An international group of scientists studying the tendency of intrinsically disordered proteins to form biomolecular condensates report that they have found that the mechanisms involved in condensation could be exploited to inhibit androgen receptor activity in prostate cancer.

“The rationale we have followed to optimize an androgen receptor inhibitor could be exploited to inhibit other transcription factors, thus opening up new opportunities to address unmet medical needs,” said Xavier Salvatella, PhD, research professor and group leader, laboratory of molecular biophysics, Institute for Research in Biomedicine (IRB) in Barcelona.

The research team published its study “Rational optimization of a transcription factor activation domain inhibitor” in Nature Structural and Molecular Biology.

“Transcription factors are among the most attractive therapeutic targets but are considered largely ‘undruggable’ in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens,” write the investigators.

“On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo.

“These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.”

Proteinaceous blobs

Under a microscope, biomolecular condensates resemble proteinaceous blobs floating on water. The condensates form in a process called liquid-liquid phase separation, similar to how oil droplets coalesce when mixed in water.

Xavier Salvatella, PhD, research professor and group leader, laboratory of molecular biophysics, Institute for Research in Biomedicine (IRB) in Barcelona. [IRB]
Xavier Salvatella, PhD, research professor and group leader, laboratory of molecular biophysics, Institute for Research in Biomedicine (IRB) in Barcelona. [IRB]
“We had previously observed that the androgen receptor forms biomolecular condensates when even a tiny amount of an activating molecule, such as testosterone, is added to cells,” notes Shaon Basu, PhD, a computational biologist at the Charité in Berlin and one of the study’s first authors, together with Paula Martínez-Cristobal, PhD, at IRB Barcelona.

The scientists hypothesized that there could be a link between the activation of the androgen receptor and its propensity to form droplets. The researchers used nuclear magnetic resonance techniques to identify several short pieces within the intrinsically disordered activation domain that are essential for phase separation. Moreover, the same short pieces also turned out to be necessary for the gene-activating function of the receptor.

“We discovered short sequences in the activation domain that tend to be disordered when the protein is soluble and, surprisingly, these regions seem to form more stable helices when the protein is concentrated in condensates,” explains Denes Hnisz, PhD, at the Max Planck Institute for Molecular Genetics, adding that the short helices create transient binding pockets that can be targeted with inhibitors when the receptor is in condensates.

Treating prostate cancer

Along with Antoni Riera, PhD, professor at the University of Barcelona and Marianne Sadar, PhD, BC Cancer, at the University of British Columbia, the team improved an experimental small molecule inhibitor to fit almost perfectly into the transient binding pocket. They then tested whether the changes introduced increased efficacy in an aggressive, late-stage form of prostate cancer in cell and mouse models.

prostate cancer
Super-resolution (τ-STED) imaging of the androgen receptor in human prostate adenocarcinoma cells. The nucleus of one cell is shown, and the nuclear contour is highlighted with a white dashed line. The androgen receptor was labelled with an antibody probe. The receptors form intense nuclear clusters with an average size ~100nm. Large scale bar: 5 μm. Scale bar in τ-STED inset: 300 nm [Max Planck Institute for Molecular Genetics]
“We modified the chemical features of the compound to match the features of androgen receptor condensation, resulting in a tenfold increase in the potency of the molecule in castration-resistant prostate cancer,” said Paula Martínez-Cristobal, PhD, IRB, also first author of the study. “This is really important because castration-resistant prostate cancer is an extremely aggressive cancer that is resistant to current first-line therapeutics.”

However, the authors agree that more research is needed before these findings can be translated into new and safe therapeutics. The team hopes that the basic mechanisms they have discovered may be applicable to other transcription factors, thereby paving the way to target these important molecules in many different diseases.

“We believe that the idea that there are certain sequences within intrinsically disordered protein domains that adopt a transiently stable structure in condensates is universal and likely to be generalizable to transcription factors,” said Hnisz.

The findings published in Nature Structural & Molecular Biology laid the groundwork for the foundation of Nuage Therapeutics in 2021, which develops drug screening assays to target intrinsically disordered proteins that undergo biomolecular condensation, thus providing new treatments for diseases currently considered difficult to treat.

Salvatella, Hnisz, Mateusz Biesaga, PhD, and Judit Anido, are company founders.

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