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

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.