Structure-Based Design of Xe-129 NMR Biosensors for Multiplexed Cancer Detection

 描述(申请人提供)：129Xe核磁共振生物传感器代表了一类全新的生物物理探头，具有作为癌症诊断试剂的巨大潜力。拟议的研究建立在Xe生物传感器项目的基础上，该项目在过去10年里一直由国防部、NIH R21、R33和R01拨款资助(PI：Dmochowski)。现在需要NIH R01续订资金，以继续这一充满活力和高生产率的计划。这项研究计划的一个重点是开发用于改善肺癌诊断的129Xe磁共振造影剂。到目前为止，我们在氙气亲和性、超极化(HP)~(129Xe)核磁共振波谱和生物应用等方面取得了重要进展。由于最近129Xe超极化技术的改进，新一代129Xe磁共振造影剂的发展正在迅速推进。一个“开源”系统会产生人体肺部成像所需的接近统一偏振的~1-L的量。在S10基金(PI：RIZI)的支持下，Dmochowski实验室将在未来两年内获得最先进的氙气偏振器。这项建议的重点是采用化学交换饱和转移(‘Hyper-CEST’)的129Xe核磁共振技术，该技术是由伯克利的Pines实验室于2006年率先使用隐形原子作为氙气主体的，并纳入了由Mugler和Ruppert于2000年在弗吉尼亚州首次描述的氙极化转移对比度(XTC)的概念。2012年，我们的实验室使用Hyper-CEST核磁共振显示，1微微克分子加密粉提供了有用的对比度，其灵敏度是标准MRI造影剂的109倍。这在伯克利报告的原始5 NM密文检测灵敏度的基础上有所提高，仍然大约是原来的100倍 比法国和德国的研究人员对单点密文实体进行的Hyper-CEST测量更敏感。我们只能将Hyper-CEST效率的这些差异中的一部分归因于我们的三官能化、水溶性隐语的更大的Xe亲和力和更快的Xe交换动力学。这提出了几个重要的问题：小分子介导的129Xe磁化转移的操作机制是什么？能否对这些过程进行优化以实现毫微摩尔(或更高)的检测灵敏度？小分子和基因编码的氙气结合CEST试剂能否被开发出来，广泛分布到对分子成像感兴趣的实验室？为了解决第一个问题，我们假设了一个Xe“气泡”包围着隐形原子，许多弱缔合的外部Xe原子在短距离内与内部单个Xe原子进行快速磁化转移。这一假设将通过与宾夕法尼亚大学化学合作者Saven合作的Aim 1.1中的计算和实验方法进行严格测试。虽然密码使氙气生物传感的探索成为可能，但它们的稀缺性限制了全世界少数几个实验室的使用。因此，在目标1.2中，我们建议开发可广泛分布于生物医学研究的新的小分子Hyper-CEST制剂。我们实验室最近发现，通过Hyper-CEST核磁共振，可以在1皮摩尔浓度下检测到商业上可获得的葫芦脲CB[6]，类似于水溶性隐丹。此外，我们还确定通过129Xe核磁共振可以在细胞和细胞裂解物中检测到CB[6]。CB[6]的一个缺点是难以用单一的靶向基团来官能化这种主体分子。为了解决这个问题，我们将开发“开启”CB[6]氙气生物传感器，利用CB[6]对许多有机小分子的亲和力。与密文一样，我们将通过计算和实验方法来澄清和改进CB[6]Hyper-CEST对比度。我们的实验室将开发用于靶向肺癌细胞的水溶性隐语和CB[6]溶液，并进行Hyper-CEST核磁共振波谱和成像研究。在目标2中，我们建议开发绿色荧光蛋白(GFP)和颜色变体的遗传编码的“MRI类似物”，这是目前通过荧光显微镜显示许多细胞过程的标准。GFP的细胞生产增加了这种荧光团编码的空间和时间信息，并绕过了许多细胞递送、定位和降解的问题。同样，基于蛋白质的氙气生物传感器将扩大细胞和体内研究的范围，同时利用磁共振成像相对于光学显微镜更大的组织穿透性。最近关于气泡(GV)蛋白达到超CEST的报道提供了有用的先例。然而，GVS由8-14种不同的蛋白质组成，这些蛋白质在细菌中自我组装，但不能在真核细胞中表达。因此，我们致力于开发更通用的单蛋白Hyper-CEST试剂。盖斯勒实验室发表的MD模拟让我们正确地假设，β-内酰胺酶应该能够实现Hyper-CEST对比度，因为它有大量隐秘的变构位点，在Xe可能短暂驻留的蛋白质内部提供了~1纳米的疏水口袋。在Aim 2.1中，我们将与Temple的合作者(Carnevale，Klein)合作，使用几种计算方法研究Xe与β-内酰胺酶的相互作用，并开发能够提高CEST对比度的β-内酰胺酶变体，同时也能够进行多路实验(类似于荧光显微镜的CFP、GFP、YFP、RFP)。在AIM 2.2中，我们将使用β-内酰胺酶变体进行Hyper-CEST核磁共振波谱和成像研究。

 DESCRIPTION (provided by applicant): 129Xe NMR biosensors represent a fundamentally new class of biophysical probes with tremendous potential as cancer diagnostic agents. The proposed studies build on a Xe biosensor program that has been continuously funded (PI: Dmochowski) for the past 10 years by DoD, NIH R21, R33, and R01 grants. NIH R01 renewal funding is now requested to continue this dynamic and highly productive program. A focus of this research program is the development of 129Xe MRI contrast agents for improved diagnosis of lung cancer. To date, we have made key advances in the synthesis, xenon affinity, hyperpolarized (hp) 129Xe NMR spectroscopy, and biological application of Xe biosensors utilizing a cryptophane moiety for Xe encapsulation. The development of next-generation 129Xe MRI contrast agents is rapidly advancing, now propelled by recent improvements in 129Xe hyperpolarization technology. An 'open source' system produces near-unity polarization in ~1-L quantities required for human lung imaging. The Dmochowski laboratory will gain access to a state-of the-art xenon polarizer within the next two years, with support from S10 funding (PI: Rizi). This proposal focuses on a 129Xe NMR technique employing chemical exchange saturation transfer ('Hyper-CEST'), which was pioneered using cryptophane as the xenon host by the Pines lab at Berkeley in 2006, and incorporates concepts of xenon polarization transfer contrast (XTC) first described by Mugler and Ruppert at Virginia in 2000. In 2012, our laboratory showed that 1 picomolar cryptophane provides useful contrast using Hyper-CEST NMR, a 109-fold sensitivity enhancement over standard MRI contrast agents. This improved upon the original 5 nM cryptophane detection sensitivity reported at Berkeley, and is still roughly 100-fold more sensitive than Hyper-CEST measurements performed for single-site cryptophane entities by researchers in France and Germany. We have been able to attribute only some of these differences in Hyper-CEST efficiency to the greater Xe affinity and faster Xe exchange kinetics of our trifunctionalized, water-soluble cryptophanes. This raises several important questions: What is the operative mechanism for small molecule-mediated 129Xe magnetization transfer? Can these processes be optimized to achieve femtomolar (or better) detection sensitivity? Can small molecule and genetically encoded xenon-binding CEST agents be developed for wide distribution to labs interested in molecular imaging? To address the first question, we hypothesize that a Xe "bubble" surrounds the cryptophane, with many weakly-associated, exterior Xe atoms undergoing rapid magnetization transfer at short-range with the single interior Xe atom. This hypothesis will be rigorously tested by computational and experimental approaches in Aim 1.1, working with UPenn Chemistry collaborator Saven. While cryptophanes enable explorations of xenon biosensing, their scarcity limits use to a handful of labs worldwide. Thus, in Aim 1.2 we propose to develop new small-molecule Hyper-CEST agents that can be widely distributed for biomedical research. Our lab made the recent discovery that commercially available cucurbituril CB[6] can be detected at 1 picomolar concentration via Hyper-CEST NMR, similar to water-soluble cryptophane. Moreover, we determined that CB[6] can be detected by 129Xe NMR in cells and cell lysate. One shortcoming of CB[6] is the difficulty of functionalizing this host molecule with single targeting moieties. To overcome this problem, we will develop "turn on" CB[6] xenon biosensors that exploit the affinity of CB[6] for many organic small molecules. As with cryptophane, we will seek to elucidate and improve upon CB[6] Hyper-CEST contrast by computational and experimental approaches. Our lab will develop water-soluble cryptophane and CB[6] solutions for targeting lung cancer cells, and perform Hyper-CEST NMR spectroscopy and imaging studies. In Aim 2, we propose the development of genetically encoded "MRI analogs" of green fluorescent protein (GFP) and color variants, which are the current standard for visualizing many cellular processes by fluorescence microscopy. Cellular production of GFP increases the spatial and temporal information encoded by this fluorophore, and also circumvents many problems of cell delivery, localization, and degradation. Similarly, protein-based xenon biosensors will expand the repertoire of cellular and in vivo studies, while taking advantage of the much greater tissue penetration of MRI relative to light microscopy. A recent report of gas vesicle (GV) proteins that achieve Hyper-CEST provides useful precedent. GVs, however, are composed of 8-14 different proteins that self-assemble in bacteria but cannot be expressed in eukaryotic cells. Thus, we are focused on developing more versatile single-protein Hyper-CEST agents. MD simulations published by the Geissler laboratory led us to hypothesize correctly that beta-lactamase should enable Hyper-CEST contrast, based on its large number of cryptic allosteric sites that provide ~1-nanometer hydrophobic pockets in the protein interior where Xe may transiently reside. In collaboration with Temple collaborators (Carnevale, Klein), in Aim 2.1, we will study Xe interactions with beta-lactamase using several computational approaches, and develop variants of beta-lactamase that increase CEST contrast, while also enabling multiplexing experiments (similar to CFP, GFP, YFP, RFP for fluorescence microscopy). In Aim 2.2, we will perform Hyper-CEST NMR spectroscopy and imaging studies using beta-lactamase variants.