Nat Chem Biol Small Molecules Targeting Biomolecular Agglomerates: Jidong Zhu/Guangya Zhu team discovered a new strategy for targeting AR phase separation for prostate cancer

Prostate Cancer (PCa) is one of the
most common malignancies in men worldwide.
Androgen and androgen receptors (AR) play a key role
in driving the development and progression of prostate cancer.
Patients with early-stage prostate cancer can eliminate tumor cells through surgery or radiotherapy, and androgen deprivation therapy (ADT) is the most important treatment for patients with relapse or metastasis
.
Patients treated with ADT develop treatment resistance and develop castration-resistant prostate cancer (CRPC)
after early remission.
The activation of AR signaling pathway is still an important factor in the development of CRPC, and antagonists (or antiandrogens) targeting the AR ligand-binding domain (LBD) are currently an important means of
treating prostate cancer.
However, AR antagonists widely used in clinical practice, such as enzalutamide, will inevitably cause drug
resistance.
Among them, the important resistance mechanism is that the LBD domain is mutated, or the AR spliceosome AR-V7
that is missing the LBD domain is expressed.
Therefore, studying the resistance mechanism of prostate cancer cells to AR antagonists and finding new targeting strategies is of great clinical significance
for the treatment of advanced prostate cancer patients.

In recent years, researchers have found that transcription factors, coactivators and RNA polymerase II can mediate liquid-liquid phase separation ( IDRs) through their own disordered regions (IDRs).
LLPS), forming liquid transcriptional agglutination collectives involved in the transcriptional regulation
of downstream genes.
Therefore, liquid-liquid phase separation targeting transcription factors is expected to be an effective strategy
for the development of small molecule drugs for "difficult-to-target" proteins such as transcription factors.
More and more studies have shown that abnormal liquid-liquid phase separation of proteins is closely related
to the occurrence and development of major human diseases including tumors, metabolic diseases, and neurodegenerative diseases.
However, the role of liquid-liquid phase separation of proteins in the process of drug resistance has not been well studied
.
Elucidating the biological function of biomolecular coagulation in the occurrence and development of diseases and even drug resistance will open up a new direction
for us to develop small molecule drugs with new mechanisms.

On October 13, 2022, the Jidong/Zhu Guangya team of the Interdisciplinary Research Center of Biology and Chemistry, Chinese Academy of Sciences, and Shanghai Itto Pharma published a paper entitled Targeting androgen receptor phase separation to overcome antiandrogen resistance online in Nature Chemical Biology research papers
.
This study reveals that liquid-liquid phase separation of AR plays a key role in the mechanism of anti-androgen resistance, and proposes a new strategy
to overcome anti-androgen resistance by targeting AR liquid-liquid phase separation.

In this study, the researchers found that the androgen dihydrotestosterone (DHT) can induce liquid-liquid phase separation of AR nucleation and form activated transcriptional agglutination (Figure 1a).

。 OptoIDR experiments show that the phase separation ability of AR is mainly driven
by the NTD domain.
Since AR-V7 has a complete NTD and DBD domain, AR-V7 can enter the nucleus and form transcriptional agglutinators even in the absence of androgens (Figure 1b).

Figure 1: Liquid-liquid phase separation of AR and AR-V7

The liquid-liquid phase separation of AR is closely related to the efficacy and resistance of AR antagonists
.
For example, the AR antagonist Enzalutamide (Enza) can significantly inhibit agglomerates produced by AR stimulated by the androgen DHT
.
However, in clinical practice, when AR developed drug-resistant mutation AR (F877L/T878A) to enzalutamide, the phase separation inhibition of enzalutamide on AR (F877L/T878A) was significantly weakened
.
More interestingly, enzalutamide can even induce liquid-liquid phase separation of AR (F877L/T878A) and activate AR transcriptional activity, meaning that when AR develops drug-resistant mutations, enzalutamide changes from an AR antagonist to an AR agonist (Figure 2).

Figure 2: Effect of Enza on phase separation and transcriptional activity of AR wild-type and drug-resistant mutant forms

Since AR promotes its transcriptional activity through NTD-mediated phase separation, the authors hope to inhibit the transcriptional activity of AR through liquid-liquid phase separation of small molecules targeting AR and overcome the problem of resistance to AR antagonist drugs caused by mutations or deletions of LBD (Figure 3).

The researchers selected AR (F877L/T878A) cell lines expressing enzalutamide resistance as a phenotypic screening system for AR liquid-liquid phase separation, combined with AR transcription reporter gene and cell activity detection, and performed compound library screening
for targeted AR phase isolation.
The authors found that a small molecule compound ET0516 can effectively inhibit the phase separation formation
of wild-type AR and drug-resistant mutation AR.
The compound binds to the NTD domain of AR, which can specifically reduce AR transcriptional activity and significantly inhibit the proliferation of prostate cancer cells and tumor growth
.

Figure 3: Screening of small molecule inhibitors for AR liquid-liquid phase separation

In summary, the authors reveal the important role
of liquid-liquid phase separation for AR transcription machine formation and AR transcriptional activity.
At the same time, liquid-liquid phase separation also plays a key role
in the resistance process of AR antagonists.
AR drug-resistant mutations found in clinical practice undergo compound-induced liquid-liquid phase separation in the presence of AR antagonists, transforming AR antagonists into AR agonists
.
The authors further screened by compound and found that small molecule inhibitors of AR liquid-liquid phase separation can effectively inhibit the growth of
prostate cancer cells expressing drug-resistant mutations AR or AR-V7.
This work has laid a solid foundation
for the development of small molecule drugs targeting "difficult-to-druggable" targets by targeting protein liquid-liquid phase separation.

Dr.
Guangya Zhu and Dr.
Jidong Zhu of Shanghai Ittop Pharma are the co-corresponding authors of this paper, and Dr.
Jingjing Xie and Dr.
Hao He of Shanghai Ittop Pharma are the co-first authors
of the paper.