The PROTAC technology in drug development

Currently, a new technology termed PROTAC, proteolysis targeting chimera, has been developed for inducing the protein degradation by a targeting molecule. This technology takes advantage of a moiety of targeted protein and a moiety of recognizing E3 ubiquitin ligase and produces a hybrid molecule to specifically knock down a targeted protein. During the first decade, three pedigreed groups worked on the development of this technology. To date, this technology has been extended by different groups, aiming to develop new drugs against different diseases including cancers. This review summarizes the contributions of the groups for the development of PROTAC. Significance of the study This review summarized the development of the PROTAC technology for readers and also presented the author's opinions on the application of the technology in tumor therapy.

proteins including ER, HER-2, Raf-1, IGFR1R, mutated v-Src, Brc-Abl, and p53. Therefore, a rational strategy for reducing the toxicity of geldanamycin was to link it to estradiol so that it could be able to target ER specifically. 21 Similarly, geldanamycin was considered to connect to testosterone for targeting androgen receptor (AR). 22 These studies originally proposed a concept that a hybrid molecule could be able to mediate specific degradation of the targeted proteins. 20 Alternatively, attempts were made to use chimeric proteins from the SCF proteolytic machinery, a multimeric E3 ubiquitin ligase complex. 23

| PEPTIDE-BASED PROTAC TECHNOLOGY
Kathleen M. Sakamoto reported the first bifunctional or hybrid molecule named PROTAC, which recruits the ubiquitin-proteasome system, where an E3 ubiquitin ligase is linked to target proteins for degradation. 2 This collaborative group designed a chimeric molecule based on the angiogenesis inhibitor ovalicin, by linking to the IκB-α phosphopeptide. Since oyalicin covalently binds MetAP-2 (methionine aminopeptidase-2) and the phosphopeptide is recognized by the F-box, cMetAP-2 could be targeted by this hybrid molecule that recruits the E3 ubiquitin ligase β-TRCP. As was expected, their results showed that MetAP-2 was tethered to SCF complex (β-TRCP) and ubiquitinated for degradation. 2 Soon later, this group continued to employ this concept to design chimeric molecules to target ER and AR. 25 They synthesized a 10-aa IκB-α peptide covalently linked to estradiol (E2) or dihydroxytestosterone (DHT) and confirmed both hybrid molecules functioned in vitro and in vivo in cells. 25 These pioneer studies started the era for the peptide-based PROTAC technology ( Figure 3).
After the studies on ER and AR, Montrose and his colleagues used peptide-based PROTAC to target the cancer-forming X-protein from HBV. 26 The X-protein is essential for viral replication, with 154 aa residues, and is a major risk for patients with hepatocellular carcinoma (HCC) developed from chronical infection with HBV. They intended to induce a poly-arginine cell-penetrating peptide (CPP) so that the PROTAC is cell-permeable. They provided evidence that the homo peptide-based PROTAC destroyed the X-protein in HepG2 cells effectively. 26 Perseveringly, Crews group designed PROTACs using FKBP12 ligand and dihydrotestosterone to target FKBP12 and AR in a model cell. 27 They proved that this PROTAC-mediated protein degradation could be a general strategy to deplete proteins, which they called "chemical knockouts" of proteins. 27 At the same time, Kim group took advantage of the interaction between pVHL (von Hippel-Lindau) and HIF-1α (hypoxia-inducible factor 1α), and designed a PROTAC based on a peptide from HIF-1α. 28,29 They synthesized estradiol-HIF-1α octapeptide (Met-Leu-Ala-Pro OH -Tyr-Ile-Pro-Met) to successfully target ER in living cells. 28,29 They subsequently confirmed that this PROTAC targeted ER and was able to inhibit the differentiation of endothelial cells in a threedimensional angiogenic sprouting assay. 30 Kim group claimed their first report on the PROTAC that is permeable to cells since Crews group used microinjection to deliver the primary PROTACs. 2,25 The same year, Crews group searched for seven amino acids from HIF1-α that recognizes VHL, aimed to overcome the obstacle of membrane permeability. 27 For this purpose, a poly-D-arginine tag derived from HIV TAT was used to merge to the carboxyl terminus of the peptide to allow the hybrid macromolecule to confer cell permeability and prevent nonspecific proteolysis. 27 Interestingly, Kim group extended their study into using apigenin, which is a low estrogenic flavonoid phytochemical found in some special diets with anticancer features. 31 Their design consisted of apigenin, a linker, and an E3 ubiquitin ligase recognition motif (H2N-Leu-La-Pro OH -Tyr-Il2-OBn). They demonstrated that this apigenin-based PROTAC effectively degraded aryl hydrocarbon receptor (AHR) in living cells. 31,32 Beside the usage of E3 ubiquitin ligase for ubiquitin-mediated degradation by proteasome, Bauer et al subsequently adopted chaperone-mediated autophage (CMA), by synthesis of a pentapeptide (KFERQ) to link two different HSP70 binding motifs to direct mutant huntingtin protein for degradation. 33 Later on, Fan et al tried to recruit autophage system by a full peptide for the protein degradation. 34 They took advantage of CMA and designed PROTACs against death associated protein kinase 1 (DAPK1), scaffolding protein PSD-95, and a-synuclein. Their design included a pentapeptide CMA-targeting motif that recognizes autophage system, a linker containing cell membrane-penetrating domain (CMPD), and a peptide for recognition of targeted proteins. They confirmed that this homo multiple-peptide efficiently knocked down the targeted protein not only in the cultured cells but also in the brains of intact rats because of CMPD, which made the peptide permeable to plasma membrane and the blood brain barrier. 34 Obviously, these initial PROTAC technologies were based on the short peptide sequence to recognize an E3 ubiquitin ligase. Therefore, researchers named this PROTAC peptide-based PROTAC. 6,35 To date, different peptides were examined to recruit E3 ubiquitin ligases including PROTACs proved that induction of the targeted protein degradation is a potent way to inhibit the activity of the targeted proteins. However, the problem for these peptide-based PROTCs was due to their difficulty to permeate the cell membrane. That is the reason why Crews group initially used microinjection to deliver the PROTAC into living cells. 2,25 Sooner, Crews group used HIV tag to fuse the peptide and could be able to allow the PROTAC being transferred into the cell, 27 and Kim group directly synthesized a cell-permeable PROTAC. 28,29 Afterwards, the peptide-based PROTACs always recruited CPPs. 26,33,36 To show the biological function of the peptide-based PROTACs, Crews group examined their PROTACs on targeting ER (named Protac-B for ERα) and AR (named Protac-A for AR). Intriguingly, the designed two PROTACs demonstrated a great accuracy to AR and ER, as both Protac-A and Protac-B did not affect the proliferation of cells lacking ERα and AR. 37 Tang et al later on demonstrated that the DHT-PROTAC promoted AR degradation in LNCaP cells, confirmed the role of the PROTAC on ERα or AR positive cells. 38 They investigated the degradation of AR for the effect on cell proliferation and viability for prostate cancer cells sensitive to androgen. As expected, the DHT-PROTAC worked specifically on the androgen positive cells. 38 The peptide-based PROTAC against ERα was further designed effectively in a MCF-7 mouse xenograft model. 39 However, the activity of these peptide-based PROTACs was low and remained at the micromolar range. The main obstacle may be the poor cell permeability. It seems that the homo peptide based PROTACs, for instance PROTACs targeting Tau, were able to transport into the cell membrane because the addition of CPP (D-Arg [8][9] ). 36,40 Another problem for these peptide-based PROTACs is the size of the chimeric molecule, which could be recognized by immune system to produce antibodies.
This may damper the clinical applications in human as the produced antibodies may neutralize the effect of the molecule in vivo. Fortunately,

| SMALL MOLECULE-BASED PROTAC
The peptide-based PROTAC takes advantage of a specific peptide on the reorganization of a specific E3 ubiquitin ligase. The peptide is called a moiety of E3 ubiquitin ligase. It immediately draws the attention that a small molecule could be used as a moiety for recognizing an E3 ubiquitin ligase. Using small molecules as moiety of an E3 ubiquitin ligase led to the development of small molecule-based PROTAC ( Figure 4). 35 Small molecule-based PROTAC has many advantages over peptide-based PROTAC. 11 Most importantly, a small molecule-based PROTAC has more potential of being developed into a drug because a small molecule is easier for human body to absorb than a peptide.
Crews group turned their attention to generate this new generation of PROTAC technology. They were the first to link a nonsteroidal AR ligand (called selective AR modulator, SARM) to nutlin (a MDM2 ligand) by a Polyethylene Glycol (PEG)-based linker. 41 The MDM2 ligand nutlin is a set of compounds of imidazoline derivatives, which bound to MDM2 to block the interaction of MDM2 with p53. The synthesized SARM-nutlin PROTAC was shown to induce strong degradation of AR in HeLa cells 41 and in LNCaP cells. 38 Considering using hydropholic tags (HyTs) to make the binding protein denatured for degradation, several HyTs were synthesized to examine their effects on different Halo tag fusion proteins. This HyTs proved a concept that small a molecule may bind to a protein and makes the protein in a denatured state, which is then degraded by ubiquitin proteasome or autophage. 42 Simultaneously, Crews group replaced the HIF1α peptide with a small-molecule ligand, the hydroxyproline moiety, which retains a high affinity and is critical for VHL binding. 43 They synthesized PROTACs against ERRα, by incorporating a thiazolidndione-based ligand specifically binding to ERRα into the hydroxyproline moiety (selected one from five hybrid molecules). They next synthesized a PROTAC against the serine-threonine kinase RIPK2, by using the inhibitor vandetanib and the hydroxyproline moiety with a 12-atom linker. They assessed the PROTACs on the expression of ERRα and RIPK2 in MCF-7 breast cancer cells and human THP-1 monocytes and proved that one PROTAC molecule FIGURE 3 A schematic diagram of a peptide-based proteolysis targeting chimera (PROTAC). This PROTAC is composed of a short peptide that binds to E3 ubiquitin ligase and a small molecular that binds to target protein, respectively, followed by polyubiquitination and proteasome degradation of target FIGURE 4 A schematic diagram of the small molecule-based PROTACs. This proteolysis targeting chimera (PROTAC) consists of a ligand on an E3 ubiquitin ligase, a linker, and a ligand on targeted protein could be able to mediate the degradation of multiple molecules of RIPK2 via ubiquitin-proteasomal pathway. 43 Furthermore, this hydroxyproline derivatives were further used for the synthesis of HaloPROTACs to target HaloTag7 fusion proteins, by developing chloroalkane-containing PROTACs against Halo Tag7 fusion protein using the acyl amine moiety for recognizing VHL. 44 Many of the small molecule-based PROTACs have been developed intensively to target the BET family proteins. BRD4 inhibitors have been extendedly studied and shown promises in anticancer therapy against MYC-driven malignancies. The first effort was to link BET inhibitor JQ1 to a moiety of VHL. 45 The designed PROTAC named MZ1 dramatically induced degradation of BRD4. 45 In another test, BRD4 inhibitors were used to design a PROTAC named ARV-825, which links a BRD4 binding The small molecule-based PROTAC was further extended to the design of a PROTAC against oncogenic kinase BCR-ABL. 49 Inhibitors including imatinib, bosutinib, and dasatinib were linked to VHL E3 ubiquitin ligase ligand or pomalidomide (to recruit cereblon E3 ubiquitin ligase). 49 For targeting kinases, a PROTAC against CDK9 was designed by using CDK9 inhibitor and thalidomide for targeting cereblon. 50 To date, small molecule-based PROTACs have been generated to recruit MDM2, cellular inhibitor of apoptosis protein 1 (cIAP1), CRBN (cereblon), and, of course, VHL (for review, see Toure and Crews 51 ). 15,52 To overcome the shortage of insufficient membrane permeability and stability of the peptide-based PROTACs, Hashimoto group focused on using cIAP1, which promotes ubiquitination and proteasomal degradation of interacting proteins. [53][54][55] They recruited a class of bestatin ester analogues (MeBS, methyl bestatin), a ligand binding to the baculoviral IAP repeat domains of cIAP1, to all-trans retinoid acid to target CRABP-I and II (cellular retinoic acid binding proteins-I and II). 55 Thus, the cIAP-1-based PROTAC could be able to induce the ubiquitination and degradation of the intracellular CRABP-I/II proteins. Other cIAP1-based PROTACs were designed to cross-link inhibitor bestatin to small molecules of multiple targets, including Retinoic Acid Receptor (RAR), ER, AR, and TACC3. 54,56 However, because bestatin itself is not highly selective and lacks activity, the activity of multiple reagents of PROTAC is not high enough, and no candidate has entered animal experiments. Interestingly, Naito and Hashimoto named their designs SNIPER (Specific and Nongenetic IAPs-dependent Protein ERaser). 57 They kept SNIPER for their following studies on designing different hybrid molecules to target different proteins. [58][59][60][61][62][63][64][65] Unexpectedly, they deciphered that one of their PROTAC based on bestatin did not recruit cIAP-1 but instead APC/C CDH1 complex. 59 In summary, to date, different sets of small molecules have been developed as the moiety of E3 ubiquitin ligases including SCF, VHL, cereblon, MDM2, APC/C, and cIAP1. 35,51,56 For the limited space in this review, the discovery of the small molecules as the moiety of E3 ubiquitin ligases could not be able to descript in this review.

| TARGETING DIFFERENT PROTEINS FOR ANTICANCER DRUG DEVELOPMENT
To date, more than 30 proteins critical for the development of diseases were targeted, with a major effort on the proteins for cancer

| Targeting nuclear receptors
Both peptide-based and small molecule-based PROTACs were designed to target ER, AR, and later on RAR, which pioneered the field. 2,25,29, 54 We have described the detailed designs of the PROTACs against ER and AR in the above sections. 25,[27][28][29]37,41 Here, we intend to address some promising progress on the development of the PROTACa against ER or AR. One progress was to use 4-hydroxy tamoxifen (4-OHT) to link to methyl bestatin. 58 46 The effect of ARV-825 was further investigated to induce more apoptosis in CD34+ post-MPN sAML cells. 47 Specifically, ARV-825 treatment led to robust and sustained depletion of BRD4 downstream genes including c-Myc, CDK4/6, JAK2, pSTAT3/5, PIM1, and BclxL, but stronger increases of the levels of p21 and p27. 47 These results suggested that PROTAC against BRD4 functions much better and the inhibitor of BRD4.
Slightly differently, Zengerle et al tethered JQ1, another inhibitor for BET family proteins, to a ligand for VHL. They observed that this PROTAC triggered in the intracellular destruction of BET proteins, preferably to BRD4. Intriguingly, the PROTAC not only led to a rapid, effective, and prolonged degradation of the BET family proteins but also caused the change of MYC, p21, and AREG, downstream of BRD4. 45 Since BET proteins are critical for the expression of NF-kB activated genes, a group reported that PROTACs against BET proteins dampened the pro-inflammatory response in microglia after Lipopolysaccharide (LPS) challenge. 69 Raina and his colleagues demonstrated that ARV-771, another small molecule-based PROTAC using pan-BET inhibitors, dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. 68 Interestingly, ARV-771 suppressed both AR protein level and AR signalling. This study provided evidence that a small molecule-based PROTAC functions in a solid-tumour malignancy of CRPC. 68 Further studies showed that PROTAC ARV-771 treatment reduced leukaemia burden and improved survival of HEL92.1.7 cellsengrafted NSG mice, better than the effect from OTX015. 47 The effects of PROTACs-based on BETi, ARV-825, and ARV-771  The group reported that their PROTACs specifically decreased BRD4 downstream genes, including c-MYC and N-MYC. Notably, they showed that PROTACs overcame the drug resistance from bortezomib, dexamethasone, lenalidomide, and pomalidomide. 73 They finally showed that PROTACs were able to induce a rapid loss of viability of primary cells from myeloma patients and inhibited the growth of MM1.S-based xenografts in mouse. 73 The PROTACs against BRD4 could also be improved by modification of hydroxylation of proline, which resulted in a PROTAC with over 100-fold activity compared with conventional one. 74 However, off-targets were reported recently. 75 A recent study extended designs of PROTACs against TRIM24, another bromodomain-containing transcriptional regulator. 76 This again encouraged to search for new path to undruggable targets.

| Targeting protein kinases
Other proteins other than the BET family proteins have been also targeted by PROTACs. In 2016, a PROTAC was designed to target Akt using protein catalysed capture (PCC) agents to target a cellpenetrating enzyme (Botulinum Neurotoxin Serotype A). They conjugated the PCC agent to a cell penetrating peptide HIV TAT peptide to allow an effective intake by cells. They further inserted two PEG spacers on both sides of a protected-lysine residue. Basically, this PROTAC used 7 aa from HIF-1α degradation peptide, ALAPYIP. This PROTAC was shown to promote the rapid degradation of Akt in live cancer cells. 77 Next, Lai designed a PROTAC to target c-ABL and BCR-ABL by recruiting either cereblon or Von Hippel Lindau E3 ubiquitin ligases.
They used inhibitors imatina, bosutinib, and dasatinb. During their study, Lai optimized the PROTAC development and proposed that both the target ligand and the recruited E3 ubiquitin ligase should be varied. 49 In 2017, Robb successfully targeted CDK9, a ubiquitously expressed kinase that contributes to a variety of malignancies. This PROTAC used cereblon (CRBN) to mediate proteasomal degradation of CDK9. The authors examined this PROTAC in HCT116 cells and observed that it selectively degrades CDK9 without affecting other CDK family members. 50 More PROTACs on CDK9 were developed by using a natural product Wogonin, which is similar to CDK9 inhibitor Flavopiridol. 78 In 2018, Zhang reported their design of a PROTAC against ALK by using ALK inhibitors. These PROTACs against ALK were named MS4077 and MS4078. They showed that the PROTACs significantly decreased cellular levels of ALK fusion proteins in different cell lines including SU-DHL-1 (lymphoma) and NCI-H2228 (lung cancer). 79 Another group reported their design on ALK PROTAC using small molecule as ligand to connect E3 ubiquitin ligase. 80 Kang et al later on proved that a synthesized PROTACs against ALK (based on VHL) worked in vivo. 81 It seemed that the PROTACs are good for mouse pharmacokinetic study for in vivo efficacy test. 73 The designed PROTAC against ALL also promoted the degradation of other kinase such as PTK2, Aurora A, FER, and RPS6KA1. 80 To date, PROTACs targeting RAR, 54 PI3K, 82 91 and TBK1 92 are also reported. 93 Most of the proteins are cellular located or nuclear located. However, for the receptors such as tyrosine kinase receptors (EGFR), it remains to question whether a PROTAC works or not. To examine this possibility, Crews group conjugated an EGFR binding element (Iapatinib) to a ligand of VHL for targeting EGFR, HER2, and c-Met. 94 Interestingly, the PROTAC mediated the internalization of EGFR and sorted to lysosomal degradation, 94 although the RTKs usually prefer to internalize into a recycle endosome. 95

| REMARKS
Although it is very promising to use PROTAC for drug development, it remains of many concerns about the clinical application. These concerns include the off-target, cellular permeability, stability, and large molecular weight. Another problem is the difficulty of synthesis of the hybrid molecule, including optimizing the linker length and composition. The good news is that many groups started to overcome these problems by different ways. 7,11,35,96 A new strategy for shortening the PROTAC, click-formed PROTAC (TCLIPTAC), is to separate the macrohybrid molecule into two parts, a tetrazine tagged ligand for target and a trans-cyclo-octene tagged ligand for E3, which are able to be "clicked" together in the cells to form a PROTAC. 97 This click reaction also provides an easier way to the synthesis of PROTACs in vitro. 98 A plausible feature for the PROTAC technology is its potential for development of drugs on the undruggable proteins. 14,93 However, the current successful PROTACs still largely used small molecules to target the druggable proteins with their inhibitors or ligands. This is mainly because the small molecules have good features of binding the targeted proteins. To date, it remains of an obstacle for the discovery of small molecule moiety to different targets. One direction is to find a peptide epitope based on protein-protein interaction. This will open a broad way for the discovery of new drugs.