Design and optimization of PROTAC linking groups

Since the first proof of concept in 2001, targeted protein degradation induced by small molecules has gradually become a popular direction in drug development. There are two main strategies for inducing degradation. One is to use small molecules that can change the E3 ligase substrate recognition domain, the so-called molecular glue, which allows the recruitment of new substrates for proteolysis, the other is to use chimeric small molecule that is composed of an E3 ligase binding part connected to a module which binds to the target protein.

Proteolysis Targeting Chimera (PROTAC, also known as SNIPER, uSMITE, or degradation agent) and bifunctional small molecule degradation agent are widely known due to their catalytic activity to induce isozyme selectivity through protein-protein interactions. PROTAC can simultaneously bind to the substrate recognition domain of E3 ligase and the protein of interest (POI), thereby inducing non-homologous ubiquitination and subsequent POI degradation. There are currently six unique chimeric degradant molecules whose effects in humans are currently studied in clinical trials. The unique metabolic activity of PROTACs comes from their ability to appropriately use RING ubiquitin ligase from the ubiquitin-proteasome system (UPS), which regulates protein homeostasis by labeling proteins with polyubiquitin chains, thereby labeling them for proteolysis.

The potency and isozyme selectivity of PROTAC can be optimized by the structure-activity relationship (SAR) within the linker. The length and the chemical composition of the connecting part have been shown to affect the structural rigidity, hydrophobicity, and solubility of PROTAC. At present, the SAR research on the length of the connection part is mainly empirical, which is time and labor-intensive work. Although great progress has been made in rational PROTAC design through structural biology and computational research, the design and synthesis of linking groups is still an arduous task. Michael D. Burkart et. al recently published an article in the Journal of Medicinal Chemistry, explaining the role of linker design in the PROTACs.

Ways to improve synthesis throughput

Current methods for simplifying SAR research on linker variants include solid-phase synthesis of bifunctional linkers using orthogonal protection, copper-catalyzed click chemistry, activated esters, and Staudinger ligation chemistry to increase synthesis throughput.

Reasonable design of PROTAC linking group

* Use X-ray crystallographic data and calculation model to rationally design PROTAC.

* In order to rationally design the de novo PROTAC development using the Molecular Operating Environment (MOE) and the open source Rosetta software suite, more rigorous calculation methods have been developed.

Empirical SAR of the linking group

The data does not necessarily indicate that any linker larger than the optimal length will produce a successful degrading agent, but generally speaking, since there is no spatial conflict, a longer linker will increase the chance of finding a degrading agent.

The empirical nature of PROTAC development activities is gradually replaced by chimeric small molecule synthesis methods, structural characterization of ternary complexes, advances in computational protocols, and balanced development of systematic analysis of previous studies. PROTAC development provides a dramatic detour for drug design, as the focus has shifted to small molecules capable of stabilizing non-homologous protein interactions long enough for polyubiquitination. This event-driven pharmacology makes it possible to reuse previously discarded ligands that have been put on hold due to lack of efficacy or lack of selectivity. It also provides an effective way to target non-catalytic protein targets, significantly expanding potential lead compounds.