实验成本。本文介绍了Autodock Vina和Discovery Studio在基于分子对接法的虚拟筛选中的使用,并对比分析其对β-内酰胺酶活性位点的筛选结果。希望通过这种比较促进虚拟筛选在药物设计领域中的应用,提高耐药基因抑制剂的发现速度。     

血管紧张素转换酶抑制剂的分子设计

目的:以血管紧张素转换酶(ACE)为受体靶蛋白,设计相应的抑制剂分子。方法:采用计算机辅助药物分子设计的方法,在分析已有的关于ACE及其抑制剂的基础上,确定分子设计软件LigBuilder中的相关参数。结果:设计得出了1500个配体分子,并从中筛选出了1个各项得分最高的新型血管紧张素转换酶抑制剂分子,其分子式为C27H36N2O3,分子量为436,logP值为4.95,与受体靶蛋白ACE的结合力得分为11.71。结论:从计算得分来看,所设计出的分子与ACE的亲和性好,且脂溶性高,为发掘高活性的降压药物提供了线索。     

综述与专论：蛋白质错误折叠相关疾病的多肽药物研究进展

蛋白质和多肽发生错误折叠形成不可溶的淀粉样纤维的过程,与阿尔茨海默病、帕金森病等多种神经退行性疾病密切相关。这些疾病可导致认知能力下降以及运动缺陷等症状。虽然已有多种相关治疗方案处于临床试验中,但目前仍无明确有效的方法可治愈或长期减缓疾病的进展。探寻和研究抑制淀粉样聚集、识别并促进毒性聚集物清除的抑制剂分子是药物研发的重要策略之一。在不同类型的抑制剂中,多肽类抑制剂因具有高特异性、低毒性、多样性,以及修饰后的抗水解稳定性和血脑屏障通透性,有望成为候选药物分子。本文总结了针对阿尔茨海默病相关的Aβ和Tau蛋白以及帕金森病相关的α-synuclein蛋白淀粉样纤维化的多肽抑制剂研究进展。基于淀粉样纤维化核心序列及纤维核心结构进行合理设计,或通过随机筛选,均可获得多肽抑制剂。这些天然和非天然的多肽分子大多具有抑制淀粉样纤维化、解聚成熟纤维和降低细胞毒性的作用,其中一些多肽在退行性疾病动物模型实验中,显示出降低脑损伤和缓解认知及运动障碍的效果。这些研究揭示了多肽作为蛋白质错误折叠和聚集相关疾病药物的特点,为研发一类新的有效药物奠定了基础。     

蛋白质错误折叠相关疾病的多肽药物研究进展

蛋白质和多肽发生错误折叠形成不可溶的淀粉样纤维的过程,与阿尔茨海默病、帕金森病等多种神经退行性疾病密切相关。这些疾病可导致认知能力下降以及运动缺陷等症状。虽然已有多种相关治疗方案处于临床试验中,但目前仍无明确有效的方法可治愈或长期减缓疾病的进展。探寻和研究抑制淀粉样聚集、识别并促进毒性聚集物清除的抑制剂分子是药物研发的重要策略之一。在不同类型的抑制剂中,多肽类抑制剂因具有高特异性、低毒性、多样性,以及修饰后的抗水解稳定性和血脑屏障通透性,有望成为候选药物分子。本文总结了针对阿尔茨海默病相关的Aβ和Tau蛋白以及帕金森病相关的α-synuclein蛋白淀粉样纤维化的多肽抑制剂研究进展。基于淀粉样纤维化核心序列及纤维核心结构进行合理设计,或通过随机筛选,均可获得多肽抑制剂。这些天然和非天然的多肽分子大多具有抑制淀粉样纤维化、解聚成熟纤维和降低细胞毒性的作用,其中一些多肽在退行性疾病动物模型实验中,显示出降低脑损伤和缓解认知及运动障碍的效果。这些研究揭示了多肽作为蛋白质错误折叠和聚集相关疾病药物的特点,为研发一类新的有效药物奠定了基础。     

Insights into integrin-ligand binding and activation from the first crystal structure

Integrin receptors transduce bidirectional signals between extracellular adhesion molecules and intracellular cytoskeletal and signalling molecules. The structural basis of integrin signalling is unknown, but the recent publication of the first crystal structure of the extracellular domain of integrin alphaVbeta3 has provided a number of insights. In this review, previous structure-function analyses of integrins that have employed biochemical and molecular biological approaches are placed in the context of the crystal structure, and novel routes to the development of integrin antagonists are discussed.     

��v��3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.     

Intermolecular Transmembrane Domain Interactions Activate Integrin αIIbβ3

Integrins are the major cell adhesion molecules responsible for cell attachment to the extracellular matrix. The strength of integrin-mediated adhesion is controlled by the affinity of individual integrins (integrin activation) as well as by the number of integrins involved in such adhesion. The positive correlation between integrin activation and integrin clustering had been suggested previously, but several trials to induce integrin clustering by dimerization of the transmembrane domain or tail region of integrin α subunits failed to demonstrate any change in integrin activation. Here, using platelet integrin αIIbβ3 as a model system, we showed that there is intermolecular lateral interaction between integrins through the transmembrane domains, and this interaction can enhance the affinity state of integrins. In addition, when integrin clustering was induced through heteromeric lateral interactions using bimolecular fluorescence complementation, we could observe a significant increase in the number of active integrin molecules. Because the possibility of intermolecular interaction would be increased by a higher local concentration of integrins, we propose that integrin clustering can shift the equilibrium in favor of integrin activation.     

Integrins: regulators of embryogenesis

Integrins are heterodimeric transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix adhesion. They also participate in cytoskeletal rearrangements, co-regulation of growth factor activities and activation of signal transductions. This review describes experimental approaches that have given new insights into the integrin functions during embryogenesis. Using anti-functional antibodies, peptide inhibitors of integrin-ligand interactions and genetic ablation of integrins results, this review will show that integrins are key molecules during early development of both invertebrates and vertebrates.     

Intracellular signalling controlling integrin activation in lymphocytes

Since the discovery that integrins at the surface of lymphocytes undergo dynamic changes in their adhesive activity after stimulation through the T-cell receptor or stimulation with chemokines, intensive research has been carried out in an attempt to clarify the signalling events that lead to the activation of integrins. Whereas structural studies have provided us with a vivid picture of the conformational flexibility of integrins, the signalling pathways that regulate these conformational changes (known as inside-out signalling) have been elusive. However, as I discuss here, recent studies have provided new insight into the pathways that control the regulation of integrin activity and the coordination of complex cellular functions, such as the homing of lymphocytes and the formation of an immunological synapse.     

Discovery of +(2-{4-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy]phenyl}-cyclopropyl)acetic acid as potent and selective alphavbeta3 inhibitor: design, synthesis, and optimization

The integrin alpha(v)beta(3) is expressed in a number of cell types and is thought to play a major role in several pathological conditions. Various small molecules that inhibit the integrin have been shown to suppress tumor growth and retinal angiogenesis. The tripeptide Arg-Gly-Asp (RGD), a common binding motif in several ligands that bind to alpha(v)beta(3), has been depeptidized and optimized in our efforts toward discovering a small molecule inhibitor. We recently disclosed the synthesis and biological activity of several small molecules that did not contain any peptide bond and mimic the tripeptide RGD. The phenethyl group in one of the lead compounds was successfully replaced with a cyclopropyl moiety. The new lead compound was optimized for potency, selectivity, and for its ADME properties. We describe herein the discovery, synthesis, and optimization of cyclopropyl containing analogs that are potent and selective inhibitors of alpha(v)beta(3).     

Structures of integrin domains and concerted conformational changes in the bidirectional signaling mechanism of alphaIIbbeta3

Integrins are heterodimeric type I transmembrane cell-adhesive receptors whose affinity for ligands is regulated by tertiary and quaternary conformational changes that are transmitted from the cytoplasmic tails to the extracellular ectodomains during the transition from the inactive to the active state. Receptor occupancy initiates further structural alterations that transduce signals across the plasma membrane and result in receptor clustering and recruitment of signaling molecules and cytoskeletal rearrangements at the integrin's cytoplasmic domains. The large distance between the intracellular cytoplasmic domains and the ligand-binding site, which in an extended conformation spans more that 200 A, imposes a complex mechanism of interdomain communication for the bidirectional information flow across the plasma membrane. Significant progress has recently been made in elucidating the crystal and electron microscopy structures of integrin ectodomains in its unliganded and liganded states, and the nuclear magnetic resonance solution structures of stalk domains and the cytoplasmic tails. These structures revealed the location of sites that are functionally important and provided the basis for defining new models of integrin activation and signaling through bidirectional conformational changes, and for understanding the structural basis of the cation-dependent ligand-binding specificity of integrins. Platelet integrin alphaIIbbeta3 has served as a paradigm for many aspects of the structure and function of integrins The aim of this minireview is to combine recent structural and biochemical studies on integrin receptors that converge into a model of the tertiary and quaternary conformational changes in alphaIIbbeta3 and other homologous integrins that propagate inside-out and outside-in signals.     

Macrophage-enriched myristoylated alanine-rich C kinase substrate and its phosphorylation is required for the phorbol ester-stimulated diffusion of beta 2 integrin molecules

An early event of beta(2) integrin activation is the increased diffusion rate of this molecule on the cell surface, thereby providing integrin molecules with a better chance to meet the ligands. The activation of protein kinase C (PKC) stimulates integrin diffusion by releasing the cytoskeletal constraint on integrin molecules. We report here that macrophage-enriched myristoylated alanine-rich C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in integrin activation, is required for this PKC-stimulated diffusion of integrin molecules. Using the single-particle tracking technique, we observed that the activation of PKC stimulated an 11-fold increase in the diffusion rate of beta(2) integrins in wild type J774 macrophage cells but not in those expressing mutant MacMARCKS. Further evidence is provided from a MacMARCKS-deficient cell line in which phorbol esters failed to stimulate the diffusion of integrin. Transfection of wild type MacMARCKS into these cells restored the rapid diffusion rate of the beta(2) integrins. The phosphorylation of MacMARCKS is important because transfection of a nonphosphorylatable MacMARCKS mutant or the addition of staurosporine eliminates the rapid diffusion rate of integrin. Furthermore, adding cytochalasin D bypasses the MacMARCKS deficiency and stimulates beta(2) integrin diffusion, suggesting that MacMARCKS's involvement in integrin activation is prior or at the site of cytoskeleton. Therefore, we conclude that MacMARCKS is required for releasing the cytoskeletal constraint on integrin molecules during PKC-mediated integrin activation.