以EGFR为靶点的肿瘤分子靶向药物研究进展

随着分子生物学研究的进展,分子靶向治疗已成为除手术、放疗、化疗之外的第4种治疗方法,越来越多的用于临床治疗恶性肿瘤。分子靶向药物进入体内能够特异地选择致癌位点,杀伤肿瘤细胞,而不会波及周围正常的组织细胞,因此分子靶向治疗又被称为"生物导弹"。与传统化疗药物相比,分子靶向药物具有特异性强、疗效明显、副作用少等优点。按照分子靶向药物的性质主要归为两大类:一类是单克隆抗体,如西妥昔单抗等;另一类是单靶点或多靶点的小分子抑制剂,如吉非替尼等。表皮生长因子受体(EGFR)对肿瘤的生长、发展以及肿瘤干细胞的维持都有着非常重要的作用,并且在多种实体瘤中存在过表达或异常表达,因此在肿瘤治疗中,EGFR成为一个非常重要的用药靶点。现主要对目前国内已上市的针对EGFR的分子靶向药物最新的临床研究进展作一简要综述。     

靶向EGFR家族的抗肿瘤药物研究进展

表皮生长因子受体（EGFR,ErbB）家族在肿瘤的发生、发展中具有重要的作用。很多实体肿瘤中存在EGFR家族受体过表达或异常激活。靶向EGFR家族的抗肿瘤药物研发已经成为一个热点领域,并且成功地应用于临床。靶向EGFR家族的抗肿瘤药物可以分为单克隆抗体和小分子酪氨酸激酶抑制剂两大类。单克隆抗体与受体胞外区结合阻止配体．受体的结合或者阻止配体结合引起的受体活化;而小分子酪氨酸激酶抑制剂则结合于胞内激酶区,抑制激酶自磷酸化和下游信号通路激活。     

非小细胞肺癌EGFR-TKIs治疗及PET分子成像的研究进展

表皮生长因子受体(epithelial growth factor receptor,EGFR)信号转导通路在非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)中发挥重要作用,尤其胞内酪氨酸激酶结构域的突变状态决定了目前NSCLC的靶向治疗。针对EGFR突变的分子靶向药物表皮生长因子受体酪氨酸激酶抑制剂(epithelial growth factor receptor tyrosine kinase inhibitors,EGFR-TKIs)已开发并应用于NSCLC的治疗。在治疗过程中,EGFR的突变状态随时间发生动态变化,因此精准掌握EGFR的突变状态是靶向治疗方案制定、优化的关键。PET分子成像可在细胞和分子水平,对在体生物活动的发生、发展过程进行实时成像,使实时、在体揭示EGFR的突变状态成为可能。因此,多种以TKIs为前体标记放射性核素作为靶向肿瘤突变EGFR胞内段分子成像探针的研究逐渐增多。本文就EGFR-TKIs在NSCLC治疗及相关PET分子成像方面的研究进展进行综述。     

从噬菌体文库中筛选与表皮生长因子结合的多肽(英)

与许多疾病相关的血管生成作用是由一些血管生成因子介导的,其中就包括表皮生长因子.在肿瘤生长、关节炎等疾病中,表皮生长因子参与了其中的血管生成作用,拮抗表皮生长因子介导的血管生成就有可能对与其相关的疾病起到治疗作用,因此,表皮生长因子的拮抗剂可能具有重要的临床价值.拮抗表皮生长因子的作用可以通过许多途径,其中之一就是找到能与表皮生长因子结合并能干预其与受体结合的分子,因而表皮生长因子可作为药物靶分子.从噬菌体文库中筛选药物靶分子的拮抗剂和激动剂已被证明是一种有效的方法.以表皮生长因子作为药物靶分子,从多肽噬菌体文库中筛选与表皮生长因子结合的噬菌体多肽,这些潜在的表皮生长因子拮抗剂先导分子经过优化可能具有重要的临床价值.     

从噬菌体文库中筛选与表皮生长因子结合的多肽

与许多疾病相关的血管生成作用是由一些血管生成因子介导的 ,其中就包括表皮生长因子 .在肿瘤生长、关节炎等疾病中 ,表皮生长因子参与了其中的血管生成作用 ,拮抗表皮生长因子介导的血管生成就有可能对与其相关的疾病起到治疗作用 ,因此 ,表皮生长因子的拮抗剂可能具有重要的临床价值 .拮抗表皮生长因子的作用可以通过许多途径 ,其中之一就是找到能与表皮生长因子结合并能干预其与受体结合的分子 ,因而表皮生长因子可作为药物靶分子 .从噬菌体文库中筛选药物靶分子的拮抗剂和激动剂已被证明是一种有效的方法 .以表皮生长因子作为药物靶分子 ,从多肽噬菌体文库中筛选与表皮生长因子结合的噬菌体多肽 ,这些潜在的表皮生长因子拮抗剂先导分子经过优化可能具有重要的临床价值 .     

EGFR靶向的PET/SPECT分子成像研究进展

恶性肿瘤的发病率及死亡率逐年递增,分子靶向治疗为癌症治疗带来了新的革命,表皮生长因子受体(EGFR)在癌症发生、发展中发挥重要作用,针对EGFR的分子靶向治疗已成为近年研究热点。目前,已有多种EGFR分子靶向药物应用于临床,但总体有效率偏低。研究表明EGFR过表达和/或突变对治疗效果影响显著,因此治疗前准确评价肿瘤EGFR表达水平及突变状态显得尤为重要。分子成像能够实现活体细胞及分子水平生物学过程成像,并进行定性定量研究,使在体揭示EGFR表达状态成为可能。本文简述EGFR靶向分子成像的研究进展并对不同分子探针成像结果进行比较分析,对不同分子成像探针的功能进行评价,以期有益于EGFR靶向分子成像探针的研发及EGFR靶向分子成像研究。     

EGF类生长因子来源的新型靶向肽在抗肿瘤药物蛋白中的应用

许多肿瘤细胞表面表皮生长因子受体EGFR都存在过表达现象。考察了牛痘病毒生长因子(VGF)中的EGFR结合域(S3)与人的肝素样表皮生长因子(HB-EGF)来源的肝素结合域(命名为HE)重组后对肿瘤细胞的选择性。通过重组表达带有靶向和穿膜结构域的EGFP-S3-HE和EGFP-S3-HE-TATm两种融合蛋白与正常细胞和肿瘤细胞的共孵育实验来研究其对肿瘤细胞的特异性靶向吸附和穿膜效应。进一步将S3-HE-TATm靶向穿膜序列与苦瓜来源的核糖体失活蛋白MAP30融合,可显著提高MAP30对肿瘤细胞的抑杀作用,但这种抑杀作用却对正常细胞仍保持在较低水平。由此表明S3-HE-TATm是一种新型优异的肿瘤细胞靶向药物运输载体,可用于肿瘤治疗的进一步开发研究。     

PEG修饰对PLL-EGF基因载体靶向结合作用的影响

聚阳离子基因载体系统由于安全性好和便于设计等优点,近年来在基因治疗中的应用发展迅速.在进行基因药物的体内靶向输送时,目前国际上主要通过在基因输送系统中修饰聚乙二醇(PEG)和靶向分子来提高体内输送的稳定性和靶向性.PEG的修饰可能会遮蔽靶向分子的功能呈现,因此建立定量分析方法评价PEG修饰对靶向结合作用的影响非常重要.将连接有表皮生长因子(EGF)的聚赖氨酸(PLL)基因载体作为研究模型,建立BIAcore检测方法,比较PLL-EGF,PEG7000修饰的PLL-EGF,PEG20000修饰的PLL-EGF对表皮生长因子受体(EGFR)的结合和解离速率,评价PEG修饰对PLL-EGF靶向功能呈现的影响.结果表明,PEG7000的修饰降低了EGF和EGFR之间的结合速率,提高了解离速率,整体减弱了靶向分子的靶向结合能力.PEG20000的修饰进一步减弱靶向分子功能的呈现.因此在进行靶向型聚阳离子基因输送系统设计时,考察PEG修饰对靶向结合能力的影响程度非常重要.该研究结果也对其他基因载体系统的设计提供必要的参考.     

肺癌的表皮生长因子受体分子靶向治疗与基因突变

肺癌分子靶向治疗近年来取得较大进展,特别是针对表皮生长因子受体（EGFR）分子靶向药物表现出确定的临床效果。临床应用表明,EGFR基因酪氨酸激酶域体细胞突变与非小细胞肺癌患者对酪氨酸激酶抑制剂吉非替尼的敏感性相关,本文就相关的研究进行了简述。     

EGFR耐药突变及其小分子抑制剂研究进展

表皮生长因子受体(epidermal growth factor receptor,EGFR)是一种具有酪氨酸激酶活性的重要跨膜受体,在多种恶性肿瘤中异常表达,与肿瘤细胞增殖、分化、转移等生命活动密切相关。EGFR已成为治疗肿瘤的靶点,针对该靶点设计的药物主要分为单克隆抗体和小分子抑制剂两大类,小分子抑制剂易导致EGFR出现突变而产生耐药现象,从而影响其临床应用。突变主要发生在酪氨酸激酶区域ATP结合位点附近,主要为19号外显子上的缺失突变,18号和21号外显子上的点突变。针对近几十年来国内外研究者对EGFR出现的耐药突变类型,及其与小分子抑制剂的相互作用方式进行综述,以期为后续EGFR靶点药物的研发提供参考。     

Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

Background

Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells.

Methodology/Principal Findings

The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength.

Conclusions/Significance

We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.     

Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, Z(EGFR) and Z(HER2), were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric Z(EGFR) and Z(HER2) ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents..     

Synthesis of galactosyl compounds for targeted gene delivery

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.     

Protein–protein interactions between SWCNT/chitosan/EGF and EGF receptor: a model of drug delivery system

Epidermal growth factor (EGF) was used as the targeting ligand to enhance the specificity of a cancer drug delivery system (DDS) via its specific interaction with the EGF receptor (EGFR) that is overexpressed on the surface of some cancer cells. To investigate the intermolecular interaction and binding affinity between the EGF-conjugated DDS and the EGFR, 50 ns molecular dynamics simulations were performed on the complex of tethered EGFR and EGF linked to single-wall carbon nanotube (SWCNT) through a biopolymer chitosan wrapping the tube outer surface (EGFR·EGF-CS-SWCNT-Drug complex), and compared to the EGFR·EGF complex and free EGFR. The binding pattern of the EGF-CS-SWCNT-Drug complex to the EGFR was broadly comparable to that for EGF, but the binding affinity of the EGF-CS-SWCNT-Drug complex was predicted to be somewhat better than that for EGF alone. Additionally, the chitosan chain could prevent undesired interactions of SWCNT at the binding pocket region. Therefore, EGF connected to SWCNT via a chitosan linker is a seemingly good formulation for developing a smart DDS served as part of an alternative cancer therapy.     

Targeted drug delivery in pancreatic cancer

Effective drug delivery in pancreatic cancer treatment remains a major challenge. Because of the high resistance to chemo and radiation therapy, the overall survival rate for pancreatic cancer is extremely low. Recent advances in drug delivery systems hold great promise for improving cancer therapy. Using liposomes, nanoparticles, and carbon nanotubes to deliver cancer drugs and other therapeutic agents such as siRNA, suicide gene, oncolytic virus, small molecule inhibitor, and antibody has been a success in recent preclinical trials. However, how to improve the specificity and stability of the delivered drug using ligand or antibody directed delivery represent a major problem. Therefore, developing novel, specific, tumor-targeted drug delivery systems is urgently needed for this terrible disease. This review summarizes the current progress on targeted drug delivery in pancreatic cancer and provides important information on potential therapeutic targets for pancreatic cancer treatment.     

A novel small peptide as a targeting ligand for receptor tyrosine kinase Tie2

Tie2 is an endothelium-specific receptor tyrosine kinase known to play an important role in tumor angiogenesis. We sought to identify a small peptide ligand against Tie2 for developing a delivery targeting agent. We used hydrophobic analysis and comparative sequence/structure analysis to select a minimal peptide based on angiopoietin-2 amino acid sequence. The resulting peptide named GA3(WTIIQRREDGSVDFQRTWKEYK) was synthesized and labeled with iodine-125 at the C-terminal tyrosine residue to characterize its binding capability. In in vitro binding assays, GA3 can not only specifically bind to SMMC7721-Tie2 but also compete with angiopoietin-2 in binding. Via mouse tail vein injection, 125I-labeled GA3 was found to favorably accumulate in SPC-A1 xenograft tumor tissues which positively express Tie2. These results demonstrated that GA3 may be useful as a drug or gene delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.     

Exploration of the conformational landscape in pregnane X receptor reveals a new binding pocket

Ligand‐regulated pregnane X receptor (PXR), a member of the nuclear receptor superfamily, plays a central role in xenobiotic metabolism. Despite its critical role in drug metabolism, PXR activation can lead to adverse drug‐drug interactions and early stage metabolism of drugs. Activated PXR can induce cancer drug resistance and enhance the onset of malignancy. Since promiscuity in ligand binding makes it difficult to develop competitive inhibitors targeting PXR ligand binding pocket (LBP), it is essential to identify allosteric sites for effective PXR antagonism. Here, molecular dynamics (MD) simulation studies unravelled the existence of two different conformational states, namely “expanded” and “contracted”, in apo PXR ligand binding domain (LBD). Ligand binding events shifted this conformational equilibrium and locked the LBD in a single “ligand‐adaptable” conformational state. Ensemble‐based computational solvent mapping identified a transiently open potential small molecule binding pocket between α5 and α8 helices, named “α8 pocket”, whose opening‐closing mechanism directly correlated with the conformational shift in LBD. A virtual hit identified through structure‐based virtual screening against α8 pocket locks the pocket in its open conformation. MD simulations further revealed that the presence of small molecule at allosteric site disrupts the LBD dynamics and locks the LBD in a “tightly‐contracted” conformation. The molecular details provided here could guide new structural studies to understand PXR activation and antagonism.     

Ligand-based virtual screening,molecular docking,QSAR and pharmacophore analysis of quercetin-associated potential novel analogs against epidermal growth factor receptor

The present study was to explore expectation and examination of therapeutic potential quercetin analogs as efficient anticancer agents against human epidermal growth factor receptor (EGFR), which is a consistent hallmark for moderating the non-small-cell lung carcinoma (NSCLC). Here, ligand-based virtual screening, pharmacophore approach and molecular docking were established as rational strategies for recognition of small analogs against the ligand binding domain of EGFR (PDB code: 1XKK). Adverse effects, toxicogenomics and pharmacokinetics reported that 10 candidates showed reliable consequences with less side effects and more efficient for target receptor. Protein–ligand interaction profiles revealed that the probable H-bonds, atomic-π contacts, salt bridges and van der Waals interactions sustain the complexity and stability of receptor structure; thus, they could complicate to generate single alteration acquired for drug resistance. In silico anticancer properties explain the lead scaffolds which are assumed to be flexible and experimentally proved chemicals. The overall consequences indicated that recognized leads could be utilized as reference skeletons for new inhibitors envisaging toward EGFR to ameliorate NSCLC and other malignant disorders.