PROTAC Linker

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PROTAC^® consists of three key components: the protein of interest (POI) binding ligand, the E3 ligase binding ligand, and the linker linking them. To date, the design of most PROTAC molecules has relied on a combination of a known target protein-binding ligand and a small number of E3 ligase-binding ligands.

The intermediate linker is usually an ether (eg, PEG) or a variant of a hydrocarbon chain linked to a POI ligand through an amide, ether, or amine. BRD4 has received a lot of attention from the academic and pharmaceutical industries due to its huge potential as a new target in a variety of disease environments, especially in cancer. Optimization of linker and small modification of von Rippel-Lindau (VHL) binding ligands resulted in an effective PROTAC molecule 9 (ARV-771), which is effective in degrading BRD4 protein in rodent cells. Linkers from different parts of the ligands bound to the bromo-based domains of the target protein can also modulate the selectivity of Protacs (MZP54) to degrade BRD3/4.

More recently, Zhang et al. have shown that PROTAC molecule RC32, designed on the basis of rapamycin, can promote protein degradation in mouse cells through oral administration (60mg/Kg, BID), and that protein degradation has been observed when injected in rats, pigs, and raccus monkeys. As in the case of VHL-PROTAC, linker consists mainly of alkyl or alkoxy groups, and usually contains additional functional groups to attach a POI binding ligand to an E3 ligand, such as an amide or triazole. The exception is compound QCA-570, which has alkynyl groups on both sides of the pyrazole moiety in linker.

Bruton's tyrosine kinase (BTK) is encoded by the BTK gene and is expressed in a variety of blood cells. As a tyrosine kinase, BTK plays an important role in physiological processes such as B lymphocyte differentiation, maturation, and signaling. Its key unction in B cell tumors has also been noted. Adelajda Zorba's team designed a targeted degradation of BTK's Protacs molecules with linkers of different lengths, and used Western Blot to test the Protacs' degradation efficiency of BTK at the cell level. It was found that Protacs molecules can begin degrading BTK in about an hour, and the degradation of BTK had a significant dose-dependent effect on Protacs molecules. In addition, they also studied the relationship between the length of the linker and the efficiency of the Protacs molecule to bind to the target protein and E3 ligase to form a ternary complex. It was found that within a certain range, longer linkers were more favorable for the formation of ternary complexes.

In addition, linker with linear fatty or ether chains is expected to be at risk for oxidative metabolism, as such linker is easily accessible to the oxidase catalytic site. Therefore, in order for PROTAC molecules to achieve sufficient oral bioavailability, the empirical reduction in clearance is important, which can be done by in vitro testing to reduce unbound clearance in liver microsomes or hepatocytes before entering in vivo pharmacokinetics studies.

References:

1. Edmondson, S. D. , Yang, B. , & Fallan, C. . (2019). Proteolysis targeting chimeras (Protacs) in 'beyond rule-of-five' chemical space: recent progress and future challenges. Bioorganic & Medicinal Chemistry Letters.
2. Zorba A, et al.Delineating the role of cooperativity in the design of potent Protacs for BTK.Proc Natl Acad Sci U S A. 2018 Jul 31;115(31):E7285-E7292.

* PROTAC^® is a registered trademark of Arvinas Operations, Inc., and is used under license.