Bioorthogonal Chemistries Targeting 5-hydroxytryptophan for Biological Discovery and Biologics Development

在美国国家科学基金会化学部的支持下，波士顿学院的Abhishek Chatterjee、Tim van Opijnen和Eranthie Weerapana组成的多学科团队正在开发技术，研究使致病菌逃避抗生素治疗并产生抗生素耐药性的过程，这是我们这个时代的一个决定性挑战。为了了解抗生素耐药性是如何产生的，有必要在生理相关的活体动物感染模型中研究细菌对抗生素治疗的反应。这类研究需要从受感染的动物组织中提取细菌（或其细胞成分）。然而，从如此复杂的环境中分离细菌成分是一项重大的技术挑战。研究小组将开发一种基因编码技术，用于选择性地标记细菌细胞中产生的蛋白质，从而使它们能够有效地从感染部位分离出来。利用这项技术，将有可能监测细菌蛋白质组在感染和抗生素治疗期间的变化，揭示抗生素耐药性出现的潜在过程。此外，研究小组开发的精确蛋白质标记技术将用于将生物活性小分子附着在抗体上。这些研究旨在证明这种新的靶向生物正交化学在“抗体-药物偶联物”领域的应用原理。如果成功，这些应用将对生物医学治疗的基础科学产生潜在的深远的长期科学影响。该集团希望在这一领域建立工业伙伴关系。主要研究人员正在开发一种针对非规范氨基酸5-羟色氨酸（5HTP）的新型生物正交偶联反应。他们将运用这种化学方法，进一步利用他们最近开发的一种工程细菌色氨酸- trna合成酶（trpr），将5HTP整合到活细胞的蛋白质中。第一个应用程序将创建一个完全遗传编码的系统来标记和纯化在致病菌中表达的新合成蛋白质。它将涉及：(i)一个可诱导的生物合成途径，在细胞中按需从色氨酸中生成5HTP； （ii）一个工程trpr，以促进生物合成的5HTP随机结合到新合成的蛋白质中，以响应色氨酸密码子；（iii）使用5HTP选择性生物偶联化学来标记和纯化5HTP标记的蛋白质，以进行蛋白质组学表征。该平台将用于在活体动物感染模型中研究抗生素治疗后致病菌的蛋白质组学变化。第二个应用将使用5htp选择性化学来生产均匀的“抗体-药物偶联物”。还将开发增加有效载荷和每个抗体附加两个不同的生物活性小分子的策略。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the NSF Division of Chemistry through the Molecular Foundations for Biotechnology (MFB) Solicitation, the multi-disciplinary team of Abhishek Chatterjee, Tim van Opijnen and Eranthie Weerapana of Boston College are developing technology to study the processes that allow pathogenic bacteria evade antibiotic treatment and develop antibiotic resistance, a defining challenge of our time. To understand how antibiotic resistance emerges, it is essential to study bacterial response to antibiotic treatment in physiologically relevant live-animal infection models. Such studies require retrieving the bacteria (or its cellular components) from the infected animal tissue. However, isolating bacterial components from such a complex milieu poses a significant technical challenge. The research team will develop a genetically encoded technology for selectively tagging proteins made in the bacterial cells, which will enable their efficient isolation from the infection site. Using this technology, it will be possible to monitor how the bacterial proteome changes during infection and upon antibiotic treatment, revealing the processes underlying the emergence of antibiotic resistance. In addition, the precise protein labeling technology developed by the team will be exploited to attach bioactive small molecules to antibodies. These studies are designed to demonstrate proof of principle for the use this new targeted bio-orthogonal chemistry in the field of 'antibody-drug conjugates.' If successful, such applications would constitute a potentially far reaching long term scientific broader impact of this fundamental science on biomedical therapeutics. The group hopes to engage in industrial partnerships in this space.The principal investigators are developing a new class of bioorthogonal conjugation reactions targeted to the noncanonical amino acid 5-hydroxytryptophan (5HTP). They will apply this chemistry, which further leverages an engineered bacterial tryptophanyl-tRNA synthetase (TrpRS) they recently developed, for incorporating 5HTP into proteins in living cells. The first application will create a fully genetically encoded system to tag and purify newly synthesized proteins expressed in pathogenic bacteria. It will involve: (i) an inducible biosynthetic pathway to generate 5HTP in cells from tryptophan on demand, (ii) an engineered TrpRS to facilitate stochastic incorporation of biosynthesized 5HTP into newly synthesized proteins in response to tryptophan codons, and (iii) the use of 5HTP-selective bioconjugation chemistry to tag and purify the 5HTP-labeled proteins for proteomic characterization. This platform will be used to investigate the proteomic changes in pathogenic bacteria upon antibiotic treatment in live-animal infection models. The second application will use the 5HTP-selective chemistry to produce homogeneous 'antibody-drug conjugates.' Strategies for increasing payload loading and attaching two distinct bioactive small molecules per antibody will also be developed.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.