Metalloprotein MRI Contrast Agent...for Targeted Imaging

金属蛋白 MRI 造影剂...用于靶向成像

基本信息

批准号：
8536630
负责人：
Zhan Wang
金额：
$ 4.79万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-06 至 2014-07-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8536630
关键词：
Active Sites Address Affinity Arachidonic Acids Binding Binding Sites Brain Carrier Proteins Catechols Chemicals Chimera organism Complex Contrast Media Cytochrome P450 Development Dopamine Electron Transport Complex III Engineering Event Gadolinium Goals Heme Image Imaging Techniques Ions Iron Lead Magnetic Resonance Imaging Manganese Mass Spectrum Analysis Measurement Measures Medicine Melatonin Metalloproteins Metals Methods Monitor Mutagenesis Mutation Norepinephrine Point Mutation Porphyrins Process Protein Engineering Proteins Reading Research Resolution Scaffolding Protein Scheme Serotonin Signal Transduction Signaling Molecule Specificity Structure System Techniques Time Variant Water Work analog base directed evolution flexibility gadolinium oxide high throughput screening improved in vivo nanoparticle nervous system disorder neurochemistry novel diagnostics overexpression scaffold sensor small molecule theories thermostability

项目摘要

DESCRIPTION (provided by applicant): Metalloprotein scaffolds are attractive candidates for MRI contrast agents because the interaction of paramagnetic metals with water molecules within the active site provides an easy read out for MRI. The protein scaffold itself is highly tunable and can be engineered to bind reversibly and specifically to one target in the presence of many structurally related compounds. However, the current methods developed using this strategy is limited by the inherent relaxivity of the agents. As improved relaxivity corresponds to better signal and resolution and may increase the scope of small molecule targets available for imaging, engineering protein sensors with higher relaxivity is important for realizing the full potential of this approach. Our proposal addresses this goal. Water exchange rate and innate spin of the metal both have profound effects on the relaxivity of the contrast agent and we will target both of these avenues for exploration. The cytochrome p450 BM3h platform has proved to be highly tunable and amenable to the directed evolution approach and we will construct sensors from this scaffold. Preliminary studies in the Arnold lab have identified mutation can lead to better flexibility and improved water access to the binding site. We believe that a targeted screen of specific active site residues via iterative saturation mutagenesis will lead to improvements in relaxivity of Mn(III) based sensors. As a complementary approach, we will also synthesize gadolinium(III) porphyrins for incorporation into BM3h. In both cases, directed evolution will be used to produce sensors with good thermostability, improved relaxivity, and high specificity for a neurochemical target like dopamine or norepinephrine. The sensors will be fully characterized by absorbance, mass spectrometry, MRI measurements, and other techniques previously used.

描述（由申请人提供）：金属蛋白支架是MRI对比剂的有吸引力的候选者，因为顺磁金属与活性位点中水分子的相互作用可为MRI读取。蛋白质支架本身是高度可调的，可以在存在许多结构相关的化合物的情况下可逆地与一个靶标结合。但是，使用此策略开发的当前方法受代理人的固有放松性的限制。随着改善的松弛性对应于更好的信号和分辨率，并可能增加可用于成像的小分子靶标的范围，具有更高松弛性的工程蛋白传感器对于实现这种方法的全部潜力很重要。我们的建议解决了这一目标。金属的水汇率和先天自旋都对造影剂的松弛性具有深远的影响，我们将以这两种途径进行探索。细胞色素P450 BM3H平台已被证明是高度可调的，可以适合定向进化方法，我们将从该支架中构造传感器。阿诺德实验室的初步研究已经确定突变可以提高灵活性，并改善对结合位点的水接收能力。我们认为，通过迭代饱和诱变的特定活性位点残基的靶向屏幕将改善基于MN（III）的传感器的松弛性。作为一种互补方法，我们还将合成gadolinium（iii）卟啉以掺入BM3H中。在这两种情况下，定向进化都将用于产生具有良好热稳定性，提高宽松性和高特异性的传感器，以产生多巴胺或去甲肾上腺素等神经化学靶标的传感器。传感器将充分以吸光度，质谱，MRI测量和其他先前使用的技术为特征。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

P450-catalyzed asymmetric cyclopropanation of electron-deficient olefins under aerobic conditions.

DOI：
10.1039/c4cy00633j
发表时间：
2014-10-02
期刊：
Catalysis science & technology
影响因子：
5
作者：
Renata H;Wang ZJ;Kitto RZ;Arnold FH
通讯作者：
Arnold FH

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

扩展酶的宇宙：通过机制引导的定向进化来访问非自然反应。

DOI：
10.1002/anie.201409470
发表时间：
2015-03-09
期刊：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
影响因子：
16.6
作者：
Renata, Hans;Wang, Z. Jane;Arnold, Frances H.
通讯作者：
Arnold, Frances H.

Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran.

DOI：
10.1002/anie.201402809
发表时间：
2014-06-23
期刊：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
影响因子：
16.6
作者：
Wang, Z. Jane;Renata, Hans;Peck, Nicole E.;Farwell, Christopher C.;Coelho, Pedro S.;Arnold, Frances H.
通讯作者：
Arnold, Frances H.

Cytochrome P450-Catalyzed Insertion of Carbenoids into N-H Bonds.