多肽类药物研究的新进展 潘婷婷

多肽药物定义： 通常将含有的氨基酸少于 10 个的肽称为寡肽，超过的就 称为多肽。所以多肽药物可以这样说： 从生物化学本质上说是一种肽，具有 10 个氨基酸以上； 从功能上讲具有药物的功能，能用于疾病的预防、治疗与 诊断。

多肽药物的作用 多肽类药物习惯上常指多肽类激素。多肽类激素是细胞 自制的，含有调节生理和代谢效能的微量有机物质。多肽 激素的分子较大，不直接进入靶细胞里，而是首先与分布 在细胞表面的特异性受体结合，这样，激活了与受体相连 接的效应器。受体和效应器都在细胞表面的质膜上，通过 某种方式相连接。活化的效应器起作用后产生 “ 第二信使 ” 而传递激素的信息，在细胞内激活一些酶系，从而促进中 间代谢或膜的通透性，或通过控制 DNA 转录或翻译而影 响特异的蛋白质合成，最终导致特定的生理效应或发挥其 药理作用。

多肽类药物由于其分子不大，吸收的优点十分显著， 主要为： （ 1 ）不需要消化，直接吸收； （ 2 ）吸收快； （ 3 ）吸收完全，几乎 100% ； （ 4 ）吸收不耗能，可减轻胃肠道功能负担。

Selective killing of cancer cells by peptide-targeted delivery of an anti-microbial peptide 1.Introduction One of the major problems in cancer chemotherapy is the severe toxic side effects of anti-cancer drugs designed to destroy dividing cells, including those found in healthy tissues. Such unwanted side effects often result in dose reduction, treatment delay or discontinuance of therapy. Moreover, all successful cancer therapies are limited by intrinsic or acquired drug resistance. Hence, some necessary steps need to be taken to improve the selectivity and overcome the predictable resistance that develops with standard cancer therapeutics by moving towards novel targeting strategies and therapies.

In the search for novel anticancer therapeutics, positively charged anti-microbial peptides have emerged as promising agents offering several advantages over other therapies. they damage cell membrane within minutes which would hinder formation of resistance. these lytic peptides do not interact with mammalian cell exterior membrane, but they can kill the cells if internalized via targeting moieties such as antibodies and peptides. Within the cytosol they act by irreversibly damaging the mitochondrial membrane, resulting in cell death by apoptosis. Since their activity is not dependent on rapid cell proliferation, they do not have many of the undesirable side effects of other chemothera- peutic drugs.

The LTVSPWY peptide (LTV peptide) is a part of a new generation of small targeting peptides capable of delivering antisense oligonucleotides and small molecules to cancer cells in vitro. In the current study, we designed a new lytic fusion peptide composed of LTV peptide and KLA peptide and investigated its in vitro cytotoxicity and mechanism of action. Furthermore, we have included a gastrin-releasing peptide (GNHWAVGHLM, GR peptide) as targeting moiety whose receptor is expressed by most cancer types, including breast, ovarian, prostate and lung cancer.

The two targeting peptides were chosen because they were found to internalize consequent to binding their cognate receptors. For the pro-apoptotic domain, we selected the synthetic KLA peptide because it killed bacteria at concentrations 1% of those required to kill mammalian cells.

2. Methods and materials 1.1Cell lines Human breast MCF-7 MDA-MB-231 cancer cells Normal human mammary epithelial cells (HMEC) Human peripheral blood mononuclear cells 1.2Peptides  LTV peptide: LTVSPWYC-NH2  GR peptide: CGNHWAVGHLM-NH2  KLA peptide: KLAKLAKKLAKLAKC-NH2  LTV-KLA fusion peptide: LTVSPWYGCGKLAKLAKKLAKLAK- NH2  GR-KLA fusion peptide: KLAKLAKKLAKLAKGCGNHWAVGHLM-NH2  Scrambled LTV-KLA peptide: YLSWVPTGCGKLAKLAKKLAKLAK-NH2  Scrambled GR-KLA peptide: KLAKLAKKLAKLAKGCGLGAWNV-HMH-NH2

3.Results 3.1Efficient binding of fusion peptides to cancer cells The cells were seeded onto 24-well plate and incubated overnight at 37 ℃. Subsequently, they were incubated with 6-iodacetamidofluorescein (6-IAF)- conjugated peptides (1 mM) for 30 min at 37 ℃, gently scraped, washed 3 times with PBS buffer and then analyzed by flow cytometry. To improve the specificity and potency of lytic peptides, we selected two promising targeting moieties; LTV and GR peptide. We coupled the targeting and pro-apoptotic domain with GCG linker to minimize potential steric hindrance that would prevent binding and to allow the conjugation of 6-iodacetamidofluorescein (6-IAF) to the thiol group of cysteine residue.

Collectively, the data indicate that the binding of KLA peptide to cancer cells is significantly enhanced by conjugation to either LTV or GR peptide.

To investigate the uptake and internalization of the peptides by breast cancer cells, confocal microscopic images of the cells incubated with either LTV-KLA or sc-LTV-KLA peptide (1 mM) were taken 30 min after peptide addition and incubation at 37 ℃.

3.2Toxicity of the fusion peptides on cancer cells To evaluate the cytotoxic effect of the fusion peptides on cancer cells, we initially examined their ability to inhibit cell proliferation and viability. Cells were treated with various concentrations of the test molecules for 24 h and then cell viability was determined by the MTT assay using CellTiter 96R Aqueous One Solution Reagent.

MTT 比色法原理： MTT 比色法，是一种检测细胞存活和生长的方 法。其检测原理为活细胞线粒体中的琥珀酸脱氢 酶能使外源性 MTT 还原为水不溶性的蓝紫色结晶 甲瓒（ Formazan ）并沉积在细胞中，而死细胞无 此功能。二甲基亚砜（ DMSO ）能溶解细胞中的 甲瓒，用酶联免疫检测仪在 490nm 波长处测定其 光吸收值，可间接反映活细胞数量。在一定细胞 数范围内， MTT 结晶形成的量与细胞数成正比。 该方法已广泛用于一些生物活性因子的活性检测、 大规模的抗肿瘤药物筛选、细胞毒性试验以及肿 瘤放射敏感性测定等。它的特点是灵敏度高、经 济。

microscopic analysis of MCF-7 cancer cells indicated that the loss of cell viability in the presence of lytic fusion peptides was accompanied by cell fragmentation.

3.3Fusion peptides disrupted cytoplasmic membrane The destruction of the cells observed with the fusion peptides raised the question of the mechanism(s) by which cancer cells were killed. First, we have investigated the effect of the conjugates on integrity of cell membranes using propidium iodide (PI) exclusion assay. PI can permeate unhealthy/damaged membranes, so positive PI fluorescence indicates compromised cell membranes. Cells were treated with the fusion peptides (10 mM) for 2 h, incubated with PI and immediately analyzed by flow cytometry.

Kinetics studies indicated that the exposure of the cells to the fusion peptides resulted in time- dependent loss of cell viability.

These results suggest that the increase in cytotoxicity that occurs when the targeting peptides were fused to KLA peptide is dependent on the targeting cell surface receptors.

4. Discussion The use of peptides and/or monoclonal antibodies with high specificity towards receptors expressed by cancer cells constitutes a promising approach in cancer diagnosis and treatment. Although antibodies are more stable than peptides in biological environments, peptide targeting should be superior to monoclonal antibodies as targeting agents because the antibody molecules are rather large and might not get to the interior of large tumors.

Improvement:  Serum stability of the peptides is an important issue when considering potential in vivo applications. One strategy used to overcome this problem is the use of both D-amino acid replacement, polymer conjugation and/or disulfide bridges.  All our experiments were done in complete medium supplemented with 10% FCS and the peptides used in this study are not modified; therefore, there is room for improvement.  Also, small peptides are expected to be less immunogenic, cost-effective, and easy to conjugate to lytic domains.