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The Research of Proteolytic Targeting Chimeras (II)

BRD4 (bromodomain-containing protein 4) belongs to the BET family. The bromodomain and extra-terminal domain contains 4 proteins, namely BRD2, BRD3, BRD4 and BRDT. Among them, BRD4 is a very popular anti-tumor target in the field of epigenetics. The Crews team linked the target drug candidate OTX-015 to a small molecule PROTA by linking the polyethylene glycol chain with pomalidomide. The small molecule PROTAC ARV-825 was obtained by linking the polyethylene glycol chain with pomalidomide. ARV-825 was able to completely degrade BRD4 protein within 10 nmol·L-1 and 6 h, and its duration lasted longer than 24 h. Further tests confirmed that ARV-825 has the dual advantages of anti-tumor cell proliferation and induction of apoptosis compared with BRD4 inhibitors JQ1 and OTX-015, indicating that PROTAC can be more thorough than OTX-015 compared with small molecule inhibitors. It has the dual advantages of anti-tumor cell proliferation and induction of apoptosis, which shows that PROTAC can inhibit target protein activity more thoroughly and long-lasting than small molecule inhibitors. Winter et al. linked BET inhibitor JQ1 to thalidomide derivatives to obtain dBET1, which can induce 87% of BRD4 protein degradation in human leukemia cell line AML at 100 nmol·L-1. As expected, dBET1 also caused degradation of other BET family members (BRD2, BRD3) due to the lack of binding specificity of JQ1. The pharmacological effects of dBET1 were further evaluated in the anti-tumor cell proliferation test, and the results showed that the induction of BRD4 degradation could significantly inhibit the growth of the tested lymphoma cells, and its activity was better than JQ1. In addition, in a xenograft mouse model of human mv4-11 leukemia cells, continuous administration for 14 days can make the tumor regression. 24 hours after stopping the administration, BRD4 protein level began to recover. Interestingly, although ARV-825 is similar in structure to dBET1, it has a 10-fold stronger effect than dBET1 in inducing the degradation of BRD4, which may be mainly due to the difference between the two molecular linker chains, indicating the composition of the linker protein and its length is a key variable that determines PROTAC activity. The linker chain also affects the fat solubility and cell membrane permeability of PROTAC, which is an important module for PROTAC design and optimization. The highly active BRD4 inhibitor HJB97 was linked to a lenalidomide analog through a fatty chain to obtain a small molecule PROTAC ZBC260, which can degrade the BRD4 protein of the RS: 11 leukemia cell line at 30 pmol·L-1, which inhibits this leukemia. The IC50 value of cell proliferation was 51 pmol·L-1. Intravenous administration of small molecule PROTAC ZBC260 can make tumors of tumor-implanted mice regress.

PROTAC, a small molecule obtained by linking thalidomide and its derivatives with a suitable linker, can also successfully degrade target proteins. These targets mainly include anaplastic lymphoma kinase (ALK), cycle protein-dependent kinase CDK8 and cyclin-dependent kinase CDK9, etc.

Figure 4. Recruiting CRBN’s small-molecule PROTAC

Recruiting E3 ubiquitin ligase VHL

VHL is another E3 ubiquitin ligase widely used in PROTAC designs. Recent studies have shown that some small molecules can competitively bind to major HIF binding sites on VHL. The Crews team developed a small molecule PROTAC for targeted degradation of the serine / threonine kinase RIPK2. They linked the VHL ligand and the RIPK2 inhibitor via a polyethylene glycol chain to obtain the PROTAC-RIPK2 molecule that degrades the RIPK2 protein. The DC50 (chimeric dose required to reduce protein levels by 50%) was 1.4 nmol·L-1, and the DC95 value was as low as 10 nmol·L-1. Because the VHL ligand has strict spatial conformational restrictions, its epimer PROTAC-RIPK2-epi has no degradation activity. The sub-stoichiometric catalytic properties of PROTAC-RIPK2 were confirmed by in vitro reconstruction of ubiquitination experiments. The results showed that 1 pmol·L-1 of PROTAC-RIPK2 could induce 3.4 pmol·L-1 of RIPK2 to degrade, proving that PROTAC can function in the cycle.

Molecular VHL ligands have also been used in the design of PROTACs that target the BRD4 protein. Zengerle et al. linked the BRD4 inhibitor JQ1 to a VHL ligand and obtained two small molecules, PROTAC MZ1 and PROTAC MZ2. When both can induce the degradation of BRD4 within 24 hours, MZ1 has a shorter polyethylene glycol linking chain and shows a higher degradation effect. This finding once again proves that the composition and length of the linker chain is a key variable that determines the activity of PROTAC, and is an important module for PROTAC design and optimization. Interestingly, partial degradation of BRD2 and BRD3 was observed after 24 hours of use of these compounds, while PROTAC ARV-825 and dBET1 based on E3 ligase CRBN targeting BRD4 could be observed within 24 hours of use degradation. PROTAC has dual functions, which should meet the target protein and E3 ligase match, so as to effectively ubiquitinate and degrade the target protein, indicating that the type of E3 ligase can affect the sensitivity and selectivity of the target protein to PROTAC-induced degradation.

Figure 5. Recruiting VHL’s small-molecule PROTAC

In theory, PROTAC only provides binding activity, and the mechanism that exerts its effect is event-driven. It is different from the traditional occupancy-driven drug model and does not need to directly inhibit the functional activity of the target protein. Drugs do not need to bind to the target protein for a long time and high intensity, so they can target targets that are difficult to be drugged, such as proteins with smooth surfaces but lacking small molecule binding regions. PROTAC’s related technologies can selectively degrade target proteins. Therefore, many targets that cannot be regulated by small molecules or cannot be reached by antibodies can be regulated using this technology. In addition, this process is similar to a catalytic reaction. PROTAC can be recycled and reused to degrade the target protein, so there is no need to wait for the amount of PROTAC. Therefore, it is expected to obtain a highly active drug. Therefore, the industry believes that the requirements for the activity of PROTAC and target protein binding activity the requirements may not need to be high. Most small-molecule drugs exert their effects, and they need to maintain a certain concentration in the cells. Higher concentrations of small-molecule drugs can cause adverse reactions due to off-target, which greatly slows down the process of drug discovery, and PROTAC can overcome the above disadvantages.

PROTAC is a combination of the advantages of small molecule compounds and small molecule nucleic acids to a large extent. It can not only effectively target the target protein, but also degrade and clear it. It has a very broad application and development space. In theory, PROTAC only needs a catalytic number of drugs to achieve the purpose of degrading proteins, and it can also selectively degrade different proteins expressed by the same gene after protein expression and modification, so it has certain theoretical advantages.

Although PROTACs have broad prospects for drug development, existing problems hinder their clinical application. These problems include off-target, low cell permeability, instability, difficulty in synthesis, and large molecular weight. Traditional small- and large-molecule drugs that target proteins, even small nucleotides, generally only inhibit the activity of the protein, and in most cases do not affect the expression of the protein, which of course increases resistance. Probability of occurrence, but at the same time, residual activity may also protect normal cells, and in most cases will not affect protein expression. This, of course, increases the probability of drug resistance, but at the same time, residual activity may also protect normal cells, the basic physiological activities of cells, tissues and organs, on the other hand, may accidentally injure other off-target proteins. Even targets that have been previously verified may also cause more serious toxicity, which needs to be closely monitored in future clinical experiments. Another hidden danger is that the off-target effect of degraded proteins is not easy to detect and track in the preclinical toxicity screening, which increases the risk in the later development of drugs. In vitro and in vivo research data on PROTAC show that this technology has obtained satisfactory initial therapeutic effects in cellular and in vivo systems, but its wider use and application in clinical settings have yet to be tested.

References

[1] Mares Alina, Miah Afjal H, Smith Ian E D et al. Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2[J]. CommunBiol, 2020, 3: 140.

[2] Sun Ning, Ren Chaowei, Kong Ying et al. Development of a Brigatinib degrader (SIAIS117) as a potential treatment for ALK positive cancer resistance[J]. Eur J Med Chem, 2020, 193: 112190.

[3] Mukhamejanova Zere, Tong Yichen, Xiang Qi et al. Recent Advances in the Design and Development of anticancer molecules based on PROTAC technology[J]. Curr. Med. Chem., 2020, undefined: undefined.

[4] He Ling, Chen Chen, Gao Guoyu et al. ARV-825-induced BRD4 protein degradation as a therapy for thyroid carcinoma[J]. Aging(Albany NY), 2020, 12: undefined.

[5] Vogelmann Anja, Robaa Dina, Sippl Wolfgang et al. Proteolysis targeting chimeras (PROTACs) for epigenetics research[J]. Curr Opin Chem Biol, 2020, 57: 8-16.

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