Periodic Reporting for period 4 - CONCERT (Description of information transfer across macromolecules by concerted conformational changes) Reporting period: 2020-01-01 to 2021-06-30 Summary of the context and overall objectives of the project The goals of the project were to develop a method to characterise large scale motions in multi-domain proteins rich in regions that are intrinsically disordered and apply it to the study of the androgen receptor, a drug target for castration resistant prostate cancer.Motions of this type are key for the transfer of information in biology but are very challenging to characterise both experimentally and computationally. The experimental challenge is associated with the paucity of experimental observables that are sensitive to the degree of correlation of large scale motions. The computational challenge is instead associated with simulating large multi-domain proteins with motions in relatively slow timescales that cannot be studied with even the fastest computers available. Characterising this type of motions is important because interfering with information transfer processes in biology offers opportunities for developing drugs. This is especially the case when tumour cells acquire mutations that render them insensitive to the treatments currently in the clinic. Understanding how the targets of these drugs play a role in information transfer processes can reveal novel ways of targeting them that can be exploited by scientists experts in discovering drugs.An example of the problem of resistance is a late stage of prostate cancer known as castration resistant prostate cancer in which patients cease to respond to hormone therapy, the first line treatment for this very common disease. An understanding of the role of large scale, concerted, motions in the function of the receptor has the potential to uncover new ways to inhibit its function in patients that have become resistant.By using a multidisciplinary approach we have uncovered the precise mechanism that connects androgen binding with the activation of transcription and, perhaps most importantly, identified a way to target the process with small molecules. The technology has been transferred to a spin off company of ICREA and IRB Barcelona, Nuage Therapeutics, that is using it for drug discovery for castration resistant prostate cancer and for other diseases involving intrinsically disordered drug targets.A key discovery has been that the activation domain of the androgen receptor, that is intrinsically disordered, has to undergo liquid liquid phase separation to gain the ability to activate transcription. This process is mediated by interactions between aromatic residues in the activation domain, leads to its partial folding, and facilitates its interaction with the transcription machinery; most importantly this process provides a mechanism to target the domain with small molecules. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The first goal of the project was the study of the structural properties of the activation domain of the receptor (AR), that is intrinsically disordered. This involved detailed studies using nuclear magnetic resonance spectroscopy, that allowed uncovering that the regions of sequence known as activation are partially folded, that was expected, but also that the polyglutamine tract of the androgen receptor was highly helical and that its helical content correlated with its degree of expansion, that was not. The work was published in ACS Chemical Biology (2016) and Biophysical Journal (2016) and led to a secondary but highly interesting research line on the study of the structural properties of polyglutamine sequences (see below).Next came the study of how these regions of sequence interact with other AR domains, with different proteins and with small molecules discovered by phenotypic screening. We first studied the interaction between this domain of the C-terminal domain of sub-unit II of TFIIF, a general transcription factor, which allowed us to identify that it is mediated by a key motif in the AD, 433WHTLF437, putting forward a new therapeutic target for castration resistant prostate cancer. The work was published in Structure in 2018 and was highlighted by the editors of the journal with a 'highlight' piece by AR expert Marianne Sadar. In a related work we studied the interaction with Bag-1, that interacts with the region known as Tau-5, and reported the finding in eLife (2017).The project reached a turning point when we discovered that the activation domain undergoes liquid liquid phase separation and other scientists working on activation domains put forward the hypothesis that this is a general mechanism for transactivation. We published our findings in a collaborative paper in Molecular Cell (2018) and have since then carried out work to understand the precise steps by which this takes place in AR, that we have now fully characterized and will reveal in an upcoming publication, still in preparation, that we consider will result in a milestone paper, that main one coming out of the CONCERT project, that will have impact in different fields.We discovered that prior to activation the activation domain forms a complex with molecular chaperones such as Hsp40 and Hsp70, that bind to the motif 23FQNLF27, and that hormone binding to the ligand binding domain leads to its interaction with the same motif, displacing the chaperones and leading to liquid liquid phase separation. This phase transition is driven by interactions between aromatic residues in the domain and is indispensable for the function of the receptor: mutations of the residues to Ser, that is not aromatic, leads to a loss of ability to translocate to the nucleus and activate transcription - some of these findings were presented in a paper published in Nature Communications (2019) but the majority of the findings are not yet in the public domain.Our findings on the structural properties of the polyglutamine tract of AR led to discovery that the helices that they form are stabilized by glutamine side chain to main chain interactions. This finding could explain why the transcriptional activity of the receptor depends on the length of the polyglutamine tract and, most importantly, why expansion of the tracts beyond 37 residues leads to spinobulbar muscular atrophy (SBMA), also known as Kennedy disease, a rare neuromuscular disease that remains not cured. This finding was reported in a second Nature Communications paper (2019), that has had substantial impact in the field (60 citations in 2 years) and was highlighted in Faculty of 1000.The translational potential of the project is reflected in us filing a number of patents for the exploitation (three: 2016, 2021, 2022) of the results obtained and the creation of the company Nuage Therapeutics in 2021, that will carry out drug discovery for castration resistant prostate cancer. In addition the secondary project on polyglutamine helices has also clear translational potential as we have used it to design peptides that are fully helical and that we envisage to use for the inhibition of protein protein interactions in a paper that is under review in Nature Communications and that is already available as a preprint in Biorxiv. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The project is now finished and we believe the results both in terms of basic and applied science are at the level that we expected at the beginning of the project because we have, on the one hand, uncovered the mechanism by which androgen binding to the androgen receptor activates transcription, and, on the other hand, used the knowledge for drug discovery for castration resistant prostate cancer. how androgen binding changes the multimerization of androgen receptor
