Designed hydrophilic and charge mutations of the fibronectin domain: towards tailored protein biodistribution
Designed hydrophilic and charge mutations of the fibronectin domain: towards tailored protein biodistribution
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DOI：
10.1093/protein/gzs036
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发表时间：
2012-10-01
影响因子：
2.4
通讯作者：
Gambhir, Sanjiv S.
中科院分区：
文献类型：
--
作者：
Hackel, Benjamin J.;Sathirachinda, Ataya;Gambhir, Sanjiv S.
工程化蛋白质是分子成像和药物递送的有吸引力的亲和支架。尽管可以设计精确的结合特异性和亲和力，但许多蛋白质表现出来自生理效应的脱靶摄取，特别是在肾脏和肝脏中。我们量化了通过亲水性和电荷突变改变肾脏和肝脏摄取的能力。作为模型蛋白，我们使用了人纤连蛋白的第10个III型结构域，该结构域已被工程化以结合许多靶标，并已被验证用于分子成像。我们筛选了合理的突变体，确定的结构和系统发育分析，产生8个突变，共同大幅增加蛋白质的亲水性。两个亲本克隆的突变产生了具有一定范围亲水性的四个结构域。这些蛋白质用Cu-64标记，静脉注射到nu/nu小鼠（每只n 35）中，并通过正电子发射断层扫描进行评价。肾吸收与亲水性强相关（Pearson相关系数0.97），范围从29 11至100 22 ID/g在1小时。肝摄取与亲水性呈负相关（Pearsons相关系数0.92），范围为30 7至3 1 ID/g。因此，肾和肝摄取可通过亲水性突变直接调节，可通过结构和系统发育分析鉴定。为了研究电荷，我们突变了两个亲本克隆中的酸性和碱性残基，并在nu/nu小鼠（n 57）中评估了Cu-64标记的突变体。选择性电荷去除降低肾脏信号：亲水性克隆为78 13至51 8ID/g（P 0.0001），疏水性克隆为32 10至21 3（P 0.0005）。亲水性和电荷的阐明使得能够调节背景信号，从而增强蛋白质支架作为用于分子成像和治疗的可翻译靶向剂的效用。
Engineered proteins are attractive affinity scaffolds for molecular imaging and drug delivery. Although exquisite binding specificity and affinity can be engineered, many proteins exhibit off-target uptake, particularly in the kidneys and liver, from physiologic effects. We quantified the ability to alter renal and hepatic uptake via hydrophilic and charge mutations. As a model protein, we used the 10th type III domain of human fibronectin, which has been engineered to bind many targets and has been validated for molecular imaging. We screened rational mutants, identified by structural and phylogenetic analyses, to yield eight mutations that collectively substantially increase protein hydrophilicity. Mutation of two parental clones yielded four domains with a range of hydrophilicity. These proteins were labeled with Cu-64, injected intravenously into nu/nu mice (n 35 each) and evaluated by positron emission tomography. Renal uptake strongly correlated with hydrophilicity (Pearsons correlation coefficient 0.97), ranging from 29 11 to 100 22 ID/g at 1 h. Hepatic uptake inversely correlated with hydrophilicity (Pearsons correlation coefficient 0.92), ranging from 30 7 to 3 1 ID/g. Thus, renal and hepatic uptake are directly tunable through hydrophilic mutation, identifiable by structural and phylogenetic analyses. To investigate charge, we mutated acidic and basic residues in both parental clones and evaluated Cu-64-labeled mutants in nu/nu mice (n 57). Selected charge removal reduced kidney signal: 78 13 to 51 8ID/g (P 0.0001) for the hydrophilic clone and 32 10 to 21 3 (P 0.0005) for the hydrophobic clone. Elucidation of hydrophilicity and charge enabled modulation of background signal thereby enhancing the utility of protein scaffolds as translatable targeting agents for molecular imaging and therapy.