PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands. It contains an “anchor” binding to an E3 ubiquitin ligase, a “warhead” binding to a POI, a chemical linker.
How PROTACs work?
Upon entry into the cell, the PROTAC molecule has a target POI ligand that binds specifically to the corresponding target protein. While the other end can recruit E3 ligase to form a POI-Linker-E3 ligase ternary complex. E3 ligase mediates the ubiquitinylation of POI by ubiquitin-binding enzyme E2. The ubiquitin-labeled POI is recognized and degraded by the proteasome. This process eliminates the need for the target protein ligand to occupy the binding site for a long period of time. And it requires only a brief formation of the ternary complex to ubiquitinate the target protein instantaneously. PROTAC can be recycled multiple times in the cell.
Advantages of PROTACs
PROTAC technology combines the advantages of gene therapy’s ability to degrade disease-causing proteins. target non-druggable proteins, potential tissue-specific targeting. And small molecule drugs that can be administered by multiple routes, broad tissue permeability, and ease of preparation.
- A broader range of action, higher activity, and the ability to address “non-druggable” targets
- Improved selectivity, activity and safety
- Overcome drug resistance
What Are PROTAC Linkers?
PROTAC linker plays a vital role in efficient ubiquitination of the target protein and its ultimate degradation. The structure and length of the linker play a crucial role. Because it can influence the overall PROTAC conformation and binding orientation as well as the formation of ternary complexes. Thus, the role of the linker goes far beyond simply connecting two molecular entities. It directly affects the activity, selectivity and physicochemical properties of PROTAC. To date, there are several kinds of linkers in PROTAC ternary complexes formation. Such as PEG linker, Alkyl linker, alkyne linkers, and click chemistry linker, etc.
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Clinical Research on PROTACs
Currently, several PROTACs have entered clinical trials, and some of them have shown encouraging results, such as ARV-110 and ARV-471.
Introduction of ARV-110
ARV-110, an oral protein degradation agent, binds AR specifically and mediates its degradation (Neklesa et al., 2018). ARV-110 completely degraded AR (DC50 < 1 nM) in all tested cell lines (Neklesa et al., 2019) and Oral ARV-110 (10 mg/kg) significantly inhibited the growth of enzalutamide-insensitive tumors in the PDX model (Wang et al., 2020b). ARV-110 degrades clinically relevant mutant AR proteins and retains activity in a hyperandrogen environment.
The early reported data (by January 2020) from the first-in-human. Phase I study of ARV-110 demonstrated its safety and tolerability in patients with metastatic castrate-resistant prostate cancer (mCRPC). Administer ARV-110 to 18 patients at four doses. Including 35mg (N=3), 70mg (N=4), 140mg (N=8) and 280mg (N=3). Among them, 12 patients received ARV-110 combined with enzalutamide (ENZ)/abiraterone (ABI), and 14 patients received prior chemotherapy. One patient administered ARV-110 280 mg experienced Grade (GR) 4 elevated AST/ALT. Followed by an acute renal failure while taking together with rosuvastatin (ROS). Similarly, another patient developed GR3 AST/ALT while taking ROS. Their plasma concentrations of ROS were increased accompanied by AST/ALT elevations, suggesting that concurrent ROS could produce toxic side effects. For other patients, there are no related GR 3/4 adverse events reported. Generally, ARV-110 possesses an acceptable safety profile. 15 of 18 patients were evaluable for prostate specific antigen (PSA) response. Of these, two patients had a PSA reduction of more than 50% (140 mg dose group). And both of them received prior therapy including ENZ and ABI, chemotherapy, bicalutamide, radium-223 and others.
Results of ARV-110
According to the recent interim clinical data released by Arvinas (https://ir.arvinas.com/). In the phase 1 clinical trial, ARV-110 shows promising activity in a very late-line mCRPC patients. With PSA reductions over 50% at doses greater than 280 mg. Previous studies have shown that multiple pre-treatments will lead to tumor resistance to targeted AR therapy. And improve the heterogeneity of tumor, resulting in a decreased efficacy of AR targeted therapy. Molecular biological analysis of patients treated with ARV-110 showed that 84% of patients carried non-AR gene mutations. Among the highly heterogeneous phase 1 patients, Arvinas has identified an advanced population. A molecular definition that has a particularly strong response to ARV-110. Of the five patients with T878 or H875 mutations in AR, two (40%) had a PSA reduction of more than 50%, including one with PR confirmed by RECIST and tumor size reduction of 80%. Additionally, two of 15 patients (13%) with wild-type AR also had PSA reductions over 50%. These results suggest ARV-110 has great potentials in molecularly defined population (T878/H875) and in wild-type patients.
What is ARV-471?
ARV-471 is an estrogen receptor (ER) alpha PROTAC molecule that degrades ER in ER-positive breast cancer cell lines with DC50 around 1 nM. It can decrease the expression of classically regulated ER-target genes and suppress the growth of ER-dependent cell lines (including cell lines expressing ESR1 variants such as Y537S and D538G) via degradation of ER. Oral administration of single agent ARV-471 (3, 10, and 30 mpk/day) shows significant anti-tumor activity in estradiol-dependent MCF7 xenografts along with ER protein reductions of over 90%. Excitingly, we observed more pronounced tumor growth inhibition (131% TGI) in the MCF7 xenograft model, accompanied by significant reductions in ER protein levels when combined with a CDK4/6 inhibitor palbociclib. Furthermore, the combination of ARV-471 and CDK4/6 inhibitor palbociclib showed great superiority over the combination of fulvestrant with palbociclib in tumor regressions. Also, ARV-471 (10 mpk) completely inhibited growth and markedly reduced mutant ER protein levels in ESR1 mutant hormone-independent PDX model (Flanagan et al., 2019).
Results of ARV-471
These promising results translate well into clinical trials. Recently, Arvinas has also announced ARV-471 for the treatment of locally advanced or metastatic ER-positive/HER2-negative breast cancer. And its phase I clinical trials have started in the second half of 2019 (https://ir.arvinas.com/). Analysis of the mid-term trial showed that ARV-471 could significantly reduce ER expression level in tumor tissues, with an average of 62% and a maximum of 90%. Moreover, ARV-471 could degrade both wild-type ER and mutant ER. According to the RECIST evaluation, one patient (a total of 21 adult patients) in the ARV-471 trial had a 51% reduction in target lesion size (confirmed PR). Two patients had unconfirmed PRs, and one additional patient showed stable disease, with a target lesion reduction of more than 50%. In the clinical benefit rate (CBR) evaluation, five of the 12 patients (42%) achieved CBR (defined as PRS + complete response + stable disease at 6 months).
Conclusion
Different from the traditional SMIs, PROTAC is a new strategy of inducing the ubiquitination degradation of target proteins. However, it is worth noting that up to now, less than 10 of more than 600 E3 ubiquitin ligases have been used to degrade target proteins, which reminds scholars to expand their knowledge in this area.
Perspectives
Although the PROTAC technology has made remarkable achievements since its development in 2001, there are still some problems in the process of design and application of PROTAC. For example, the effectiveness of PROTAC depends not only on the ligands of POIs and E3 ligases but also on the length and chemical properties of the linkers connecting the ligands. In addition, the binding strength of ligands, spatial orientation, cell permeability, and other factors will have important impacts on the efficacy of PROTACs.
Therefore, A key scientific question that remains to be addressed is how these factors work together to achieve maximum efficiency. Although the PROTACs can target protein for degradation, they cannot actively locate the target tumor tissue and may have off-target effects. It takes much time and manpower. So the application of new design strategies or technologies (e.g., CADD and AI) has huge importance in the rational design of PROTACs.
