Targeted protein degradation: elements of PROTAC design
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.
Section snippets
Current status of the PROTAC technology
In the past several years, targeted protein degradation has generated excitement in both academic and industrial settings where POIs ranging in protein class, function, and/or subcellular localization have been successfully degraded (Table 1) [8, 9, 10, 11,35•,36, 37, 38, 39, 40, 41, 42,43••,44, 45, 46, 47,48••,49••,50••,51, 52, 53,54••,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80••]. In contrast to this wide range of targeted POIs,
What does the future hold for PROTACs?
Irrespective of the mechanistic insight acquired, the full potential of PROTAC technology remains untapped. We have learned a lot from using tool compounds that target kinases and BET proteins, but it remains to be determined how transferable these discoveries are to other protein families and/or classes.
In addition to exploring other protein types, it is imperative that we explore the ‘PROTACability’ of other E3 ligases given observed discrepancies in POI degradation depending on which E3
Conclusions
Most research efforts highlighted here demonstrate that we have yet to furnish a plug-and-play approach for PROTAC development. However, we can now appreciate that binary target engagement affinities are not indicative of degradation efficiencies for PROTACs [49••,50••,56]. Meanwhile, the importance of POI ubiquitination versus ternary complex formation and stability for efficient degradation has been uncovered, using well-established target proteins and tool compounds [102••]. Given these
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Doris Hellerschmied-Jelinek, Mariell Pettersson and John Hines for helpful discussions and reading of the manuscript.
CMC gratefully acknowledges support from the N.I.H. (R35CA197589) and Arvinas, Inc. CMC is a founder, consultant, and shareholder in Arvinas, Inc.
References (116)
- et al.
A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes
Biochem Biophys Res Commun
(1978) - et al.
Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown
J Biol Chem
(1983) - et al.
Targeted protein degradation: from chemical biology to drug discovery
Cell Chem Biol
(2017) - et al.
Targeted protein knockdown using small molecule degraders
Curr Opin Chem Biol
(2017) - et al.
Targeted protein degradation and the enzymology of degraders
Curr Opin Chem Biol
(2018) - et al.
U box proteins as a new family of ubiquitin-protein ligases
J Biol Chem
(2001) - et al.
The von Hippel-Lindau tumor suppressor protein
Curr Opin Genet Dev
(2001) - et al.
Dissecting fragment-based lead discovery at the von hippel-lindau protein:hypoxia inducible factor 1α protein-protein interface
Chem Biol
(2012) - et al.
Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells
Leukemia
(2017) - et al.
SNIPER(TACC3) induces cytoplasmic vacuolization and sensitizes cancer cells to Bortezomib
Cancer Sci
(2017)
Development of BCR-ABL degradation inducers via the conjugation of an imatinib derivative and a cIAP1 ligand
Bioorg Med Chem Lett
Identification and characterization of von Hippel-Lindau-recruiting proteolysis targeting chimeras (PROTACs) of TANK-binding kinase 1
J Med Chem
Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway
Eur J Med Chem
Targeting the C481S Ibrutinib-resistance mutation in Bruton’s tyrosine kinase using PROTAC-mediated degradation
Biochemistry
Delineating the role of cooperativity in the design of potent PROTACs for BTK
Proc Natl Acad Sci U S A
Development of the first small molecule histone deacetylase 6 (HDAC6) degraders
Bioorg Med Chem Lett
An oral androgen receptor PROTAC degrader for prostate cancer
J Clin Oncol
Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation
Nat Commun
Epigenetic protein families: a new frontier for drug discovery
Nat Rev Drug Discov
Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system
Proc Natl Acad Sci U S A
Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis
Proc Natl Acad Sci U S A
Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP
Proc Natl Acad Sci U S A
The ubiquitin system for protein degradation
Annu Rev Biochem
Protacs: chimeric molecules that target proteins to the Skp1-cullin-F box complex for ubiquitination and degradation
Proc Natl Acad Sci U S A
Small-molecule modulation of protein homeostasis
Chem Rev
The ubiquitin code
Annu Rev Biochem
Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways
Nat Rev Drug Disc
Building ubiquitin chains: E2 enzymes at work
Nat Rev Mol Cell Biol
Ubiquitin ligases: structure, function, and regulation
Annu Rev Biochem
RING domain E3 ubiquitin ligases
Annu Rev Biochem
RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis
Nat Rev Cancer
HECT and RING finger families of E3 ubiquitin ligases at a glance
J Cell Sci
Physiological functions of the HECT family of ubiquitin ligases
Nat Rev Mol Cell Biol
RING-between-RINGs—keeping the safety on loaded guns
EMBO J
RING-between-RING E3s ligases: emerging themes amid the variations
J Mol Biol
The N-end rule: functions, mysteries, uses
Proc Natl Acad Sci U S A
N-degron and C-degron pathways of protein degradation
Proc Natl Acad Sci U S A
The N-end rule pathway and regulation by proteolysis
Protein Sci
Building better vasculature
Genes Dev
Structure of an HIF-1alpha-pVHL complex: hydroxyproline recognition in signaling
Science
Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction
J Am Chem Soc
Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1α
Angew Chem Int Ed
Catalytic in vivo protein knockdown by small-molecule PROTACs
Nat Chem Biol
Cancer drug resistance: an evolving paradigm
Nat Rev Drug Disc
Molecular mechanisms of drug resistance
J Pathol
The advantages of targeted protein degradation over inhibition: an RTK case study
Cell Chem Biol
Chemical approaches to targeted protein degradation through modulation of the ubiquitin–proteasome pathway
Biochem J
Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation
J Biol Chem
Development of a peptide-based inducer of protein degradation targeting NOTCH1
Bioorg Med Chem Lett
Development of a small hybrid molecule that mediates degradation of his-tag fused proteins
J Med Chem
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