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。但也有人 他指出，许多蛋白质药物的成本极高，使其难以广泛使用， 实现.为了提高这些分子的效用，降低生产它们的成本至关重要。 蛋白质偶联是许多基于蛋白质的药物（包括抗体-药物偶联物）的基本特征 和PEG化蛋白质。酶促方法提供了解决以下问题的巨大潜力 特异性和简化蛋白质缀合的过程。事实上任何蛋白质都可以 通过掺入与所述四肽识别序列偶联的四肽识别序列， 这种酶能够耐受类异戊二烯结构中的过多修饰， 是进行酶促蛋白质标记的有吸引力的选择。在上一个融资周期，我们 证明PFTase可用于制备位点选择性修饰的蛋白质，并采用 该方法将荧光标记和PEG基团掺入蛋白质中用于治疗 在细胞培养和动物模型中评估的应用。共发表论文21篇，获专利1项 这一工作已经出版，目前正在审查另外两个。在下一个融资周期，我们 假设PFT酶催化酶标记将极大地促进新的 通过追求以下目标，开发用于治疗应用的“生物制剂”： PFT酶改变酶的特异性，并扩大类异戊二烯底物的大小， 转移.这将被追求以产生完全正交形式的PFT酶，其可以转移新的 类异戊二烯转化为特定的蛋白质。扩大类异戊二烯结合位点将允许有效掺入较大的类异戊二烯结合位点。 （2）使用聚合物引发剂制备蛋白质-聚合物缀合物 通过位点特异性酶标记安装在蛋白质上。这里，连接聚合物的标准方法 通过引入位点选择性定位的引发剂， 各种类型的聚合物链;（3）使用酶促蛋白质标记来制备基于 用于成像和治疗应用的纤连蛋白支架。纤连蛋白（Fn）支架靶向 癌细胞上的EGF受体将被DOTA配体修饰用于PET成像应用或药物 用于小鼠异种移植模型中的治疗实验;（4）采用三正交PFT酶底物， 创建化学自组装纳米环结构（CSAN）， 治疗应用。用白喉毒素或穿孔素功能化的纳米结构将用于靶向 CD 3 + 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 这些非常重要和创新的目标可能会在蛋白质领域产生重大影响， 缀合物及其在促进人类健康中的用途。

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.