Science depth: a new idea for cancer targeted therapy "degrading waste protein"

Release date: 2017-04-12

Among the current cancer-targeted drugs, small molecules or monoclonal antibody inhibitors against oncogenic proteins are dominant. However, over time, many cancer cells become resistant to such drugs, such as generating new mutations or activating other oncogenic proteins. Over the past 15 years, some researchers have targeted the “cleaners” of the cell, the ubiquitin-proteasome system, when looking for other anticancer pathways. The ubiquitin-proteasome system is responsible for cleaning up unwanted or harmful proteins in cells. By activating this system-specific cleanup of oncogenic proteins, it is expected to restore protein homeostasis and prevent cancer. Recently, Science published a long article, reviewing and looking forward to the development of anticancer drugs targeting ubiquitin-proteasome.

A new model of anticancer drugs: induced protein degradation

Many cancer-targeted new drugs target oncogenic proteins. These small molecule drugs usually bind to the active sites of those oncogenic proteins to inhibit their activity. For this mode of drug, continued binding to the active site of the protein is necessary to maintain its efficacy. Many of these oncogenic proteins are kinases with structurally similar active sites, and the use of high doses of small molecule drugs is likely to cause non-specific side effects.

â–²Anticancer drugs acting on the mechanism of protein degradation (Source: Science)

Induced protein degradation is a new anti-cancer mechanism that has recently emerged. In theory, only small molecule drugs need to be combined with oncogenic proteins for a short time, and the oncogenic protein should be labeled as “need to clean up”. These drugs do not require high concentrations, can be recycled, and proteins need to be re-synthesized to restore function after degradation, which greatly delays the development of resistance.

The ubiquitin-proteasome system degrades the protein into several steps. The E3 ligase first binds the target protein with a ubiquitin protein tag, and then the protein undergoes multiple rounds of ubiquitination with multiple ubiquitin tags. The protein after polyubiquitination is recognized by the 26S proteasome and is degraded.

Small molecule drugs that simultaneously inhibit protein activity and promote protein degradation

Studies have shown that some small molecule drugs used to inhibit the activity of oncogenic proteins can also promote the degradation of target proteins. For example, canertinib is an inhibitor of the tyrosine kinase ErbB-2, and ErbB-2 is expressed in a variety of cancers, while canertinib can increase the polyubiquitination of ErbB-2 to promote its degradation. The fulvestrant, an inhibitor of ERα, is another example that also promotes the degradation of ERa. In breast cancer patients with ERa, fulvestrant is more effective than another ERα inhibitor, tamoxifen, which does not promote protein degradation. The arsenic trioxide, a very effective drug for acute promyelocytic leukemia, can also promote the degradation of the fusion oncogenic protein PML-RARα, which is common in patients with this disease.

â–²Canertinib (left), fulvestrant (middle) and lenalidomide (right) molecular structure

(Source: Wikipedia)

The recently developed immunotherapy drug lenalidomide for multiple myeloma has also been found to induce ubiquitination and degradation of kinase CK1α and two transcription factors Ikaros and Aiolos, which are important in multiple myeloma. The success of these drugs has sparked interest in the industry as a whole for drugs that promote protein degradation. However, people accidentally found that these drugs have the function of increasing protein degradation. How to design drugs specifically for protein degradation systems remains a challenge.

Protein cleavage targeting chimera (PROTAC) technology

In the natural case, E3 ubiquitin ligase requires a special recognition signal to recruit and ubiquitinate its target protein. The emergence of PROTAC technology has made E3 ubiquitination of any protein possible. This technique designs a dual-function molecule that binds to the target protein at one end and E3 ligase at the other end, and forms a polymer. At this point E3 is able to ubiquitinate the target protein and direct it into the degradation pathway.

â–²E3 ligase complex (Source: "Biochemical Journal")

The first generation of PROTAC molecules successfully ubiquitinated and degraded estrogen and androgen receptors, which play important roles in breast and prostate cancer, in in vitro experiments. However, the first generation of PROTAC is a polypeptide that is not very effective due to its low efficiency in penetrating cells. Subsequent research focused on the development of the true small molecule PROTAC, an effort to achieve this through a small molecule ligand, nutlin, of E3 ligase. The androgen receptor was selectively degraded in the cervical cancer cell line HeLa by linking nutlin to a small molecule modulator of the androgen receptor. Other research teams have successively synthesized small PROTAC molecules that selectively degrade oncogenic proteins HIFα, ERRα and RIPK2.

Future development of PROTAC

In summary, the PROTAC molecule has three basic components: a ligand for the target protein, a ligand for the E3 ligase, and a linker. A successful PROTAC molecule needs to optimize all three components. Although there are more than 700 different E3 ligases in humans, several of the major enzymes in cancer cells already have small molecular ligands. For example, the ligands for CRBN include thalidomide, lenalidomide, and pomalidomide, ligands for cIAP1 and MDM2. There are methyl bestatin (MeBS) and nutlin.

In addition to the ligand of the target protein and the ligand of the E3 ligase, the linker between the two is also very important, and its length, degree of hydrophobicity and rigidity are important for the function of PROTAC. Recent studies have shown that a small change in the linker can also have a large impact on the function of the entire PROTAC.

â–²PROTAC technology basic model (Source: "ACS Chemical Biology")

BCR-ABL is the leading cause of chronic myeloid leukemia, a carcinogenic tyrosine kinase produced by fusion. Although small molecule drugs including imatinib, bosutinib, and dasatinib have been shown to inhibit their activity, some patients develop resistance. Therefore, based on these drugs, researchers have attempted to manufacture PROTAC by linking these drugs with the ligand pomalidomide of CRBN or another small ligand of ligase VHL. They tested four different binders, one of which failed to affect binding to BCR-ABL, and the proatinb-based PROTAC successfully incorporated BCR-ABL but did not induce degradation. Dasatinib-CRBN and bosutinib-CRBN achieved the best results and successfully degraded BCR-ABL, but bosutinib-VHL failed to do so. These data show that changes in any of the three components of PROTAC change the function of the entire molecule.

Other strategies for inducing protein degradation

In addition to PROTAC, there are potential strategies for developing small molecule drugs that promote protein degradation. One of them is an analog of PROTAC, a small molecule called SNIPER PROTAC that directly fuses the ligand of the target protein with the ligand of the E3 ligase. An example of this is a hybrid based on the cIAP1 ligand MeBS and the retinoic acid receptor ligand all-trans retinoic acid (ATRA). It can direct cIAP1 ubiquitination and degrade intracellular retinoic acid-binding proteins, which play important roles in acute promyelocytic leukemia and neuroblastoma. Similarly, using SNIPER technology, targeting ERα in breast cancer by tamoxifen has also achieved good results in in vitro experiments.

In addition, it is also an option to attract a ubiquitin-proteasome system based on hydrophobic groups on the protein. Usually the hydrophobic part of the protein is embedded inside the protein, and the hydrophobic group will only be exposed when the protein is not properly folded. E3 ligase is able to automatically recognize these hydrophobic groups and degrade these abnormally folded proteins. Thus, by adding a hydrophobic group to a small molecule ligand of a target protein, an E3 ligase can be introduced. Research teams have applied this idea to create small molecule drugs that degrade HER3 and androgen receptors.

â–²A variety of PROTAC small molecules and analogues (Source: "Biochemical Journal")

Candidate drugs in preclinical experiments

Efforts to specifically and efficiently degrade key oncogenic proteins have made great strides in the past two years. Proteins of the BET family, including BRD4, play an important role in the development of a variety of cancers including acute myeloid leukemia, multiple myeloma, ovarian cancer, and prostate cancer. Multiple BET small molecule inhibitions have entered the clinic, but these drugs are generally effective due to insufficient inhibition of downstream signaling pathways and feedback mechanisms that up-regulate the expression of the BRD4 gene.

A team at Yale University in the United States designed a small molecule PROTAC drug, ARV-825, which completely degrades BRD4 protein in cell experiments by linking BRD4 and CRBN. The team and a biotechnology company, Arvinas, also developed ARV-771, which degrades the BET family of proteins through VHL. Another team from the Dana-Farber Cancer Institute in the United States designed another small molecule PROTAC drug, dBET1, based on the BET-based small molecule inhibitor JQ1, which is used in the mouse acute myeloid leukemia model. Shows better results than JQ1.

Conclusion

The most attractive aspect of targeted protein degradation is that it can target protein targets that are traditionally considered to be inoperable, and these proteins may account for more than 80% of the human proteome. Since the targeted protein degradation strategy can achieve the goal of selectively degrading proteins by binding to almost any site on the protein rather than the active site, in theory this strategy can be applied to any protein. In addition, another advantage of this strategy is that it can work on tumors that have developed resistance. Finally, targeted protein degradation has considerable potential in diseases other than cancer.

We wish this new treatment idea to bring innovative drugs to the benefit of the majority of cancer patients.

Reference material

[1] Waste disposal—An attractive strategy for cancer therapy

[2] Stocking oncology's medicine cabinet

Source: WuXi PharmaTech (WeChat: WuXiAppTecChina)

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