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Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy Philipp Ottis† and Craig M. Crews*,†,‡,§ Department of Molecular, Cellular and Developmental Biology, ‡Department of Chemistry, and §Department of Pharmacology, Yale University, New Haven, Connecticut 06511, United States
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ABSTRACT: Until recently, the only ways to reduce specific protein signaling were to either knock down the target by RNAi or to interfere with the signaling by inhibiting an enzyme or receptor within the signal transduction cascade. Herein, we review an emerging class of small molecule pharmacological agents, called PROTACs, that present a novel approach to specifically target proteins and their respective signaling pathways. These heterobifunctional molecules utilize endogenous cellular quality control machinery by recruiting it to target proteins in order to induce their degradation.
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bifunctional molecules consist of three components: a target protein-binding moiety, a degradation machinery recruiting unit, and a linker region that couples these two functionalities. Typically, the utilized degradation machinery is the ubiquitin− proteasome system (UPS) by the recruitment of an E3 ubiquitin ligase followed by ubiquitylation of the target protein and its subsequent degradation by the proteasome (Figure 1). However, preliminary approaches to recruit heat-shock proteins to enter the chaperone-mediated autophagy system (CMA) have been reported as well. Here, we review the development of this potentially new class of drugs from early peptide-based degraders to highly potent small-molecule PROTACs, which are now highly advanced and poised to enter the clinic as therapeutics. Peptide-Based PROTACs. The first PROTAC was described 15 years ago and consisted of the methionine aminopeptidase-2 (MetAP-2) binding small molecule ovalicin attached via an aminohexanoic acid linker to an IκBα-derived phospho-decapeptide recognition motif for the E3 ubiquitin ligase SCFβ‑TRCP (Figure 2). Ubiquitylation and degradation of MetAP-2 by the covalently binding PROTAC was demonstrated in Xenopus egg extracts using recombinant SCFβ‑TRCP and substrate proteins.5 Shortly after, in vitro ubiquitylation of the estrogen receptor (ERα) by SCFβ‑TRCP was successfully induced with an analogous estradiol-based phosphopeptide PROTAC.6 To address the lack of cell-permeability of the highly polar IκBα-phosphopeptide, Sakamoto and co-workers subsequently injected HEK293 cells with a dihydrotestosterone (DHT)-based phosphopeptide PROTAC and demonstrated the ability of PROTACs to degrade the androgen receptor (AR) in living mammalian cells.6
or decades, drug development has had the aim of inhibiting aberrant and disease-promoting protein function by utilizing the pharmacological paradigm of occupancy-driven inhibition based on small molecules occupying and blocking active or regulatory sites of enzymes or receptors. To establish and maintain a level of efficacious inhibition (IC90), high concentrations of drug are required, which can lead to off-target effects. Accordingly, drug development research has evolved to focus strongly on proteins with accessible, “druggable” active and regulatory sites. This definition of the “druggable” proteome, however, drastically limits potential drug targets: less than 20% of the approximately 20,300 known human proteins are currently considered as drug targets.1,2 In an effort to expand upon this traditional paradigm of occupancy-driven protein inhibition, a new class of small molecules, called proteolysis-targeting chimeras (PROTACs), has emerged. Unlike traditional drugs, PROTACs aim to eliminate the aberrantly functioning protein rather than to inhibit it. PROTACs follow an event-driven rather than an occupancy-driven pharmacological paradigm and act catalytically to degrade superstoichiometric amounts of the target protein. Earlier attempts to similarly downregulate target proteins using RNA interference-based approaches were hampered by high metabolic instability, off-target effects, and low bioavailability of the oligonucleotide-based drugs and, therefore, failed to demonstrate a broader therapeutic applicability.3,4 With better pharmaceutical properties, e.g., higher cell permeability and greater metabolic stability, than nucleic acids, PROTACs offer a significantly broader therapeutic applicability for protein knockdown than RNAi. This holds particularly true for allsmall molecule PROTACs.
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PROTACS The PROTAC Technology. PROTACs are small molecules capable of recruiting the cellular proteostasis apparatus to degrade intracellular disease-causing proteins. These hetero© 2017 American Chemical Society
Received: December 1, 2016 Accepted: March 6, 2017 Published: March 6, 2017 892
DOI: 10.1021/acschembio.6b01068 ACS Chem. Biol. 2017, 12, 892−898
Reviews
ACS Chemical Biology
Figure 1. Scheme of PROTAC mechanism of action utilizing the E3 ubiquitin ligase VHL. (A, B) The PROTAC binds the target protein with its substrate binding functionality (gray square) and VHL with its E3-ligand (orange wedge), thereby bridging the E3 substrate-recognition subunit and the target protein. Upon target engagement, the functional E3-ligase complex consisting of VHL, Elongin B and C, together with Rbx, assembles on the Cullin 2 scaffold protein and recruits an E2 ubiquitin-conjugating enzyme. (C) The E2 transfers multiple ubiquitins (Ub; red) onto the presented target, creating a polyubiquitin chain. (D) The polyubiquitylated target is recognized as a substrate by the proteasome and is subsequently degraded. The PROTAC remains unmodified and, hence, can initiate a new targeted degradation event.
Figure 2. The first PROTAC was described by Sakamoto et al.5 in 2001. This hybrid PROTAC consists of the MetAP-2 binding molecule ovalicin linked by an aminohexanoic acid linker and six glycine residues to the phospho-decapeptide recognition motif for SCFβ‑TRCP. Asterisks indicate phosphorylated residues.
the HIF1α peptide and polyarginine tail to a phosphorylation substrate-targeting peptide to create a conditional “phosphoPROTAC”. By choosing the phosphorylation motifs of either TrkA or ErbB3 as the targeting peptides, the phosphoPROTACs would remain “silent”, solely bound to VHL, until either nerve growth factor (NGF) or neuregulin are recognized by the receptors TrkA or ErbB2/ErbB3, respectively. Recognition of the growth factors results in auto- and transphosphorylation of the phosphorylation motifs of these receptors and hence in the phosphorylation of the conditional phosphoPROTACs as well. Phosphorylation of the targeting peptides rendered them ligands for either FRS2α (TrkA-based PROTAC) or phosphatidylinositol-3-kinase (ErbB3-based PROTAC). Both described phosphoPROTACs conditionally induced degradation of their respective targets with the ErbB3-based PROTAC even being demonstrated to reduce tumor growth in mice, which is the first described PROTAC application in animals.16 In another study, using peptides to compensate for suitable small molecules, the X-protein of hepatitis B virus could be successfully degraded by fusion of the oligomerization domain to the VHL recognition peptide. A poly-D-arginine tail aided cell permeability.17 Furthermore, by fusing a peptide derived from β-tubulin to the poly-D-Arg-tagged HIF1α heptapeptide, levels of the Alzheimer’s Disease (AD)-associated protein tau could be reduced in primary neurons and in a mouse model of AD.18 Similar to the peptide-based approaches described above, Henning and co-workers achieved the post-translational
By replacing the IκBα-phosphopeptide component with the 5−8 amino acid HIF1α-derived substrate motif of another E3 ubiquitin ligase, the Von Hippel−Lindau (VHL) protein,7−9 creating cell-permeable PROTACs became feasible. PROTACmediated degradation of proteins in living cells by merely adding the PROTAC to the culture medium was demonstrated by Schneekloth and colleagues, wherein they targeted both a mutant FKBP12 protein and the androgen receptor using a poly-D-arginine-tagged VHL substrate heptapeptide coupled to a mutant-specific analogue of rapamycin and DHT, respectively.10 In parallel, Zang et al. reported the induced intracellular degradation of MetAP-2 and ERα utilizing the full HIF1α octapeptide without any additional permeabilityaiding sequences.11 Shortening of the VHL recognition sequence to a pentapeptide subsequently allowed for degradation of ERα in human endothelial cells12 The incorporation of apigenin as a targeting moiety for the pentapeptide PROTACs led to inducible degradation of the aryl hydrocarbon receptor (AHR).13,14 Beyond the role of the targeting ligand and the E3 enzyme-binding peptide, the importance of linker-position in the design of PROTACs was illustrated by a study on ERα degraders.15 Recent approaches have also highlighted the ability of allpeptide PROTACs to overcome a lack of target-binding small molecules, to recruit other proteins of the quality control machinery or to link target degradation to intracellular signaling. In an elegant approach, Hines and colleagues linked 893
DOI: 10.1021/acschembio.6b01068 ACS Chem. Biol. 2017, 12, 892−898
Reviews
ACS Chemical Biology
(PEG) linker to nutlin. With degradation of AR in HeLa cells at concentrations ≥10 μM, the potency of this first generation small molecule degrader, however, resembled that of peptide PROTACs.23 Alternatively, the research groups of Yuichi Hashimoto and Mikihiko Naito incorporated the aminopeptidase inhibitor bestatin24 into their small molecule degraders. As part of a heterobifunctional molecule, this Actinomyces-derived natural compound can recruit the cellular inhibitor of apoptosis protein 1 (cIAP1) E3 ubiquitin ligase to a target protein. Knockdown of the retinoic acid binding proteins CRABP I and II, the retinoic acid receptor (RAR), ERα, and AR at micromolar concentrations was described.25−28 Degradation of the spindle regulatory protein “transforming acetic coiled coil 3” (TACC3) with similarly designed bestatin-based degraders, however, turned out to depend on the recruitment of the unrelated E3 ubiquitin ligase APC/CCDH‑1.29 This observation and the moderate efficacies yielded with bestatin-based degraders can most likely be linked to the low binding specificity of bestatin for cIAP1, the destabilization, and the high off-target effects of this parent compound.30 Cereblon. Most recently, the immunomodulatory drug (IMiD) thalidomide and its derivatives lenalidomide and pomalidomide have been revealed to bind to the Cul4− Rbx1−DDB1−cereblon E3 ubiquitin ligase complex. In their binding to cereblon, these IMiDs modulate the specificity of the E3 ligase to induce degradation of the IKAROS family transcription factors IKZF1 and IKZF3, which forms the basis of their anticancer effect.31−34 Within the past two years, three reports have demonstrated successful incorporation of thalidomide or its derivatives into PROTACs. Two of these independent, yet simultaneously conducted, studies describe the targeting of the transcription factor BRD4, a member of the bromodomain and external domain (BET) family. The first study utilized the BRD4 inhibitor OTX015 fused to pomalidomide to create the PROTAC ARV-825 (Figure 4).35 This PROTAC induced approximately 50% BRD4 degradation observed after 2 h treatment of Burkitt’s lymphoma (BL) cells with 100 nM. In overnight treatment, ARV-825 yielded an astonishing DC50 of
