Targeting androgen receptor phase separation to overcome antiandrogen resistance.

Patients with castration-resistant prostate cancer inevitably acquire resistance to antiandrogen therapies in part because of androgen receptor (AR) mutations or splice variants enabling restored AR signaling. Here we show that ligand-activated AR can form transcriptionally active condensates. Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role. AR liquid-liquid phase separation behaviors faithfully report transcriptional activity and antiandrogen efficacy. Antiandrogens can promote phase separation and transcriptional activity of AR-resistant mutants in a ligand-independent manner. We conducted a phase-separation-based phenotypic screen and identified ET516 that specifically disrupts AR condensates, effectively suppresses AR transcriptional activity and inhibits the proliferation and tumor growth of prostate cancer cells expressing AR-resistant mutants. Our results demonstrate liquid-liquid phase separation as an emerging mechanism underlying drug resistance and show that targeting phase separation may provide a feasible approach for drug discovery.

The novel nematicide chiricanine A suppresses Bursaphelenchus xylophilus pathogenicity in Pinus massoniana by inhibiting Aspergillus and its secondary metabolite, sterigmatocystin.

Introduction: Pine wilt disease (PWD) is responsible for extensive economic and ecological damage to Pinus spp. forests and plantations worldwide. PWD is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted into pine trees by a vector insect, the Japanese pine sawyer (JPS, Monochamus alternatus). Host infection by PWN will attract JPS to spawn, which leads to the co-existence of PWN and JPS within the host tree, an essential precondition for PWD outbreaks. Through the action of their metabolites, microbes can manipulate the co-existence of PWN and JPS, but our understanding on how key microorganisms engage in this process remains limited, which severely hinders the exploration and utilization of promising microbial resources in the prevention and control of PWD. Methods: In this study we investigated how the PWN-associated fungus Aspergillus promotes the co-existence of PWN and JPS in the host trees (Pinus massoniana) via its secondary metabolite, sterigmatocystin (ST), by taking a multi-omics approach (phenomics, transcriptomics, microbiome, and metabolomics). Results: We found that Aspergillus was able to promote PWN invasion and pathogenicity by increasing ST biosynthesis in the host plant, mainly by suppressing the accumulation of ROS (reactive oxygen species) in plant tissues that could counter PWN. Further, ST accumulation triggered the biosynthesis of VOC (volatile organic compounds) that attracts JPS and drives the coexistence of PWN and JPS in the host plant, thereby encouraging the local transmission of PWD. Meanwhile, we show that application of an Aspergillus inhibitor (chiricanine A treatment) results in the absence of Aspergillus and decreases the in vivo ST amount, thereby sharply restricting the PWN development in host. This further proved that Aspergillus is vital and sufficient for promoting PWD transmission. Discussion: Altogether, these results document, for the first time, how the function of Aspergillus and its metabolite ST is involved in the entire PWD transmission chain, in addition to providing a novel and long-term effective nematicide for better PWD control in the field.

The nematicide emamectin benzoate increases ROS accumulation in Pinus massoniana and poison Monochamus alternatus.

Pine wilt disease (PWD) is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted by a vector insect, the Monochamus alternatus. The PWN has caused much extensive damage to pine-dominated forest ecosystems. Trunk injection of emamectin benzoate (EB) has been found to be the most useful protective measure against the PWN, due to its low effective dose and long residence time in the field. However, the interactions between EB and the host or the environment remain largely unknown, which limits the efficacy and stability of EB in practical field settings. In this study, we investigated the impact on PWN from EB injection for both adult and young host plants (Pinus massoniana) by taking a multi-omics (phenomics, transcriptomics, microbiome, and metabolomics) approach. We found that EB injection can significantly reduce the amount of PWN in both living adult and young pine trees. Additionally, EB was able to activate the genetic response of P. massoniana against PWN, promotes P. massoniana growth and development and resistance to Pine wilt disease, which requires the presence of PWN. Further, the presence of EB greatly increased the accumulation of reactive oxygen species (ROS) in the host plant in a PWN-dependent manner, possibly by affecting ROS-related microbes and metabolites. Moreover, we uncovered the function of EB limiting the consumption of P. massoniana by the JPS. Based on biochemical and gut microbial data, we found that EB can significantly reduces cellulase activity in JPS, whose transcription factors, sugar metabolism, and the phosphotransferase system are also affected. These results document the impact of EB on the entire PWD transmission chain through multi-omics regarding the dominant pine (P. massoniana) in China and provide a novel perspective for controlling PWD outbreaks in the field.

Structure-Guided Design of EED Binders Allosterically Inhibiting the Epigenetic Polycomb Repressive Complex 2 (PRC2) Methyltransferase.

PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.

Discovery of First-in-Class, Potent, and Orally Bioavailable Embryonic Ectoderm Development (EED) Inhibitor with Robust Anticancer Efficacy.

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound 43 (EED226). Guided by X-ray crystallography, compound 43 was discovered by fragmentation and regrowth of compound 7, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of 43. Compound 43 induces robust and sustained tumor regression in EZH2MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy.