Sulfatase Activated Fluorescent Probes for In Vivo Diagnostic Imaging of Cancer

描述（申请人提供）：硫酸酯酶，催化硫酸酯水解，改变许多底物，这些底物在发育，信号传导，降解，发病和疾病中具有关键功能。最近表征的磺化酶HSulf-1和HSulf-2的表达水平在某些癌症（如乳腺癌、胰腺、结肠癌）中升高，通过肿瘤细胞中磺化酶活性的量化和可视化，可以实现癌症的早期诊断。对细胞外磺化酶HSulf-1和HSulf-2的灵敏和选择性检测需要设计高亲和力的荧光探针。本提案描述了在细胞外硫酸酯酶水解后，在量子产率或发射方面应发生重大变化的荧光团的设计。由于一种酶可以水解许多探针，因此硫酸盐酶激活的荧光信号将被放大，从而能够灵敏地检测异常的癌症相关活性。具有红色或近红外发射的荧光团将用于实现更深的组织穿透并减少组织自身荧光。将评估不同的可水解荧光基团支架，包括硫酸二甲基吖啶酮、间苯二酚、苝二酮和致光香豆素-镧系化合物缀合物。将在体外、癌细胞系和小鼠肿瘤模型中确定每种探针的光化学和动力学性质。虽然已知磺化酶可以水解多种芳基硫酸盐，但将研究对染料支架进行轻微修饰（例如氟化）以改善其结合亲和力或荧光特性。通过筛选DMA适体、多肽或类肽文库，通过与阴离子分子结合，探针-酶的亲和力将进一步增强。综上所述，磺胺氧化酶激活探针将能够对活体动物体内的磺胺氧化酶活性进行敏感和选择性的荧光成像，用于癌症的诊断。硫酸盐酶在某些癌症中含量升高，包括乳腺癌、胰腺癌和结肠癌。通过它们的活性，这些酶可能通过影响细胞表面的相互作用来控制癌症的严重程度。所描述的探针被细胞外硫酸酯酶激活，对癌细胞发出独特的荧光信号。这些探针的设计是为了能够早期和准确地诊断癌症。

DESCRIPTION (provided by applicant): Sulfatase enzymes, which catalyze the hydrolysis of sulfate esters, alter many substrates which have key functions in development, signaling, degradation, pathogenesis, and disease. The expression levels of the recently characterized sulfatases HSulf-1 and HSulf-2 are increased in certain cancers (e.g. breast, pancreas, colon), making early diagnosis of cancer achievable by the quantification and visualization of sulfatase activity in tumor cells. Sensitive and selective detection necessitates the design of high affinity fluorescent probes for the extracellular sulfatases HSulf-1 and HSulf-2. This proposal describes the design of fluorophores which should undergo significant alterations in quantum yield or emission after hydrolysis by extracellular sulfatases. Because one enzyme can hydrolyze many probes, the sulfatase-activated fluorescent signal will be amplified, enabling sensitive detection of abnormal, cancer-associated activity. Fluorophores with red or near-infrared emission will be targeted to enable deeper tissue penetration and to reduce tissue autofluorescence. Distinct hydrolyzable fluorophore scaffolds will be evaluated, including sulfated dimethylacridinones, resorufins, perylene diones, and luminogenic coumarin-lanthanide conjugates. The photochemical and kinetic properties will be determined for each probe in vitro, in cancer cell lines, and in mouse tumor models. Although it is known that sulfatases can hydrolyze diverse aryl sulfates, slight modifications to the dye scaffolds (e.g., fluorination) will be examined to improve the binding affinity or fluorescent properties. The probe-enzyme affinity will be further enhanced by conjugation to anionic molecules identified by screening libraries of DMA aptamers, peptides, or peptoids. In summary, the sulfatase activated probes will enable sensitive and selective fluorescence imaging of sulfatase activity inside living animals for the diagnosis of cancer. ***Sulfatases have elevated levels in certain cancers, including breast, pancreatic, and colon cancer. Through their activity, these enzymes may be controlling the severity of cancers by influencing interactions at the cell surface. The described probes are activated by extracellular sulfatases to give a distinct fluorescent signal at cancer cells. The probes are designed to enable early and accurate cancer diagnosis.