Metalloprotein MRI Contrast Agent...for Targeted Imaging

金属蛋白 MRI 造影剂...用于靶向成像

• 批准号: 8397007
• 负责人: Zhan Wang
• 金额: $ 4.71万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-06 至 2014-08-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8397007
• 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

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. PUBLIC HEALTH RELEVANCE: The ability to monitor neurochemical fluctuations in the brain in real time is essential to the next generation of diagnostic medicine and treatment neurological disease yet very few imaging techniques are capable of this feat. The development of MRI contrast agents that can selectively bind one molecule and relay the binding event via changes in MRI signal is a promising avenue of research toward this goal. By engineering protein-based sensors with good target selectivity and higher innate relaxivity, we will improve the signal and resolution of existing agents, which will allow us to examine small changes in concentrations of important signaling molecules like dopamine, serotonin and norepinephrine.

描述（由申请人提供）：金属蛋白支架是 MRI 造影剂的有吸引力的候选者，因为顺磁性金属与活性位点内的水分子的相互作用为 MRI 提供了轻松的读数。蛋白质支架本身是高度可调的，并且可以被设计为在许多结构相关的化合物存在的情况下可逆地、特异性地结合到一个靶标上。然而，当前使用这种策略开发的方法受到代理固有的弛豫性的限制。由于弛豫度的提高对应于 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，设计具有更高弛豫度的蛋白质传感器对于实现这种方法的全部潜力非常重要。我们的提案旨在解决这一目标。水交换率和金属的固有自旋都对造影剂的弛豫率产生深远的影响，我们将针对这两个途径进行探索。细胞色素 p450 BM3h 平台已被证明具有高度可调性，并且适合定向进化方法，我们将利用该支架构建传感器。阿诺德实验室的初步研究发现，突变可以带来更好的灵活性并改善水进入结合位点的机会。我们相信，通过迭代饱和诱变对特定活性位点残基进行靶向筛选将改善基于 Mn(III) 的传感器的弛豫率。作为补充方法，我们还将合成钆(III)卟啉并入 BM3h。在这两种情况下，定向进化将用于生产具有良好热稳定性、改善弛豫性以及对多巴胺或去甲肾上腺素等神经化学目标具有高特异性的传感器。这些传感器将通过吸光度、质谱、MRI 测量和之前使用的其他技术进行全面表征。 公共健康相关性：实时监测大脑神经化学波动的能力对于下一代诊断医学和治疗神经系统疾病至关重要，但很少有成像技术能够实现这一壮举。开发可选择性结合一个分子并通过 MRI 信号变化传递结合事件的 MRI 造影剂是实现这一目标的一条有前途的研究途径。通过设计具有良好目标选择性和更高先天弛豫性的基于蛋白质的传感器，我们将改善现有药物的信号和分辨率，这将使我们能够检查多巴胺、血清素和去甲肾上腺素等重要信号分子浓度的微小变化。