In 2001, chemists K. Barry Sharpless, Hartmuth C. Kolb, and M. G. Finn introduced click chemistry, a concept in which organic molecules can be rapidly and reliably joined to form more complex structures. They recognized that many natural compounds are assembled through efficient carbon–heteroatom (C–X) bond formation, particularly with nitrogen, oxygen, and sulfur, and they sought to replicate this in the laboratory.
Since its introduction, click chemistry has transformed the field and was later recognized with the Nobel Prize in Chemistry. One of its most influential reactions is the copper-catalyzed azide–alkyne cycloaddition (CuAAC), where an azide (N3) reacts with an alkyne to create a stable triazole ring. Click reactions are now widely used in drug discovery and in bioconjugation, allowing researchers to attach probes, such as fluorescent dyes or radioactive tags, to biomolecules, aiding in diagnosis and therapy. These tools have helped scientists uncover how drugs interact with their biological targets.
Using the principles of click chemistry, researchers from Tokyo University of Science (TUS), Japan, have developed an efficient strategy to synthesize proteolysis-targeting chimeras (PROTACs). These therapeutic molecules selectively eliminate disease-causing proteins by recruiting the cell's own protein-degradation machinery.
The study, published in the journal Bulletin of the Chemical Society of Japan on November 28, 2025, was led by Associate Professor Suguru Yoshida of the Department of Life Systems Engineering, TUS. The research team also included Ms. Yuri Taninaga, a second-year Master's student, Ms. Maho Miyamoto from Yokohama City University, former Master's students Mr. Gaku Orimoto, Ms. Kaho Yamada, Dr. Hidetomo Yokoo, and Dr. Yosuke Demizu from the National Institute of Health Sciences, Japan.
"We demonstrated that PROTACs with proteolytic activity can be rapidly synthesized by assembling functional molecules through three consecutive click reactions. This method allows easy introduction of ligand components with suitable linkers, physical-property tuning units, and probe functionalities, and is therefore expected to accelerate PROTAC discovery," says Dr. Yoshida.
PROTACs consist of two bioactive ligands connected by a linker. One ligand binds to the protein of interest, and the other recruits an E3 ubiquitin ligase, which marks the target protein for destruction by the cell's proteasome.
To construct their PROTACs, the team began with VH032, a small substrate molecule that binds the VHL E3 ubiquitin ligase. They introduced a small alkyne group into VH032 so that it could participate in click reactions. Using this modified VH032, they assembled PROTACs through three sequential click reactions: CuAAC, a Michael addition, and sulfur (VI) fluoride exchange (SuFEx).
The first step, a CuAAC reaction, attached VH032 to a specially designed three-part scaffold. The second step used SuFEx chemistry to add a second ligand that binds to the target protein, such as an EGFR inhibitor. The final step, a Michael addition, served as the customization stage. Here, the researchers could fine-tune the PROTAC's properties by attaching units, such as polyethylene glycol, to improve water solubility or by adding fluorescent tags to create a traceable molecular probe.
Using this three-step modular approach, the team rapidly generated fully assembled PROTAC candidates that joined the VHL-binding ligand with a variety of bioactive molecules. Because the method tolerates many functional groups and does not require protecting groups, it can be used to quickly create structurally diverse PROTACs that retain biological function.
The newly assembled PROTACs—identified as compound 17, which targets EGFR, and compound 23, which is a fluorescent-tagged version—showed clear EGFR degradation activity in HeLa cells. Both compounds reduced EGFR protein levels in a dose-dependent manner, meaning higher concentrations produced greater degradation.
Although these initial click-assembled PROTACs showed lower potency compared to some traditionally synthesized versions, the researchers highlight that their straightforward construction dramatically simplifies the development process. This enables faster synthesis, optimization, and testing of new therapeutic candidates.
"We anticipate that this modular, click-based strategy will accelerate the development of pharmaceuticals and other bioactive products by enabling efficient creation of protein-degrading compounds from readily available and easily synthesized components, enabling a 'Direct-to-Biology' strategy," says Dr. Yoshida.
