Reversible Covalent PROTACs: Novel and Efficient Targeted Degradation Strategy

The proteolysis targeting chimeras (PROTACs), which are composed of a target protein binding moiety, a linker, and an E3 ubiquitin ligase binder, have been a promising strategy for drug design and discovery. Given the advantages of potency, selectivity, and drug resistance over inhibitors, several PROTACs have been reported in literature, which mostly focus on noncovalent or irreversible covalent binding to the target proteins. However, it must be noted that noncovalent or irreversible PROTACs have several drawbacks such as weak binding affinity and unpredictable off-target effects. Reversible covalent PROTACs, with properties of enhanced potency, selectivity, and long duration of action, have attracted an increasing amount of attention. Here, we propose a comparison between these three patterns and highlight that reversible covalent PROTACs could pave the way for a wide variety of challenging target degradations.

Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 691093 2 CRBN ligand to design a PROTAC that could simultaneously degrade multiple proteins of the BRD family, including BRD2, BRD3, and BRD4 (Lu et al., 2015). Research from the Bondeson group used foretinib as the target protein binding part and VHL as the E3 ubiquitin ligase ligand, respectively, to design a PROTAC that can degrade a total of nine kinases simultaneously ). An explanation is that reversible noncovalent PROTACs could recruit multiple proteins and E3 ligases and then form ternary complexes to make protein ubiquitination and degradation. Due to the strong affinity and potent occupancy ability, irreversible covalent PROTACs have also successfully degraded target proteins such as HaloTag-fused cAMP-responsive element-binding protein 1 (HaloTag-CREB1), HaloTag-fused c-jun (HaloTag-c-jun) (Tomoshige et al., 2016), recombinant methionyl aminopeptidase 2 (MetAP-2) (Sakamoto et al., 2001), and Bruton's tyrosine kinase (BTK) (Xue et al., 2020). Nevertheless, as shown in Figure 1, once the irreversible covalent PROTACs form a ternary complex with the target protein and E3 ubiquitin ligase, they will be directly degraded by the proteasome and cannot be recycled. Undoubtedly, irreversible covalent binding may reduce potency by negating the catalytic nature of the PROTAC's activity (Bondeson et al., 2015;Lebraud et al., 2016). Furthermore, some studies reported that irreversible covalent PROTACs inhibited the degradation of target proteins and even irreversibly bound to other biomolecules to cause off-target toxicity (Dahal et al., 2013;Burslem et al., 2017;Tinworth et al., 2019).
Reversible covalent PROTACs are theorized to combine the benefits of covalent bond formation with the substoichiometric target turnover achieved by reversible PROTACs, which is unattainable for covalent PROTACs (excluding PROTACs with covalent ligands for the E3 ligase) (KielyCollins et al., 2021). Compared with the other two types of PROTACs, reversible covalent PROTACs have better target selectivity and lower potential toxicity (Gabizon et al., 2020a;Guo W. H. et al., 2020;KielyCollins et al., 2021). As a possible mechanism of action, which is described in Figure 1 (blue), in vitro/vivo, the ligand parts of reversible covalent PROTACs bind to the target proteins or E3 ubiquitin ligases through reversible covalent bonds, thereby forming stable ternary complexes. Ubiquitin located on the E2 ubiquitinconjugating enzymes is then transferred to the target protein, which leads to the ubiquitination of the target proteins and degradation by the proteasome. PROTACs are released from the target protein or the E3 ligase, and a new ternary complex is formed again.
As far as known to the authors, researches in this area are scarce. We have listed some reported reversible covalent PROTACs, as shown in Figure 2. Maimone and his cooperators were the first to design a reversible covalent PROTAC CDDO-JQ1 based on bardoxolone, which successfully degraded BRD4. The reversible covalent moiety of CDDO-JQ1 was the E3 ligase recruiter Tong et al., 2020). Following this, Jin Wang and Nir London designed reversible covalent PROTACs, which both used the first FDA-approved covalent kinase inhibitor ibrutinib as the target protein moiety and chose pomalidomide as the CRBN E3 ligase binder, successfully degrading Bruton's tyrosine kinase (BTK). Most PROTACs have poor cell uptake capacity and membrane permeability due to their large molecular weight. The Jin Wang research group certified through SPPIER imaging that reversible covalent PROTAC RC-1 is more efficient in inducing BTK-PROTAC-CRBN ternary complexes formation in living cells compared to the other two types PROTACs. They enhanced the accumulation of PROTACs in cells and their binding ability by introducing reversible covalent groups. Inspired by Jin Wang's idea, another target protein (Fms-like tyrosine kinase 3) had been degraded by this same strategy (Guo W. H. et al., 2020). The Nir London group's research proved that reversible covalent PROTACs based on dimethylated cyanoacrylamide could form covalent complexes more rapidly and validated reversible binding by the ibrutinib competition assay. Moreover, they made a comparison among these three types and found that only the reversible covalent PROTAC RC-3 degraded a known ibrutinib off-target BLK (a covalent off-target of ibrutinib) with no activity against the noncovalent off-targets CSK and LYN, representing enhanced selectivity. In essence, their research suggested that degradation by reversible covalent PROTACs was driven by covalent engagement and exhibited enhanced selectivity toward BTK compared to noncovalent and irreversible covalent PROTACs (Gabizon et al., 2020b). The aforementioned PROTACs may form reversible covalent complexes with the target proteins or E3 ubiquitin ligases. The PROTACs are then released and form a ternary complex again, thus inducing protein degradation in a substoichiometric/catalytic manner. The advantages of reversible covalent PROTACs are evident in Table 1. Furthermore, many reports had proven that covalent enzyme inhibitors displayed powerful therapeutics and exquisite selectivity by using reversible covalent warheads in drug design and discovery (Bandyopadhyay and Gao, 2016).
In conclusion, reversible covalent PROTACs present a very promising and powerful approach for current drug discovery and tool development in biology with better selectivity, degradation activity, and longer duration of action compared to noncovalent and irreversible covalent PROTACs. Reversible covalent PROTACs can overcome the drawbacks of the other two types by avoiding a permanent protein complex and maintaining the catalytic nature of PROTACs. Currently, keys to designing a reversible covalent PROTAC are to discover a reversible covalent E3 recruiter or introduce a reversible covalent ligand binding to the target protein (KielyCollins et al., 2021), such as cyanoacrylamide (Guo W. H. et al., 2020) and dimethylated cyanoacrylamide (Gabizon et al., 2020b). On one hand, more target binders and E3 ligases applicable in the development of PROTACs are awaiting to be discovered. On the other hand, extra efforts are required to gain deeper insight into the clinical effectiveness and safety of PROTACs.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
MY contributed to conception and design of the study and wrote the first draft of the manuscript. YD wrote sections of the manuscript. YC was involved in revising the grammar and drawing the diagram. All authors contributed to manuscript revision, read, and approved the submitted version.