While VHL and CRBN represent the majority of E3 ligases regularly utilized for targeted protein degradation, MDM2 and cIAP are also frequently employed. Other E3 ligases have been liganded (detailed below) but are still in earlier development.

Thalidomide, lenalidomide and pomalidomide are the common IMiD drugs that can be used as recruiters of the cereblon E3 ligase. It was shown that the glutarimide of the IMiDs goes into the binding site of CRBN and interacts [16,17] strongly with Trp380 and His378 (Figure 5a). Mutations of Trp380 resulted in no binding and phthalimides without glutarimide also showed no binding to CRBN [19]. Furthermore, methylation of the glutarimide moiety in heterobifunctional degraders reduced binding with CRBN and degradation of BRD4. Thus, the glutarimide was shown to be essential for CRBN recruitment. The phenyl ring of the IMiDs is pointing out of the cereblon binding site and is perfectly suited for exit vectors to conjugate target binding warheads through a linker or even directly.

Recently, several CRBN-recruiting degraders were developed using phenyl glutarimides where glutarimides are directly attached to the aryl rings and then conjugated with target warheads [20]. CRBN-based degraders are small in size considering their molecular weight and tPSA, and they have lower cLogP and improved solubility. While adding linker and warhead comes with a gain in size, these degraders often still have better physicochemical properties compared with the VHL-, IAP- or MDM2-recruiting degraders. The relative hydrophilicity of CRBN recruiters not only facilitates solubility but also reduces non-specific interactions/plasma protein binding-thus improving bioavailability.

Von Hippel-Lindau (VHL) is also a heavily researched and promising E3 ligase in the development of proximity-induced degraders [21]. The most commonly used recruiter of VHL, VH032, mostly comprises five main fragments (Figure 5b). The thiazole part of the recruiter seems to be necessary, though other substituents seem possible and the methyl substitution at the 4-position of the thiazole ring seems to be pointing outside the E3 binding pocket. This allows for the use of these residues as exit vectors for degrader design.

The phenyl group adjacent to the thiazole is not being extensively explored, but the phenolic replacement is widely known and the ethers with the phenolic group are often used conjugate the linkers carrying the warhead [22]. The hydroxyproline part of the recruiter plays a crucial role in binding with the ligase and the hydroxy group appears buried in the protein site. Epimers here were shown to be inactive, demonstrating that the stereochemistry is unalterable. But there seems to be a fluoro-substitution possibility in place of the hydroxyl group [23] and the possibility of capping the OH to generate pro-drug esters [16]. Stereochemistry is crucial for the amine/amide functional end of the molecule and many substitutions were employed here as this part is the most commonly used exit vector. Though the branched benzylamino fragment does not seem crucial and remains unmodified even with unsubstituted benzylamine, it can be used as an exit vector with the desired stereochemistry for warhead conjugation as it is pointing out of the binding domain. VHL is a promising E3 ligase; however, the binding site is unfortunately rather hydrophobic.

The resulting heterobifunctional degraders, thus mostly hydrophobic as well, might therefore interact with plasma proteins through non-specific interactions and eventually result in poor bioavailability. VHL degraders are usually higher in molecular weight, topological polar surface area and hydrophobicity and hence need to be highly potent to degrade the target protein within the limits of their reduced bioavailability.

E3 ubiquitin-protein ligase MDM2 mediates ubiquitination and subsequent degradation of p53 and inhibits p53-mediated cell cycle arrest and apoptosis. MDM2 acts as an E3 ligase for itself and promotes proteasomal degradation. MDM2 also facilitates proteasomal degradation of DYRK2, IGF1R and SNAI1. MDM2 inhibitors trigger loss of mitochondrial membrane potential, caspase activation and DNA fragmentation. MDM2 is involved in normal hematopoiesis and its inhibition can lead to hematopoietic defects. The main toxicity observed by using MDM2 inhibitors are gastrointestinal and bone marrow [24] disorders. MDM2 is highly expressed in most tissues as well as in several cancers, likely due to its role as a proto-oncogene. Nutlin-based binders imidazole, pyrrolidine and piperidinone (AMG-232) bind to MDM2 amino acid residues Trp23 (p-chloro phenyl goes into the groove and binds), Leu26, His96 (with the acetic acid), Phe19 and Gly58. Nutlins have a central imidazoline ring with two p-chloro phenyl rings and a third phenyl ring with alkoxy groups (Figure 5c). The N1 of the imidazole connects to the piperzine ring via a urea linkage. The compound idasanutlin has a pyrrolidine ring replacing the central imidazole ring, and AMG-232 on the other hand has piperidine ring. All these inhibitor derivatives have the room for linker attachment at the piperazine or phenyl carboxylic acid, which points out of the MDM2 binding site. As an exemplary target using an MDM2 degrader strategy, BCR-ABL was degraded by recruitment of MDM2 through nutlin by means of an alkoxy linker. [25,26]. In general, the MDM2 recruiters have greater molecular weight and lipophilicity, and this further increases upon the degrader designs. However, the recruiters have low tPSA and this also contributes to the relatively lower tPSA of MDM2 ligase-recruiting degraders.

Cellular inhibitor of apoptosis 1 (c-IAP1), also known as Baculoviral IAP repeat-containing protein 2 (BIRC2), regulates caspases and apoptosis and modulates inflammatory signaling and immunity. IAP family proteins contain a 70 amino acid motif referred to as the baculovirus IAP repeat (BIR) domain and there are three such BIR domains along with ubiquitin binding and RING E3 ligase activity domains (Figure 6a). It functions as an E3 ligase regulating the canonical NF-kappa-B signaling pathway and suppressing the non-canonical NF-kappa-B. The target proteins for its E3 ubiquitin-protein ligase activity include a number of RIP/MAP kinases [27] (p. 2). Specific and nongenetic IAP-dependent protein erasers (SNIPERs) induce degradation of the target protein by forming the ternary complex between the cIAP1 BIR domain and the target protein, which then facilitates ubiquitination of the target with cIAP1-bound E2. Several Ala-Val-Pro-containing SMAC mimetics have been explored as the binders of IAPs. Most of the binders except bestatin have proline analogues as a fixed backbone while bestatin features a serine amino acid instead [28]. Some recruiters have a six-membered nitrogen heterocycle or a fused nitrogen heterocycle replacing the proline.

X-linked inhibitor of apoptosis protein (XIAP) also regulates caspases and apoptosis, inflammatory signaling and immunity, copper homeostasis, mitogenic kinase signaling and cell proliferation. It acts as an E3 ligase in regulating NF-kappa-B signaling and the target proteasome-degraded proteins include RIP kinases and caspases. Piperazine remains at the central core for binding and the indole group is included as a mean for conjugating the target warhead and for capping. One prominent example of a successful XIAP-based degrader was used for the degradation of BCR–ABL using dasatinib as the warhead [30].