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PROTACs: Induced Protein Degradation as a Therapeutic Strategy Philipp Ottis, and Craig M Crews ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01068 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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ACS Chemical Biology

ToC graphic 75x29mm (300 x 300 DPI)

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PROTACs – Induced Protein Degradation as a Therapeutic Strategy

Author List: Philipp Ottis Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

Craig M. Crews Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. Department of Chemistry, Yale University, New Haven, Connecticut, USA. Department of Pharmacology, Yale University, New Haven, Connecticut, USA.


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Abstract Until recently, the only ways to reduce

specific

protein Target

Ub Ub Ub

signaling were to either knock

Target

down the target by RNAi or to interfere with the signaling by inhibiting

an

receptor

within

enzyme the

or

Ub Ub Target

Proteasome

Ub

Ub

E3

E3

signal

transduction cascade. Herein, we review an emerging class of small molecule pharmacological agents, called PROTACs, that present a novel approach to specifically target proteins and their respective signaling pathways. These hetero-bifunctional molecules utilize the endogenous cellular quality control machinery by recruiting it to target proteins in order to induce their degradation.

Introduction For decades, drug development has aimed to inhibit aberrant and disease-promoting protein function by utilizing the pharmacological paradigm of occupancy-driven inhibition, based on small molecules occupying and blocking active or regulatory sites of enzymes or receptors. To establish and maintain a level of efficacious inhibition (IC90), high concentrations of drug are required, which can lead to off-target effects. Accordingly, drug development research has evolved to focus strongly on proteins with accessible, “druggable” active and regulatory sites. This definition of the “druggable” proteome, however, drastically limits potential drug targets: less than 20% of the approximately 20,300 known human proteins are currently considered as drug targets.1, 2 In an effort to expand upon this traditional paradigm of occupancy-driven protein inhibition, a new class of small molecules, called proteolysis-targeting chimeras (PROTACs), has emerged. Unlike traditional drugs, PROTACs aim to eliminate the aberrantly functioning protein rather than to inhibit it. PROTACs follow an event-driven, rather than an occupancy-driven, pharmacological paradigm, and act catalytically to ACS Paragon Plus Environment


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degrade super-stoichiometric amounts of the target protein. Earlier attempts to similarly downregulate target proteins using RNA interference-based approaches were hampered by high metabolic instability, off-target effects and low bioavailability of the oligonucleotide-based drugs and, therefore, failed to demonstrate a broader therapeutic applicability.3, 4 With better pharmaceutical properties, e.g., higher cell permeability and greater metabolic stability, than nucleic acids, PROTACs offer a significantly broader therapeutic applicability for protein knock-down than RNAi. This holds true, particularly, for all-small molecule PROTACs.

PROTACs The PROTAC Technology PROTACs are small molecules capable of recruiting the cellular proteostasis apparatus to degrade intracellular disease-causing proteins. These hetero-bifunctional molecules consist of 3 components: a target protein-binding moiety, a degradation machinery recruiting unit and a linker region that couples these two functionalities. Typically, the utilized degradation machinery is the ubiquitin–proteasome system (UPS) by the recruitment of an E3 ubiquitin ligase, followed by ubiquitylation of the target protein and its subsequent degradation by the proteasome (Figure 1). However, preliminary approaches to recruit heat-shock proteins in order to enter the chaperone-mediated autophagy system (CMA) have been reported as well. Here, we review the development of this potentially new class of drugs, from early peptide-based degraders to highly potent small-molecule PROTACs, which are now highly advanced and poised to enter the clinic as therapeutics.


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A)

PROTA

B) C

VHL

Ub

Cullin 2

Rbx

E2

Cullin 2

C)

D) VHL

Target Protein

Elongin B

Elongin C

VHL

Rbx

Elongin B

Elongin C

Target Protein

Target Protein

Ub

Rbx

Elongin B

Ub

Elongin C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


t ge Tar tein Pro

Ub

Ub Ub

Ub

Ub Ub

Proteasome

E2

Cullin 2

Figure 1 Scheme of PROTAC mechanism of action utilizing the E3 ubiquitin ligase VHL. A) and B) The PROTAC binds the target protein with its substrate binding functionality (grey square) and VHL with its E3-ligand (orange wedge), thereby bridging the E3 substrate-recognition subunit and the target protein. Upon target engagement, the functional E3-ligase complex consisting of VHL, Elongin B and C, together with Rbx, assembles on the Cullin 2 scaffold protein, and recruits an E2 ubiquitin-conjugating enzyme. C) The E2 transfers multiple ubiquitins (Ub; red) onto the presented target, creating a poly-ubiquitin chain D) The poly-ubiquitylated target is recognized as a substrate by the proteasome and is subsequently degraded. The PROTAC remains unmodified and, hence, can initiate a new targeted degradation event.

Peptide-based PROTACs The first PROTAC was described 15 years ago and consisted of the methionine aminopeptidase-2 (MetAP-2) binding small molecule ovalicin attached via an aminohexanoic acid linker to an IκBα-derived phospho-decapeptide recognition motif for the E3 ubiquitin ligase SCFβ-TRCP (Figure 2). Ubiquitylation and degradation of MetAP-2 by the covalently binding PROTAC was demonstrated in Xenopus egg extracts using ACS Paragon Plus Environment


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recombinant SCFβ-TRCP and substrate proteins.5 Shortly after, in vitro ubiquitylation of the estrogen receptor (ERα) by SCFβ-TRCP was successfully induced with an analogous estradiol-based phosphopeptide PROTAC.6 To address the lack of cell-permeability of the highly polar IκBα-phosphopeptide, Sakamoto and coworkers subsequently injected HEK293 cells with a dihydrotestosterone (DHT)-based phosphopeptide PROTAC and demonstrated the ability of PROTACs to degrade the androgen receptor (AR) in living mammalian cells.6

Figure 2 The first PROTAC, described by Sakamoto et al.5 in 2001. This hybrid PROTAC consisted of the MetAP-2 binding molecule ovalicin linked by an aminohexanoic acid linker and 6 glycine residues to the phospho-decapeptide recognition motif for SCFβ-TRCP. Asterisks indicate phosphorylated residues.

By replacing the IκBα-phosphopeptide component with the 5-8 amino acid HIF1αderived substrate motif of another E3 ubiquitin ligase, the Von Hippel-Lindau (VHL) protein,7-9 creating cell-permeable PROTACs became feasible. PROTAC-mediated degradation of proteins in living cells by mere addition of the PROTAC to the culture medium was demonstrated by Schneekloth and colleagues, wherein they targeted both a mutant FKBP12 protein and the androgen receptor using a poly-D-arginine tagged VHL substrate heptapeptide coupled to a mutant-specific analog of rapamycin and DHT, respectively.10 In parallel, Zang et al. reported the induced intracellular degradation of MetAP-2 and ERα utilizing the full HIF1α-octapeptide without any additional permeability-aiding sequences.11 Shortening of the VHL-recognition sequence to a pentapeptide subsequently allowed for degradation of ERα in human endothelial cells12 The incorporation of apigenin as a targeting moiety for the pentapeptide PROTACs led to inducible degradation of the aryl hydrocarbon receptor (AHR).13, 14 Beyond the role of the targeting ligand and the E3 enzyme-binding peptide, the importance of linkerposition in the design of PROTACs was illustrated by a study on ERα degraders.15


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Recent approaches have also highlighted the ability of all-peptide PROTACs to overcome a lack of target-binding small molecules, to recruit other proteins of the quality control machinery or to link target degradation to intracellular signaling. In an elegant approach, Hines and colleagues linked the HIF1α-peptide and poly-arginine tail to a phosphorylation substrate-targeting peptide to create a conditional “phosphoPROTAC”. By choosing the phosphorylation motifs of either TrkA or ErbB3 as the targeting peptides, the phosphoPROTACs would remain ‘silent’, solely bound to VHL, until either nerve growth factor (NGF) or neuregulin are recognized by the receptors TrkA, or ErbB2/ErbB3, respectively. Recognition of the growth factors results in auto- and transphosphorylation of the phosphorylation motifs of these receptors, and hence in the phosphorylation of the conditional phosphoPROTACs as well. Phosphorylation of the targeting peptides rendered them ligands for either FRS2α (TrkA-based PROTAC), or the

phosphatidylinositol-3-kinase

(ErbB3-based

PROTAC).

Both

described

phosphoPROTACs conditionally induced degradation of their respective targets, with the ErbB3-based PROTAC even being demonstrated to reduce tumor growth in mice – the first described PROTAC application in animals.16 In another study, using peptides to compensate for suitable small molecules, the X-protein of hepatitis B virus could be successfully degraded by fusion of the oligomerization domain to the VHL recognition peptide. A poly-D-arginine tail aided cell permeability.17 Also, by fusing a peptide derived from β-tubulin to the poly-D-Arg tagged HIF1α-heptapeptide, levels of the Alzheimer’s Disease (AD)-associated protein tau could be reduced in primary neurons and in a mouse model of AD.18 Similar to the peptide-based approaches described above, Henning and co-workers achieved the post-translational knock-down of the protein kinase Akt with a branched, trimeric protein catalyzed capture agent (PCC) attached to the HIF1α-sequence and a cell penetrating TAT-peptide.19 Finally, others have sought to recruit different components of the cellular quality control machinery to target proteins for degradation. Bauer and colleagues utilized the CMA pathway via a peptidic recognition motif to direct target proteins to the lysosome for degradation. One study targeted mutant huntingtin protein by combining two polyglutamine binding peptides with two recognition motives for the heat shock protein HSC70. In an expression-vector based delivery approach, mutant huntingtin could be



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reduced by lysosomal degradation and a murine Huntington’s Disease phenotype could be ameliorated.20 In a similar approach, but utilizing the TAT peptide delivery system, Fan et al. demonstrated the induced lysosomal degradation of PSD-95, and α-synuclein in HEK293 cells and cultured primary neurons, as well as in vivo ischemic stimulationdependent degradation of “death associated protein kinase 1” (DAPK1) in rat brains.21 Thereby, they comprehensibly illustrated similar modularity and generalizability of CMA peptide degraders as compared to UPS peptide PROTACs.

Small Molecule PROTACs With the discovery of small molecule ligands for E3 ubiquitin ligases (Figure 3), the E3recruiting peptide moiety in PROTACs could be substituted with a non-peptidic ligand to create more drug-like small molecule degraders, that possess physicochemical properties fully enabling their therapeutic potential. This new class of PROTACs now is capable of rapid and unaided diffusion into live cells, thereby displaying substantially lower toxicity, high affinity target engagement and increased metabolic stability, allowing for catalytic, super-stoichiometric target degradation. A number of E3 ligases have now been shown to be accessible for the small-molecule PROTAC technology:

MDM2 and cIAP1 Utilizing the development of nutlin, an inhibitor of the p53-degrading Mouse Double Minute 2 (MDM2) E3 ubiquitin ligase22, Schneekloth et al. presented the first all-small molecule PROTAC.23 This PROTAC was designed to degrade the androgen receptor (AR) and consisted of a non-steroidal AR ligand fused via a polyethylene-glycol (PEG) linker to nutlin. With degradation of AR in HeLa cells at concentrations ≥ 10 µM, the potency of this first generation small molecule degrader, however, resembled that of peptide PROTACs.23 Alternatively, the research groups of Yuichi Hashimoto and Mikihiko Naito incorporated the aminopeptidase inhibitor bestatin24 into their small molecule degraders. As part of a hetero-bifunctional molecule, this Actinomyces-derived natural compound can recruit the ACS Paragon Plus Environment

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cellular inhibitor of apoptosis protein 1 (cIAP1) E3 ubiquitin ligase to a target protein. Knock-down of the retinoic acid binding proteins CRABP I and II, the retinoic acid receptor (RAR), ERα, and AR at micromolar concentrations was described.25-28 Degradation of the spindle regulatory protein “transforming acetic coiled coil 3” (TACC3) with similarly-designed bestatin-based degraders, however, turned out to depend on the recruitment of the unrelated E3 ubiquitin ligase APC/CCDH-1.29 This observation and the moderate efficacies yielded with bestatin-based degraders can most likely be linked to the low binding specificity of bestatin for cIAP1, the destabilization and the high off-target effects of this parent compound.30

Figure 3 Small molecule ligands E3 ubiquitin ligases. Nutlin derivatives bind MDM2, while bestatin engages cIAP1. The IMiDs thalidomide and its derivatives pomalidomide and lenalidomide bind cereblon. On the lower right the ligand to the VHL E3 ligase is depicted.



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Cereblon Most recently, the immunomodulatory drug (IMiD) thalidomide, and its derivatives lenalidomide and pomalidomide, have been revealed to bind to the Cul4–Rbx1–DDB1– cereblon E3 ubiquitin ligase complex. In their binding to cereblon, these IMiDs modulate the specificity of the E3 ligase to induce degradation of the IKAROS family transcription factors IKZF1 and IKZF3, which forms the basis of their anti-cancer effect.31-34 Within the past two years, three reports have demonstrated successful incorporation of thalidomide or its derivatives into PROTACs. Two of these independent, yet simultaneously conducted, studies describe the targeting of the transcription factor BRD4, a member of the bromodomain and external domain (BET) family. The first study utilized the BRD4 inhibitor OTX015 fused to pomalidomide to create the PROTAC ARV-825 (Figure 4).35 This PROTAC induced about 50% BRD4 degradation observed after 2 h treatment of Burkitt’s lymphoma (BL) cells with 100 nM. In overnight treatment, ARV-825 yielded an astonishing DC50 of < 1 nM. Considering the affinity of ARV-825 for BRD4 (Kd ≥ 28 nM), this strongly suggests a super-stoichiometric degradation of BRD4 due to catalytic activity of this PROTAC.36 Strikingly sustainable effects of ARV-825 compared to BRD4-inhibition were demonstrated. Whereas PROTAC-mediated BRD4-degradation and the associated suppression of c-myc expression persisted for up to 24 h after washout, rapid recovery of c-myc levels following 24 h of inhibitor treatment and washout was observed. Moreover, inhibition of cell proliferation and induction of apoptosis in BL cells was shown to be more pronounced and sustained upon PROTAC treatment as compared to the inhibitors JQ1 and OTX015. Consistent with the employment of a pan-BET inhibitor for the development of ARV-825, this PROTAC was found to also degrade the other BET family members BRD2 and BRD3.35 The second approach targeting BRD4 by use of cereblon describes the creation of the PROTAC dBET1 by linking the BET inhibitor JQ1 to a thalidomide derivative (Figure 4).37 This degrader proved to be fast-acting with 95% BRD4 degradation achieved after 2 h at 100 nM in acute myeloid lymphoma (AML) cells. Partial recovery of BRD4 levels ACS Paragon Plus Environment

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was observed 4 – 24 h following start of treatment, possibly attributable to the low stability of thalidomide38; this instability likely also explains the observed half-maximal degradation (DC50) at 430 nM after 18 h treatment. Consistent with the utilization of the panBET inhibitor JQ1, quantitative proteomics analysis demonstrated near equal knockdown of BRD2, 3 and 4 by dBET1. In line with the PROTAC-mediated knockdown of BRD4, levels of the proliferation-driving transcription factor c-myc as well as the oncoprotein

PIM1

were

found

diminished.

Analyses

revealed

the

observed

downregulation of c-myc, PIM1 and BRD2 to occur at the transcriptional level following treatment with dBET1, similar to the effect of JQ1 alone. Finally, it was tested whether the observed dBET1-induced apoptosis in cultured AML cells would translate to an in vivo model. Therefore, mice with xenografted MV4;11 leukemia tumors were treated with the BRD4-degrader, and significant inhibition of tumor growth compared to vehicletreated control was detected. A generally broad application spectrum for cereblon-based degraders was highlighted by the additional demonstration of FKBP12 degradation upon exchange of the target ligand.37 Lai and coworkers described additional pomalidomide-based PROTACs, targeting the kinase c-Abl and its oncogenic fusion BCR-Abl for degradation.39 As this study directly compares cereblon- and VHL-recruiting PROTACs, it shall be discussed in the following section.



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Table 1 Overview of successfully degraded proteins along with respective PROTAC compositions. peptide PROTACs target

target ligand

α-synuclein AHR

a

AR

e

ERα ERα

DHT

estradiol

FKBP12 FRS2a

j

MetAP-2

f

MetAP-2

ovalicin

h

PI3K

PSD-95 k

tau

l

X-protein 13, 14

19

6

β-tubulin peptide oligomerization peptide

VHL

10

11

;c ;d ;e ;f ;g

12, 15

JQ1

cereblon

q

JQ1

VHL

r

OTX015

cereblon

s

OTX015

VHL

bosutinib, dasatinib

cereblon

dasatinib

VHL

all-trans retinoic acid

cIAP1

estrone

cIAP1

c-Abl

t

CRABP I/II ERα

m;u

ERRα RAR

thiazolidinedionebased ligand Ch55

cIAP1

v

inhibitor

VHL

w

KHS101

v

m

RIPK2

VHL CMA

cereblon

o

β-TRCP

peptide

bosutinib, dasatinib

c-Abl

VHL

ErbB3 peptide a

MDM2

BRD4

CMA SCF

non-steroidal ligand p

o

VHL

ovalicin

cIAP1

BRD4

β-TRCP

VHL

TrkA peptide poly-Q binding peptide

i

DHT

BRD4

VHL

rapalog

h

Huntingtin

a ;b

SCF

recruited

BRD4

CMA

estradiol

f;g

BCR-Abl

VHL

peptide

d

e

21

SCF

n o

β-TRCP

target ligand

m

AR

VHL

DHT

DAPK1

AR

VHL

PCC

a

target

CMA

apigenin

Akt AR

recruited

peptide

b

c

d

small-molecule PROTACs

TACC3

VHL

APC/C

CDH-1

VHL

16

20

5

18

17

26

23

39

37

40

35

41

25, 27

;h ;i ;j ;k ;l ;m ;n ;o ;p ;q ;r ;s ;t

28

36

;u ;v ;w

29

VHL Owing to the success of peptidic VHL PROTACs, a small molecule binding to the HIF1αpeptide recognition site with similarly high specificity (but with better pharmaceutical properties) was desired. In 2012, we published the first small molecule inhibitors of the VHL–HIF1α interaction.42-44 The high specificity of this class of molecules was maintained by inclusion of the characteristic hydroxyproline pharmacophore of the HIF1α-peptide. Galdeano et al. have further optimized the ligand to yield a potent VHL binder.45 One of the first studies incorporating this class of small molecule VHL ligands into PROTACs describes targeting of estrogen related receptor (ERRα) and the serine/threonine kinase RIPK2. For the ERRα-PROTAC, a VHL ligand was linked to a thiazolidinedione-based small molecule to yield a degrader capable of reducing ERRα ACS Paragon Plus Environment

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levels in cells with a DC50 of 100 nM, and maximal degradation of 86%. Quantitative proteomics indicated high specificity for ERRα. Importantly, no stabilization of HIF1α was detected with up to 30 µM of the ERRα-PROTAC, indicating bio-orthogonality of the VHL-ligand. In vivo experiments in mice with this PROTAC demonstrated a broad tissue distribution and significant target knock-down in heart and kidney as well as in xenografted MDA-MB-231 tumors. In order to target RIPK2, the VHL ligand was attached to a RIPK2-inhibitor, with the resulting PROTAC showing a remarkable DC50 of 1.4 nM, and a maximal degradation of 95% at 10 nM. Also, by radioactively labeling recombinant RIPK2 and subsequent quantification of ubiquitylated species, Bondeson and colleagues experimentally validated the hypothesized substoichiometric, catalytic action of PROTACs, determining the stoichiometry of degradation for the RIPK-VHL PROTAC as greater than three-fold.36 BRD4 was also successfully targeted for degradation using PROTACs that hijack VHL. The BRD4-VHL PROTAC MZ140 (Figure 4) was based on the JQ1 inhibitor, which had been employed previously to create the cereblon-recruiting degrader dBET137. With complete degradation of BRD4 after 3 h, and sustained knock-down out to 24 h, the JQ1-VHL PROTAC MZ1 appears to be more stable than the thalidomide-based dBET1. However, most remarkable is that MZ1, despite it’s construction from a promiscuous pan-BET inhibitor, shows preferential degradation kinetics of BRD4 over knock-down of BRD2 and BRD3.40 This observation hints at the potential of PROTACs to add a new layer of specificity to a promiscuous binding ligand; a concept that has been explored further by Lai and coworkers39, reviewed at the end of this section. Differing from the other BRD4-PROTAC approaches described above, the most recent study on targeted BRD4 degradation applied pharmacokinetic considerations when designing the PROTAC. Thus, compared to this group’s first reported BRD4 degrader – the pomalidomide-based ARV-82535 – pharmacokinetic optimization of the OTX015 inhibitor and the PROTAC linker along with an E3 recruiting element switch to the VHLbinding ligand yielded the PROTAC ARV-77141 (Figure 4). This new PROTAC degrades BRD4, together with BRD2 and BRD3, in cells with a DC50 of < 5 nM and leads to the depletion of c-myc transcript and protein with a DC50 < 1 nM. Notably, an inactive diastereomer that fails to bind to VHL (ARV-766) did not affect c-myc levels at ACS Paragon Plus Environment


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concentrations up to 1 µM, owing to decreased cell permeability compared to the OTX015 inhibitor. Also, despite possessing Kd-values similar to the JQ1-inhibitor, ARV771 demonstrated a more than 10-fold higher efficacy in reducing c-myc levels. Taken together, this strongly supports the previous finding36 that PROTAC-mediated catalytic target degradation, rather than mere stoichiometric inhibition results in the observed differential decrease in c-myc levels. A direct comparison showed that ARV-771 is 500fold higher potent41 than the dBET1 BRD4 degrader37. Furthermore, the ARV-771 PROTAC described in this study proved to cause an additional reduction in AR expression, accompanied by several downstream anti-androgenic effects.41 Consistent with signs of apoptosis induction, ARV-771 significantly inhibited prostate cancer cell proliferation with 10-500 fold higher potency than the inhibitors OTX015 and JQ1. This effect translated to in vivo models of castration-resistant prostate cancer, where ARV771 but not its inactive diastereomere ARV-766 or the inhibitor OTX015 induced regression of xenografted AR-V7 22Rv1 tumors.41


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Figure 4 PROTACs targeting BRD4. Each depicted PROTAC consists of a BET-domain binding functionality (left side of the molecules), coupled via a linker to either cereblon or VHL-recruiting ligands (right side).

Finally, Lai et al. have reported on the generation of a PROTAC capable of degrading the oncogenic fusion protein BCR-Abl. In this methodology-oriented publication, the authors comprehensively describe options currently available for the design of PROTACs and the reasoning and choices that guided them in their PROTAC design and optimization process. To target c-Abl kinase and the oncogenic form BCR-Abl, three well-characterized tyrosine kinase inhibitors (TKIs) – imatinib, bosutinib, and dasatinib – were employed. In order to create a small screening library, those TKIs were fused to either a VHL ligand or to cereblon-recruiting pomalidomide using distinct linker variations. By subsequent in vitro testing, certain linker compositions could be excluded as they prevented target binding by the TKI moieties. PROTAC composition notwithstanding, a general loss of affinity compared to the native inhibitors was reported. Using K562 chronic myelogeneous leukemia (CML) cells harboring c-Abl and BCR-Abl, no degradation by imatinib-based PROTACs was detected, despite observed downstream kinase inhibition effects. Likewise, bosutinib linked to the VHL ligand did not yield any target degradation. Interestingly, a similar dasatinib-VHL PROTAC showed > 65% degradation of c-Abl, independent of several linker-compositions tested. These observations suggested a significant influence of the target ligands on PROTAC functionality, exceeding mere binding efficiency. However, this study also shows that the E3 ubiquitin ligase choice appears to be important; by switching the E3-recruiting element of the PROTAC to pomalidomide, the bosutinib-based degrader – formerly found inactive – now degraded c-Abl and BCR-Abl by more than 80%. Similar results were observed for a PROTAC with dasatinib linked to pomalidomide, while the corresponding

VHL-recruiting

PROTAC

degraded

only

c-Abl.

The

dasatinib-

pomalidomide PROTAC was evaluated further and a reduction in cell viability of BCRAbl driven K562 cells was observed; a 1,000-fold more potent effect than observed in non-BCR-Abl driven cell lines.39 Taken together, the observations reported for the MZ1 BRD4-degrader40 as well as for the BCR-Abl PROTAC39 suggest the general potential of PROTACs to add selectivity to a promiscuous inhibitor in order to yield a selective degrader. ACS Paragon Plus Environment


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Concluding Remarks Several successful PROTAC applications have been reported in recent years revealing the technology’s underlying potential for catalytic activity that allows for greater potency and more sustainable effects than classical protein inhibition. In theory, PROTACs could induce the degradation of virtually any protein – given the availability of a specific ligand to the target protein. Thus, PROTACs represent a new therapeutic modality that can be expected to substantially expand the repertoire of “druggable” proteins.

Glossary BRD4 Member of the BET (bromodomain and extra terminal domain) family. Binds to chromatin and drives the expression of oncogenes, such as c-myc. Cereblon Substrate binding subunit of a Cullin-RING E3 ubiquitin ligase. DC50 Concentration of PROTAC at which 50% of the target protein is being degraded. Dmax Maximum level of induced degradation achievable. Event-driven pharmaceutical paradigm Efficacy arises out of single drug-binding events. A binding event causes sustainable and efficacy mediating changes to the target, such as its ubiquitylation and degradation. Occupancy-driven pharmaceutical paradigm Efficacy arises out of continuous drug-binding. A drugs efficacy is mediated by its continuous binding and occupation of a spot on the target protein, such as the inhibition of an active site by off-competing the natural ligand. PROTAC


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Proteolysis Targeting Chimera. Hetero-bifunctional molecule comprised of a targetbinding moiety fused by a linker to an E3 ubiquitin ligase-recruiting ligand. VHL Von Hippel-Lindau disease tumor suppressor. Substrate binding subunit of a CullinRING E3 ubiquitin ligase.

Acknowledgments C.M.C. gratefully acknowledges support from the NIH (R35CA197589).

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