Proteolysis targeting chimeras (PROTACs) in cancer therapy

Cancer is a multistep process in which genomic and epigenomic alterations lead to the abnormal cellular proliferation and dissemination [1]. Identification of molecular vulnerabilities that maintain the oncogenic phenotype has attracted major interest as the first step for the development of novel therapeutics [2].

A disbalance in the homeostasis of the protein production can be an oncogenic vulnerability in some tumors [3, 4], as demonstrated by the arrival of proteasome inhibitors to the oncology clinic [3, 4]. A novel class of agents that exploit the cellular protein degradation machinery with therapeutic purposes are the Proteolysis Targeting Chimeras or PROTACs [5]. These compounds can be used to facilitate proteasomal degradation of proteins that participate in the prooncogenic process. Importantly, PROTACs can be used to target a variety of proteins, including those with enzymatic activity or others difficult to target, such as those with scaffolding properties [3]. That is the case of transcription factors (TFs) (see glossary), which represent a large family of proteins against which very limited therapeutic options exist [6, 7]. TFs, as well as nuclear receptors, have been involved in the oncogenic generation of several malignancies. In fact, genomic alterations in c-MYC, FOXO1 or the androgen receptor (AR) have been described in neuroblastoma, breast or prostate cancer, respectively [6, 8]. A therapeutic strategy that has been contemplated is the reduction of the expression of these proteins by inducing their degradation. In this context, two PROTACs targeting the AR and estrogen receptor (ER) have reached the clinical setting being explored in two phase I studies in prostate and estrogen receptor-positive breast cancer [9].

In this review we will focus on strategies to increase protein degradation of druggable and undruggable targets focusing on PROTACs, describing the current stage, the main limitations and their potential for improvement.

Cell homeostasis depends on an accurate control of the quantity and quality of constituent proteins [10, 11]. This is more necessary in cells that have a high rate of turnover as they need to synthesize and consequently degrade proteins in an efficient manner [10, 11]. Protein degradation may occur in the lysosomes, by the action of acidic proteases that degrade proteins reaching these organelles [12]. Such situation is observed in the case of membrane receptors, that may increase their internalization and targeting to the lysosomes upon activation by their ligands or after binding to anti-receptor antibodies. This characteristic is exploited in the case of therapeutic antibody-drug conjugates (ADCs), a sophisticated evolution of anti-receptor antibodies. ADCs are composed of three components: an antibody against a protein expressed on the surface of tumor cells, a chemotherapeutic agent, and a linker that binds both. Upon interaction with the cell surface protein, the latter is driven to the endocytic lysosomal route. In the lysosomes, the ADC is proteolytically degraded facilitating the release of membrane-permeant cytotoxic drugs outside the lysosome to reach its cellular target. Acting on the lysosome has been less explored, although some recent preclinical studies have demonstrated the potential utility of this strategy, at least for the treatment of some proteinopathies [13].

Another major proteolytic system is the ubiquitin-proteasome degradation pathway, which relies on the ubiquitination of proteins, a process that triggers their degradation [14]. The targeting of an oncogenic protein or a protein not essential for the oncogenic phenotype but critical in the proliferation process, may also result in clinical benefit. This strategy needs ubiquitination of a target protein to subsequently be recognized by the proteasome [15].

Proteasomal-mediated protein degradation is an ordered multistep and sequential process, which requires several enzymatic reactions [16]. Ubiquitination is produced by three different steps: (i) activation by an E1 Ub-activating enzyme, (ii) conjugation mediated by the E2-conjugating enzyme, and finally (iii) ligation that is produced by the E3-protein ligases (Fig. 1) [17, 18]. In mammalian cells there are two E1 that can bind to forty E2s, which can bind with hundreds of E3s in a hierarchical way [19]. In the activation process, E1 enzymes bind both ATP and ubiquitin and catalyse the acyl-adenylation of the C-terminus of the ubiquitin molecule and transfer ubiquitin to a cysteine residue producing a thioester linkage between the C-terminal carboxyl group of ubiquitin and the sulfhydryl group of the E1 cysteine [17, 18]. E1-thioesterified ubiquitin is then ready to transfer the latter to a cysteine located in the active center of an E2 conjugating enzyme [20]. Finally, in the ligation process, E3 ligases act bridging the E2-ubiquitin and the substrate and create an isopeptide bond between a lysine of the target protein and the C-terminal glycine of ubiquitin. This last step provides the substrate specificity of the reaction as there are hundreds of E3 ligases, being the majority of them included in two families”: the HECT and the RING ligases [21, 22]. In addition, these ligases vary depending on cellular and tissues contexts, diversifying their protein substrates [23]. Rpn receptors present in the 19S unit of the proteasome help degradation of tagged proteins acting as binding sites [23]. Proteins entering the proteasome are then degraded by peptidases of the 20S region, resulting in the formation of fragmented proteins and the removal of ubiquitin from the protein being degraded [23].

PROTACs take advantage of the ubiquitin-mediated degradation system as a therapeutic strategy. A PROTAC is a molecule that consists of three parts: (i) a ligand (warhead) that interacts with the protein to be degraded, (ii) a different ligand that binds to an E3 ubiquitin ligase and (iii) a linker that connects both ligands (Fig. 1) [5]. The proximity between the E3 ligase and the protein target achieved by the heterobifunctional PROTAC facilitates ubiquitination and degradation of the protein target. First PROTAC compounds were developed more than 20 years ago using the E3 ligase TRCP [24]. Of note in this work the phosphopeptide ligand for TRCP did not penetrate the cellular membrane, limiting their development [24]. That aspect merits consideration as membrane permeation still represents a limiting step in the development of PROTACs. Later on, peptide ligands were changed to small molecules to avoid the low potency and to increase the cell permeability [25]. Nutlin-3a a ligand of the E3 ligase MDM2 was then used, showing capacity to degrade the androgen receptor [25].

Several structural studies have helped in the development of PROTACs. For instance, Van Molle and colleagues used a fragment based lead discovery approach to identify regions in VHL that are used for its interaction with the target protein hypoxia inducible factor1α (HIF1α) [26]. Crystal structures revealed a site of interaction of VHL with a 19 amino acid peptide derived from HIF1α. In silico and structural analyses identified three drugs that bound to the same site in VHL as the HIF1α peptide and acted as competitors of that protein-protein interaction [26, 27]. An ulterior study in the same experimental setting allowed development of drugs with better membrane permeabilization properties [28]. The above studies allowed identification of the groove on the surface of pVHL that was used to interact with a region of HIF1α, paving the way to the development of VHL-based PROTACs.

An important effort was made to develop warheads to target the nuclear hormone receptors AR and ER, generating a series of PROTACs which showed preclinical activity [29‐34]. Other warheads that were developed later included known tyrosine kinase inhibitors, or novel families of epigenetic agents like the Bromo and Extraterminal Domain inhibitors (BETi) [31, 33]. These proteins belong to super enhancer complexes that regulate the expression of TFs, indirectly modulating transcription initiation and elongation [35]. Most studies evaluating BET-PROTACs have been performed in leukemia and lymphoma, followed by some indications in solid tumors like prostate cancer, triple negative breast cancer or osteosarcoma (Table 1). BETi provided a therapeutic opportunity to target transcription, especially due to the difficulties in targeting TFs, which were considered as undruggable targets [43]. BETi have shown antitumoral activity in several haematological malignancies and solid tumors [43, 44], and BET-PROTACs were developed with the aim to boost and prolong the pharmacological effect of these agents, increasing their anti-tumoral activity. Beyond the activity of BET-PROTACs in different solid and hematologic tumors, these agents have also demonstrated activity in preclinical models that were resistant to BETi, suggesting that resistant tumors still depend on these proteins [38].

Additional PROTACs targeting other components of the transcription machinery include those based on inhibitors of CDK9 or SMARCA2/4 [45‐49]. PROTACs based on inhibitors targeting ALK or CDK6 kinases or based on inhibitors of the BCL6, BCL-XL or MCL1 apoptotic machinery components have also been developed [50‐55]. Supplementary Table 2 shows a complete list of PROTACs explored in preclinical studies.

Although many of these agents have been designed and evaluated preclinically, only two PROTACs, ARV-110 and ARV-471, have entered the clinical setting. ARV-110, a PROTAC designed to provoke degradation of the AR is being analysed in patients with castration resistant metastatic prostate cancer who have progressed on at least two prior therapies (enzalutamide or abiraterone, see clinicaltrials.gov reference NCT03888612). ARV-471, that provokes the degradation of the ER, is being explored in ER positive locally advanced or metastatic breast cancer (NCT04072952) [9]. Figure 2 describes all the chronological process for the development of this family of agents including information for each compound.

There is still much room to optimize this family of relatively new agents. For instance, it is relevant to mention that only 1% of the more than 500 E3 ligases have been explored for target degradation, and the selection of the E3 pairing seems to be critical [56]. Indeed, E3 ligases dictate target specificity [56]. Only few ligases have been explored, including CRBN, VHL, IAPs, MDM2, DCAF15, DCAF16 and RNF114, but only CRBN and VHL have shown activity in preclinical models [56]. Adequate optimization of the selection of the E3 ligases may increase efficacy and decrease potential toxicity of the PROTACs. One opportunity is based in their tissue specific expression. Thus, ASB9, a SOCS box E3 ligase, is only expressed in pancreatic and testis tissue, and FBXL16 is mainly found in the cerebral cortex, providing the possibility of acting on diseases affecting those tissues [56]. Similarly, if a ligase is only expressed in specific cellular components it can be used to degrade proteins located at that particular cellular site, as is the case for the use of the nuclear E3 ligase DCAF16 for nuclear targets [57]. Another example is represented by the action of CRBN against Ikaros and Aiolos, both nuclear proteins [3, 56].

Other parameters beyond the mere presence of the E3 ligase have to be taken into consideration when evaluating how active a PROTAC can be. Thus, ideally, the interaction between the ligand for the protein of interest and the E3 ligase should promote the formation of ternary complexes (Protein of interest-PROTAC-E3 ligase), leading to polyubiquitination of the protein and subsequent proteasomal degradation. However, in some circumstances such ternary complex may fail to be produced. That may happen, for example, in case the concentration of the PROTAC is in large excess with respect to the E3 ligase and the protein target. Since PROTACs are bifunctional molecules, they may independently bind to two molecules: the E3 ligase and the protein target. Desirably, one PROTAC molecule would act as a bridge between one E3 ligase and one protein target, creating a ternary complex. In case high concentrations of the PROTAC are present, it is possible the formation of binary complexes (protein target-PROTAC, or E3 ligase -PROTAC) that are ineffective [58]. In this circumstance, the right equilibrium is not achieved, since elevated PROTAC concentrations would saturate binding sites on the E3 ligase on one side and on the protein target on the other side, exhausting free forms of these proteins that could be used for ternary complexes. This process, produced when high concentrations of the PROTAC are present, is called the “hook effect” [58, 59]. It is relevant to mention that some PROTACs such as MZ1 may mitigate the hook effect as they exhibit positive cooperativity with respect to the assembly of ternary complexes.

The chemical characteristics of the linker can also affect the degradation capacity of the PROTAC. For instance the linker length can modify the degradation profile of lapatinib-based PROTACs targeting the EGFR and HER2 or only EGFR [60]. A similar finding was observed when different linkers were developed tethering JQ1 to VHL-1 showing that some PROTACs were able to degrade BRD2–4 and others were specifically selective for BRD4 [33].

Exploitation of the protein degradation machinery for therapeutic purposes opens new possibilities to target proteins involved in pathophysiological processes. Although important advances have been made using PROTAC technology, there are still many challenges for their clinical development. A crucial aspect is the selection of proteins which play a major oncogenic role in a certain tumor type, as is the case of the AR in prostate cancer or the ER in breast cancer. Optimization of the ligases used in the design of PROTACs for specific tumor tissues or cell types, or vectorization of the compounds with specific antibodies are strategies to be implemented and exploited. In addition, selection of the best combination with other therapies could reduce side effects augmenting activity. PROTACs are not limited to cancer therapy and they are under investigation in all diseases where an accumulation of proteins are important in their pathogenesis. That is the case in some neurodegenerative diseases or in conditions where degradation of a protein could have major impact than its enzymatic inhibition, as in the case of IRAK4 targeting in autoimmune diseases [81, 82]. Finally in situations where the target has a scaffolding role that cannot be inhibited by a conventional inhibitor or forms part of a hard-to-drug target, PROTACs could play a central role [82]. In conclusion, the first steps have been taken and offer hope for the incorporation of this family of agents in the clinic.