Epigenetic Proteins: The Significance of PROTAC on Diseases (II)
PROTAC for epigenetics research
Different types of epigenetic enzymes can promote different cellular processes and pathogenesis by modifying gene expression and protein function. For example, “Writers” can connect chemical groups to specific amino acid residues of histone and non-histone proteins. Basically, “Erasers” reverse this reaction by cutting off the epigenetic modification. “Readers” (such as transcription factors) will specifically recognize the modified residues and guide the protein to a specific modified area. Currently, all three epigenetic proteins have related PROTAC designs.
- PROTAC targeting “Readers”
Most PROTAC research in the field of epigenetics is about Reader protein BRD4 (member of BET protein). The BET family mainly makes these proteins specific for targets by recognizing acetylated lysine residues of histones and non-histones. Since BRD4 can cause cancer and inflammatory diseases, several BET protein inhibitors have been reported in recent years, but its poor selectivity limits its clinical application.
BRD4 is the first epigenetic protein that uses chemically induced protein degradation. Three research groups reported on three different BRD4-targeted PROTACs (dBET1, ARV-825, and MZ1), which showed similar degradation effects in the submicromolar range and all relied on JQ1 as the ligand structure for BRD4 binding. But Linker’s design is different from the recruited E3 ligase. Both dBET1 and ARV-825 target CRBN and MZ1 targets VHL, but the selectivity of dBET1 is poor. In recent years, PROTAC for BRD4 has been further optimized to make it more selective and degradable. For example, the effective degradation concentration of QCA570 has been as low as the picomolar range, and it can significantly inhibit the growth of cancer cells.
BRD9 is less studied in the Reader proteome. Apart from being a component of the self-integration barrier factor (BAF) complex (SWF/SNI complex), its function is not fully understood. Advanced BRD9 degraders can significantly inhibit the proliferation of different cancer cell lines. Zoppi et al. synthesized nanomolecule efficient dual-degradation agents for BRD7 and BRD9 based on modified BRD9/7 inhibitors and VHL ligands.
The chromatin-related Reader protein TRIM24 is a member of the TRIM/RBCC family and is an important transcriptional regulator. High levels of TRIM24 are associated with cancer, but TRIM24 inhibitors cannot affect cancer cells. Interestingly, the TRIM24-targeted PROTAC designed based on the TRIM24 inhibitor IACS-9571 and VHL ligands can effectively inhibit the proliferation of acute leukemia cells.
A dual PROTAC (ACBI1) has been released for the degradation of SMARCA2/4, which is an important member of the BAF complex containing bromodomain proteins. Ciulli’s research team used a structure-based approach to optimize its degradation molecules, which consist of SMARCA bromodomain inhibitors and VHL ligands. Contrary to SMARCA inhibitors, PROTAC can reproduce the phenotypic effects of knockout experiments, and therefore, can promote the study of the function of SMARCA and BAF in cancer cells.
- PROTAC targeting “Erasers”
To date, only PROTAC for histone deacetylase (HDAC) has been developed in the Eraser proteome. HDAC plays an important role in regulating chromatin structure, transcription factors and non-histone functions. Over the past few decades, effective HDAC inhibitors have been discovered, five of which have been approved for use as therapeutic drugs. However, most of these inhibitors are non-selective, leading to off-target toxicity and side effects.
In 2017, the team of researchers applied PROTAC technology to Sirtuin2 (Sirt2), which is a member of NADþ-dependent Class III HDAC and can participate in a variety of cellular processes, including transcription, signaling, metabolic pathways, aging, apoptosis and inflammation . Sirt2-PROTAC consists of SirReal Sirt2-based selective ligands and thalidomide-based CRBN recruitment ligands. Compared with the enzymatic inhibition of SirReal inhibitors, Sirt2-PROTAC can achieve the isoform selective degradation of Sirt2 and more significant tubulin acetylation in Hela cells.
Zinc-dependent type IIb HDAC6 is mainly located in the cytoplasm, and is mainly involved in cell movement and immune regulation by modifying non-histone proteins (such as α-tubulin and HSP90). Yang et al. first reported the targeted degradation of HDAC6 in 2018. Unexpectedly, the use of pan-HDAC inhibitors as PROTAC’s target protein binding ligands can achieve selective degradation of HDAC6 in cancer cells, which again emphasizes the importance of the stability of the ternary complex for the selective degradation of target proteins.
- PROTAC targeting “Writers”
The p300/CBP related factor (PCAF) and the key factor for histone acetylation modification GCN5 are multi-domain proteins with acetyltransferase domain and bromodomain. These proteins have important roles in DNA damage repair, cell proliferation and differentiation, and metabolism and immune pathways. The inhibitory effect of the reported inhibitors on the bromodomain of PCAF/GCN5 is insufficient to mimic the effect of protein levels in macrophages and dendritic cells, and functional analysis of these proteins in cells is not possible. The Tough team is addressing this issue by developing a PCAF/GCN5 degradation product (GSK983) that targets the bromodomain. They combined the PCAF/GCN5 inhibitor GSK4027 with the CRBN ligand to achieve a high-efficiency degradation of PCAF/GCN5 in the nanomolar range. In addition, they found that inhibiting the bromodomain with small molecule inhibitors is not sufficient to affect the differentiation of monocytes, but requires the use of PROTACs to completely degrade proteins.
Although PROTAC technology has made significant progress in recent years, it still faces many problems. Theoretically, we should continue to study the structure of Linker and the ternary complex, further clarify the protein interactions in the complex, and better evaluate the role of Linker in pharmacodynamics and pharmacokinetics. To date, the available E3 ligase is also very limited, and the expansion of E3 ligase may also lead to the creation of new selective PROTAC.
At present, many PROTACs have been designed in the field of epigenetics, and further research on the function of epigenetic eggs in diseases is conducive to the discovery of potential drug candidates.