Kacper Kossakowski, Michalina Jurga, Anna Pawełczyk

How to design PROTAC molecules? A practical guide to proteolysis-targeting chimeras

 

2025-12-05

This article provides a comprehensive guide to the design, synthesis, and biological characterization of PROTAC molecules, also known as proteolysis-targeting chimeras. The aim of the study was to compile up-to-date knowledge and present it in the form of a practical compendium that can serve both as an educational resource and as a tool to support experimental planning. The following sections discussed the mechanism of action of PROTACs and their role within the broader field of targeted protein degradation technologies. The principles for selecting the protein of interest, ligands, E3 ligases, and linkers were outlined, with emphasis on molecular modeling methods and physicochemical parameters affecting pharmacokinetic properties. Available synthetic strategies and methods for assessing PROTAC biological activity were described, including biophysical and biochemical techniques for analyzing molecular interactions, validating the mechanism of action, and interpreting data. Particular emphasis was placed on explaining key concepts such as the “hook effect”, DC50, and Dmax, as well as comparing the modes of action of classical inhibitors with those of PROTAC-based degraders. The paper also explored protein-protein interactions within ternary complexes and outlined common challenges in PROTAC design, highlighting directions for further development based on modern structural concepts and molecular innovations aimed at improving selectivity, bioavailability, and degradation efficiency. This review provides a cross-sectional overview of current topics in the design and functional evaluation of PROTAC molecules, combined medicinal chemistry, molecular biology and biophysics, bioinformatics, and pharmacology. This work may serve as a starting point for further research into targeted protein degradation technologies and support the development of experimental strategies in research projects - regardless of the stage of advancement - while also offering valuable educational support.

Keywords: drug design, medicinal chemistry, PROTAC, targeted protein degradation, proteolysis-targeting chimeras.

© Farm Pol, 2025, 81(4): 195–211