Targeted protein degradation: elements of PROTAC design

Targeted protein degradation using Proteolysis Targeting Chimeras (PROTACs) has emerged as a novel therapeutic modality in drug discovery. PROTACs mediate the degradation of select proteins of interest (POIs) by hijacking the activity of E3 ubiquitin ligases for POI ubiquitination and subsequent degradation by the 26S proteasome. This hijacking mechanism has been used to degrade various types of disease-relevant POIs. In this review, we aim to highlight the recent advances in targeted protein degradation and describe the challenges that need to be addressed in order to efficiently develop potent PROTACs.

Introduction

Protein conjugation with ubiquitin, a small protein modifier, is essential for regulated protein degradation by the 26S proteasome. Despite delineating the ATP-dependent pathway of protein degradation in the late 1970s [1, 2, 3, 4, 5, 6], the first application to exploit this system for targeted protein degradation was reported thirty years later [7]. Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules consisting of: (1) a ligand that binds a POI; (2) a ligand for recruiting an E3 ubiquitin ligase (E3 recruiting element; E3RE) to promote POI ubiquitination; and (3) a linker connecting these ligands (Figure 1a) [7, 8, 9, 10, 11]. To date, there are over 100 reports describing the use of PROTACs for targeted protein degradation (Web of Science search: February 14, 2018) and their utility in chemical biology and drug development. In this review, we describe recent advances in the targeted protein degradation field and discuss those principles underlying efficient PROTAC design that remain to be elucidated.

Ubiquitin is conjugated to a protein substrate via an enzymatic cascade [5,6,12]. First, an E1 activating enzyme primes ubiquitin via an ATP-dependent mechanism forming an E1∼ubiquitin conjugate (∼; thioester bond) [5,6,13] followed by formation of an E2∼ubiquitin conjugate via a transthiolation reaction with an E2 conjugating enzyme (Figure 1a) [5,6,14]. Finally, one of the ∼600 putative E3 ligases mediates the transfer of ubiquitin to a substrate protein [5,6,15].

E3 ligases mediate protein substrate specificity and catalyze this final transfer via a non-covalent or covalent mechanism depending on the E3 type [12,15]. The three major families of E3 ligases include the RING/U-box family [16, 17, 18] and the active-site cysteine-containing HECT [19,20] and RING-in-Between-RING (RBR) families [21,22]. Some E3 ligases function by recognizing specific degradation motifs, known as degrons [23,24]. For example, UBR E3 ligases function via the N-end rule pathway, wherein a ‘destabilizing’ N-terminal amino acid promotes UBR-mediated ubiquitination [23,25]. Meanwhile, the von Hippel Lindau (VHL) E3 ligase recognizes Hypoxia-Inducible Factor 1 α (HIF1-α) whereby hydroxylation of a key proline residue on the HIF1-α degron motif is essential for VHL-recruitment [26, 27, 28]. This degron forms the basis of one of the most widely used E3REs for PROTACs (Table 1) [29, 30, 31].

By recruiting an E3 to a POI, PROTACs hijack ligase activity for POI ubiquitination and subsequent degradation by the 26S proteasome (Figure 1a) [8, 9, 10, 11]. PROTACs induce the ternary complex (POI:PROTAC:E3 ligase) for ubiquitination, after which the POI is committed for destruction. Since the PROTAC is not degraded in this process, it can promote ubiquitination and degradation of multiple POI equivalents, thus operating substoichiometrically [32^•]. This catalytic, event-driven modality contrasts with the traditional inhibitor paradigm wherein sustained target binding is indispensable for eliciting a desired biological response. In the standard occupancy-driven paradigm of drug development, potency is dependent on binding affinity. For example, POI inhibition likely cannot influence non-catalytic target protein function(s) (Figure 1b). Additionally, sustained target engagement is difficult in cases of target overexpression, the presence of competing native ligand(s), or target protein mutations that result in loss of target engagement and subsequent resistance (Figure 1b) [33,34]. Since PROTACs inhibit protein function via degradation, this event-driven technology can be used to circumvent the common disadvantages of traditional occupancy-driven inhibitors described above.