The story continues from the last time【What is the future of PROTAC/molecular glue? Current status of new E3 ligase and E3 new ligand development】
This time, let me introduce a leader in the development of new E3 ligands in China: HitGen.
According to HitGen's earlier introduction:
The targeted protein degradation platform of HitGen utilizes the company's trillion-level DNA-encoded compound library to screen certain E3 ligases, validate the binding of novel ligand molecules to E3 ligases, and demonstrate that these ligands have good degradation capability for popular protein degradation targets.
CRBN
First is CRBN, involving 4 patents.
It can be seen that HitGen indeed obtained a new skeleton CRBN ligand through DEL and structural modification. Compared to the key group of thalidomide derivatives, the key group of this type of ligand is clearly the spirocyclic structure containing succinimide (red box in the image above).
Currently, the Kd values of the three commonly used amide compounds are all above 100 nM. In comparison, HitGen's new scaffold CRBN ligand has improved the affinity to about 10 nM.
HitGen also provided comparative data, with its representative ligand mostly being A3 from WO2024051664A1. It also presented a BRD4 PROTAC designed based on A3, which has better degradation activity compared to dBET1, consistent with its promotional claims.
However, this comparative data clearly involves some sleight of hand, turning cause into effect.
Firstly, the conformational changes of the E3 ligands and the linker will affect the degradation activity of PROTACs. The design of thalidomide-based PROTACs can also achieve excellent degradation activity through optimization (see the right side of the figure below). It is difficult to intuitively compare the degradation activities of PROTACs based on the two, not to mention that the formation of the ternary complex mediated by PROTACs does not have high requirements for the affinity of the E3 ligands.
However, what can be intuitively evaluated is the drug-like properties of the two. It is well known that currently, medicinal chemists are focusing on optimizing PROTAC by reducing molecular weight and finding a fine-tuning balance between activity and overall pharmacokinetic properties, while the molecular weight of A3 molecule 354 has no advantage compared to the current new generation of thalidomide-like CRBN ligands (the molecular weight of PG is only 189). Based on this, the overall molecular weight of PROTAC is too large, which can lead to potential solubility, metabolic absorption, and many other drug-like property issues. Personally, I am not optimistic about this.
The above viewpoint also applies to molecular adhesives, as seen from the patents published by Monte Rosa, which also choose to reduce molecular weight in drug-like optimization.
Of course, the new skeleton CRBN ligands led by HitGen are not without merit, and the new skeletal structure is bound to lead to new protein binding conformations. This may enable the design of molecular glues to recruit new targets.At the same time, these new spirocyclic structures containing succinimide may also be the core group for future rational design of molecular glues..This point can also be glimpsed from a recent article featuring many experts (https://doi.org/10.1101/2024.09.30.615685). The article reports on the ENL molecular glue dHTC1, whose CRBN recruiting fragment is the spirocyclic structure containing succinimide.
BIRC7
First, let's discuss the tissue distribution of BIRC7.
From the database, BIRC7 is indeed a very tissue-specific E3 ligase, highly expressed only in SKCM (skin melanoma) and UVM (uveal melanoma) in tumors, which is consistent with the introduction of HitGen.
In normal tissues, BIRC7 is also highly expressed in the skin, with expression levels second only to the placenta.
The data provided by HitGen also indicates that BIRC7 is highly expressed in the skin. The BRAF PROTAC designed based on BIRC7 ligands only shows BRAF degradation in cell lines with high BIRC7 expression, while it has poor degradation capability in cells with low BIRC7 expression.
These features indicate that for BIRC7 high-expressing melanoma, BIRC7-based PROTACs may have better safety and tolerability.
So, what does the BIRC7 ligand look like?
From the recently published patent by HitGen WO2024165017A1, the patent discloses a series of compounds, some of which have good BIRC7 affinity activity (0.1 ~ 1 μM). There is not much information revealed, and some representative structures are as follows.
TRIM21
TRIM21 is a broadly expressed E3 ligase, but according to HitGen's materials, TRIM21 has a higher expression level in tumors than in normal tissues, while CRBN has similar expression levels in tumors and normal tissues. This also suggests that TRIM21-based PROTACs may have better safety compared to CRBN PROTACs.
As for the statement that TRIM21 PROTAC has better degradation activity than CRBN PROTAC, I still hold a reserved attitude.
In addition, it is worth noting that TRIM21 ligand 652-NX-1 exhibits anti-tumor activity in certain tumor cell lines, which also indicates its potential molecular glue properties.
According to patent WO2024179572A1, the representative embodiment has excellent TRIM21 affinity activity (KD = 1~10 nM), as shown in the figure below.
From the database, TRIM21 has PPI with many proteins, and this type of compound has a relatively simple structure and a wide range of development directions, making TRIM21 a potential high-value E3 ligase for molecular glue development.
However, if there are readers interested in this, the primary difficulty in developing TRIM21 lies in the fact that HitGen has not provided the binding conformation of 652-NX-1 with TRIM21, as well as the specific cell lines that exhibit anti-tumor activity.
Of course, the solution is also simple: either contact HitGen to seek joint development cooperation to obtain this key information, or you can only conduct a co-crystal analysis, followed by high-throughput screening, high-resolution mass spectrometry, and proteomics analysis, which will provide some insights.
Epilogue
The currently published results from HitGen are indeed impressive, which may also be attributed to its powerful DEL screening platform. Looking forward to more outstanding progress in the future.
After all, compared to some domestic companies that are limited to targets like IKZF1/3 and GSPT1, being able to focus on basic research in the TPD field is quite rare.
In addition, according to its statement, other patents such as MDM2 (high expression in tumor tissues) ligands and ABTB1 (high expression in lymphatic and thyroid tissues) ligands have not yet been disclosed, and these can only await further reports.
Finally, I would like to clarify that this article is merely a summary of the latest advancements in E3 ligands, and HitGen has not provided any funding support for this article (since we are already at this point...).
If you’re exploring new E3 ligase binders, DEL could be the game-changer you’re looking for. We can connect you with a well-established, cost-effective DEL provider to accelerate your research. Feel free to reach out – we’re here to help.
you can comment at bottom of the article or contact the author (https://www.linkedin.com/in/haixiang-pei-1a40b82b0/) on LinkedIn
As for other developments in this field, stay tuned for the next installment.
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