Small-Molecule Modulation of Protein Homeostasis - ACS Publications

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Small-Molecule Modulation of Protein Homeostasis George M. Burslem and Craig M. Crews* Departments of Molecular, Cellular, and Developmental Biology, Chemistry, and Pharmacology, Yale University, 219 Prospect Street, New Haven, Connecticut 06511, United States ABSTRACT: Control of protein levels by nucleic-acid-based technologies has proven to be a useful research tool but lacks the advantages of small molecules with respect to cell permeability, temporal control, and the potential generation of therapeutics. In this Review, we discuss the technologies available for the control of intracellular protein levels with small molecules and compare the various systems available.

CONTENTS 1. Introduction 2. Brief Guide to the Ubiquitin/Proteasome System 3. Inhibitors of the Ubiquitin/Proteasome System 3.1. Proteasome Inhibitors 3.2. Deubiquitinase Inhibitors 3.3. Inhibitors of E1 Activation Enzymes 3.4. Inhibitors of E2 Conjugation Enzymes 3.5. Inhibitors of E3 Ligases 3.6. Inhibitors of Other Protein−Protein Interactions in the UPS System 4. Immunomodulatory Drugs 5. Proteolysis Targeting Chimera 5.1. Peptide-Based PROTACs 5.2. Realization of All-Small-Molecule PROTACs 5.3. Specific and Nongenetic Inhibitors of Apoptosis Protein-Dependent Protein Erasers 5.4. Importance of Linker Composition 5.5. Hook Effect 5.6. Summary 6. Destabilization Domains 6.1. Shield System 6.2. LID System 6.3. Other Destabilizing Domains 7. Auxin-Induced Degradation 8. Hydrophobic Tagging 8.1. Selective Estrogen Receptor Degraders 8.2. Selective Androgen Receptor Degraders 8.3. HaloTag-Based Systems 8.4. Adamantyl Small Molecules 8.5. Boc3 Arg 8.6. Other Hydrophobic Small Molecules 9. Split Ubiquitin Systems 9.1. Split Ubiquitin for the Rescue of Function 9.2. Traceless Shielding 10. Folding Modulators 10.1. Pharmacological Chaperones 10.2. Other Folding Modulators 11. Other Systems 11.1. Direct Recruitment to the Proteasome 11.2. Chemical Rescue of Structure © 2017 American Chemical Society

11.3. Folding Intermediate Selective Inhibitors 11.4. Split Tobacco Etch Virus Protease 11.5. Peptide-Directed Lysosomal Degradation 12. Conclusions Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

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1. INTRODUCTION The control of intracellular processes has long been a target for manipulation by small molecules in the study of biology and medicine. Many successes have been achieved by developing small-molecule inhibitors of enzymatic function, and these have revolutionized the medical profession. Additionally, the development of small-molecule probes has also furthered our understanding of biological systems, with new tools continually being developed, adding to the arsenal of chemical biologists. However, not every protein has an enzymatic activity that can be inhibited, and as such, these are traditionally thought of as “undruggable”.1 Molecular biology techniques such as si/ shRNA2,3 and more recently CRISPR/Cas9 systems4 can suppress the expression of these proteins at the genetic level in a research environment but are not easily translated into therapeutics. Small-molecule control of intracellular protein levels could provide both novel therapeutics and tools for the study of biological systems irrespective of the function of the proteins themselves. The advantages of small-molecule control include increased cell permeability compared to nucleic acids/ proteins, greater levels of temporal control (e.g., transient knockdown), the potential for dual directional control, and, ideally, the avoidance of any requirement for genetic modification. In this Review, we will discuss the range of tools available to exert control over cellular protein levels using small molecules. The majority of the tools available currently

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Received: February 2, 2017 Published: August 4, 2017 11269

DOI: 10.1021/acs.chemrev.7b00077 Chem. Rev. 2017, 117, 11269−11301

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Figure 1. Overview of the ubiquitin system.

isopeptide bond.17 The E3 is the enzyme that determines substrate specificity, and as such, E3s are the most diverse and numerous of the ubiquitin cascade enzymes. There are two main families of E3 ligases, RING and HECT, which function via different mechanisms.18 RING (really interesting new gene) E3 ligases catalyze the transfer of Ub directly from the E2 to the substrate, whereas HECT (homologous to E6-AP carboxy terminus) E3s transfer Ub via a transient E3 thioester (Figure 2a and b).19 RING E3 ligases can consist of a single protein

function via the modulation of the ubiquitin/proteasome system (UPS), which will be introduced in section 2. Control of genetic induction by small molecules, such as tetracyclinecontrolled transcriptional activation,5 will not be described because it has been reviewed elsewhere.6,7 Instead this Review will focus on post-translational control of intracellular protein levels.

2. BRIEF GUIDE TO THE UBIQUITIN/PROTEASOME SYSTEM Much of the turnover of protein in cells is mediated by the UPS. Ubiquitin is a small protein consisting of 76 amino acids and is implicated in many cellular functions.8 The attachment of ubiquitin (Ub) to lysine side chains via an isopeptide bond is a common post-translational modification and can control protein trafficking and cellular localization. On a larger scale, protein ubiquitination is instrumental in regulating the cell cycle;9,10 however, most importantly in the context of this Review, ubiquitination can target a protein to the proteasome for degradation. The position of a Ub modifier is important for its function, as is the differentiation between chain length.11,12 Ubiquitin itself has 7 lysine residues, which can in turn be ubiquitinated, producing poly-Ub chains. The linkage of these chains is also important in determining the fate of the tagged protein. Polyubiquitin chains linked by Lys48 are the canonical signal for degradation and induce recruitment of the substrate to the proteasome.11 The UPS regulates the intracellular concentration of many proteins and as such requires a large degree of substrate specificity. The transfer of ubiquitin to its intended substrates (Figure 1) results from a cascade of enzymes with increasing specificity. Initially, an E1-activating enzyme catalyzes the production of a ubiquitin acyl adenylate from free Ub in an ATP-dependent process.13 The resulting adenylate reacts with a cysteine side chain on the E1, producing a thioester. There are only 2 known E1 enzymes in the human proteome.14 E1 enzymes then interact with E2 enzymes, which catalyze the transfer of Ub from the E1 to a cysteine residue on the E2 via trans-thioesterification. There are ∼40 E2 enzymes in humans, so some degree of substrate specificity can be imparted at this stage. The type of ubiquitin chain linkage can also be partially determined by the E2.15,16 Finally, there are E3 ligases that catalyze the transfer of Ub from the E2 thioester to a bound substrate lysine via an

Figure 2. Subtypes of E3 ligases. (a) RING-type E3 ligase. (b) Hecttype E3 ligase. (c) Multicomponent SCF RING ligase.

(e.g., c-CBL or MDM2) or a multiprotein complex such as the Skp-1-Cullin-F-box (SCF) E3 ligases (Figure 2c).20 SCF E3 ligases consist of the RING domain containing protein RBX-1, which binds to a cullin scaffolding protein (Cul1). The other end of Cul1 binds to adapter protein SKP1, which in turn binds to F-box motifs. A range of F-box proteins can then associate with the complex and determine the specificity of the SCF complex.21 Polyubiquitination often results in the degradation of the protein via the proteasome.22,23 The 26S proteasome is a large proteolytic complex consisting of the 20S core complex and two 19S cap complexes (Figure 3). The core complex consists of two 7-membered rings of β-subunits flanked by 7-membered rings of α-subunits (shown in Figure 3 as yellow and red, respectively) that produce a narrow channel down the center.24 The β-subunits possess proteolytic activity, which can divide proteins into smaller peptides. The 19S cap complex recognizes 11270


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development and use of proteasome inhibitors have been extensively reviewed elsewhere27,31−33 and will not be discussed here save for to note that they provide a mechanism to control protein levels in a global sense, with very little specificity. Recent reports suggest that inhibition of the AAA ATPase p97, another regulator of protein homeostasis, might have similar therapeutic benefits.34,35 3.2. Deubiquitinase Inhibitors

Inhibition of the proteasome results in nonspecific inhibition of protein degradation; an alternative mechanism for proteostasis modulation is inhibition of the deubiquitinase enzymes (DUBs) associated with the proteome. Before proteins can be unfolded and enter into the pore of the 20s proteasome, the ubiquitin chains signaling for degradation must be removed. There are three proteasome associated DUBs: UCHL5, USP14, and RPN11, two cysteine proteases and a metalloprotease, respectively. In the context of protein homeostasis, these three are the most important and will be the focus of this section. Other DUBs are crucial for regulating other cellular processes and have been reviewed elsewhere.11,36 The development of specific cysteine protease DUB inhibitors has proven challenging, although there are examples of selective molecules.37 Nonspecific inhibition can be achieved via the use of maleimides to covalently label the nucleophilic cysteine residues, but this also inhibits other cellular processes dependent on cysteine nuclephiles.38 By employing activitybased chemical proteomics, the Kessler group identified a cellpermeable pan-DUB inhibitor called PR-619 (Figure 5), which inhibits the majority of cysteine protease DUBs but spares other cysteine proteases.39 PR-619 has proven to be a useful research tool to study the UPS but lacks the specificity to be useful as a potential therapeutic. Michael acceptors have proven to be successful in targeting DUBs, as well as cysteine proteases in general.40 The small molecule WP1130 (Figure 5) was derived from a JAK inhibitor and possesses antiproliferative activity.41 Analysis of the mode of activation revealed inhibition of a subset of DUBs including both of the proteasome-associated cysteine protease DUBs, UCHL5 and USP14.42 Treatment of cells with WP1130 results

Figure 3. Structure of the proteasome.

substrate proteins via their polyubiquitin chains, deubiquitinates, and unfolds the proteins such that they can enter the pore of the hydrolytic 20S core complex.25,26

3. INHIBITORS OF THE UBIQUITIN/PROTEASOME SYSTEM There are several points of potential small-molecule modulation (Figure 4) in the UPS, some of which have yielded pharmaceutical agents and some of which have proven largely intractable to inhibition thus far. As parts of the UPS are general in nature, these targets are unlikely to yield specific control over individual protein levels but may still provide useful research tools and therapeutics. The main targets and their inhibitors are discussed below. 3.1. Proteasome Inhibitors

The most therapeutically relevant and perhaps most general point of inhibition of the UPS is the proteasome itself. By inhibiting the proteasome’s proteolytic activity, degradation of ubiquitinated proteins can be halted. This approach has proven to be therapeutically useful as cancer cells are often more reliant than healthy cells on the proteasome to clear misfolded proteins.27 Indeed three proteasome inhibitors28−30 have been approved for the treatment of multiple myeloma. The

Figure 4. Potential points of inhibition in the ubiquitin/proteasome system. 11271


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of both could be expected to prevent all cellular ubiquitination.51 Nevertheless, several small-molecule inhibitors of the E1 enzymes have been reported and allow global control of proteins degraded via the UPS as well as modulation of other systems requiring ubiquitination. The first cell-permeable inhibitor of an E1 was reported in 2007 by Weissman and co-workers.52 Identified in a screen for p53 stabilizing compounds, PYR-41 (Figure 6) was shown to

Figure 5. Structures of DUB inhibitors.

in the accumulation of polyubiquitinated proteins in a similar way to proteasome inhibition. Another molecule containing three Michael acceptors, b-AP15 (Figure 5), has been identified as an inhibitor with some degree of specificity for UCHL5 and USP14.43 DUB inhibition by b-AP15 appears to have a distinct cellular response from proteasome inhibition including a lack of dependency on p53 status. Several analogues of b-AP1544 have been reported to also possess DUB inhibitory activity, including the ring-expanded analogue VLX1570 (Figure 5). VLX1570 is more soluble, more potent, and more selective for USP14 than b-AP15.45 Inhibition of USP14 is a viable strategy for the treatment of multiple myeloma patients,46 particularly those who are resistant to proteasome inhibition; and VLX1570 is currently in phase 1/2 clinical trials. Conversely, inhibition of USP14 by IU1 (Figure 5) appears to enhance the proteasomal degradation of certain substrates.47 RPN11 is the only proteasome-associated DUB that is essential for a functional proteasome, making it an excellent target for inhibition as a potential alternative to proteasome inhibition.25 As a metalloprotease, RPN11 requires different approaches to abrogate its function.25 It is likely susceptible to nonselective metal chelation; indeed, thiolutin has been reported as an inhibitor of all JAMM metalloproteases including RPN11,48 but until very recently no specific inhibitors had been reported. The Deshaies and Cohen laboratories performed high-throughput screening for RPN11 inhibitors and identified a novel fragment (8-thioquinoline) for nonspecific inhibition of metalloproteases.49 Crucially, they were able to derivatize this core to produce capzimin (Figure 5), the first selective RPN11 inhibitor.50 The DUB inhibitors discussed above provide evidence that inhibition of proteasome-associated DUBs can prove to be a viable target for small-molecule intervention, which may be particularly useful in the treatment of multiple myeloma, where proteasome inhibitors have proven therapeutically useful. However, they provide no control over the levels of specific proteins, functioning rather as proteome-wide modulators.

Figure 6. Structures of selected E1 inhibitors.

be a selective inhibitor of E1 over E2 enzymes. PYR-41 prevents the accumulation of ubiquitinated proteins and allows the accumulation of normally rapidly degraded proteins (e.g., p53), as well as a plethora of other ubiquitination-dependent process in cells. The mechanism has been postulated to involve covalent labeling of a cysteine side chain on E1. Subsequent studies have shown that PYR-41 is also a DUB inhibitor that functions via protein cross-linking.53 Another small molecule, PYZD-4409 (Figure 6), with a similar structure to PYR-41, was discovered to also inhibit E1 enzymes.54 Leukemia and multiple myeloma cells are particularly reliant on the UPS and are therefore sensitive to proteasome inhibition (discussed earlier) and E1 inhibition. PYZD-4409 is able to preferentially induce cell death in hematological malignant cell lines over normal hematopoietic cells and slow tumor progression in vivo. Studies showed similar increases in the levels of rapidly degraded proteins to

3.3. Inhibitors of E1 Activation Enzymes

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allosterically, and additional studies show that it functions by stabilizing the interaction between Cdc34 and ubiquitin.67 A pair of structurally and mechanistically similar compounds, BAY 11-708268 and NSC697923 (Figure 7),69 have been shown to inhibit a range of E2 enzymes by covalently reacting with, and therefore blocking, the Ub-accepting cysteine residue. Despite the lack of selectivity, these molecules proved to be valuable tools and can be employed in vivo with no significant side effects reported.

PYR-41 and a strong induction of endoplasmic reticulum stress consistent with the unfolded protein response. AMP analogue MLN4924 (Figure 6) (Pevonedistat) was identified as an inhibitor of the NEDD8 E1 activating enzyme and is currently undergoing clinical trials.55 It was subsequently shown to also be a potent Ub E1 inhibitor that reacts with the thioester to form a nonhydrolyzable Ub-AMP mimetic.56 An and Statsyuk were able to improve the selectivity and potency for the Ub E1 of MLN4923 via activity-based proteomics and rational design.57 MLN7243 has also entered clinical trials as an E1 inhibitor.58 A cellular screen of compounds performed to identify inhibitors of p27 proteolysis identified the relatively simple disulfide compound NSC 624206 (Figure 6), which inhibits the loading of adenylated ubiquitin onto the E1 cysteine but not the ubiquitin-activation step.59 The surprisingly simple structure appears to be at least partially selective for E1 over E2 enzymes. JS-K, a nitric oxide prodrug, also inhibits Ub loading of the E1 cysteine, presumably by thiol nitrosylation.60 Additionally several natural product inhibitors of E1-activating enzymes have been identified: largazole appears to inhibit the ubiquitin adenylation step rather than the thioester formation,61 and himeic acid A,62 panepophenanthrin,63 and the hyrtioreticulins64 have been shown to have weak E1 inhibitory activity in vitro.

3.5. Inhibitors of E3 Ligases

There are ∼600 postulated E3 ligases that act as the terminal and specificity-defining step in the human ubiquitin cascade. For many of the E3 ligases, the substrates are not known, and therefore, specific targeting of E3 ligases can prove challenging.70 However, many examples exist, some of which are highlighted below. MDM2 is a prototypical RING-type E3 responsible for the rapid degradation of p53, often referred to as the “Guardian of the Genome”.71 p53 levels are crucial to prevent the development of cancers, and as such, inhibition of the p53/ MDM2 interaction has been an area of significant research efforts.72 A plethora of compounds have been reported as inhibitors of the p53/MDM2 protein−protein interaction, and many have reached clinical evaluation, which has been reviewed elsewhere.73 It has also become a model system for the study of protein−protein interaction inhibitors.74 Several other E3 ligase substrate interactions have been extensively explored,75 including work from our laboratory on the HIF-1α/VHL interaction (discussed below), the IAP family proteins,76,77 and many others.78,79 Comprehensive discussion of all reported E3 ligase inhibitors is beyond the scope of this Review, but it should be noted that inhibition of specific E3/substrate interactions has provided small-molecule-based control of intracellular protein levels with an enhanced degree of specificity over E1 or proteasome inhibitors. A recently reported technique may facilitate the rapid discovery of new E3 ligase inhibitors.80

3.4. Inhibitors of E2 Conjugation Enzymes

Despite the potential for therapeutic intervention at the E2 stage, very few small-molecule inhibitors have been identified, possibly due to the inherent challenges involved in targeting protein−protein interactions. It could be envisioned that specific inhibition of an E2 could provide a similar biological consequence to E1 inhibition (i.e., accumulation of proteins) but of a smaller subset of cellular proteins.65 E2 enzymes play a role in the determination of Ub linkage, and hence, their selective small-molecule inhibition may allow control over differential Ub signals.15 The first E2 inhibitor, CC0651 (Figure 7), was identified in a p27 ubiquitination assay and efficiently inhibits the activity of

3.6. Inhibitors of Other Protein−Protein Interactions in the UPS System

To function, the majority of the UPS system is mediated by protein−protein interactions such as those between E1, E2, and E3 subunits, substrates, ubiquitin, and proteasome components. Development of protein−protein interaction (PPI) inhibitors in the UPS system has proved to be challenging, but several examples are discussed below. Verma et al. reported the identification of the ubistatins (Figure 8) as molecules that inhibit the interaction of select ubiquitin chains with the 19S cap complex.81 They do not inhibit the proteasome or DUBs but rather strongly inhibit the association of K48-linked, but not K29- or K63-linked, ubiquitin chains to the proteasome. NMR revealed ubistatin binding to a hydrophobic patch on the surface of K48 diubiquitins, which blocks their interactions with the ubiquitinbinding domain(s) of the proteasome. Ubistatin A was able to rescue ubiquitinated protein degradation to the same extent as proteasome inhibition. The natural product honokiol also appears to be able to prevent degradation of K48-linked substrates in a similar manner.82 Another example of a modulator of ubiquitin chain/protein interactions is withaferin A (Figure 8).83 Withaferin A appears to covalently label the zinc finger domain of NF-κB essential modulator (NEMO) protein and induce its interaction with

Figure 7. Structures of selected E2 inhibitors.

SCF complexes with several different F-box components but has no effect on HECT or RING E2 domains.66 Thermal shift, crystallographic, and mutational analysis identified the E2 Cdc34 as the target of CC0651. Knockdown of Cdc34 simulates the cellular activity of CC0651, further confirming the target. Structural studies showed that CC0651 acts 11273


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ligase.93 They initially attributed ImiD teratogenic activity to the inhibition of E3 activity because thalidomide is capable of inhibiting the autoubiquitination of cereblon. However, patients with higher levels of cereblon expression respond better when treated with these immunomodulatory drugs (ImiD’s),94 and lenalidomide resistance appeared to be related to a decrease in cereblon. Both of these findings suggested an alternative mechanism. In 2014, two groups simultaneously reported on the identification of the Ikaros family zinc finger proteins 1 and 3 (IKZF1/3) as the ImiD-induced substrates of cereblon:95,96 through a careful series of experiments, these studies conclude that the ImiD’s induce the degradation of the Ikaros transcription factors via the induction of their interaction with the E3 ligase and the subsequent ubiquitination/ proteasomal degradation of the former. An additional, structurally distinct ImiD CC-122 also induces the degradation of the Ikaros transcription factors (Figure 9).97 Further proteomic analysis resulted in the identification of casein kinase 1A1 (CK1α) as an additional substrate for lenalidomide-induced degradation but not pomalidomide/ thalidomide-induced degradation.98 Curiously, in vitro experiments reveal that both pomalidomide and thalidomide are able to induce CK1α ubiquitination, but no cellular degradation is observed.99 Further biophysical and structural characterization revealed the molecular mode of action and showed that CK1α and IKZF1 bind at the same interface of cereblon.99 Interestingly, no interactions between cereblon and its neosubstrates could be detected in the absence of ImiD’s, suggesting the molecules act as a form of “molecular glue” by reengineering the protein surface to allow neosubstrate recognition (Figure 10). Screening of a library of lenalidomide

Figure 8. Structures of UPS PPI modulators.

long K48-linked ubiquitin chains. Treatment of cells with withaferin A appeared to stabilize long K48 chains. Many of the E3 Cullin-RING ligases recruit the E2 Cdc34 via an electrostatic protein−protein interaction.84 Pan and coworkers have reported that the anthelmintic suramin is able to inhibit this E2−E3 interaction across many Cullin-RING ligases in vitro.85 Despite the poly pharmacology and poor cell penetrability of suramin, this nonetheless validates the targeting of this protein−protein interaction with small molecules. Peptoids86 and natural products87,88 have been identified as inhibitors of the Ubc/Uev, a complex responsible for the formation of Lys63 Ub linkages,89,90 and in the case of the peptoids have been shown to be active in cells and in vivo.

4. IMMUNOMODULATORY DRUGS Thalidomide and its analogues, pomalidomide and lenalidomide (known collectively as immunomodulatory drugs (ImiD’s)) (Figure 9), have been widely used in the clinic for the treatment of multiple myeloma;91 however, until relatively recently their mechanism of action was unknown.92 In 2010, Handa and co-workers identified the molecular target of thalidomide as cereblon and showed cereblon to be an E3 Figure 10. Substrate adapter mechanism of the ImiD’s.

derivatives in a cell-proliferation assay followed by target identification identified CC-885 (Figure 9) as a potent degrader of GSPT1,100,101 as well as identifying more potent degraders of IKZF1/3.102 CC-885 retains the ability to degrade IKZF1 indeed, it appears to have an increased ability to degrade IKZF1but none of the previously characterized ImiD’s induce the degradation of GSPT1. This family of compounds is able to induce the degradation of Ikaros transcription factorsotherwise undruggable proteinsand was discovered serendipitously. The identification of CC-885 poses the following question: can additional de novo complexes be induced by small molecules and elicit pharmacologically relevant degradation? Crucially, these FDA-approved entities prove that targeted protein degradation is a viable approach to the treatment of disease.

Figure 9. Structures of the immunomodulatory drugs (ImiD’s). 11274


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Figure 11. PROTAC-mediated ubiquitination and proteasomal degradation.

5. PROTEOLYSIS TARGETING CHIMERA Another method for the development of compounds capable of inducing protein degradation is proteolysis targeting chimera (PROTACs). PROTACs are heterobifunctional compounds comprising a recruiting element for a protein of interest (POI) and an E3 ligase recruiting element bound together via a linker. By bridging the gap between a POI and an E3 ligase and inducing their proximity, PROTACs can induce the ectopic ubiquitination of the POI. Polyubiquitination subsequently leads to proteasomal degradation of the POI (Figure 11).

time that PROTACs can function in the context of an intact cell, albeit via microinjection to circumnavigate issues with cell permeability associated with peptides. Crucial experiments carried out in this study show that both ends of the heterobifunctional molecule are required for activity and that the mechanism proceeds via proteasomal degradation (confirmed by protein-level rescue upon proteasome inhibition). The next generation of PROTACs employed the minimal recognition amino acid sequence for the von Hippel Lindau tumor-suppressor protein VHL, another E3 ligase,105,106 from its native substrate, HIF-1α; it also incorporated a cellpenetrating peptide sequence.107 Coupling this peptide sequence to either the ARIAD FK506- and rapamycin-binding protein (FKBP) ligand108 (Figure 13) or dihydrotestosterone allowed degradation of green fluorescent protein (GFP)-labeled FKBP or androgen receptor by simply adding the compound to cell culture media. Crucially, no genetic modification of the cells is necessary for the function of these compounds. The Kim group has also employed the HIF-1α peptide sequence to recruit VHL to degrade the estrogen receptor,109 the aryl hydrocarbon receptor,110 and MetAP2;111 however, because they did not employ the cell-penetrating peptide sequence, they required higher concentrations (100 μM) to see significant degradation. Conjugation of recruiting elements to either end of the HIF-1α peptide sequence can yield productive PROTACs, while simultaneous conjugation to both the N- and C- terminus of the peptide yields a superior degrader, presumably by taking advantage of the multivalent effect.112 The HIF-1α peptide sequence has also been employed to degrade Smad3 protein,113 incorporated into a branched unnatural peptide for the degradation of Akt 114 and incorporated into a BH3 peptide to degrade Bcl-xL115 with moderate success. A particularly exciting peptidic PROTAC is TH006, which is able to induce the degradation of tau, a protein implicated in the development of Alzheimer’s disease.116 By fusing a peptide derived from β-tubulin, which interacts with tau, to the HIF-1α

5.1. Peptide-Based PROTACs

The first PROTAC (Figure 12) was reported by the laboratories of Crews and Deshaies in 2001103 and consisted

Figure 12. Structure of the first PROTAC. Asterisk denotes serine phosphorylation.

of a phospho-peptide derived from the recognition sequence of the F-box protein β-transducing repeat-containing protein (βTRCP) from its native substrate, IκBα. The IκBα peptide was chemically coupled to ovalicin, a covalent methionine aminopeptidase inhibitor (MetAP-2) via a subsrate linker to yield PROTAC-1 (Figure 12). Initial in vitro studies revealed that PROTAC-1 could induce ubiquitination of MetAP-2, and experiments in cell lysates revealed rapid (30 min) degradation. In an extension of this early work, the same groups extended the scope of the protein targets to both estrogen and androgen receptors by employing estradiol and dihydroxytestosterone, respectively.104 Sakamoto et al. also demonstrated for the first

Figure 13. Structure of the first HIF peptide-derived PROTAC. 11275


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Figure 14. Structure of the first all-small-molecule PROTAC.

hydroxylation is key for VHL binding.105 Structure-guided drug design performed in the Crews lab led to the development of potent small-molecule inhibitors of the HIF-1α/VHL protein− protein interaction (Figure 15).121,122 Crucially, by inverting

recognition sequence and a cell-penetrating peptide, Chu et al. were able to induce the ubiquitin-mediated degradation of tau in cell culture and in vivo. The tau-degrading peptide was also able to reduce the toxicity of Aβ. Interestingly, dual-peptide PROTACs can yield conditional protein degradation control via an external stimulant.117 So called Phospho-PROTACs require activation of the POIrecruiting element and the VHL-recruiting element such that they only function in the presence of both growth factors and oxygen. A Phospho-PROTAC is composed of a receptor tyrosine kinase (RTK) trans-phosphorylation sequence coupled to the VHL-recognition sequence. Co-treatment with PhosphoPROTAC and the appropriate growth factor induces phosphorylation of the PROTAC peptide, resulting in its ability to bind SH2 or PTB domains on a POI and subsequent degradation of the POI. Phospho-PROTACs have proven to be efficient degraders of both FRS2α (containing a PTB domain), by employing a TrkA phosphorylation sequence and treatment with nerve growth factor (NGF), and PI3K (containing an SH2 domain), by employing a phosphorylation sequence from ErbB3 and treatment with neuregulin (NRG). Indeed, the PI3K Phospho-PROTAC was able to inhibit tumor growth in murine models, providing the first evidence that PROTACs can function in vivo. Other peptidic PROTAC systems have been reported more recently. Montrose and Krissansen were able to show that a peptide sequence derived from the X-protein of the hepatitis B virus is comparable to the HIF-1α peptide in inducing degradation of the X-protein transfected into HepG2 cells via the recognition of a dimerization domain by a peptide ligand.118 Kurihara and co-workers also reported a peptide-based PROTAC system for the ERα protein, albeit requiring the inclusion of some unnatural amino acids.119

Figure 15. Structures of the VHL ligand and HaloPROTAC1.

the stereochemistry of the hydroxyproline, the VHL ligand can be rendered inactive, thus providing an excellent control compound. These VHL-binding small molecules were first employed as recruiting elements for protein degradation with the development of HaloPROTACs (Figure 14).123 HaloPROTACs are composed of a VHL ligand and a chloroalkane ligand for the HaloTag system.124 Covalent labeling of HaloTag fusion proteins (EGFP, ERK1, or MEK1) with VHL ligands in this manor led to rapid and almost complete degradation. This publication also reported on the importance of linker length, which is discussed in further detail later. Subsequent optimization of VHL ligands has identified some to be inhibitors that can stabilize HIF-1α in a cellular context, providing additional control over protein levels125both POI recruited to VHL and its own native substrates. The development of potent VHL ligands has allowed the development of a wide range of small-molecule PROTACs. PROTACs that target the kinase RIPK2 have been designed with DC50 values (the concentration at which 50% of maximal degradation is observed) as low as 1.4 nM (Figure 16) despite

5.2. Realization of All-Small-Molecule PROTACs

The proof-of-principle studies with peptidic PROTACs described earlier highlighted the need for small-molecule recruiting elements. The first small-molecule PROTAC was reported in 2008 and employed a nutlin derivative to recruit the E3 ligase MDM2 (Figure 14).120 Schneekloth et al. employed a selective androgen receptor modulator ligand conjugated to nutlin-3 via a short polyethylene glycol (PEG) linker and were able to degrade the androgen receptor in HeLa cells (Figure 13). Again, the degradation could be prevented by pretreatment of the cells with a proteasome inhibitor, confirming the proposed mechanism. In an effort to expand the repertoire of small-molecule E3 ligands, research efforts focused once again on the E3 ligase, VHL, which had proven to be useful with peptide PROTACs. The hydroxyproline motif in the HIF-1α peptide portion of earlier PROTACs provided a starting point because the prolyl

Figure 16. Structure of a RIPK2 PROTAC. 11276


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panel of Burkitt lymphoma cell lines. It is important to note that inhibition of ARV-825 binding to cereblon by either imide N-methylation or cotreatment with pomalidomide blocks degradation. A parallel study conducted in the Bradner laboratory also resulted in cereblon-based PROTACs for BRD4, denoted dBET1.129 Using recombinant proteins, Winter et al. were able to confirm ternary complex formation between BRD4, cereblon, and dBET1 via AlphaScreen. The use of CRISPR to knock out cereblon inhibits the ability of dBET1 to induce the degradation of BRD4, providing more evidence for the proposed mechanism.96 To demonstrate the modular nature of ImiD-based PROTACs, the recruiting element and linker were switched to create degraders for FKBP12, which were similarly effective. Proteomic analysis of cells treated with both JQ1 and dBET1 showed a reduced relative abundance of both c-MYC and PIM, while only cells treated with dBET1 showed a reduction in BRD4 levels. Due to the lack of selectivity of JQ-1 between the BRD family proteins, BRD2 and BRD3 are also depleted after treatment with both ARV-825 and dBET1.129,130 Quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that BRD family protein loss is posttranslational while c-MYC and PIM loss is regulated at the transcriptional level as expected. Furthermore, dBET1 is able to degrade BRD4 and induce apoptosis in primary acute myeloid leukemia (AML) cells. Progression of dBET1 into in vivo studies revealed its ability to slow tumor progression and decrease the leukemic burden in bone marrow. The comparison between ARV-825 and dBET1, which are both composed of very similar recruiting elements, further highlights the importance of the linker chemistry. A direct comparison revealed that ARV-825 is more potent than dBET1 in 22Rv1 cells.131 Wang and co-workers have also developed potent BRD PROTACs employing Cereblon recruiting elements but employing an alternative BET ligand.132 ImiD-based PROTACs have also been employed by the Jung group to degrade Sirtuin 2.133 Degradation of BRD family proteins can also be mediated via the VHL E3 ligase as evidenced by MZ1134 and ARV-771 (Figures 18 and 19).131 Interestingly, MZ1 proved to be more selective for BRD4 than BRD2 and BRD3 despite broadly similar affinity between the PROTAC and the bromodomaincontaining proteins, leading the authors to speculate about the role of lysine availability and the productivity of ternary

the affinity of the recruiting element for VHL being >200-fold weaker.126 Furthermore, proteomics revealed a high level of specificity for the target proteins. Perhaps even more importantly, Bondeson et al. were able to demonstrate that these small-molecule PROTACs are catalytic. A similar in vitro approach has also been shown to be catalytic using engineered subtilisin.127 Following the discovery of the mode of action of the ImiD drugs (discussed earlier), they have also been used as recruitment elements for the E3 ligase cereblon, most notably for the degradation of BRD4, which subsequently causes loss of c-MYC.128,129 Traditional inhibition of BRD4 suppresses cMYC expression but leads to accumulation of the BRD4 protein itself, leaving an excess of protein poised to elicit its oncogenic effect upon drug withdrawal. Conjugation of a BRD4-binding molecule to pomalidomide results in rapid, dose-dependent degradation of BRD4 and concomitant loss of c-MYC. BRD4 degradation results in lower cellular levels of both c-MYC protein and RNA than BRD4 inhibition, and the effect lasts for up to 24 h after PROTAC withdrawal. The most potent of the reported BRD4 PROTACs, denoted ARV-825 (Figure 17), has a subnanomolar DC50 despite the relatively

Figure 17. Structures of cereblon BRD4 PROTACs.

low affinity of pomalidomide for cereblon (3 μM).128 ARV-825 is able to inhibit cell proliferation better and induce apoptosis more efficiently than BRD4 inhibitors OTX015 or JQ1 in a

Figure 18. X-ray crystal structure of the BRD4-MZ-1-VHL complex. (A) Overall structure of the trimeric complex. BRD4 (red), VHL (green), Elongin-B (blue), and Elongin-C (cyan). (B) Close-up of the PROTAC-mediated contacts. PDB ID: 5T35. 11277


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generation of an effective PROTAC. For example, conjugation of the Abl kinase inhibitor, bosutinib, to a VHL ligand results in a molecule with no capacity for target degradation, but conjugation of bosutinib to a cereblon ligand (while maintaining the same linker) results in an efficient degrader of Abl and BCR-Abl (Figure 20). Variability of both ends of the

Figure 20. Degradation is dependent on both recruiting elements in a PROTAC.

PROTAC, as well as within the linker, can be crucial for PROTAC efficacy, as evidenced by the fact that conjugation of dasatinib (a structurally dissimilar Abl kinase inhibitor) to a VHL ligand induced only Abl degradation but conjugation to a cereblon ligand induced degradation of both Abl and BCR-Abl. This observation may allow the development of PROTACs with enhanced specificity over their cognate POI warheads.

Figure 19. Structures of VHL-based BRD4 PROTACs.

complex formation imparting selectivity. Gene-expression analysis comparing JQ1 and MZ1 revealed some differential biology, perhaps resulting from the comparison between panBRD inhibition and selective BRD4 degradation. The Ciulli group were able to determine the structure of the trimeric complex between VHL and BRD-4 mediated via MZ-1 and therefore rationalize the observed selectivity for BRD4 over other BRD family members.135 The structure revealed new protein−protein contacts that induce cocooperativity in the formation of the ternary complex with increased cooperativity observed with BRD-4 as measured by isothermal titration calorimetry (ITC). Structural data also allowed the rational design of AT1, a PROTAC with further enhanced selectivity for BRD4, albeit along with an overall decrease in potency compared to MZ-1. Arvinas, LLC, subsequently reported on ARV-771,131 another VHL-based BRD4 PROTAC, with a subnanomolar DC50 value. ARV-771 and ARV-825 have approximately the same ability to suppress c-MYC levels, but ARV-711 has more favorable pharmacokinetic properties. Interestingly, ARV-771 treatment significantly lowers the level of androgen receptor mRNA (both the wild-type AR and a mutant splice variant) in castration-resistant prostate cancer (CRPC) cell lines more profoundly than BRD4 inhibition alone does. ARV-771 has an up to 500× more potent antiproliferative effect than JQ1 or OTX015 on CRPC cell lines and is able to induce tumor regression in xenograft models.136 Given the ability of either cereblon or VHL to degrade BRD4, it is interesting to note that this redundancy is not the case for every target protein. Lai et al. reported on the modularity of PROTAC design for the BCR-Abl oncoprotein and noted that the choice of recruiting element is crucial for the development of successful PROTACs.137 Simple conjugation of an E3 ligand to a ligand for the POI is not always sufficient for

5.3. Specific and Nongenetic Inhibitors of Apoptosis Protein-Dependent Protein Erasers

Another small-molecule strategy, referred to as specific and nongenetic inhibitors of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), has also been developed. The SNIPER approach employs bestatin esters as recruitment elements for the cellular inhibitor of apoptosis protein 1 (cIAP1). In 2008 Naito and co-workers discovered that bestatin methyl esters are able to induce the degradation of cIAP1 and therefore trigger apoptosis (Figure 21).138 More specifically, binding of the bestatin ester to the BIR3 domain of cIAP1 activates the E3 ligase, resulting in autoubiquitination and its own degradation.139 By employing bestatin derivatives as recruiting elements in PROTAC-type molecules, the activation of the cIAP1 RING domain can be repurposed to induce POI ubiquitination rather than autoubiquitination.140 This was first demonstrated by degradation of the cellular retinoic acid binding proteins (CRABPs) by employing the all-trans retinoic acid as the POI ligand (Figure 21). Itoh et al. were able to show evidence for ternary complex formation, proteasome dependence, and the requirement for conjugation rather than cotreatment with unconjugated bestatin ester and retinoic acid. Interestingly, some cIAP1 degradation is still observed with the SNIPER but to a lesser extent than with unconjugated bestatin methyl ester. CRABP−cIAP1 hybrid molecules were able to significantly reduce cell migration in IMR-32 cells, while cotreatment with both ligands resulted in increased migration. Because the “suicide” degradation of cIAP1 upon SNIPER treatment could hinder the function of these compounds, investigations into the attachment point of the linker were 11278


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increase the interaction between the two proteins, either directly or indirectly. Despite the unexpected change in mechanism, the TACC3 SNIPER was able to induce degradation of the POI and specifically reduce cell proliferation in TACC3-dependent cell lines. The SNIPER technology has been shown to be modular across a range of different targets but thus far lacks the degradation efficiency and potency of the PROTAC technology. SNIPERs often require concentrations of 10−100 μM to induce significant degradation, while some PROTACs are capable of degradation at nanomolar (and even picomolar) concentrations. Perhaps the best comparison between PROTACs and SNIPERs is their ability to degrade GFPHaloTag fusion proteins because the POI recruiting ligand is identical. A VHL-derived Halo-PROTAC has a DC50 of ∼19 nM, while a similar Halo-SNIPER has a DC50 of ∼10 μM.150 Recently, the potency of SNIPER compounds has been greatly improved by the use of LCL161, a recruiting element for IAP with higher affinity.156 The availability of a higheraffinity recruiting element has enabled SNIPERs to rival PROTACs for potency; however, they still exhibit concomitant degradation of the E3 ligase.157 LCL161-derived SNIPERs have been developed against the estrogen receptor, BRD4, PDE4, and BCR-Abl with submicromolar activity. Indeed SNIPER(ER)-87 is able to function in vivo and slow the growth of tumor in xenograft models (Figure 22).

Figure 21. Structures of a CRABP SNIPER molecules and cIAP ligands.

performed by employing CRABP as a model target.141,142 Switching from bestatin esters to bestatin amides allows the development of SNIPERs that induce the degradation of CRABP but not cIAP. The differential activity between the ester and amide is not well understood as they appear to have similar affinity for cIAP1; nonetheless, the amide compound does not induce degradation.142 Studies have shown that replacement of bestatin with MV-1, a pan-IAP ligand (Figure 21),77 yields a compound that can degrade CRABP more potently than bestatin-based compounds but still suffers from also targeting cIAP1.143 SNIPER compounds have been employed to degrade a range of proteins such as retinoic acid receptor alpha, BCR-Abl, estrogen receptor alpha, and androgen receptor as well as Histagged fusion proteins.144−148 Chloroalkane-labeled bestatin149 and MV-1 analogues150 have also been prepared for the degradation of HaloTag fusion proteins, although they are considerably less-potent degraders than the corresponding VHL-utilizing HaloPROTACs.123 Okuhira et al. have demonstrated that SNIPERs can degrade CRABP-11 in different cellular compartments when the protein is artificially localized. There appears to be some selectivity between MV-1-based compounds and bestatin-based SNIPERs, as well as some debate around their dependence on cIAP1.151 Interestingly, when Naito and co-workers developed a SNIPER for the mitogenic protein transforming acidic coiled coil-3 (TACC3)152 by employing its ligand KHS-101,153 they were able to induce degradation but not via cIAP1.154 siRNA knockdown of cIAP1 did not prevent SNIPER-mediated degradation. The authors were able to show that ubiquitination of TACC3 was dependent on the E3 ligase anaphasepromoting complex/cyclosome in complex with CDH1 (APC/C CDH1 ). APC/C CDH1 is the native regulator of TACC3.155 Although the exact mechanism of SNIPER-induced degradation of TACC3 via APC/CCDH1 has yet to be elucidated, the bivalent compound appears to specifically

Figure 22. Structures of LCL161 and SNIPER(ER)-87.

5.4. Importance of Linker Composition

Throughout the development of the PROTAC technology, it has become increasingly clear that both the length and chemical composition of the linker between the two protein-binding moieties is critical for success.126 This is presumably due to its ability to influence the formation of the functional trimeric complex between the PROTAC and both proteins. Formation of a productive trimer may be determined by a variety of factors such as target lysine presentation, depth of binding site, optimal protein−protein orientation/contacts, and vector;158,159 the linker itself can have a dramatic impact on the management of some or all of these factors. Kim and co-workers reported on linker-length optimization for the degradation of estrogen receptor with estradiol/VHL peptide PROTACs.159 Using the same model system, Cyrus et al. also experimented with moving the linker-attachment position, although variation in linker length and composition in this study makes it challenging to draw useful conclusions.158 During the early stages of RIPK2 11279


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Figure 23. “CLIPTAC” approach.

PROTAC development, a series of vandetanib-based160 molecules was prepared, showcasing the dramatic impact of linker length.126 A linker consisting of 3 ethylene glycol subunits gave a DC50 value of 0.8 μM, but extending the linker by an additional ethylene glycol subunit results in a molecule that induces no observable degradation. This is also the case when comparing ARV-825 and dBET1 (Figure 17), both consisting of very similar recruiting elements but with variation between their linkers, which results in different activities. The SNIPER system is also heavily dependent on linker length.140 When considering linkers, it is important to consider the length but also the chemical composition, as this could profoundly affect cell permeability as well as influence trimer formation. Once approach to circumnavigate the issues with cell penetration was recently published by Heightman and coworkers at Astex Therapeutics, whereby they employ inverse electron demand Diels−Alder reactions 161 to assemble PROTACs within the cell (Figure 23).162 Employing a tetrazine-labeled ImiD derivative and trans-cyclooctene-labeled ligands for either BRD4 (JQ1) or ERK1/2,163,164 Lebraud et al. were able to show that degradation of the target proteins was dependent on cotreatment with both components. Crucially, treatment with preassembled “CLIPTAC” resulted in no degradation and validated the cellular assembly approach. This may prove to be a useful tool for cellular studies but is unlikely to be amenable to in vivo applications. Wurz et al. have also employed a click chemistry approach to screening of linkers and rapid library generation.165

unproductive dimers at high concentrations, as shown in Figure 24. PROTACs are active only at concentrations where they induce ternary complex formation because the ternary complex is required for ubiquitination and therefore for degradation.

Figure 24. Hook effect. Increased concentrations of PROTACs can lead to the formation of unproductive dimers.

5.5. Hook Effect

One phenomenon that must be considered when working with PROTACs and similar targeted degraders is the so-called “Hook effect”.166,167 Protein degradation induced by PROTACs is frequently biphasic. As PROTACs are applied into a system, target degradation is proportional to the concentration of the PROTAC; however, once a critical PROTAC concentration is exceeded, the dose-dependent target degradation begins to reverse itself with further increases in PROTAC. The target abundance in a dose−response graph is observed to “hook” back upward.126 While initially counterintuitive, this observation can be rationalized by the production of

5.6. Summary

PROTACs represent a powerful technological advance with the ability to exert exogenous small-molecule control over cellular protein levels without the need for genetic modification. The wide range of targets already degraded only hints at the possible scope of these molecules, and the ability to function in vivo suggests it may be possible to develop these heterobifunctional compounds into pharmaceutical agents. To this end, several companies have been launched to exploit this technology in drug development. Additional studies are required to further 11280


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Figure 25. Ligand-controlled destabilization domains. (A) Schematic representation of the destabilization domain system. (B) Structures of the shield ligands.

system has been shown to function as either N- or C-terminal fusion proteins and works for a variety of protein targets. Egeler et al. showed that fusion protein degradation in the Shield system results from ubiquitination of the destabilizing domain, which is inherently only partially folded and as such is recognized by the protein quality-control machinery.175 Further characterization of the destabilizing domain by 15N 2D heteronuclear single-quantum correlation (HSQC)-NMR revealed that Shield1 induces additional folding of the destabilizing domain, preventing ubiquitination and degradation by the proteasome.175 Considerable effort has been spent attempting to prove that Shield1 has no inherent biological activity in wild-type cells.176 A synthetically more-accessible Shield ligand (Shield2) has also been reported (Figure 25).177 Subsequently, the Shield system has been shown to work in vivo in a dose- and time-dependent fashion.178 HCT116 cells expressing FKBP12 fused to interleukin-2 (IL2) allowed the chemical control of fusion protein secretion. IL2 could be detected in cell culture medium in a Shield1 concentrationdependent manner. Mice bearing tumors from the IL2 expressing HCT116 cells showed significant reduction in tumor size upon treatment with Shield1 (10 mg/kg every 48 h i.p.) as IL2 activated the immune system.179 Thorne and coworkers were similarly able to control the levels of TNF-α, resulting in a pronounced antitumor effect. The Shield system can also be incorporated into specific tissues in transgenic mice via the CRE/Lox approach, thereby providing a dual-inducible system.180 The Shield system has been successfully employed in a wide range of organisms including zebrafish,181 transgenic medaka,182 Plasmodium falciparum,183,184 and Toxoplasma gondii185 but has proved not to be amenable to use in Saccharomyces cerevisiae.186 TALEN and CRISPR/Cas9 have been employed to knock in the shield system destabilization domain into cancer targets (p53 and PI3Kα). These authors have shown that withdrawal of Shield1 can recapitulate the phenotypes of specific smallmolecule inhibitors.187 The Shield system can also be used to induce protein aggregation by fusing an N-terminal nuclear export signal to the destabilization domain. Withdrawal of

elucidate the requirements for PROTAC activity as it is rapidly becoming clear from the current body of work that more factors are at play than simple dimerization via a two-headed molecule. Additional detailed structural characterization will be crucial to determine the role of synergistic protein−protein interactions in successful PROTACs.

6. DESTABILIZATION DOMAINS The development of small-molecule ligands for a protein can be a challenging and time-consuming process. As such, it can be advantageous to develop a hybrid system that provides the advantages of small-molecule control but is merged with the specificity and modularity of genetic approaches. Destabilizing domains represent fusion protein components that are inherently unstable and can be genetically incorporated into various proteins to destabilize them, resulting in degradation. Early destabilization domains (DDs) were based on the N-end rule168 or were under thermal control.169 Varshavsky and coworkers identified a mutated dihydrofolate reductase (DHFR) in yeast that was stable at 23 °C but not at 37 °C.169 By observing that the DHFR ligand methotrexate could prevent degradation of the fusion protein, they described the first smallmolecule-controlled destabilization domain.170 Stankunas et al. developed an early destabilization domain system based on the FKBP-rapamycin binding (FRB) protein and MaRAP.171 However, this system was limited as it requires dimerization with FKBP to stabilize the POI.172 Nevertheless, this proved to be a crucial proof of principle for the development of further destabilizing domains discussed below. 6.1. Shield System

The Shield system developed by Wandless and co-workers provides a useful tool for small-molecule control of protein levels. Employing a destabilized version of FK506- and rapamycin-binding protein (FKBP12) generated by errorprone PCR, Banaszynski et al. were able to introduce into proteins an intrinsically unstable destabilizing domain, which leads to their rapid degradation in cells (Figure 25).173,174 Addition of a small-molecule rapamycin analogue Shield1 (Figure 25) stabilizes the fusion proteins and returns expression to a normal level in a dose-dependent manner. The Shield 11281


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Shield1 induces rapid aggregation in both human cells and C. elegans.188

blood−brain barrier, the ecDHFR system has been exploited to control protein levels in the central nervous system. The ecDHFR system is complementary and orthogonal to the Shield system.192,193 The ecDHFR DD has been employed to impart temporal small-molecule control to the CRISPR/Cas9 system194 or control the heat-shock response.195 Since the previously described destabilization domains were optimized for ligand control at 37 °C, an alternative version of the ecDHFR system was developed for use in C. elegans at 25 °C.196 Another degradation domain has been engineered from the estrogen receptor ligand-binding domain (ERLBD), which is stabilized via the addition of either CMP8 or 4-hydroxytamoxifen and has been used to control levels of YFP, H-Ras, and p21.197 More recently, a fluorescent destabilization domain (FDD) has been developed from the UnaG/bilirubin receptor−ligand pair.198 Crucially, UnaG is only fluorescent when bilirubin is bound,199 providing a rapid readout of protein level and protein localization. Degradation domains (with the exclusion of LID) work in the opposite way from ImiD- and PROTAC-based technologies: instead of ligand inducing degradation of the protein, the ligand actually stabilizes the POI. While this still provides a similar level of temporal control, constant treatment of cells with a small molecule can be costly and may interfere with other biological process. Alternatively this represents an effective small-molecule “turn-on” system for transient protein expression. Finally, also in contrast to PROTACs, the use of Shield and others is the requirement to genetically encode the destabilizing domain. Although this can impart a high level of specificity, it may also alter the intrinsic activity/interactions of the protein being studied, requiring extensive validation particularly as the proteins will likely be overexpressed A recent report from the Lin laboratory demonstrates a method for the small-molecule control of unmodified proteins via a self-immolative degradation domain.200 The smallmolecule-assisted shutoff (SMASh) technique fuses a degron to the POI via the HCV NS3 protease and its cleavage sequence. In the presence of the NS3 protease inhibitor asunaprevir, the autoproteolysis is inhibited and the full-length fusion protein is expressed and rapidly degraded (Figure 27). Withdrawal of asunaprevir allows the protease to cleave the SMASh tag from the POI, resulting in the stable expression of unmodified protein. Although yet relying on ectopic expression of the target proteins, SMASh tag provides a clear advantage over other DDs. The utility of the SMASh tag has been demonstrated in both mammalian cells and yeast, against a

6.2. LID System

The Wandless lab has been able to reverse the activity of the Shield system by engineering a cryptic short-peptide degron sequence onto the C-terminal of FKBP. Careful selection of the peptide sequence resulted in a 19-residue amino acid sequence that folds back into the rapamycin-binding site, resulting in stabilization of the FKBP domain and masking of the degron, resulting in stable fusion proteins.189 Addition of Shield1 displaces the degron, thus unmasking it and resulting in efficient degradation of the so-called “ligand-induced degradation” (LID) fusion protein (Figure 26). Fusion of just the 19-

Figure 26. Schematic representation of the LID system.

residue peptide to YFP results in efficient degradation in the presence or absence of Shield1, validating it as a degron. LID fusion proteins have been generated for a range of transcription factors and β-actin to demonstrate the generalization of this technique. Because the same small-molecule ligand is used for the LID and Shield systems, an effective protein swap can be induced in cells expressing both a repressive fusion protein (destabilization domain) and a LID fusion protein upon Shield1 treatment. Bonger et al. went on to employ a similar approach to develop a blue-light-inducible degradation (B-LID) domain employing a similar strategy requiring light rather than a small molecule to reveal the cryptic degron.190 6.3. Other Destabilizing Domains

The Wandless group has developed several additional destabilizing domains derived from a variety of ligand/receptor pairs. An E. coli dihydrofolate reductase (ecDHFR)/trimethoprim-based system proved to be advantageous over the Shield system as it offers a greater degree of destabilization and can therefore target more challenging substrates such as G-coupled protein receptors (GPCRs) (e.g., β2AR).191 It can be incorporated into either termini or even within the proteins sequence. Given the ability of trimethoprim to cross the

Figure 27. SMASh tag system. 11282


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range of proteins and functions as either N- or C-terminal fusions. Curiously, in contrast to other DDs, the SMASh tag appears to be degraded via a combination of proteasomal and lysosomal degradation.

degradation (>95%) in a dose-dependent manner. Proteasome inhibition can rescue protein levels, suggesting an “onmechanism” pathway for UPS-mediated degradation. In a different strategy, direct fusion of F-box proteins to a POI (e.g., β-catenin) can also result in the efficient ubiquitination and proteasomal degradation, albeit not under small-molecule control.204,205 Despite the fact that high concentrations (>20 μM) are required, mammalian cells appear to be relatively inert to indole-3-acetic acid (IAA) (Figure 28), the most commonly used auxin. The major advantage of AID appears to be the speed of protein degradationacute versus chronic knockdown could potentially reveal differential biology as cellular systems may develop compensatory systems during chronic genetic knockdown. However, as with many of the techniques described here, this approach requires genetic introduction of both the AID tag and the F-box protein. As with all ectopically expressed fusion proteins, another disadvantage of the application of AID to the study of the role of proteins in a cellular context is the requirement to deplete the levels of untagged protein. To this end, Kanemaki and co-workers have developed a method to insert the AID-compatible degron into the genomic copy of the gene of interest using CRISPR/ Cas9.206 The AID system has been employed to study a range of proteins207−209 and has been employed in avian and mammalian cells as well as in vivo in C. elegans210 and Drosophila melanogaster.211 Photocaging of IAA allows the spatial and temporal control of protein degradation in mammalian cells (Figure 28).212 An analogous system has recently been developed by the Lemischka group, employing the jasmonate-induced interaction between the coronatine insensitive 1 (COI1) F-box protein and proteins bearing the JAZ1 degron.213−215 The active molecule in plants is jasmonate-isoleucine (JA-Ile), but because mammalian cells lack the required conjugation enzymes to produce JA-Ile, coronatine must be used instead. Brosh et al. were able to demonstrate orthogonal control of two proteins using both the auxin system and the jasmonate system in the same cells. There may be scope to expand the scope of the plant F-box transplant techniques to include other hormones such as abscisic acid.216

7. AUXIN-INDUCED DEGRADATION The auxin-induced degradation system is found naturally in plants. It relies on the auxin family of hormones to induce protein−protein interactions between an F-Box protein and the substrate proteins,201 with auxins acting as “molecular glue” between the two proteins, in an analogous fashion to ImiD’s. In the absence of auxins, no degradation is observed, but addition of an auxin results in rapid degradation of proteins containing auxin-induced degradation (AID) degron sequences.202 Nishimura et al. have demonstrated that transplantation of the TIR1 F-box protein into nonplant cells (yeast and mammalian) can induce the degradation of AID degron-tagged proteins under auxin control.203 The plant F-box is still able to form part of the SCF complex (Figure 28 and discussed earlier) in both yeast and human cells, resulting in rapid (∼30 min) and efficient

8. HYDROPHOBIC TAGGING The major driving force for protein folding is the internalization of hydrophobicity.217 In the event of a protein only partially achieving a folded state, chaperones (e.g., heat-shock proteins) are often recruited to attempt to refold the protein. If that protein fails to fold even with the chaperone, the protein is cleared from the cell to prevent excessive aggregation caused by terminally misfolded proteins. Our group and others thought that this process could be mimicked chemically. By introducing an external hydrophobic tag to the surface of a protein, the fold of the protein could be destabilized, resulting in recognition of the partially folded protein and subsequent degradation.218 8.1. Selective Estrogen Receptor Degraders

Fulvestrant (ICI-182,780) and its predecessor ICI-164,384 (Figure 29) were initially developed as pure antiestrogen compounds for the treatment of breast cancer.219−221 These ICI compounds, derived from the endogenous 17β-estradiol ligand with a long alkyl chain, proved to be very potent inhibitors and to be advantageous over tamoxifen. Mechanistic studies revealed that ICI-164,384 destabilizes the estrogen

Figure 28. Auxin-induced degradation system. (A) Schematic representation of the auxin-induced degradation system. (B) Structures of commonly used auxin indole-3-acetic acid (IAA) and photoactivatable variant. 11283


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Figure 29. Structures of the steroidal SERDs.

receptor, resulting in a significant decrease in protein levels in cells resulting in the title selective estrogen receptor degraders (SERDs).222 Structural analysis of the estrogen receptor/ICI 164,384 complex revealed that a large hydrophobic patch had become exposed on the surface of the protein, likely resulting in the unfolded protein response.223 It was subsequently shown that the ICI-182,780-induced degradation occurs via ubiquitination and the proteasome.224 However, the efficacy of ICI182,780 cannot be entirely attributed to the induced degradation, as Wardell et al. have suggested that the conformational change induced by compound binding can be decoupled from degradation and may be the most important pharmacological characteristic.225 The recent identification of a sulfonamide series of compounds that exhibit full antagonism but limited degradation supports this theory.226 Fulvestrant (ICI-182,780) is currently used in the clinic after successfully progressing through clinical trials.227 Unfortunately, ICI182,780 has very poor pharmacological properties as it has low oral bioavailability and is rapidly metabolized, such that it currently is administered via intramuscular injection.228 Attempts have been made to produce orally bioavailable SERDs via the modification of ICI-182,780.229−231 Several nonsteroidal SERDs have also been reported and developed including the tamoxifen analogue GW-7604,232 which was subsequently optimized to give ARN-810233 and AZD9496 (Figure 30).234 GW-7604, ARN-810, and AZD9496 all feature a common cinnamic acid side chain that appears to be crucial for activity.232,235 Another nonsteroidal SERD, RAD1901, which is capable of crossing the blood−brain barrier, has been identified. These nonsteroidal SERDs are all orally bioavailable and have entered into clinical trials. Additional preclinical molecules have been reported as SERDs, including other tamoxifen derivatives236 and diphenylheptane-derived molecules.237 The SERDs demonstrate the therapeutic applicability of protein-degradation techniques once again and suggest that hydrophobic tagging may be a generally applicable technique because appending hydrophobic moieties established degraders across a range of different chemical scaffolds, albeit all for the same protein target. Additional work is required to expand the hydrophobic tagging approach to other targets and on the optimization of physicochemical properties without the loss of degradation induction.

Figure 30. Structures of the nonsteroidal SERDs.

Figure 31. Structure of the SARD AZD3514.

inhibitor that also reduces levels of cellular AR.239 The mechanism via which the AR protein levels are decreased has not been fully elucidated, but it appears to have multiple facets and may not be acting via hydrophobic tagging or protein destabilization240 as SERDs do. AZD3514 entered phase 1 clinical trials but has not progressed further due to poor pharmacokinetics (PK) and adverse effects.241 The Phase 1 trial, however, did suggest that the development of a SARD that works through a defined and well-tolerated mechanism may be a viable therapeutic strategy. Galeterone has also been shown to induce degradation of the androgen receptor.242 8.3. HaloTag-Based Systems

Work in the Crews laboratory employed HaloTag fusion proteins to validate the hydrophobic tagging approach for protein degradation (Figure 32). HaloTag is an engineered bacterial dehalogenase that forms a covalent adduct with chloroalkanes.124 By expressing a HaloTag-2 luciferase reporter fusion in HEK293 cells, Neklesa et al. were able to screen a range of hydrophobically functionalized chloroalkanes for their ability to induce degradation of the fusion protein. Their results identified a potent degrader: the adamantyl-functionalized choloroalkane HyT13 (Figure 32) .243 HyT13 can induce the degradation of HaloTag fusions in a dose-, time-, and

8.2. Selective Androgen Receptor Degraders

On the basis of the success of SERDs, attempts to develop selective androgen receptor degraders (SARDs) have been made.238 The first SARD to be developed was AZD3514 (Figure 31), an orally available androgen receptor (AR) 11284


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Figure 32. HaloTag system. (A) Schematic representation of the HaloTag hydrophobic tagging strategy. (B) Structures of HyT/HALTS system ligands.

proteasome-dependent manner and is nontoxic both in cell culture and in vivo. The HaloTag hydrophobic tagging approach can induce efficient degradation of multiple proteins in the cytosol and within the plasma membranes including 7pass GPCRs. It has also proven to be effective in zebrafish and mice. Further investigations led to the development of a moreeffective hydrophobic tagging ligand named HyT36 (Figure 32) that is able to degrade the more-stable HaloTag-7-based fusion proteins.244 The hydrophobic tagging approach has been used to induce and study the unfolded protein response (UPR) in the endoplasmic reticulum by providing a milder pharmacological tool than the previously employed toxic natural products.245 In contrast to the destabilization domains discussed above, the HaloTag-7 fusion proteins have no inherent effect on protein stability, functioning only when treated with a small-molecule stimulus. While the HaloTag hydrophobic tagging approachlike many of the systems included hererequires genetic labeling of the POI, it requires the introduction of only a single fusion protein. HyT36 provides an extra chemical tool to study the many commercially available HaloTag fusion proteins as well as enabling the use of other commercially available chloroalkane probes with one fusion protein. Interestingly, HaloTag-2 (the earlier version of the HaloTag) has a slightly destabilizing effect on proteins, resulting in lower expression levels of GFP fusion proteins than unmodified GFP, for example. Neklesa et al. were able to identify a molecule that can stabilize HaloTag-2 in an analogous fashion to the previously discussed destabilization domains.246 The HaloTag-2 stabilizer (HALTS1, Figure 32) binds to the core of HaloTag-2 and greatly increases the thermal stability, resulting in reduced ubiquitin-mediated degradation (Figure 33) and an increase in fusion protein expression level (relative to nonHALTS1 treated levels). A combination of HyT/HALTS molecules provides a bidirectional system for the stabilization or degradation of the same fusion protein, allowing the study of up- or downregulation in the same cellular system.

Figure 33. Crystal structure of HALT1 bound to HaloTag2 (PDB ID: 4KAC).

8.4. Adamantyl Small Molecules

Hydrophobic tags similar in structure to HyT13 and HyT36 can also be covalently attached to small-molecule ligands, rather than to chloroalkanes, to successfully induce degradation of a selected POI. Identification of a novel ligand for the pseudokinase Her3 followed by its conjugation to an adamantane247 resulted in small molecules (e.g., TX2-121-1, Figure 34) capable of inducing degradation of this receptor tyrosine kinase.248 Preliminary mechanistic investigations suggest that Hsp70, Hsp90, and the proteasome are all involved in the degradation pathway. Additionally in this case the bulky adamantyl group enhanced the effectiveness of the compound by inhibiting Her3 dimerization with other HER family members. In an analogous fashion to the SERDs (described earlier), Gustafson et al. revisited the notion of the selective androgen receptor degrader (SARD) by conjugating an adamantyl group to the androgen receptor agonist RU59063.249 The resultant compound, SARD279 (Figure 34), was able to efficiently degrade endogenous androgen receptor in prostate cancer cells in a dose- and proteasome-dependent manner.250 SARD279 proved to be advantageous over the parent inhibitor, as well as the recently FDA-approved enzalutamide, in cell-proliferation assaysparticularly in the presence of a competing androgen 11285


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Figure 35. Structures of (Boc)3Arg and an example (Boc)3Arg ligand.

8.6. Other Hydrophobic Small Molecules

A small-molecule inhibitor of the ANO1 ion channel has been shown to also induce the degradation of its target protein. CaCCinh-A01 (Figure 36) appears to do so by proteasomal

Figure 36. Structures of other hydrophobic tagging small molecules.

Figure 34. Structures of adamantyl-tagged small molecules.

degradation of ANO1 after translocation of the ion channel to the endoplasmic reticulum via retrograde trafficking.257 Gaither and co-workers postulated that the induced degradation may arise from the hydrophobic surface of the molecule. Small hydrophobic isothiocyanates, particularly benzyl isothiocyanate (BITC) (Figure 36), have been shown to induce the degradation of α- and β-tubulin in a relatively selective fashion as identified by proteomic analysis.258 The isothiocyanates form covalent adducts with the cysteines on the surface of the proteins, which caused aggregation followed by their ubiquitination and proteasomal degradation. Because lesshydrophobic isothiocyanates have a lesser effect on tubulin degradation, the recruitment of chaperones and subsequent Ubmediated degradation has been invoked to rationalize these observations.

receptor agonist. SARD279 was even able to inhibit cell proliferation in an enzalutamide-resistant prostate cancer cell line. A family of small molecules bearing the adamantyl motif has also been shown to prevent the accumulation of HIF-1α under hypoxic conditions in a post-translational manner.251 Under hypoxic conditions, HIF-1α should accumulate because the oxygen-dependent process resulting in its degradation is halted.252 In the presence of IDF-11774 (Figure 34), HIF-1α is still degraded even under hypoxic conditions; yet in the presence of an adamantyl-lacking IDF-11774 analogue, HIF-1α accumulates under hypoxic conditions. The mechanism of action is still unclear, but a recent photo-cross-linking experiment identified the molecular target of IDF-11774 to be the chaperone protein, HSP-70.253

9. SPLIT UBIQUITIN SYSTEMS The split ubiquitin system was initially developed as a proximity sensor for protein−protein interactions.259 By exploiting the fact that the ubiquitin domain is rapidly cleaved from ubiquitinfusion proteins by ubiquitin-specific proteases (USPs),260 Johnsson and Varshavsky were able to develop a split ubiquitin domain that resulted in the cleavage of a reporter when reconstituted by a protein−protein interaction.259 By splitting ubiquitin into two subdomains and introducing a point mutation, the components could only recapitulate the ubiquitin fold required for USP activity upon their reassembly concurrent with dimerization of their respective fused domains.

8.5. Boc3 Arg

The attachment of triply Boc (t-butoxycarbamate)-protected arginine (Boc3Arg, Figure 35) to a ligand appears to be a general approach for hydrophobic tagging. Hedstrom and coworkers have reported on the ligand-mediated degradation of GST-1 and DHFR proteins via Boc3Arg conjugation.254 Interestingly, the Boc3Arg approach to hydrophobic tagging appears to result in degradation in an ubiquitin-independent fashion and not from destabilization of the protein (as confirmed by HSQC NMR). It appears that Boc3Arg activates and localizes tagged complexes to the 20S proteasome.255 Notably, Boc3Arg binds to ubiquilins 1, 2, and 4, which contain both ubiquitin-like and ubiquitin association domains and are known themselves to associate with the proteasome.256

9.1. Split Ubiquitin for the Rescue of Function

The Muir group developed the split ubiquitin for the rescue of function (SURF) approach by combining split ubiquitin with 11286


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the FRB destabilization domains described above.261 By creating two fusion proteins (Figure 37)one containing the

and Pratt were able to access protein levels over a 130-fold range by dual small-molecule control.263

10. FOLDING MODULATORS The adoption of the correct tertiary structure is crucial for protein homeostasis, and several techniques have been developed to control this process via small molecules. In previous sections we discussed the use of engineered proteins to provide a general approach; here we describe techniques that have proven successful with endogenous proteins. 10.1. Pharmacological Chaperones

Chaperones aid in the folding of proteins within the secretory system and allow proteins to progress, often via the endoplasmic reticulum, to their subcellular locale.264 Proteins that are incorrectly folded may be retained in the ER and subsequently degraded. Under normal conditions, the disposal of incorrectly folded proteins is advantageous; however, some diseases result from protein misfolding. Several groups have developed small-molecule pharmacological chaperones that aid in the folding of specific proteins and can even induce the correct folding of mutated proteins, thus restoring normal phenotypes. It is important to note the distinction between chemical chaperones and pharmacological chaperones; chemical chaperones (e.g., glycerol and trimethylamine-N-oxide) are general folding aides while pharmacological chaperones are specific for individual proteins.265 Early work on the development of pharmacological chaperones employed P-glycoprotein mutants as a model system.266 Two misfolded mutants, which do not reach the mature glycosylated form, can be restored to full processing by the secretory system upon treatment with the known Pglycoprotein binders capsaicin, cyclosporin, verapamil, and vinblastine in a dose-dependent fashion. The rescued mutants are then capable of migrating to the cell surface and functioning as wild type. The identification of pharmacological chaperones led to the extension of this technology to the mutated protein responsible for cystic fibrosis. The cystic fibrosis transmembrane conductance regulator (CFTR) modulates the hydration state of the mucus layer in airway epithelial cells.267 Cystic fibrosis presents when this protein is unable to function adequately, often due to deletion of phenylalanine 508 (ΔF508).268 This mutation results in misfolding and retention in the ER. Lowtemperature culture of cells expressing ΔF508 CFTR269 or treatment with chemical chaperones results in the rescue of the mature cell surface phenotype, suggesting it may be a suitable target for pharmacological chaperones. A high-throughput screen resulted in the identification of CFcor-325 (Figure 38), a compound capable of restoring function and processivity to ΔF508 CFTR.270−272 Additional screening identified further examples of compounds capable of restoration of the functional protein, such as Corr-4a;273 however, many of the early pharmacological chaperones also identified restored function of P-glycoprotein mutants, bringing into question their role as specific pharmacological chaperones.274 VRT-532 was identified as specific for CFTR and shown to directly interact with ΔF508 CFTR, validating it as a true pharmacological chaperone.275 Several other pharmacological chaperones for C FT R h av e b e e n r e p o rt e d i n c l u d i n g be n z o ( c) quinoliziniums,276,277 RDR1,278 genistein,279 and sildenafil (Viagra).280

Figure 37. Split ubiquitin for rescue of function system.

POI, the C-terminal fragment of ubiquitin, and the FRB degron, and the other containing the N-terminal fragment of ubiquitin and FKBPa small-molecule-dependent stabilization and release of the POI is created. In the absence of rapamycin, the FRB degron-tagged fusion protein is degraded; however, upon the rapamycin-induced dimerization of FRB and FKBP, the destabilization domain is stabilized, resulting in the stabilized N-terminal portion of ubiquitin coming into close proximity with its C-terminal counterpart on the other fusion protein. The resultant reconstitution of the ubiquitin fold results in the ubiquitin cleavage by endogenous proteases, thereby releasing the POI from the degron and ubiquitin fusion proteins. The SURF system is dose- and time-dependent and is advantageous in that it has the ability to release untagged proteins into the cell. However, SURF requires genetic modification of the POI, is relatively slow, and only has one directional control. Withdrawal of the ligand prevents the accumulation of additional protein but does not actively degrade the POI already extruded from the fusion proteins. 9.2. Traceless Shielding

Pratt and co-workers have built upon the SURF system to develop a technique they termed traceless shielding (TShld).262 By employing the Shield system destabilization domain (discussed earlier) in place of the FRB destabilization domain used in SURF and essentially flipping the dimerization domains required for reconstitution of the split ubiquitin, they were able to develop a system with the same advantages as SURF but enhanced ease of use and an improved range of protein abundance. Furthermore, by putting the TShld under the control of the tetracycline responsive promoter (Tet-On), Lin 11287


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Figure 39. Structures of pharmacological chaperones for galactosidase enzymes.

in free enzyme, which is able to remain folded and function.288 DGJ was recently approved for the treatment of Fabry disease under the name Migalastat (Galafold). Similarly, inhibitors of β-galactosidase such as N-nonyldeoxynojirimycin (NN-DNJ) were identified as pharmacological chaperones for treatment of Gauchers disease.289 These compounds function via the stabilization of the folded state in the ER allowing the protein to evade endoplasmic-reticulumassociated degradation (ERAD). A shorter-chain analogue of NN-DNJ, named Miglustat, has been approved for use in Gauchers disease and appears to operate as both an inhibitor of glucosyltransferase and a pharmacological chaperone for βgalactosidase.290 Isofagomine also functions as a pharmacological chaperone for β-galactosidase,291 and a cocrystal structure has been solved, confirming the direct interaction and stabilization of protein tertiary structure.292 Subsequent optimization of isofagomine has led to the discovery of analogues that exhibit a 7.2-fold increase of β-galactosidase levels compared to 2.3-fold observed with NN-DNJ.293 Another target where pharmacological chaperones have proven successful is the GPCR V2 vasopressin receptor (V2R).294 Patients with mutations in V2R suffer from nephrogenic diabetes insipidus; >90% of these mutations result in retention of the protein in the endoplasmic reticulum. Treatment of cells expressing mutant V2R with small-molecule antagonists, such as SR121463A,295 induces release from the ER and subsequent maturation, presumably by stabilization of the correctly folded conformation. These pharmacological chaperones effectively restore function to mutant receptors, which are otherwise unable to reach the cell surface. The pharmacological chaperone paradigm has also been leveraged against a wide range of other targets including opioid receptors,296 hERG,297 α-crystallin,298 β-N-acetyl hexosaminidase,299 and retromer,300 among others.301,302 Pharmacological chaperones have proven to be useful tools, and in some cases therapeutics, for protein-misfolding diseases; however, their paradoxical nature and requirement for ERlocalized mutants prevents them from being generally applicable in the use of small molecules to control proteostasis.

Figure 38. Structures of pharmacological chaperones for ΔF508 CFTR.

A high-throughput campaign followed by extensive medicinal chemistry efforts led to the identification of VX-809 (Figure 38)281 as a bona fide pharmacological chaperone for ΔF508 CFTR.282 VX-809 was approved by the FDA in 2015 as part a combination therapy for cystic fibrosis.283,284 This approval validates the use of pharmacological chaperones for the smallmolecule control of homeostasis and their use as therapeutics. Another significant target for the pharmacological chaperones are the galactosidase enzymes associated with Fabry disease and Gauchers disease, α-galactosidase A and βgalactosidase, respectively. These, and other, lysosomal storage disorders arise from the necessity for enzymes to fold at nearneutral pH in the endoplasmic reticulum (ER) and Golgi but then function enzymatically at low pH in lysosomes. Mutations in both of these proteins result in accumulation in the ER, leading to ER stress and an accumulation of their respective substrates in the lysosome. Pioneering work from Fan et al. showed that treatment of Fabry disease patients cells with an α-galactosidase A (α-Gal A) inhibitor (1-deoxygalactonojirimycin, DGJ) resulted in an increase of cellular enzymatic activity (Figure 39).285 Paradoxically, DGJ acts an inhibitor in vitro but an enhancer in cellulo and in vivo, at least at moderate concentrations.286 Careful studies to elucidate the mechanism underlying this paradox revealed that DGJ increased the levels of α-Gal A by facilitating its localization from the ER/Golgi to lysosomes. Crucially, the pH differential between the ER and the lysosome is responsible for the paradoxical activity; in the ER DGJ binds to α-Gal A, stabilizes the folded state, and allows it to traffic to the lysosomes.287 In the acidic lysosomes the nitrogen becomes protonated, reducing the affinity for α-Gal A 80-fold, resulting 11288


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10.2. Other Folding Modulators

conceivably be applied, to the control of intracellular protein levels via small-molecule modulation.

Distinct from pharmacological chaperones are small molecules that can stabilize endogenous proteins, without having an impact on their trafficking, in a similar fashion of the Shield or HALTs systems described earlier. The Kelly laboratory has pioneered this technique to stabilize the transthyretin tetramer. Transthyretin (TTR) is a protein present in human plasma that is important in the transport of thyroxine. However, TTR is implicated in amyloidosis in familial amyloid polyneuropathy (FAP). Under normal conditions TTR exists as a stable tetramer but can rearrange and dissociate to amyloidogenic monomers under acidic conditions or in certain mutational states.303 These monomers self-assemble to form amyloid fibrils, resulting in FAP. The native substrate, thyroxine, is able to stabilize the native tetramer, preventing amyloid formation, suggesting that small-molecule stabilization of the tetramer could prove to be a useful therapeutic strategy. Employing pharmacophore models, screening,304 and rational structure-based design,305 Kelly and co-workers have developed a wide range of compounds capable of stabilizing the tetrameric species, resulting in reduced amyloid deposition.306 Initial studies focused on analogues of the nonsteroidal anti-inflammatory drug diclofenac,307 but several other chemotypes have been identified including bisaryloxime ethers308 and dibenzofurans.309 The most successful stabilizing ligands for TTR are derived from benzoxazoles,310,311 which have been shown to function in vivo and eventually led to the development and approval of Tafamidis (Vyndaqel) as a treatment for FAP (Figure 40).312 This type of protein-

11.1. Direct Recruitment to the Proteasome

Chemical-induced dimerization has been employed to recruit proteins directly to the proteasome, which results in ubiquitinindependent protein degradation.313 Employing the rapamycin dimerization system314 in yeast, Janse et al. were able to show that colocalization with the proteasome is sufficient for degradation; however, additional studies have suggested that an unstructured region is also required to allow the initiation of proteasome subunit-mediated substrate unfolding.315,316 Proteasome adaptor proteins containing ubiquitin-like domains (UBLs) and ubiquitin-associated domains (UBDs) can bridge the gap between the proteasome and ubiquitinated proteins, respectively.317,318 Small-molecule-controlled artificial proteasome adaptor proteins in mammalian cells have been reported by the Matouschek group.319 By adapting the FRB/rapamycin/ FKBP314 destabilizing domain system, a UBL-FRB-fusion protein can mediate recruitment of an FKBP-fusion protein to the proteasome, resulting in the ubiquitin-independent degradation of the FKBP-fusion (providing it has a proteasomeinitiation site) but not the artificial adapter protein. The GID1/ Gibberellin/GAI320 dimerization system can also be employed but requires much higher concentrations of ligand. A similar approach can be employed in E. coli, employing rapamycincontrolled recruitment of substrates to the ClpXP protease via the SspB adaptor.321,322 11.2. Chemical Rescue of Structure

The chemical rescue of structure technique, developed by Karanicolas and co-workers, exploits the structural cavity resulting from a mutation from tryptophan to glycine.323 Employing a split repressor assay in E. coli, Xia et al. were able to identify protein homodimers that lost function upon single W-to-G mutations (Figure 41). Addition of very high

Figure 40. Structures of some TTR tetramer stabilizers.

stabilization effect may be applicable to a subset of other proteins with defined structures that can be stabilized to prevent aggregate formation, although identification of both proteins of this type and small-molecule stabilizers may prove to be challenging.

Figure 41. Chemical rescue of structure system.

concentrations of indole (1 mM) could partially restore function in a subset of these mutants by replacing the absent indole side chain of tryptophan, thus allowing the smallmolecule control of EGFP expression. Interestingly, the same system can be applied to a GFP mutant to impart smallmolecule control of both its fluorescent properties and

11. OTHER SYSTEMS The previous sections dealt with systems deliberately designed and validated for the small-molecule control of intracellular protein levels. In this section, we aim to highlight some recently developed technologies that have been applied, or could 11289


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Genetic modulation of the sequence of a target protein to engineer in the TEV cleavage sequence allows site-specific cleavage upon treatment with rapamycin (Figure 43).330 Wells

proteolytic stability in vitro, hinting at future applications for cellular control of protein levels. 11.3. Folding Intermediate Selective Inhibitors

Some kinases have been shown to have crucial folding intermediates with a distinct kinase activity.324 For example, GSK3β, a trans-serine/threonine kinase, autoactivates by phosphorylation at a tyrosine residue during its chaperonemediated folding.325 Both GSK3β and DYRK2 have been shown to have folding intermediates with differential inhibition profiles. DYRK2 intramolecular tyrosine kinase activity is not inhibited by tetrabromobenzotriazole while its serine/threonine kinase activity is, suggesting differential ATP binding sites between the two folding states.326 The Hagiwara group has developed substrate phosphorylation by sequential induction of kinase and substrate (SPHINKS) assays, which employ the tet operator and the Shield system to screen for selective inhibitors of transitional folding intermediates.327 Using DYRK1A as a model system, they were able to identify a folding intermediateselective inhibitor of DYRK1A (FINDY, Figure 42). FINDY

Figure 43. Rapamycin-mediated split TEV protease.

and co-workers employed this approach to control the intracellular levels of activated caspases. Subcellular protein localization has also been controlled by small-molecule control of split TEV.331 Incorporation of TEV sites into other proteins in the future may enable small-molecule control of protein levels by cleavage of degrons/destabilization domains or by introducing critical cleavage sites. Such a system has already been demonstrated in yeast.332 11.5. Peptide-Directed Lysosomal Degradation

Peptides composed of a chaperone-mediated autophagy targeting motif (CTM),333 a protein binding domain, and a cell-penetrating peptide (CPP) sequence are able to induce protein degradation via the lysosomal proteolytic machinery.334 The expression level of a CTM-tagged GFP protein is significantly lower than that for a wild-type GFP or an inactive CTM mutant-tagged GFP protein but can be elevated to wildtype level upon treatment with ammonium chloride (an inhibitor of lysosomal degradation) or pepstatin A (an inhibitor of the lysosomal proteases cathepsin D and E). Inhibition of the proteasome by MG132 had no effect on expression levels, nor did inhibition of macro-autophagy with 3-methyladenine. Taken together, these experiments provide strong evidence that the degradation occurs via the lysosome. Having demonstrated the efficacy of direct tagging, Fan et al. incorporated a protein-binding domain, a CTM, and a CPP sequence into synthetic peptides and were able to show doseand time-dependent downregulation of POI levels, which could be rescued by lysosomal degradation inhibitors. This approach has been utilized to significantly reduce levels of α-synuclein, PSD-95, and DAPK1 in cell culture and DAPK1 in vivo. The majority of the techniques discussed in this article relied on the UPS for control of protein levels, but this last approach highlights the opportunity to exploit other proteolysis pathways. Although an exciting proof of principle, the poor pharmacological properties of peptides means this approach is unlikely to be developed into a therapeutic approach unless a small-molecule CTM can be developed. The successful development of PROTACs from peptidic to small molecules suggests there may be scope for small-molecule-directed lysosomal degradation.

Figure 42. Structure of FINDY and schematic of folding intermediate selective inhibitors.

has no inhibitory activity against the final folded state of DYRK1A but is able to bind and stabilize an intermediate state. This stalling of the folding process results in a dose-dependent decrease in DYRK1A protein levels in HEK293 cells and primary neurons treated with FINDY. The protein-degradation effect was found to be selective for DYKR1A over DYKR1B and DYRK2, as well as a wide range of other kinases, and was able to restore the wild-type phenotype of zebrafish embryos that had been induced by overexpression of DYRK1A. While this technology is in its infancy, it may prove to be a very powerful technique to selectively control protein levels with traditional small molecules but without the requirement for genetic manipulation. However, as it relies on differential activity between folding intermediates and terminal state, it is unlikely to be amenable to every protein. 11.4. Split Tobacco Etch Virus Protease

The tobacco etch virus (TEV) protease is a remarkably selective protease with no cleavage sites in the human proteome.328 It can be split into two fragments that, when brought into close proximity, reconstitute its proteolytic activity.329 This approach was originally used as a reporter to study protein−protein interactions and can be activated by the rapamycin-induced interaction between FKBP and FRB.

12. CONCLUSIONS Described in the Review are a wide range of different techniques for the control of protein concentration. The choice of system can be challenging. Summarized in Table 1 are 11290


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Table 1. Some of the Major Protein Level Control Systems requires genetic modification

in vivo activity demonstrated

time to full effect

FDA approved

general applicability

no no no yes yes yes no

yes yes yes yes no yes no

In Vitro And < Em > In Vivo. Mol. Cancer Ther. 2013, 12 (9), 1715. (241) Omlin, A.; Jones, R. J.; Van Der Noll, R.; Satoh, T.; Niwakawa, M.; Smith, S. A.; Graham, J.; Ong, M.; Finkelman, R. D.; Schellens, J. H. M.; et al. AZD3514, An Oral Selective Androgen Receptor DownRegulator In Patients With Castration-Resistant Prostate Cancer − Results Of Two Parallel First-In-Human Phase I Studies. Invest. New Drugs 2015, 33 (3), 679. (242) Yu, Z.; Cai, C.; Gao, S.; Simon, N. I.; Shen, H. C.; Balk, S. P. Galeterone Prevents Androgen Receptor Binding To Chromatin And Enhances Degradation Of Mutant Androgen Receptor. Clin. Cancer Res. 2014, 20 (15), 4075. (243) Neklesa, T. K.; Tae, H. S.; Schneekloth, A. R.; Stulberg, M. J.; Corson, T. W.; Sundberg, T. B.; Raina, K.; Holley, S. A.; Crews, C. M.

Small-Molecule Hydrophobic Tagging−Induced Degradation Of Halotag Fusion Proteins. Nat. Chem. Biol. 2011, 7 (8), 538. (244) Tae, H. S.; Sundberg, T. B.; Neklesa, T. K.; Noblin, D. J.; Gustafson, J. L.; Roth, A. G.; Raina, K.; Crews, C. M. Identification Of Hydrophobic Tags For The Degradation Of Stabilized Proteins. ChemBioChem 2012, 13 (4), 538. (245) Raina, K.; Noblin, D. J.; Serebrenik, Y. V.; Adams, A.; Zhao, C.; Crews, C. M. Targeted Protein Destabilization Reveals An EstrogenMediated ER Stress Response. Nat. Chem. Biol. 2014, 10 (11), 957. (246) Neklesa, T. K.; Noblin, D. J.; Kuzin, A.; Lew, S.; Seetharaman, J.; Acton, T. B.; Kornhaber, G.; Xiao, R.; Montelione, G. T.; Tong, L.; et al. A Bidirectional System For The Dynamic Small Molecule Control Of Intracellular Fusion Proteins. ACS Chem. Biol. 2013, 8 (10), 2293. (247) Lim, S. M.; Xie, T.; Westover, K. D.; Ficarro, S. B.; Tae, H. S.; Gurbani, D.; Sim, T.; Marto, J. A.; Jänne, P. A.; Crews, C. M.; et al. Development Of Small Molecules Targeting The Pseudokinase Her3. Bioorg. Med. Chem. Lett. 2015, 25 (16), 3382. (248) Xie, T.; Lim, S. M.; Westover, K. D.; Dodge, M. E.; Ercan, D.; Ficarro, S. B.; Udayakumar, D.; Gurbani, D.; Tae, H. S.; Riddle, S. M.; et al. Pharmacological Targeting Of The Pseudokinase Her3. Nat. Chem. Biol. 2014, 10 (12), 1006. (249) Teutsch, G.; Goubet, F.; Battmann, T.; Bonfils, A.; Bouchoux, F.; Cerede, E.; Gofflo, D.; Gaillard-Kelly, M.; Philibert, D. NonSteroidal Antiandrogens: Synthesis And Biological Profile Of HighAffinity Ligands For The Androgen Receptor. J. Steroid Biochem. Mol. Biol. 1994, 48 (1), 111. (250) Gustafson, J. L.; Neklesa, T. K.; Cox, C. S.; Roth, A. G.; Buckley, D. L.; Tae, H. S.; Sundberg, T. B.; Stagg, D. B.; Hines, J.; Mcdonnell, D. P.; et al. Small-Molecule-Mediated Degradation Of The Androgen Receptor Through Hydrophobic Tagging. Angew. Chem., Int. Ed. 2015, 54 (33), 9659. (251) Lee, K.; Lee, J. H.; Boovanahalli, S. K.; Jin, Y.; Lee, M.; Jin, X.; Kim, J. H.; Hong, Y.-S.; Lee, J. J. (Aryloxyacetylamino)Benzoic Acid Analogues: A New Class Of Hypoxia-Inducible Factor-1 Inhibitors. J. Med. Chem. 2007, 50 (7), 1675. (252) Wang, G. L.; Jiang, B. H.; Rue, E. A.; Semenza, G. L. HypoxiaInducible Factor 1 Is A Basic-Helix-Loop-Helix-PAS Heterodimer Regulated By Cellular O2 Tension. Proc. Natl. Acad. Sci. U. S. A. 1995, 92 (12), 5510. (253) Ban, H. S.; Naik, R.; Kim, H. M.; Kim, B.-K.; Lee, H.; Kim, I.; Ahn, H.; Jang, Y.; Jang, K.; Eo, Y.; et al. Identification Of Targets Of The HIF-1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes. Bioconjugate Chem. 2016, 27 (8), 1911. (254) Long, M. J. C.; Gollapalli, D. R.; Hedstrom, L. Inhibitor Mediated Protein Degradation. Chem. Biol. 2012, 19 (5), 629. (255) Shi, Y.; Long, M. J. C.; Rosenberg, M. M.; Li, S.; Kobjack, A.; Lessans, P.; Coffey, R. T.; Hedstrom, L. Boc3Arg-Linked Ligands Induce Degradation By Localizing Target Proteins To The 20S Proteasome. ACS Chem. Biol. 2016, 11 (12), 3328. (256) Coffey, R. T.; Shi, Y.; Long, M. J. C.; Marr, M. T.; Hedstrom, L. Ubiquilin-Mediated Small Molecule Inhibition Of Mammalian Target Of Rapamycin Complex 1 (Mtorc1) Signaling. J. Biol. Chem. 2016, 291 (10), 5221. (257) Bill, A.; Hall, M. L.; Borawski, J.; Hodgson, C.; Jenkins, J.; Piechon, P.; Popa, O.; Rothwell, C.; Tranter, P.; Tria, S.; et al. Small Molecule-Facilitated Degradation Of ANO1 Protein: A NEW TARGETING APPROACH FOR ANTICANCER THERAPEUTICS. J. Biol. Chem. 2014, 289 (16), 11029. (258) Mi, L.; Gan, N.; Cheema, A.; Dakshanamurthy, S.; Wang, X.; Yang, D. C. H.; Chung, F.-L. Cancer Preventive Isothiocyanates Induce Selective Degradation Of Cellular A- And B-Tubulins By Proteasomes. J. Biol. Chem. 2009, 284 (25), 17039. (259) Johnsson, N.; Varshavsky, A. Split Ubiquitin As A Sensor Of Protein Interactions In Vivo. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (22), 10340. (260) Baker, R. T.; Tobias, J. W.; Varshavsky, A. Ubiquitin-Specific Proteases Of Saccharomyces Cerevisiae. Cloning Of UBP2 And UBP3, 11298


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