Enzymatic Protein labeling

最近有报道称，目前最畅销的8种药物中有7种是蛋白质类药物。然而，它也一直是 注意到许多以蛋白质为基础的药物的成本极高，使其难以广泛使用 实施。为了提高这种分子的效用，降低它们的生产成本至关重要。 蛋白质偶联是许多蛋白质类药物的基本特征，包括抗体-药物偶联物 和聚乙二醇化的蛋白质。酶法提供了巨大的潜力来解决这个问题 特异性和流线型的蛋白质结合过程。这一发现表明几乎任何蛋白质都可以 通过掺入四肽识别序列来呈现PFTase底物 该酶耐受类异戊二烯结构中过多的修饰的能力使PFTase 一个有吸引力的选择进行酶蛋白标记。在上一个资助期，我们 证明了PFTase可用于制备位点选择性修饰的蛋白质，并应用于 这种将荧光标记和聚乙二醇基结合到蛋白质中进行治疗的方法 在细胞培养和动物模型中进行评估的应用。共有21篇论文和1项专利 这项工作已经出版了，目前还有两本正在审查中。在下一个资助期，我们将 假设PFTase催化的酶标记将极大地促进新的 用于治疗应用的“生物制品”通过追求以下目标：(1)创造突变形式的 PFTase可以改变酶的专一性并扩大类异戊二烯底物的大小 调走了。这将被追求以产生完全正交的形式的PFTase，该PFTase可以转移新的 类异戊二烯与特定蛋白质的关系。扩大异戊二烯结合部位将允许更大的 包括整个高分子链的部分；(2)使用聚合物引发剂制备蛋白质-聚合物偶联物 通过定点酶标记法安装在蛋白质上。这里，连接聚合物的标准方法 通过加入可用于生长的位置选择性定位的启动子，将逆转TO蛋白质 各种类型的聚合链；(3)利用酶标记法制备蛋白质偶联物 用于成像和治疗应用的纤维连接蛋白支架。纤维连接蛋白(FN)是靶向的支架 癌细胞上的EGF受体将被用于PET成像的DOTA配体或药物修饰 用于小鼠异种移植模型的治疗实验；(4)使用三正交PFTase底物来 创建包含蛋白质毒素的化学自组装纳米结构(CSAN) 治疗应用。用白喉毒素或穿孔素功能化的纳米结构将被用于靶向 CD3+T细胞用于治疗1型糖尿病和其他自身免疫性疾病。成功完成 这些具有重大意义和创新性的目标可能会对蛋白质领域产生重大影响 关于结合物及其在促进人类健康方面的用途。

It was recently reported that 7 of the current top 8 selling drugs are proteins. However, it has also been noted that the cost of many protein-based drugs is extremely high, making their widespread use difficult to implement. To increase the utility of such molecules, it is essential that the cost of producing them be reduced. Protein conjugation is an essential feature of many protein-based drugs including antibody-drug conjugates and PEGylated proteins. Enzymatic methods offer tremendous potential to solve the problem of specificity and streamline the process of protein conjugation. The discovery that virtually any protein can be rendered a PFTase substrate by incorporation of a tetrapeptide recognition sequence coupled with the ability of the enzyme to tolerate a plethora of modifications within the isoprenoid structure has made PFTase an attractive choice for performing enzymatic protein labeling. In the previous funding period, we demonstrated that PFTase could be used to prepare site-selectively modified proteins, and employed this method to incorporate fluorescent labels and PEG groups into proteins for therapeutic applications that were evaluated in cell culture and animal models. A total of 21 papers and one patent from this work have been published with two more currently under review. In this next funding period, we hypothesize that PFTase-catalyzed enzymatic labeling will greatly facilitate the preparation of new “biologics” for therapeutic applications by pursuing the following Aims: (1) Create mutant forms of PFTase that alter enzyme specificity and enlarge the size of the isoprenoid substrate that can be transferred. This will be pursued to generate a completely orthogonal form of PFTase that can transfer novel isoprenoids to specific proteins. Enlarging the isoprenoid binding site will allow efficient incorporation of larger moieties including whole polymer chains; (2) Prepare protein-polymer conjugates using polymer initiators installed on proteins via site-specific enzymatic labeling. Here, the standard approach of linking polymers to proteins will be reversed by incorporating site-selectively positioned initiators that can be used to grow polymeric chains of various types; (3) Use enzymatic protein labeling to prepare protein conjugates based on fibronectin scaffolds for imaging and therapeutic applications. Fibronectin (Fn) scaffolds that target EGF receptors on cancer cells will be modified with either DOTA ligands for PET imaging applications or drugs for therapeutic experiments in mouse xenograft models; (4) Employ triorthogonal PFTase substrates to create chemically self-assembled nanoring structures (CSANs) that incorporate protein toxins for therapeutic applications. Nanostructures functionalized with diphtheria toxin or perforin will be used to target CD3+ T-cells for the treatment of Type 1 Diabetes and other autoimmune disorders. Successful completion of these highly significant and innovative Aims could have a major impact in the field of protein conjugates and on their use in promoting human health.