Pathogen specific imaging of endocarditis

描述（由申请人提供）：急性心内膜炎是一种常见且致命的心脏瓣膜感染（死亡率高达47%），其临床管理仍然极具挑战性，且通常不成功1，2。急性心内膜炎最常见的病原体是金黄色葡萄球菌，其次是链球菌2，3。未满足的临床需求包括：（i）可靠的诊断或排除心内膜炎，（ii）特异性鉴定病原体，告知抗生素的选择，和（iii）获得定量数据以指导外科手术干预。在这里，我们的目标是开发一种临床上可行的，新的方法，病原体特异性成像的金黄色葡萄球菌心内膜炎。我们的策略是基于细菌分泌的毒力因子葡萄球菌凝固酶对凝血酶原（ProT）的亲和力（17 pM）5，并利用这种相互作用开发特异性成像探针6。葡萄球菌凝固酶的NH 2-末端D1和D2-结构域结合凝血酶（或ProT成像平台），而COOH-末端重复区域同时结合纤维蛋白原。因此，葡萄球菌凝固酶牢固地将成像探头锚定在心内膜赘生物中。我们的初步数据显示，利用ProT和葡萄球菌凝固酶之间的高亲和力用于工程化成像剂是可行的（Nature Med.2011）6。我们使用凝血酶原的活化口袋作为亲和配体，该活化口袋通过称为“分子性”的机制与葡萄球菌凝固酶紧密结合（Bode和Huber 1976）7。用荧光染料标记ProT的丝氨酸蛋白酶活性位点，使我们能够检测和监测S。金黄色葡萄球菌心内膜炎的小鼠模型与光学成像6.此外，我们合成了一个PET报告S。金黄色葡萄球菌（64 Cu-iProT），并发现葡萄球菌凝固酶的敏感PET成像也是可行的。在第一个目标中提出的研究是基于这种已经建立的成像剂，但也将寻求开发针对其他细菌菌株的替代成像剂。第二个目标描述了使用点击化学开发具有优化的药代动力学的18 F化合物，我们最近采用点击化学来简单合成PET试剂8，9。将在心内膜炎的哥廷根小型猪模型中测试两种先导化合物。我们将使用这些探头通过混合ECG触发的PET/CT和PET/MRI对小鼠心内膜炎进行成像，从而将敏感的分子模式（PET）与用于评估左心室功能的主要模式（也可以检测瓣膜功能不全（MRI））相结合。制剂开发将侧重于临床可行性，以解决上述紧急医疗需求，最终目标是使用PET/MRI检测瓣膜病变中的细菌，以诊断患者的急性心内膜炎。

DESCRIPTION (provided by applicant): Clinical management of acute endocarditis, a frequent and deadly infection of the heart valves (mortality of up to 47%), remains highly challenging and often unsuccessful1,2. The most common pathogen in acute endocarditis is Staphylococcus aureus, followed by streptococcus species2,3. Unmet clinical needs include: (i) reliable diagnosis or exclusion of endocarditis, (ii) specific identification of the pathogen informing selection of antibiotics, and (iii) acquisition of quantitative data to guide surgical intervention. Here we aim to develop a clinically viable, novel method for pathogen-specific imaging of Staphylococcus aureus endocarditis. Our strategy is based on the ultrahigh affinity (17pM)5 of the virulence factor staphylocoagulase, which is secreted by the bacteria, to prothrombin (ProT) and the use of this interaction to develop specific imaging probes6. Staphylocoagulase's NH2-terminal D1 and D2-domains bind to thrombin (or the ProT imaging platform), while the COOH-terminal repeats region binds to fibrinogen at the same time. Therefore, staphylocoagulase firmly anchors the imaging probe in endocarditic vegetations. Our preliminary data show that harnessing the high affinity between ProT and staphylocoagulase for engineering imaging agents is feasible (Nature Med. 2011)6. We used prothrombin's activation pocket that tightly binds to staphylocoagulase by a mechanism dubbed "molecular sexuality" (Bode and Huber 1976)7 as an affinity ligand. Labeling ProT's serine protease active site with a fluorochrome allowed us to detect and monitor S. aureus endocarditis in a mouse model with optical imaging6. Furthermore, we synthesized a PET reporter for S. aureus (64Cu-iProT) and found that sensitive PET imaging of staphylocoagulase is also feasible. The studies proposed in the first aim are based on this already-established imaging agent but will also pursue development of alternative imaging agents targeted to other bacterial strains. The second aim describes developing 18F compounds with optimized pharmacokinetics using click chemistry, which we recently adopted for facile synthesis of PET agents8,9. Two lead compounds will be tested in a G¿ttingen minipig model of endocarditis. We will employ these probes to image murine endocarditis by hybrid ECG- triggered PET/CT and PET/MRI, thereby combining a sensitive molecular modality (PET) with a leading modality for assessing left ventricular function which can also detect valvular insufficiency (MRI). Agent development will focus on clinical feasibility to address the aforementioned urgent medical needs, with the ultimate goal of using PET/MRI detection of bacteria in valve lesions for the diagnosis of acute endocarditis in patients.