Discovery of small molecule inhibitors that interact with γ-tubulin

Recent studies have shown an overexpression of γ-tubulin in human glioblastomas and glioblastoma cell lines. As the 2-year survival rate for glioblastoma is very poor, potential benefit exists for discovering novel chemotherapeutic agents that can inhibit γ-tubulin, which is known to form a ring complex that acts as a microtubule nucleation center. We present experimental evidence that colchicine and combretastatin A-4 bind to γ-tubulin, which are to our knowledge the first drug-like compounds known to interact with γ-tubulin. Molecular dynamics simulations and docking studies were used to analyze the hypothesized γ-tubulin binding domain of these compounds. The suitability of the potential binding modes was evaluated and suggests the subsequent rational design of novel targeted inhibitors of γ-tubulin.     

Fatty acyl moieties: improving Pro‐rich peptide uptake inside HeLa cells

Abstract: In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro‐rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro‐rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.     

Physicochemical and Biological Characterization of Pep‐1/Elastin Complexes

Transdermal drug delivery of proteins is challenging because the skin acts as a natural and protective barrier. Several techniques including using the cell‐penetrating peptides have been studied to increase the penetration of therapeutic proteins into and through the skin. Cell‐penetrating peptides facilitate and improve the transduction of large and hydrophilic cargo molecules through plasma membrane. We have recently reported an efficient skin delivery of elastin protein in complex with a cell‐penetrating peptide called Pep‐1. As the biophysical characteristics of cell‐penetrating peptide/protein complexes have been linked with their biological responses, in this study, we investigated biophysical properties of Pep‐1/elastin complexes (ratio 10:1) stored in three temperatures (?20 °C, 4 °C and 25 °C) by photon correlation spectroscopy, circular dichroism and isothermal denaturation. We also evaluated the ability of transduction of this complex into cells and skin tissue using both fluorescence microscopy and Kodak In‐Vivo FX Pro Imaging System.     

细胞穿膜肽在癌症治疗中的应用

细胞膜有选择渗透性,能阻止大多数外来因子进入细胞。然而许多治疗因子必须穿过细胞膜才能进入细胞内起作用。细胞穿膜肽是一组简短的阳离子序列,它具有很强的穿膜能力。自从1988年发现细胞穿膜肽后,细胞穿膜肽被用于携带各种物质,如小分子、核酸、抗体、纳米粒等进入细胞膜。本文重点介绍了近几年来关于细胞穿膜肽穿膜机制的研究进展,以及细胞穿膜肽作为运载体携带抗癌药物靶向进入肿瘤细胞,从而实现对肿瘤细胞的靶向性,同时可克服肿瘤的多药抗药性等研究进展。     

多肽类药物在新生儿缺氧缺血性脑损伤中的研究进展

新生儿缺氧缺血性脑损伤是新生儿死亡和婴幼儿神经系统功能障碍的主要原因,目前唯一公认有效的治疗方法是亚低温治疗。近年来,随着新技术的发展,多肽类药物由于具有高选择性、高效性、耐受性好、制备简单、成本低等优点,得到了广泛关注。利用新生儿缺氧缺血性脑损伤模型的研究发现,多种多肽类药物可直接作用或通过与细胞穿膜肽结合进入神经细胞,通过抑制细胞凋亡、降低神经兴奋性毒性、改善线粒体通透性等机制发挥脑保护作用,作为一种新的新生儿缺氧缺血性脑损伤治疗途径有着巨大的潜力。本文就这些多肽类药物的最新研究进展做一综述,为新生儿缺氧缺血性脑损伤的治疗提供新思路。     

Cationic Amphiphilic Polyproline Helices: Side‐Chain Variations and Cell‐Specific Internalization

Cell‐penetrating peptides present an attractive and efficient tool for the delivery of a variety of cell impermeable cargoes across the cellular membrane. Cell‐penetrating peptides usually consist of short basic peptide sequences that are internalized by a variety of cell lines. Most cell‐penetrating peptides lack cell specificity, however, which greatly limits their use as efficient therapeutic agents. Herein, we present two cell‐penetrating peptides displaying a type II polyproline helical backbone that are functionalized to contain six cationic moieties and two distinctive hydrophobic functionalities, namely isobutyl or benzyl groups. The uptake efficiency of these cationic amphiphilic polyproline helices was studied in seven different cell lines, six cancerous (MCF‐7, HOS, HT1080, HeLa, KB‐FD, KB3‐1) and one non‐cancerous (WI 38). The cationic amphiphilic polyproline helix P11LRR at 50 μm showed high specificity toward MCF‐7 breast cancer cells. Co‐culture experiments with P11LRR demonstrated almost exclusive internalization by MCF‐7 cells and not WI38. The replacement of the isobutyl hydrophobic group with a benzyl moiety resulted in a shift in uptake efficiency and specificity across some cell lines. These results demonstrate that the type of hydrophobic residues utilized in the creation of cell‐penetrating peptides can strongly influence the extent and specificity of cellular internalization.     

Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58)

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.     

Design of a new pH‐activatable cell‐penetrating peptide for drug delivery into tumor cells

Cell‐penetrating peptides (CPPs) have been considered as potential drug delivery vectors due to their remarkable membrane translocation capacity. However, lack of specificity and extreme systemic toxicity hamper their successful application for drug delivery. Here, we designed a new pH‐activatable CPP, LHHLLHHLHHLLHH‐NH₂ (LH), by substitution of all lysines and two leucines of LKKLLKLLKKLLKL‐NH₂ (LK) with histidines. As expected, histidine‐rich LH could be activated and penetrate into cells at pH 6.0, whereas its membrane transduction activity could be shielded at pH 7.4. In contrast, LK showed no obviously different cellular uptake at both pH conditions. Importantly, LH was significantly less cytotoxicity compared with LK at both pH values, suggesting a better safety for further application. In addition, after conjugation of camptothecin (CPT) with LH, this conjugate displayed remarkably pH‐dependent antitumor activity than free CPT and LK‐CPT. This study provides a new tumor pH‐responsive CPP with low toxicity for selective anticancer drug delivery.     

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

Recent advances in the use of cell-penetrating peptides for medical and biological applications

The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.     

Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives

Cell penetrating peptides, generally categorized as amphipathic or cationic depending on their sequence, are increasingly drawing attention as a non-invasive delivery technology for macromolecules. Delivery of a diverse set of cargo in terms of size and nature ranging from small molecules to particulate cargo has been attempted using different types of cell penetrating peptides (CPPs) in vitro and in vivo. However, the internalization mechanism of CPPs is an unresolved issue to date, with dramatic changes in view regarding the involvement of endocytosis as a pathway of internalization. A key reason for the lack of consensus on the mechanism can be attributed to the methodology in deciphering the internalization mechanism. In this review, we highlight some of the methodology concerns, focus more on the internalization pathway and also provide a novel perspective about the intracellular processing of CPPs, which is a crucial aspect to consider when selecting a cell penetrating peptide as a drug delivery system. In addition, recent applications of cell penetrating peptides for the delivery of small molecules, peptides, proteins, oligonucleotides, nanoparticles and liposomes have been reviewed.     

Cell Penetrating Peptides in Drug Delivery

Protein transduction domains (PTDs) are small cationic peptides that can facilitate the uptake of large, biologically active molecules into mammalian cells. Recent reports have suggested that PTDs may be able to mediate the delivery of cargo to tissues throughout a living organism. Such technology could eliminate the size restrictions on usable drugs, enabling previously unavailable large molecules to modulate in vivo biology and alleviate disease. In this article, we review the evidence that PTDs can be used both to deliver active molecules to pathological tissue in vivo and to treat models of disease such as ischemia, inflammation, and cancer.     

Functionalised nanostructures for transdermal delivery of drug cargos

Nanotechnology has burgeoned over last decade exploring varieties of novel applications in all areas of science and technology. Utilisation of bio-friendly polymers for engineering nanostructures (NS) improves safety and efficacy in drug delivery. Biopolymers not merely employed for fabricating drug carriers but also leveraged for surface functionalisation of other NS to impart bio-mimicking properties. Biopolymer functionalised NS garnered researcher’s attention because of their potential to enhance skin permeability of drug cargo. Biopolymers, i.e. cell-penetrating peptides (CPP), chitosan and hyaluronic acid not only enhance skin permeability but also add multiple functions due to their intrinsic biomimetic properties. This multifunctional drug delivery system is a promising tool to achieve skin delivery of large number of therapeutic agents. In this review, functionalisation of NS with biopolymers particularly polysaccharides and polypeptides is discussed in detail. In particular, applications of these functionalised NS for TDDS is elaborated. Moreover, this review provides framework for elaborating importance of functionalisation of NS to enhance skin permeability and depicts advantages of biopolymers to construct more biocompatible carriers for drug cargos.     

糖类高渗化合物对TAT穿膜效率的影响

目的研究糖类高渗化合物葡萄糖、蔗糖或甘露醇预处理细胞对TAT穿膜效率的影响。方法人工合成荧光标记多肽TAT-FITC和无意义肽NCO-FITC,将其作用于体外培养的人宫颈癌细胞株Siha、小鼠成纤维细胞L929及人肝癌细胞株HepG2。荧光酶标仪定量检测不同预处理对各细胞摄取荧光标记短肽的影响及内吞抑制药阿米洛利对不同预处理后TAT穿膜的影响;荧光显微镜观察不同预处理后,TAT-FITC的穿膜效率及其在Siha细胞内的定位;MTT检测不同预处理对Siha细胞存活率的影响。结果糖类高渗化合物预处理细胞后,TAT-FITC可高效穿膜进入细胞内,且在胞浆、胞核中均匀分布,胞核浓度高于胞浆浓度;未见NCO-FITC穿膜进入细胞。0.6mol·L-1葡萄糖预处理后,细胞摄取TAT-FITC的荧光强度较0.6mol·L-1蔗糖预处理后相近且较对照组明显增强(P<0.01),0.5mol·L-1甘露醇预处理的荧光强度次之。加入阿米洛利后,糖类高渗化合物预处理的细胞摄取TAT-FITC荧光强度均明显减弱(P<0.01)。MTT数据显示适当浓度的糖类高渗化合物对细胞的活力有轻微影响。结论0.6mol·L-1葡萄糖或0.6mol.L-1蔗糖或0.5mol·L-1甘露醇预处理均可提高TAT对培养细胞的穿膜效率,但对细胞活力影响较小;预处理后细胞可能以内吞方式摄取TAT-FITC短肽。     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.