Proteolysis-targeting chimeras for targeting protein for

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Proteolysis-targeting chimeras for targeting protein for degradation Jianguo Qi1 & Gang Zhang*,2 1 Key Laboratory of Natural Medicine & Immuno-Engineering of Henan Province, Henan University Jinming Campus, Kaifeng 475004, PR China 2 State Key Laboratory of Bioactive Substances & Function of Natural Medicine, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, PR China *Author for correspondence: [email protected]

Proteolysis-targeting chimeras (PROTACs) are an emerging tool for therapeutic intervention by reducing or eliminating disease-causing proteins. PROTACs are bifunctional molecules that consist of a target protein ligand, a linker and an E3 ligase ligand, which mediate the polyubiquitination of the target protein, ultimately leading to the target protein degradation by the ubiquitin–proteasome pathway. We review some of the main PROTACs that have been reported recently and discuss their potential therapeutic benefits over classical enzyme inhibition. Future research is expected to focus on the delivery and bioavailability of PROTACs due to their high molecular weight (700–1000 Da). First draft submitted: 13 November 2018; Accepted for publication: 14 January 2019; Published online: 1 February 2019 Keywords: E3 ligase • protein degradation • proteolysis-targeting chimeras

For decades, drug development has focused on identifying small molecules that inhibit the function of diseasecausing proteins based on occupancy-driven pharmacology, via blocking the active or regulatory sites of receptors. However, this approach requires high drug doses to maintain high target occupancy at equilibrium and this may cause side effects due to off-target interactions. In addition, there are many undruggable targets, including transcription factors, scaffold proteins and nonenzymatic proteins, which are disease-related targets for which it is difficult or impossible to develop suitable small-molecule inhibitors. Recently, monoclonal antibodies, RNAi, and CRISPR-Cas9 technology have been used to inhibit target protein functions by degrading target proteins, inhibiting the synthesis of target proteins or knocking out genes, respectively. However, monoclonal antibodies are not cell-permeable [1], and RNAi and CRISPR-Cas9 technology is limited due to lack of temporal control or off-target effects [2,3]. Proteolysis-targeting chimeras (PROTACs), which hijack the ubiquitin–proteasome system, show promise in overcoming these disadvantages. The ubiquitin–proteasome pathway (UPP) is a main pathway for degrading cellular protein. Ubiquitin is a small polypeptide containing 76 amino acids residues and is present in all eukaryotic cells. The UPP degrades specific proteins via tagging ubiquitin to the protein for proteasomal degradation (Figure 1) [4,5]. First, ubiquitinactivating enzyme (E1) activates free ubiquitin in the cytoplasm to form a highly reactive ubiquitin acyl adenylate intermediate which subsequently reacts with a nucleophilic residue, most typically cysteine, to form E1-ubiquitin via a thioester bond. Then, ubiquitin is transferred from E1-ubiquitin to ubiquitin transferase (E2) through trans-thioesterification. Finally, a ubiquitin-conjugating enzyme (E3) catalyzes the transfer of ubiquitin from E2ubiquitin to a lysine residue of the specific substrate via an isopeptide bond. Each ubiquitin has seven lysine residues that can also be ubiquitinated to form polyubiquitin chains by the same process. The substrate tagged with the special polyubiquitin is recognized and recruited to the proteasome for degradation. The E3 ligases are the most heterogeneous class of enzymes in the ubiquitination pathway. The E3 ligases regulate the homeostasis, cell cycle and DNA repair pathways, which involve many proteins, the most well-known of which are MDM2 homolog, BRCA1 susceptibility protein and von Hippel–Lindau (VHL) tumor suppressor [6]. The PROTACs are heterobifunctional molecules responsible for the target protein degradation, which consist of an E3 ligase ligand, a linker and a target protein ligand [5,7–15]. The PROTACs promote the formation of a

C 2019 Newlands Press 10.4155/fmc-2018-0557

Future Med. Chem. (Epub ahead of print)

ISSN 1756-8919

Review

Qi & Zhang

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target protein-E3 ligase complex and ultimately lead to degradation of the target protein via the UPP (Figure 2). The PROTACs destroy disease-causing proteins rather than inhibiting them like traditional inhibitors. Only a low dosage is required due to the catalytic nature of PROTACs, which are recycled, and this may prevent the off-target effects of traditional inhibitors. The PROTACs combine aspects of small-molecule inhibitors and largemolecule regulators of protein levels (antibodies, RNAi and CRISPR-Cas9) to achieve good tissue distribution and bioavailability [14,16]. In addition, PROTACs degrade the target protein specifically, avoiding the unintentional degradation by RNAi of mRNA that overlaps with target mRNA. Herein, we review the development of PROTAC technology and some of the main PROTACs that have been recently reported. We also discuss the promising therapeutic benefits of PROTACs over classical enzyme inhibition and the future prospects for this field. PROTACs with peptide-based E3 ligands PhosphoPROTACs

The first PROTAC consisted of a NFKB1 enhancer in B cells inhibitor alpha (IκBα)-derived phosphodecapeptide that recruits the E3 ligase, hetorotetrameric Skp1-cullin-F-box (SCFβ-TrCP ), linked by an aminohexanoic acid to a small-molecule ligand ovalicin, which covalently inhibits MetAP-2 (Figure 3, P1) [17]. The MetAP-2 as a cytosolic metalloenzyme catalyzes the cleavage of methionine at N terminal of nascent polypeptides and plays a key role in angiogenesis, which is essential for the progression of solid tumors and rheumatoid arthritis [18,19]. Thus, ovalicin is a potent antiangiogenesis agent [20]. The PROTAC P1 hijacks SCFβ-TrCP to ubiquitinate and degrade of MetAP-2 [17]. Sakamoto et al. reported two PROTACs with a phosphopeptide moiety as E3 ligase ligand, targeting the estrogen receptor (ER) and androgen receptor (AR) responsible for the progression of breast and prostate cancers, respectively. (Figure 3, P2 & P3). The two PROTACs also hijack SCFβ-TrCP via an IκBα-derived phosphodecapeptide, and the

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Proteolysis-targeting chimeras for targeting protein for degradation

Review

Target Protein Ligands-Linkers-E3 Ligase Ligands

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Chemical structures of phosphoproteolysis-targeting chimeras.

ER and AR via estradiol and dihydrotestosterone (DHT), respectively [21]. A polyglycine sequence was attached to the N-terminal of the IκBα-derived phosphodecapeptide used for recruiting E3 ligase in PROTACs P1, P2 and P3. Hines et al. [22] developed two phosphoPROTACs, which consisted of the tyrosine-phosphorylated sequences derived from the nerve growth factor receptor TrkA or the neuregulin receptor ErbB3, respectively, and a peptide ligand for the E3 ubiquitin ligase, VHL. The phosphoPROTAC, TrkA PPFRS2α , comprised a decapeptide sequence (IENPQYFSDA) derived from TrkA with an autophosphorylation site on the central tyrosine to recruit the FRS2α, and a heptapeptide sequence derived from hypoxia-inducible factor (HIF)-1 for binding to the E3 ligase, CRL2VHL (Figure 3, P4). The knockdown of FRS2α by TrkA PPFRS2α occurred in a time- and dose-dependent manner. After PC12 cells incubate with nerve growth factor and TrkA PPFRS2α for 60 min, FRS2α level is decreased by about 90%, which suggests that the efficiency of this PROTAC was high [22]. The ErbB2/ErbB3-PI3K-AKT pathway is activated in many human cancers, which plays an important role in mitogenesis and apoptosis [23]. The ErbB2, which functions as a co-receptor with ErbB3, is overexpressed in breast and ovarian cancers and phosphorylated in the absence of neuregulin to activate AKT. Thus, the ErbB2/ErbB3-PI3K-AKT pathway is essential for tumor cell growth. The PROTAC ErbB2 PPPI3K as a degrader targeted the ErbB2/ErbB3-PI3K-AKT pathway and eliminated the subunit p85 of PI3K in a dose-dependent manner in MCF7 cells, which led to the inactivation of AKT without any effect on total AKT (Figure 3, P5). Moreover, tumor size in a mouse xenograft model was reduced to about 40% by daily treatment with ErbB2 PPPI3K , suggesting that this phosphoPROTAC has promising therapeutic effects in vivo [22]. Although phosphoPROTACs have shown excellent efficacy in degrading target proteins, these molecules are difficult to develop as drug candidates due to their high molecular weight, poor cell permeability and metabolic instability in cells. Cell-permeable PROTACs

The first cell-permeable PROTAC was reported by Schneekloth et al. [24] in 2004. The heptapeptidesequence (ALAPYIP), the minimum recognition domain for E3 ligase VHL, was used as the ligand for E3 ligase VHL. A poly-D-arginine tag was introduced to the C-terminus of the peptide sequence in order to enhance cell permeability and resist nonspecific proteolysis [25]. The PROTACs employed a rapamycin analogue and DHT as the ligands for mutant FKBP12 and AR, respectively (Figure 4, P6 & P7). The PROTACs caused a loss of green fluorescence in Hela cells, which stably express EGFP-(F36V)FKBP12. However, the fluorescence was still retained in 786-O cells without the VHL protein. The PROTAC mediated and degraded (F36V)FKBP12, which was also confirmed by western blotting. Montrose et al. [26] designed a PROTAC through fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein. The researcher also worked to improve the cell permeation of the PROTAC through installing a polyarginine cell-penetrating peptide (Figure 4, P8 & P9). The PROTAC simultaneously induced the degradation of the X-protein of the hepatitis B virus and antagonized its function.


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Chemical structures of cell-permeable peptide-based proteolysis-targeting chimeras.

Recently, Lu et al. reported a peptide PROTAC which hijacked Keap1 to target Tau for degradation [27], which comprised of two peptide sequences derived from Keap1 and Tau, respectively (Figure 4, P10). Aggregation of the microtubule-associated protein Tau involved in the neurodegenerative diseases [28]. The PROTAC P10 showed strong binding affinity with Keap1 and Tau in vitro, which could promote the Keap1-dependent polyubiquitination and proteasome-dependent degradation of Tau. Jiang et al. identified a stabilized peptide-based PROTAC targeting ER-α by tethering an N-terminal aspartic acid cross-linked stabilized peptide ER-α modulator (TD-PERM) with a pentapeptide ligand of E3 ligase VHL (Figure 4, P11) [29]. The stabilized peptides constructed by N-terminal aspartic acid cross-linking strategy (TD strategy) showed good stability and cell permeability [30]. The PROTAC selectively degraded ER-α in a proteasome-dependent manner and significantly inhibited the proliferation of ERα-positive breast cancer cells compared with control peptides. In addition, the PROTAC led to tumor regression in the MCF-7 mouse xenograft model. Zhang et al. [31] reported two cell-permeable PROTACs that used ovalicin and estradiol as ligands for MetAP-2 and ER, respectively, aminohexanoic acid as the linker and the HIF-1α-derived octapeptide, MLAPOH YIPM, as a ligand for recruiting VHL (Figure 4, P12 & P13). Interestingly, these two PROTACs destroyed the target proteins in cells without permeability-aiding sequences. Furthermore, in the ER degradation assay to determine the minimum peptide size, the pentapeptide-based PROTAC (Figure 4, P14) showed the most effective growth inhibition of MCF-7 tumor cells [32].

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Lee et al. [33] reported a cell-permeable PROTAC that induced AHR degradation in viable cells (Figure 4, P15). The PROTAC consisted of the AHR ligand apigenin and an E3 recognition motif, which was a pentapeptide residue derived from HIF-1α protein, linked by an alkyl linker. The PROTAC was designed specifically to deconvolute the role of modulated AHR in tumor development and progression, and allowed the researcher to gain important insight into this important target. In 2010, Cyrus et al. [34,35] reported a two-headed PROTAC with two ER ligands that showed improved binding affinity and superior ER degradation compared with the one-headed PROTAC (Figure 4, P16). PROTACs with small-molecule E3 ligands The first-generation PROTACs showed poor physicochemical properties due to the highly peptidic nature, such as bad intracellular stability and poor cell permeability, which limited their application as chemical probes and their therapeutic development. The PROTACs with small-molecule E3 ligands could be used to overcome these limitations. This type of PROTAC consists of small molecules for targeting the proteins and E3 ligases to improve the cell permeability, distribution and metabolic stability. Recently, various PROTACs that destroy different diseasecausing proteins were reported. These PROTACs employed small-molecule ligands for several E3 ligases, such as MDM2, cIAP1, cereblon (CRBN) and VHL. MDM2-based PROTACs

In 2008, Schneekloth et al. [36] reported the first entirely small-molecule PROTAC, which consisted of a SARM, a polyethylene glycol linker and a nutlin for recruiting E3 ligase MDM2 (Figure 5, P17). The ARs are responsible for several male diseases, such as androgen insensitivity syndrome and prostate cancer. Dysregulation of AR inhibits prostate cancer growth [37]. Nutlins are a class of potent, selective small-molecule antagonists of MDM2 that induce ubiquitination and degradation of tumor suppressor P53 and its overexpression in many human tumors [38]. The PROTAC P17 recruiting E3 ligase MDM2 led to ubiquitination and its subsequent degradation of the AR [36]. The SARM-nutlin PROTAC bound AR with high affinity and a Ki of 4 nM [39], and the PROTAC destroyed AR in HeLa cells at a concentration of 10 μM. This PROTAC provided proof-of-concept that PROTACs can be developed as small-molecule drugs [36]. Rao et al. identified a degrader targeting PARP1 which is a major member of the PARP super family and involved in DNA damage repair and other important cellular processes (Figure 5, P18) [40,41]. The PROTAC P18, consisting of a PARP1 ligand, a nutlin and a polyethylene glycol linker, could induce PARP1 cleavage and cell apoptosis in the MDA-MB-231 cells. The PROTAC P18 also showed fivefold more potent than niraparib, olaparib and veliparib. However, it exhibited no cytotoxicity to the normal breast epithelial cells. cIAP1-based PROTACs

In 2010, Itoh et al. [42] developed PROTACs that consisted of all-trans retinoic acid and methyl bestatin linked by polyethylene glycol linkers of different lengths (Figure 5, P19). The all-trans retinoic acid is an endogenous ligand of CRABP-I and -II, which is associated with Alzheimer’s disease and some human tumors, including neroblastoma, Wilms tumor, and head and neck squamous cell carcinoma. The methyl bestatin binds to the BIR3 domain of the E3 ligase cIAP1 and leads to autoubiquitination of cIAP1 [43,44]. The formation of a complex of cIAP1 and CRABP-II in vitro was induced by the PROTAC P19, which destroyed CRABPs via the UPP and inhibited migration of neuroblastoma IMR-32 cells. However, these PROTACs led to undesired degradation of cIAP1, which made the protein knockdown unsustainable [42,45]. In subsequent work, the ester bond in the linker was replaced with an amide bond and the PROTAC induced degradation of CRABP-II specifically without inducing cIAP1 degradation (Figure 5, P20) [46]. Itoh et al. [47] designed PROTACs consisting of BE04 as the cIAP1 ligand and a ligand for the target protein, namely, Ch55, DHT and estrone for retinoic acid receptor, AR and ER, respectively (Figure 5, P21–P23). Demizu et al. [48] also synthesized ER degraders that contained a tamoxifen derivative as the ER protein ligand and a bestatin (BS) moiety that bound to cIAP1 (Figure 5, P24). In 2014, Ohoka et al. [49] developed a PROTAC called SNIPER(TACC3) consisting of BS as a ligand for E3 ligase recruitment and a small-molecule ligand for the spindle regulatory protein, TACC3, which is overexpressed in cancer cells (Figure 5, P25). The PROTAC led to the degradation of TACC3 and cancer cell death by expressing an abnormally high level of TACC3 protein. Mechanistic analysis showed that SNIPER(TACC3) recruited APC/CCDH1 E3 ligase but not cIAP1.


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Chemical structures of MDM2- and cIAP1-based proteolysis-targeting chimeras.

CRBN-based PROTACs

Research on the mechanism of the immunomodulatory drug thalidomide and its derivatives lenalidomide and pomalidomide has demonstrated that they bind to CRBN, which could form an E3 ligase CRL4CRBN complex with DNA binding protein 1, cullin-4A and regulator of cullins 1. The immunomodulatory drugs bound to CRBN and formed CRL4CRBN , leading to the degradation of the transcription factors IKZF1 and IKZF2 and casein kinase 1A1, which is important for treatment of myelodysplastic syndrome [50,51]. In 2015, Lu et al. [52] reported the first CRBN-based PROTAC (ARV-825), consisting of the BRD4 ligand OTX015, pomalidomide, and a polyethylene glycol linker (Figure 6, P26). The BRD4 is a member of the bromodomain and extra-terminal domain (BET) family [53] and contains two bromodomains that recognize acetylated lysine residues [54]. The BRD4 plays a critical role in many hematological and solid tumors by acting as a coactivator for the expression of proliferative genes, such as c-Myc, B-cell lymphoma-extra large and BCL6 protein gene [55]. BRD4 inhibition is promising in cancer treatment by targeting c-Myc expression in Myc-driven oncogenesis [56,57]. The BRD4 inhibitors, such as JQ1 and OTX015, induce significant BRD4 accumulation over time in cell lines, leading to incomplete suppression of c-Myc. The ARV-825 degraded BRD4 and suppressed c-Myc expression persistently, resulting in robust proliferation inhibition and apoptosis induction. In addition, ARV-825 showed potent degradation of BRD4 with DC50 below 1 nM compared with OTX015 with a binding

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affinity of ∼10 nM [52]. Importantly, ARV-825 also showed promising activity in preclinical models of multiple myeloma, which support its application to the patients with relapsed or refractory disease [58,59]. Winter et al. [60] reported another BRD4 degrader, dBET1, which was composed of the thalidomide derivative, JQ1, and a hydroxyacetamide linker (Figure 6, P27). The PROTAC induced the degradation of BRD4 with DC50 of 430 nM, and also decreased the protein levels of BRD2, BRD3 and Myc. In the aggressive disseminated leukemia animal model, dBET1 showed comparable activity to JQ1. The kinetics study showed that BRD4 was completely degraded when treated with 100 nM dBET1 for 2 h. However, the partial recovery in BRD4 abundance at 24 h indicated the instability of the PROTAC molecule. The PROTAC also dampened the proinflammatory responses in lipopolysaccharide-stimulated microglia due to degradation of BRD2 and BRD4, which are associated with some proinflammatory genes [61]. A BET degrader, QCA570, was reported by Qin et al. [62] in 2018, and consisted of the potent BET inhibitor, thalidomide derivative QCA276, and an alkyne linker (Figure 6, P28). QCA570 reduced the levels of BRD3 and BRD4 proteins at 10 pM and that of BRD2 protein at 30–100 pM, as well as reducing c-Myc levels at 10 pM. QCA570 showed potent cell growth inhibitory activity in various cell lines. In RS4;11 xenograft models, dosing with QCA570 resulted in total inhibition of tumor growth, however it did not result in tumor regression at weekly doses of 5 mg/kg over 3 weeks. Thus, QCA570 is the most potent and efficacious reported PROTAC targeting BET to date and has showed promising therapeutic potential for acute leukemia. Zhou et al. [63] reported the BET degrader, ZBC260 (BETd-260), which exhibited an IC50 value of 51 pM for the inhibition of RS4;11 cell growth and led to rapid tumor regression in vivo against RS4;11 xenograft tumors (Figure 6, P29). Wang et al. [64]


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also reported a BRD degrader in which the BET inhibitor, BI2536, was connected to thalidomide as ligand for CRBN/cullin4A via a linker. This PROTAC exhibited potent BRD4 inhibition with an IC50 value of 9.4 nM, and potent inhibition of cell proliferation in the BRD4-sensitive cell line, RS4;11, with an IC50 value of 27.6 nM. The PROTAC also induced degradation of BRD4 protein at 0.5–1.0 μmol/L in RS4;11 cells (Figure 6, P30). PROTACs have been used to degrade the CDK, CDK9, which is involved in transcription regulation (Figure 6, P31 and P32). The CRBN-based PROTAC P31 was composed of an aminopyrazole analog as the ligand for CDK9 and thalidomide as the ligand for CRBN/cullin4A linked with an alkyl linker. The PROTAC selectively degraded CDK9 while sparing other CDK family members in HCT116 cells [65]. Bian et al. designed and synthesized the Wogonin-based PROTAC P32 targeting CDK9 by recruiting the E3 ligase CRBN [66]. P32, bearing a triazole functional group, can selectively downregulate the levels of the intracellular CDK9 in a dose-dependent manner. Furthermore, both the proteasome-dependent and CRBN-dependent degradation have been confirmed through the use of the proteasome inhibitor MG132 as well as selective CRBN siRNA silencing. P32 selectively inhibited the proliferation of cancer cells overexpressed with CDK9, which was clearly consistent with the degradation of the CDK9 protein by the aforementioned PROTAC. Ibrutinib, a covalent inhibitor of Bruton’s tyrosine kinase (BTK), has been a transformative treatment option for chronic lymphocytic leukemia (CLL) and other B-cell malignancies [67]. As with many kinase inhibitors, many patients develop resistance, in this case due to the mutation of a cysteine to serine at the site to which ibrutinib binds covalently (C481S) [68]. Buhimschi et al. [69] developed PROTAC MT-802, which had an ibrutinib-based scaffold conjugated to pomalidomide by a linker (Figure 6, P33). The PROTAC induced the degradation of BTK with a DC50 value of 9.1 nM and showed enhanced kinase selectivity compared with ibrutinib. In addition, MT-802 degraded wild-type and C481S mutant BTK. Sun et al. [70] also reported a similar BTK degrader composed of an ibrutinib derivative and pomalidomide (Figure 6, P34), which degraded both wild-type and the ibrutinib-resistant C481S kinases in lymphoma cell lines. PROTAC P34 significantly inhibited the proliferation of BTK C481S mutant HBL-1 cells in vitro, without degradatory effects on off-targets of ibrutinib, including ITK, EGFR and TEC family kinases. Yang et al. [71] reported the first HDAC6-selecitve degrader by linking a nonselective HDAC inhibitor with a thalidomide-type E3 ligase ligand by various linkers (Figure 6, P35). It was observed that PROTAC P35 could

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Phosphorated peptides

Small molecules Figure 7. The development of VHL ligands on proteolysis-targeting chimeras.

Cell-permeable peptides

selectively degrade HDAC6 over other HDACs in cell-based assays. Zhang et al. [72] developed two PROTACs composed of ceritinib and pomalidomide linked by two different linkers, and the PROTACs degraded anaplastic lymphoma kinase in a concentration- and time-dependent manner in lymphoma and lung cancer cells (Figure 6, P36 and P37). Li et al. [73] designed a PROTAC to target MDM2 protein, which acts as the primary endogenous cellular inhibitor of the tumor suppressor p53 (Figure 6, P38) [74]. The PROTAC P38 efficiently degraded MDM2 at concentrations below 1 nM in human leukemia cells. It was also shown to induce complete and long lasting tumor regression in RS4:11 xenograft tumor mice model under well-tolerated dose schedules. Recently, McCoull et al. reported a BCL6 PROTAC based on their developed BCL6 inhibitor (Figure 6, P39) [75]. However, both the PROTAC and the inhibitor showed weak antiproliferative activity which may ascribe to the residual BCL6 population. The PCAF and GCN5 are epigenetic proteins with the ability to selectively modify and recognize specific marks on histone tails [76]. Tough et al. identified the first PCAF/GCN5 PROTAC P40 (Figure 6, P40), which is able to degrade PCAF/GCN5 by recruiting the E3 ligase CRBN and to potently modulate the expression of multiple inflammatory mediators in lipopolysaccharide-stimulated macrophages and dendritic cells [77]. The PROTAC P40 induced concentration-dependent degradation of PCAF and GCN5 in THP1 cells, with a DC50 value (concentration required to degrade 50% of the target protein) of 1.5 and 3 nM, respectively. It also could cause robust and concentration-dependent degradation of PCAF and GCN5 in both macrophages and DCs. Studies showed that most of activity was retained by the isomer cis-(R,R)-enantiomer of P40. VHL-based PROTACs

The VHL-based PROTACs are reliably developed from peptidic VHL ligands, including phospholated peptides and peptide conjugates with cell-permeable sequences or tags, to small-molecule VHL ligands aiming to improve cell permeability and stability (Figure 7). Due to the limitations of peptidic VHL PROTACs, it is necessary to develop small-molecule ligands for recruiting VHL. In 2015, Bondeson et al. reported two PROTACs involving smallmolecule VHL ligands [78,79], targeting ERRα (Figure 8, P41) and serine/threonine kinase RIPK2 (Figure 8, P42). The ERRα is an orphan nuclear hormone receptor that is involved in regulating cellular energy homeostasis [80]. The PROTAC ERRα reduced ERRα levels with a DC50 value of 100 nM and maximal degradation of 86%. It degraded ERRα specifically and did not target other ERR isoforms. Moreover, the PROTAC produced an efficient in vivo knockdown in mice and had broad tissue distribution [81]. The RIPK2 plays an important role in innate immune signaling by activating the NF-κB and MAPK signaling pathways [82]. Dysregulation of RIPK2 is related to autoinflammatory diseases, such as Blau syndrome [83] and early-onset sarcoidosis [84]. The PROTAC P42 comprised a RIPK2 inhibitor, a small-molecule VHL ligand and a linker. The PROTAC degraded RIPK2 with a DC50 of 1.4 nM and a maximal degradation of 95% at 10 nM. Furthermore, the degradation induced by PROTAC RIPK2 was highly specific and mediated by catalytic ubiquitination [81]. Zengerle et al. [85] developed the BRD4 PROTAC, MZ1, hijacking VHL with small-molecule ligands. The MZ1 consisted of a moiety derived from pan-BET-selective bromodomain inhibitor JQ1, a small-molecule VHL ligand and a polyethylene glycol linker (Figure 8, P43). The MZ1 degraded 90% of all BET proteins at concentrations down to 1 μM, which could selectively degrade BRD4 over BRD2 and BRD3 compared with pan-selective inhibitor JQ1. In addition, MZ1 induced rapid degradation of target proteins and did not interfere with the normal endogenous levels of both VHL and HIF-1α. After 2 years, the same research group [86] solved the crystal structure of MZ1 in complex with VHL and the BRD4 bromodomain ternary complexes for the first time (PDB code 5T35). This work probed pivotal in the elucidation of the mechanism of action of PROTAC mediated protein–protein interactions. There appeared to be more cooperative interactions between the side chains on BRD4 and VHL than that of the binary complex formed by MZ1 with BRD4 or VHL. Additionally, a highly selective


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BRD4 degrader was designed based on the crystal structure via redirection of linker junction on VHL ligand and modification of linker. Raina et al. [87] reported BET PROTAC AVR-771, which had the same BET binding ligand but different VHL binding ligands and linker lengths (Figure 8, P44). In cellular models of castration-resistant prostate cancer, AVR-771 led to rapid BET protein degradation with a DC50 value of 85% degradation of c-ABL and >60% degradation of BCR-ABL at 1 μM, and showed highly selective inhibition of growth of the BCR-ABL-driven cells. In 2018, Bondeson et al. [90] reported a further study of the specificity and selectivity of different E3 ligaserecruiting PROTACs. The multiple kinase inhibitor, foretinib, which binds to >130 kinases, retained binding to 52 kinases or 62 kinases when conjugated to a VHL or CRBN ligand, respectively. However, the VHL and CRBN PROTACs degraded 9 and 14 kinases, respectively (Figure 8, P46 & P47). This demonstrated that the PROTACs were more specific than the inhibitors and different E3 ligases led to selective degradation of target proteins. Furthermore, the stability of the target:PROTAC:ligase trimer complex rather than the target:PROTAC binding affinity correlated with the degradation potency. This is consistent with the results reported by Chan et al. [91]. Two BET inhibitors, triazolodiazepine JQ1 or the more potent tetrahydroquinoline I-BET726, were used as BET ligands conjugated to the same VHL ligand (Figure 8, P48 & P49). However, the JQ1 PROTACs were more potent than the I-BET726 PROTACs because the JQ1 PROTACs formed trimer complexes that were more stable. It was observed that BET-degrading and c-Myc-driven antiproliferative activities were dependent greatly on linker length in acute myeloid leukemia cell lines. The FLT-3 is expressed in most acute myeloid leukemia cases (41/44 tested) [92], and is frequently mutated in acute myeloid leukemia patients. Several FLT-3 kinase inhibitors in clinical trials have demonstrated only limited efficacy [93]. Burslem et al. identified a PROTAC targeting FLT-3, using the clinical candidate quizartinib as the FLT-3 ligand and VHL as the E3 ligase, which induced degradation of FLT-3 ITD mutant at low nanomolar concentrations (Figure 8, P50). The PROTAC could inhibit cell growth more potently than the warhead alone while no degradation was found against a large panel of other kinases. Importantly, the PROTAC displays acceptable pharmacokinetic properties and could degrade FLT-3 in tumor-bearing mice [94]. Cromm et al. [95] reported the first PROTAC outperformed an optimized kinase inhibitor with respect to autophosphorylation and inhibition of downstream signaling to date, which could degrade fak (Figure 8, P51).


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Fak is a cytoplasmic tyrosine kinase which plays an important role in tumor progression via kinase-dependent and -independent mechanisms. Phosphorylation at Y397 induces Fak to form a complex with Src-family kinases and results in full activation of Fak, which has been attributed to poor overall patient survival [96]. Additionally, Fak is considered as a valuable target for cancer immunotherapy as its activation is associated with CD8 T-cell exhaustion. The PROTAC induces 95% degradation of Fak at 50 nM in human prostate tumor (PC3) cells, which results in reduced phosphorylation levels downstream of the protein, p-paxillin and p-Akt. TBK1 is implicated in many cellular functions, such as innate immune response as well as tumorigenesis and progression [97,98]. Crew et al. reported a selective PROTAC for degradation of TBK1 by employing a nonselective TBK1 inhibitor and recruiting the E3 ligase VHL (Figure 8, P52) [99]. The PROTAC is a potent degrader (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKK. Furthermore, it was used to assess TBK1 as a target in mutant K-Ras cancer cells. Click-formed proteolysis-targeting chimeras Most PROTACs have poor drug-like properties, such as cellular permeation, solubility and absorption, due to their high molecular weight. Recently, Lebraud et al. [100] developed a kind of PROTACs that were formed by the bioorthogonal click combination of two smaller precursors in cells, named in-cell click-formed proteolysis-targeting chimeras (CLIPTACs). The two small precursors were a tetrazine-tagged thalidomide derivative and a trans-cyclooctene-tagged ligand of the target protein, respectively. The CLIPTACs were used to degrade two important targets associated with cancer, BRD4 and ERK1/2 in cells. However, the CLIPTACs did not degrade the target proteins in cells when they were formed before they were added to the cells. This study provided an alternative strategy to degrading a target protein in cells by click chemistry of a tagged ligand for the target protein and a tagged E3 ligase ligand (Figure 10). Wang et al. [101] developed ER degraders with the ER ligand bearing a destabilizing amino acid recognized by the ubiquitin proteasome system (Figure 11, P54) [102]. PROTAC P54, with Boc-Trp as the degron, exhibited potent

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ER degradation activity (DC50 = 0.5 nM). The removal of the Boc group in P39 completely abolished its selective estrogen receptor degrader (SERD) activity. However, the mechanism of action is still unknown. Homo-PROTACs Homo-PROTACs are a special type of PROTACs, which hijack E3 ligase to induce auto degradation. Ciulli et al. [103] reported the first Homo-PROTAC P55 (Figure 12, P55) that could dimerize VHL and induce potent, rapid and proteasome dependent self-degradation of VHL in cells. PROTAC P55 was composed of two identical VHL ligands linked by a polyethylene glycol chain. This Homo-PROTAC could find wide use amongst biologists who are interested in expounding the pleiotropic biological functions of pVHL, and affords a chemical tool alternative to RNAi or gene editing. Steinebach et al. [104] reported the Homo-PROTACs for degradtion of CRBN, one of which exhibt highly potent degradation of CRBN with minimal effects on IKZF1 and IKZF3 (Figure 12, P56). It is once again a useful chemical tool to unravel endogenous substrates and physiological roles of CRBN.


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Chemical structures of selected homo-proteolysis-targeting chimeras.

Future perspective PROTACs are a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges that drug development programs currently face. PROTACs have been developed for degrading target proteins and are composed of peptidic- or small-molecule-based ligands for E3 ligase, small-molecule ligands for the target proteins, and a linker. PROTACs have some advantages over traditional target protein inhibitors, including the ability to degrade undruggable proteins, and the catalytic and specific degradation of target proteins. PROTACs are more potent than traditional inhibitors and overcome drug resistance. However, there is still no clinical research; PROTACs have high molecular weights that impair drug-like properties. New strategies, such as CLIPTACs and altering degrons, have been developed to overcome the drawbacks of PROTACs. In the future, PROTACs with improved drug-like properties will be developed. Further studies on the mechanism of action and safety evaluation should be carried out to support their transfer to clinical use. In addition, it is essential to develop design and synthesis strategies for rapid chemical diversification to optimize the ADMET properties of PROTACs. Acknowledgements The authors thank all of the researchers working on PROTAC research field for their contribution. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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Review

Executive summary • The ubiquitin–proteasome pathway could recognize and degrade target proteins by tagging ubiquitin. • Proteolysis-targeting chimeras (PROTACs) degrade target proteins by recruiting an E3 ligase, which are composed of a ligand for recruiting an E3 ligase, a linker and a ligand for binding to the target protein. PROTACs with peptide-based E3 ligands • PhosphoPROTACs were comprised of a phosphorylated peptidic sequence derived from endogenous substrate of E3 ligases or target proteins. • Cell-permeable PROTAC consisted of a ligand for target protein, a linker and an E3 peptidic ligand with cell-permeable property. PROTACs with small-molecule E3 ligands • PROTACs employed small molecules as the ligands for E3 and target proteins, respectively, which were mainly classified to MDM2-based PROTACs, cIAP1-based PROTACs, CRBN-based PROTACs and von Hippel–Lindau-based PROTACs. Click-formed PROTACs • Click-formed PROTACs were formed in cell by the bio-orthogonal click combination of two smaller precusors used as the ligands for E3 and target proteins, respectively, which avoided poor drug-like properties of most PROTACs, such as cell permeability and solubility. Homo-PROTACs • Homo-PROTACs could degrade an E3 ligase to arrogate its functions.

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•

The first phosphoPROTAC was presented in this paper.

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The first cell-permeable PROTAC was presented in this paper.

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The first all-small-molecule PROTAC recruiting E3 ligase mouse double minute 2 was reported in this paper.

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Review

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The first CRBN-based PROTAC with small-molecule ligands was reported in this paper.

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Review

Qi & Zhang

70. Sun Y, Zhao X, Ding N et al. PROTAC-induced BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell malignancies. Cell Res. 28(7), 779–781 (2018). 71. Yang K, Song Y, Xie H et al. Development of the first small molecule histone deacetylase 6 (HDAC6) degraders. Bioorg. Med. Chem. Lett. 28(14), 2493–2497 (2018). 72. Zhang C, Han XR, Yang X et al. Proteolysis targeting chimeras (PROTACs) of anaplastic lymphoma kinase (ALK). Eur. J. Med. Chem. 151, 304–314 (2018). 73. Li Y, Yang J, Aguilar A et al. Discovery of MD-224 as a first-in-class, highly potent, and efficacious proteolysis targeting chimera murine double minute 2 degrader capable of achieving complete and durable tumor regression. J. Med. Chem. doi:10.1021/acs.jmedchem.8b00909 (Epub ahead of print). 74. Moll UM, Petrenko O. The MDM2-p53 interaction. Mol. Cancer Res. 1(14), 1001–1008 (2003). 75. McCoull W, Cheung T, Anderson E et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC to provide insight into small molecule targeting of BCL6. ACS Chem. Biol. 13(11), 3131–3141 (2018). 76. Nagy Z, Tora L. Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene 26(37), 5341–5357 (2007). 77. Bassi ZI, Fillmore MC, Miah AH et al. Modulating PCAF/GCN5 immune cell function through a PROTAC approach. ACS Chem. Biol. 13(10), 2862–2867 (2018). 78. Galdeano C, Gadd MS, Soares P et al. Structure-guided design and optimization of small molecules targeting the protein–protein interaction between the von Hippel–Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J. Med. Chem. 57(20), 8657–8663 (2014). 79. Buckley DL, Van Molle I, Gareiss PC et al. Targeting the von Hippel–Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1alpha interaction. J. Am. Chem. Soc. 134(10), 4465–4468 (2012). •

One von Hippel–Lindau-based PROTAC with small-molecule ligands for von Hippel–Lindau and target protein were reported.

80. Zhang Z, Teng CT. Interplay between estrogen-related receptor alpha (ERRalpha) and gamma (ERRgamma) on the regulation of ERRalpha gene expression. Mol. Cell. Endocrinol. 264(1–2), 128–141 (2007). 81. Bondeson DP, Mares A, Smith IED et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11(8), 611–617 (2015). 82. McCarthy JV, Ni J, Dixit VM. RIP2 is a novel NF-κB-activating and cell death-inducing kinase. J. Biol. Chem. 273(27), 16968–16975 (1998). 83. Miceli-Richard C, Lesage S, Rybojad M et al. CARD15 mutations in Blau syndrome. Nat. Genet. 29(1), 19–20 (2001). 84. Kanazawa N, Okafuji I, Kambe N et al. Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome. Blood 105(3), 1195–1197 (2005). 85. Zengerle M, Chan KH, Ciulli A. Selective small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem. Biol. 10(8), 1770–1777 (2015). 86. Gadd MS, Testa A, Lucas X et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13(5), 514–521 (2017). 87. Raina K, Lu J, Qian Y et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113(26), 7124–7129 (2016). 88. Zoppi V, Hughes SJ, Maniaci C et al. Iterative design and optimization of initially inactive proteolysis targeting chimeras (PROTACs) identify VZ185 as a potent, fast and selective von Hippel–Lindau (VHL)-based dual degrader probe of BRD9 and BRD7. J. Med. Chem. doi:10.1021/acs.jmedchem.8b01413 (2018) (Epub ahead of print). 89. Lai AC, Toure M, Hellerschmied D et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew. Chem. Int. Ed. 55(2), 807–810 (2016). 90. Bondeson DP, Smith BE, Burslem GM et al. Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem. Biol. 25(1), 78–87.e75 (2018). 91. Chan KH, Zengerle M, Testa A, Ciulli A. Impact of target warhead and linkage vector on inducing protein degradation: comparison of bromodomain and extra-terminal (BET) degraders derived from triazolodiazepine (JQ1) and tetrahydroquinoline (I-BET726) BET inhibitor scaffolds. J. Med. Chem. 61(2), 504–513 (2018). 92. Birg F, Courcoul M, Rosnet O et al. Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lympoid lineages. Blood 80(10), 2584–2593 (1992). 93. Grunwald MR, Levis MJ. FLT3 inhibitors for acute myeloid leukemia: a review of their efficacy and mechanisms of resistance. Int. J. Hematol. 97(6), 683–694 (2013). 94. Burslem GM, Song J, Chen X et al. Enhancing antiproliferative activity and selectivity of a FLT-3 inhibitor by proteolysis targeting chimera conversion. J. Am. Chem. Soc. 140(48), 16428–16432 (2018).

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Review

95. Cromm PM, Samarasinghe KTG, Hines J, Crews CM. Addressing kinase-independent functions of Fak via PROTAC-mediated degradation. J. Am. Chem. Soc. 140(49), 17019–17026 (2018). 96. Sulzmaier FJ, Jean C, Schlaepfer DD. FAK in cancer: mechanistic findings and clinical applications. Nat. Rev. Cancer 14(9), 598–610 (2014). 97. Clark K, Plater L, Peggie M, Cohen P. Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. J. Biol. Chem. 284(21), 14136–14146 (2009). 98. Yu T, Yang Y, Yin DQ et al. TBK1 inhibitors: a review of patent literature (2011–2014). Expert Opin. Ther. Pat. 25(12), 1385–1396 (2015). 99. Crew AP, Raina K, Dong H et al. Identification and characterization of von Hippel–Lindau-recruiting proteolysis targeting chimeras (PROTACs) of TANK-binding kinase 1. J. Med. Chem. 61(2), 583–598 (2018). 100. Lebraud H, Wright DJ, Johnson CN, Heightman TD. Protein degradation by in-cell self-assembly of proteolysis targeting chimeras. ACS Cent. Sci. 2(12), 927–934 (2016). •

The click-formed PROTAC in cell was reported in this paper.

101. Wang L, Guillen VS, Sharma N et al. New class of selective estrogen receptor degraders (SERDs): expanding the toolbox of PROTAC degrons. ACS Med. Chem. Lett. 9(8), 803–808 (2018). 102. Dougan DA, Micevski D, Truscott KN. The N-end rule pathway: from recognition by N-recognins, to destruction by AAA+proteases. Biochim. Biophys. Acta 1823(1), 83–91 (2012). 103. Maniaci C, Hughes SJ, Testa A et al. Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat. Commun. 8(1), 830–843 (2017). •

The first homo-PROTAC was reported in this paper.

104. Steinebach C, Lindner S, Udeshi ND et al. Homo-PROTACs for the chemical knockdown of cereblon. ACS Chem. Biol. 13(9), 2771–2782 (2018).


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