Fine-tuning a drug for applications in the clinic is a dramatically prolonged process. Although a compound might show initial promise, it can hit roadblocks related to the body’s ability to absorb it or the compound’s solubility, side effects, pharmacokinetic profile—or all of the above. Modern science can tackle many of these chemical puzzles, but adding specific chemical groups to particular positions on a molecule—and only on that position and without changing other parts of your compound—can be a daunting and sometimes impossible task.
Medicinal chemistry takes compounds and figures out ways of modifying them or creating new versions with specific properties, and each new element that’s added to a molecule can be the result of one or many steps conducted across a range of time spans and temperatures.
But where possible, why reinvent the wheel? Enzymes are biological machines that are already optimized to carry out specific reactions and so can serve as biocatalytic tools in the medicinal chemist’s arsenal. Such biocatalytic tools have a wide range of applications, from modifying peptides for therapeutic applications to facilitating research by helping scientists create custom molecules, but the use of many enzymes as biocatalysts can be limited by their narrow scope of acceptable substrates and the often-unknown need for regulatory or accessory factors.
Adding a chlorine, bromine, or other halogen atom to a molecule (halogenation) is one tack that scientists can use to try to improve a compound’s characteristics and make them more appropriate for clinical use. The lab of Doug Mitchell, the William Kelly Warren Sr. Professor of Biochemistry in the School of Medicine Basic Sciences, recently reconstituted and characterized a versatile tryptophan halogenase—an enzyme that halogenates tryptophan residues on its substrates. Improving the field’s understanding of this enzyme brings it a step closer to seeing use as a biocatalytic tool in basic and translational research. The Mitchell group’s research was published in Angewandte Chemie in August 2025.
The enzyme in question, ChlH, halogenates tryptophan residues on peptides, short amino acid chains, and proteins, longer and more complex amino acid chains. Its normal cognate substrate is a peptide called ChlA, but first authors Andrew Rice and Mayuresh Gadgil found that, unlike most other known enzymes, ChlH can modify an incredibly broad array of peptide and protein substrates.
“ChlH has early potential as a broadly useful biocatalyst, especially for the modification of peptide drugs, which have recently seen major successes in treating diabetes and obesity,” Rice said.
Although Mitchell’s team, which moved with him from the University of Illinois Urbana-Champaign to Vanderbilt in the beginning of 2025, had made significant progress on this project before their arrival, the lab of Professor of Molecular Physiology and Biophysics Hassane Mchaourab was critical for informing the team’s understanding of the enzyme-substrate interactions in their system. According to Rice, Mchaourab’s lab provided “incredible” molecular dynamics data that pushed the project to completion. Mchaourab is the director of the Center for AI in Protein Dynamics.
The devil is in the details
Among the proteins and peptides that Rice and Gadgil attempted to halogenate with ChlH was GLP-1, the peptide that serves as the basis for popular weight-loss and type 2 diabetes-related drugs. Although ChlH could only modify a trace amount of some of the tested substrates (including GLP-1), it could more effectively halogenate slightly modified peptides, thereby helping the team develop a fuller picture of the conditions that allow ChlH to function.
Molecular dynamics results depicting a tryptophan residue bound within the active site of ChlH. Rice, Gadgil et al. Angewandte Chemie, 2025.
Rice believes that, because ChlH has such broad catalytic activity on a wide variety of substrates, it could theoretically be modified through directed evolution so it can selectively and robustly modify any tryptophan residue of interest (provided there is at least some catalytic activity observed in the first place). Directed evolution is a protein engineering method in which researchers can artificially evolve proteins to arrive at a researcher-defined endpoint.
Existing biocatalytic tools for post-translationally adding halogens to tryptophan residues in peptides and proteins have been limited to tryptophans located at the beginning or end of an amino acid chain. This work identified ChlH as a possible alternative considering its incredibly broad substrate scope. Although a lot of pieces must fall in precisely the right place, it’s possible that ChlH could one day be used to selectively modify peptide drugs to increase their efficacy, stability, or solubility. “That would be incredibly satisfying to see,” Rice said.
In the meantime, he hopes that other groups will leverage ChlH as a tool for the halogenation of tryptophan residues or simply to better understand the complex mechanism of this enzyme class.
Go deeper
The paper “Peptidic Tryptophan Halogenation by a Promiscuous Flavin-Dependent Enzyme” was published in Angewandte Chemie in August 2025. Andrew Rice and Mayuresh Gadgil are co-first authors.
The study was published open access through a transformative agreement negotiated by Vanderbilt University’s Jean and Alexander Heard Libraries. Transformative agreements eliminate traditional paywalls and remove the obstacle of article processing charges, ensuring immediate and unrestricted access to research worldwide. Vanderbilt authors can learn more about the Heard Libraries’ agreements supporting open access publishing in this research guide.
Funding
This research used funds from the National Institute of General Medical Sciences.
School of Medicine Basic Sciences shared resources and co-authors
This research was supported by the Center for AI in Protein Dynamics and the Mass Spectrometry Research Center. SOMBS co-authors include Mitchell, Mchaourab, Paola Bisignano and Richard Stein, research associate professors of molecular physiology and biophysics.