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

在NSF化学部通过生物技术分子基金会(MFB)征求意见的支持下，由波士顿学院的Abhishek Chatterjee、Tim van Opijnen和Eranthie Weerapana组成的多学科团队正在开发技术，以研究允许病原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，有必要在生理学上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带来了巨大的技术挑战。研究小组将开发一种基因编码技术，选择性地标记细菌细胞中制造的蛋白质，这将使它们能够有效地从感染部位分离出来。使用这项技术，将有可能监测细菌蛋白质组在感染期间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，该团队开发的精确蛋白质标记技术将被用于将生物活性小分子附着在抗体上。这些研究旨在证明将这种新的靶向生物正交化学用于“抗体-药物结合物”领域的原则证据。如果成功，这种应用将构成这一基础科学对生物医学疗法的潜在深远的长期科学更广泛的影响。该组织希望在这一领域建立产业合作伙伴关系。主要研究人员正在开发一类新的生物正交结合反应，目标是非规范氨基酸5-羟色氨酸(5htp)。他们将应用这种化学，进一步利用他们最近开发的一种工程细菌色氨酰-tRNA合成酶(TrpRS)，将5htp整合到活细胞的蛋白质中。第一个应用将创建一个完全遗传编码的系统，以标记和纯化在病原菌中表达的新合成的蛋白质。它将涉及：(I)根据色氨酸在细胞中产生5htp的可诱导生物合成途径，(Ii)工程TrpRS，以促进生物合成的5htp随机掺入新合成的蛋白质中，以响应色氨酸密码子，以及(Iii)使用5htp选择性生物结合化学来标记和纯化5htp标记的蛋白质，用于蛋白质组学研究。该平台将用于在活体动物感染模型中研究抗生素治疗后病原菌的蛋白质组学变化。第二个应用将使用5HTP选择性化学来产生均一的“抗体-药物结合物”。还将开发增加有效载荷并为每个抗体连接两个不同的生物活性小分子的策略。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.