DETERMINATION OF INTERACTIONS BETWEEN RIBONUCLEASE 1 AND CHEMICAL LIGANDS

核糖核酸酶 1 和化学配体之间相互作用的测定

基本信息

  • 批准号:
    8361216
  • 负责人:
  • 金额:
    $ 0.2万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-03-01 至 2012-02-29
  • 项目状态:
    已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Onconase, a ribonuclease (RNase) from Northern Leopard Frogs, has shown promise as a selective chemotherapeutic and is currently undergoing Phase IIIb clinical trials for malignant mesothelioma (1,2). This member of the RNase A superfamily of ribonucleases cleaves tRNA leading to cell death via apoptosis (3,4). However, treatment with Onconase has been observed to induce some renal toxicity (5). Pancreatic RNases are small highly cationic proteins found ubiquitously across species and function to degrade RNA for digestion, gene regulation, and viral immunity. Drug design research in this area has uncovered the use of other ribonucleases in this protein family as potential cancer therapeutics (6,7). Human ribonuclease (RNase 1) and the bovine homologue (RNase A) are under inquiry as chemotherapeutics as variants selectively target cancer cells while inducing less toxic renal effects as seen with Onconase (7). The mechanism of RNase into the cell consists of the steps: binding to the cell membrane, internalization, translocation out of the endosome, escaping inhibitor binding, and finally RNA cleavage leading to cell death (8). Previous research in our lab has looked at internalization of RNases among cells of varying surface groups, yet there is no definitive data that indicates how RNase variants bind onto the surface of cancerous cells and internalize (9). However, RNase cytotoxicity has been correlated to overall cationicity (10,11) and more recently the specific distribution of positive charges (8). As such binding and internalization has been linked to the negatively charged molecules on the outer membrane of the cell. Variation of the composition on cancer cell membranes compared to normal cells might target ribonucleases selectively to cancer cells (7, 12), adding to the encouraging therapeutic index observed in animal models and human trials (13,14). The surface of cancer cells frequently display changes in glycosaminoglycan profile (15), phospholipid composition (16,17) and ganglioside array (18). Specific residues of interest include heparan sulfates, charged sugar groups such as sialic acid, and lipids such as phosphotidylserine as recent research has indicated this importance by possible therapeutic targeting (19). We hypothesize that ribonuclease binding to these specific residues on the cell surface may be a factor of protein internalization and thus affect efficiency of cytotoxicity as well as selectivity of cancerous cell lines. The structure of human RNase 1 was recently determined using NMR (20). Other recent papers have also used NMR as a method to look at specific interactions between residues of proteins (21). In order to determine RNase interactions with cell plasma components, we want to similarly label RNase 1 with 15N isotope and titrate cell membrane components including but not limited to phosphatidylserine, heparan sulfate, and sialic acid. We will then look at 2D NOESY (mixing time = ms) spectra recorded on the 600 MHz NMR spectrometer as described by the methods that solved the RNase1 structure (20). Comparison of the shifts in the spectra to the structure solved in the PDB (accession number 2K11) will indicate the location and a broad degree of binding affinity of specific interactions between the RNase 1 and plasma membrane components. The results will establish how RNase1 is bound to the cell surface components and may elucidate the mechanism of internalization. We can then repeat examination using the 15N labeled RNase A and cytotoxic variants to observe a possible correlation between cell membrane interactions and RNase cytotoxicity. Also a broad range of cell membrane components can be further investigated. This may allow for developing of RNases with higher internalization due to increased binding or targeting towards cell markers that are over-expressed on cancerous cells.
该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和子项目主要研究者的主要支持可能由其他来源提供,包括其他NIH来源。 为子项目列出的总成本可能代表子项目使用的中心基础设施的估计数量,而不是NCRR赠款向子项目或子项目工作人员提供的直接资金。 Onconase是一种来自北方豹蛙的核糖核酸酶(RNase),已显示出作为选择性化疗药物的前景,目前正在进行恶性间皮瘤的IIIb期临床试验(1,2)。 核糖核酸酶的RNase A超家族的这一成员切割tRNA,导致细胞通过凋亡死亡(3,4)。 然而,已观察到Onconase治疗可诱导一些肾毒性(5)。 胰腺RNA酶是一种小的高度阳离子蛋白,在物种中普遍存在,其功能是降解RNA以进行消化、基因调控和病毒免疫。 在这一领域的药物设计研究已经揭示了使用该蛋白质家族中的其他核糖核酸酶作为潜在的癌症治疗剂(6,7)。 人核糖核酸酶(RNase 1)和牛同源物(RNase A)正在研究作为化疗药物,因为变体选择性靶向癌细胞,同时诱导较少的毒性肾效应,如Onconase所见(7)。 RNA酶进入细胞的机制包括以下步骤:与细胞膜结合,内化,易位出内体,逃避抑制剂结合,最后RNA切割导致细胞死亡(8)。 我们实验室以前的研究已经研究了不同表面基团的细胞中RNA酶的内化,但没有确切的数据表明RNA酶变体如何结合到癌细胞的表面并内化(9)。 然而,RNA酶的细胞毒性与总体阳离子性相关(10,11),最近与正电荷的特异性分布相关(8)。 因此,这种结合和内化与细胞外膜上的带负电荷的分子有关。 与正常细胞相比,癌细胞膜上组成的变化可能选择性地将核糖核酸酶靶向癌细胞(7,12),增加了在动物模型和人体试验中观察到的令人鼓舞的治疗指数(13,14)。 癌细胞的表面经常显示糖胺聚糖谱(15)、磷脂组成(16,17)和神经节苷脂阵列(18)的变化。 感兴趣的特定残基包括硫酸乙酰肝素、带电糖基(如唾液酸)和脂质(如磷脂酰丝氨酸),因为最近的研究已经通过可能的治疗靶向表明了这一重要性(19)。 我们推测,核糖核酸酶结合到这些特定的残基上的细胞表面可能是一个因素的蛋白质内化,从而影响效率的细胞毒性以及癌细胞系的选择性。 最近使用NMR确定了人RNase 1的结构(20)。 最近的其他论文也使用NMR作为一种方法来研究蛋白质残基之间的特定相互作用(21)。 为了确定RNA酶与细胞血浆组分的相互作用,我们希望用15 N同位素类似地标记RNA酶1,并滴定细胞膜组分,包括但不限于磷脂酰丝氨酸、硫酸乙酰肝素和唾液酸。 然后,我们将查看在600 MHz NMR光谱仪上记录的2D NOESY(混合时间= ms)光谱,如解决RNase 1结构的方法所述(20)。 将光谱中的位移与PDB(登录号2K 11)中解析的结构进行比较,将表明RNase 1和质膜组分之间特异性相互作用的位置和广泛程度的结合亲和力。 结果将确定RNase 1是如何结合到细胞表面成分,并可能阐明内在化的机制。 然后,我们可以使用15 N标记的RNA酶A和细胞毒性变体重复检查,以观察细胞膜相互作用和RNA酶细胞毒性之间可能的相关性。 还可以进一步研究广泛的细胞膜组分。 这可以允许开发具有更高内化的RNA酶,这是由于针对在癌细胞上过表达的细胞标志物的结合或靶向增加。

项目成果

期刊论文数量(0)
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Ronald T Raines其他文献

Hydrolytic Stability of Hydrazones and Oximes** Hydrazones and Oximes (c
腙和肟的水解稳定性** 腙和肟 (c
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
    J. Kalia;Ronald T Raines;J. Kalia;R T Raines;R T Raines;W. W. F. Cleland;Nelsen;T. J. Shoulders;Rutkoski
  • 通讯作者:
    Rutkoski
n→π* interactions in proteins
蛋白质中的 n→π* 相互作用
  • DOI:
    10.1038/nchembio.406
  • 发表时间:
    2010-07-11
  • 期刊:
  • 影响因子:
    13.700
  • 作者:
    Gail J Bartlett;Amit Choudhary;Ronald T Raines;Derek N Woolfson
  • 通讯作者:
    Derek N Woolfson
Jeremy R. Knowles (1935-2008).
杰里米·R·诺尔斯 (1935-2008)。
  • DOI:
    10.1021/cb800099n
  • 发表时间:
    2008
  • 期刊:
  • 影响因子:
    4
  • 作者:
    Ronald T Raines
  • 通讯作者:
    Ronald T Raines

Ronald T Raines的其他文献

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{{ truncateString('Ronald T Raines', 18)}}的其他基金

Protein Chemistry
蛋白质化学
  • 批准号:
    10552380
  • 财政年份:
    2023
  • 资助金额:
    $ 0.2万
  • 项目类别:
Biochemical Strategy to Avert Microbial Drug Resistance
避免微生物耐药性的生化策略
  • 批准号:
    10645218
  • 财政年份:
    2022
  • 资助金额:
    $ 0.2万
  • 项目类别:
Biochemical Strategy to Avert Microbial Drug Resistance
避免微生物耐药性的生化策略
  • 批准号:
    10510991
  • 财政年份:
    2022
  • 资助金额:
    $ 0.2万
  • 项目类别:
Esterase Specificity for Pharmacology and Chemical Biology
药理学和化学生物学的酯酶特异性
  • 批准号:
    10017292
  • 财政年份:
    2019
  • 资助金额:
    $ 0.2万
  • 项目类别:
Chemistry and Biology of Collagen
胶原蛋白的化学和生物学
  • 批准号:
    9101491
  • 财政年份:
    2015
  • 资助金额:
    $ 0.2万
  • 项目类别:
TRAINING IN THE USE OF BRUKER AND VARIAN SPECTROMETERS AND NMR
布鲁克和瓦里安光谱仪和核磁共振的使用培训
  • 批准号:
    8361163
  • 财政年份:
    2011
  • 资助金额:
    $ 0.2万
  • 项目类别:
ROUTINE NMR FOR CHARCATERIZATION OF ORGANIC CHEMICALS
用于表征有机化学品的常规 NMR
  • 批准号:
    8361152
  • 财政年份:
    2011
  • 资助金额:
    $ 0.2万
  • 项目类别:
NMR INVESTIGATIONS BY THE RAINES LABORATORY 2
由 Raines 实验室进行的 NMR 研究 2
  • 批准号:
    8361217
  • 财政年份:
    2011
  • 资助金额:
    $ 0.2万
  • 项目类别:
TRAINING IN THE USE OF BRUKER AND VARIAN SPECTROMETERS AND NMR
布鲁克和瓦里安光谱仪和核磁共振的使用培训
  • 批准号:
    8168959
  • 财政年份:
    2010
  • 资助金额:
    $ 0.2万
  • 项目类别:
ROUTINE NMR FOR CHARACTERIZATION OF ORGANIC COMPOUNDS
用于表征有机化合物的常规 NMR
  • 批准号:
    8168934
  • 财政年份:
    2010
  • 资助金额:
    $ 0.2万
  • 项目类别:

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