From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation

Abstract

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional degraders that specifically eliminate targeted proteins by hijacking the ubiquitin-proteasome system (UPS). This modality has emerged as an orthogonal approach to the use of small-molecule inhibitors for knocking down classic targets and disease-related proteins classified, until now, as "undruggable." In early 2019, the first targeted protein degraders reached the clinic, drawing attention to PROTACs as one of the most appealing technology in the drug discovery landscape. Despite these promising results, PROTACs are often affected by poor cellular permeability due to their high molecular weight (MW) and large exposed polar surface area (PSA). Herein, we report a comprehensive record of PROTAC design, pharmacology and thermodynamic challenges and solutions, as well as some of the available strategies to enhance cellular uptake, including suggestions of promising biological tools for the in vitro evaluation of PROTACs permeability toward successful protein degradation.

Keywords: PROTAC technology; cell permeability; drug discovery; protein degradation; proteolysis targeting chimeras; ubiquitin-proteasome system.

Figure 1

Targeted protein degradation by PROTACs. A PROTAC simultaneously binds a target of interest (TOI) and an E3 ubiquitin ligase complex, leading to ubiquitination and degradation of the TOI via the UPS. E2, Ubiquitin-conjugating enzyme; Ub, Ubiquitin.

Figure 2

Different mode-of-action of small-molecule inhibitors and PROTACs. Small-molecule inhibitors often require higher concentrations to be effective, while PROTACs act through event-driven pharmacology that leads to targeted protein degradation at lower concentrations.

Figure 3

PROTAC-mediated ternary complex formation and hook effect. The hook effect is a function of PROTAC concentration (black line). A possible strategy to reduce the hook effect is increasing cooperative-binding PPIs to stabilize ternary complexes (red line).

Figure 4

The reversible cis-trans isomerization of PHOTAC general structures by irradiation with UV-VIS wavelengths (Reynders et al., 2020). Lenalidomide (in blue), linker (in red), photoswitch groups (surrounded). The structure of the TOI ligand (R) is not shown.

Figure 5

Trans-photoPROTAC and cis-photoPROTAC. trans-photoPROTAC displays an optimal distance between both warhead moieties to engage the proteins in a ternary complex; in red is shown the “pull-pull” diacid linker. cis-photoPROTAC is shorter and thus inactive (Pfaff et al., 2019).

Figure 6

LYTAC technology. LYTACs, respectively, bind the transmembrane receptor (CI-M6PR) and the extracellular target, leading to his internalization into endosomes and final degradation into lysosomes.

Figure 7

Challenges in the use of PROTAC technology. Schematic representation of the multiple questions that PROTACs need to address for affording a satisfactory TOI degradation.

Figure 8

Schematic representation of the in-cell CLIPTAC PROTACs mode of action. In the example, cells are treated with TCO-tagged ligand BRD4, followed by functionalized E3 ligase binder (thalidomide). Click reaction happens in situ by the combination of two smaller precursors, leading to the formation of the CLIPTAC degrader.

Figure 9

Mode of action of cyanoacrylamide-derivatized PROTAC after cellular internalization. The presence of an electrophilic group allows a reversible covalent bond between the degrader and a reactive Cys belonging to the protein of interest. TOI is eliminated while PROTAC regenerated.

Figure 10

Schematic representation of the NanoBret assay. The NanoLuc (nLuc) luciferase-tagged protein interacts with a fluorescent tracer to produce a bioluminescent resonance energy transfer (BRET). The addition of PROTACs to cells results in a competition between the degrader molecule and the tracer in protein binding. The recorded BRET signal decreases accordingly to PROTAC efficiency in target engagement.

Figure 11

The pulse-chase procedure for the chloroalkane penetration assay (CAPA). (i) The Halo-GFP-Mito (red) is cytosolically oriented and bind covalently to the ct-molecules (green). (ii) After washing, ct-dye (gray) chases unoccupied HaloTag and provides fluorescence upon binding. (iii) The fluorescent signal is quantified by flow cytometry and normalized fluorescence is plotted as a function of ct-molecule concentration.