Biomolecular condensates compartmentalize and regulate assemblies of biomolecules engaged in vital physiological processes in cells. Specific proteins and nucleic acids engaged in shared functions occur in any one kind of condensate, suggesting that these compartments have distinct chemical specificities. Indeed, some small-molecule drugs concentrate in specific condensates due to chemical properties engendered by particular amino acids in the proteins in those condensates. Here we argue that the chemical properties that govern molecular interactions between a small molecule and biomolecules within a condensate can be ascertained for both the small molecule and the biomolecules. We propose that learning this ‘chemical grammar’, the rules describing the chemical features of small molecules that engender attraction or repulsion by the physicochemical environment of a specific condensate, should enable design of drugs with improved efficacy and reduced toxicity.
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Wagner, R. Einige Bemerkungen und Fragen über das Keimbläschen (vesicular germinativa). Müller’s Archiv. Anat. Physiol. Wissenschaft Med. 1835, 373–377 (1835). Wagner was apparently the first to publish a description of the largest and best-characterized condensate, the nucleolus.
Valentin, G. Repertorium für Anatomie und Physiologie, Vol. 1 (Verlag von Veit und Comp., 1836).
Valentin, G. Repertorium für Anatomie und Physiologie, Vol. 4 (Verlag von Veit und Comp., 1839).
Cajal, S. R. Y. Un sencillo metodo de coloracion seletiva del reticulo protoplasmatico y sus efectos en los diversos organos nerviosos de vertebrados e invertebrados. Trab. Lab. Invest. Biol. 2, 129–221 (1903).
Vincent, W. S. The isolation and chemical properties of the nucleoli of starfish oocytes. Proc. Natl Acad. Sci. USA 38, 139–145 (1952).
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009). This pioneering study noted that the behavior of a cellular body was not that of a standard membrane-bound organelle but rather more like liquid droplets that arise by phase separation.
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).
Case, L. B., Ditlev, J. A. & Rosen, M. K. Regulation of transmembrane signaling by phase separation. Annu. Rev. Biophys. 48, 465–494 (2019).
Alberti, S. & Dormann, D. Liquid–liquid phase separation in disease. Annu. Rev. Genet. 53, 171–194 (2019).
Boija, A., Klein, I. A. & Young, R. A. Biomolecular condensates and cancer. Cancer Cell 39, 174–192 (2021).
Sabari, B. R., Dall’Agnese, A. & Young, R. A. Biomolecular condensates in the nucleus. Trends Biochem. Sci. 45, 961–977 (2020).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
Chen, X., Wu, X., Wu, H. & Zhang, M. Phase separation at the synapse. Nat. Neurosci. 23, 301–310 (2020).
Nedelsky, N. B. & Taylor, J. P. Bridging biophysics and neurology: aberrant phase transitions in neurodegenerative disease. Nat. Rev. Neurol. 15, 272–286 (2019).
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell 63, 72–85 (2016). This study provides a powerful example of combining theory and experimentation to enhance understanding of the roles of amino acid patterns in protein phase separation.
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699 (2018). A study exploring the roles of diverse amino acids in promoting phase separation and influencing the material properties of condensates formed by a class of proteins.
Martin, E. W. et al. Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. Science 367, 694–699 (2020).
Banani, S. F. et al. Composition control of phase-separated bodies. Cell 166, 651–663 (2016).
Abyzov, A., Blackledge, M. & Zweckstetter, M. Conformational dynamics of intrinsically disordered proteins regulate biomolecular condensate chemistry. Chem. Rev. 122, 6719–6748 (2022).
Greig, J. A. et al. Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation. Mol. Cell 77, 1237–1250 (2020).
Case, L. B., Zhang, X., Ditlev, J. A. & Rosen, M. K. Stoichiometry controls activity of phase-separated clusters of actin signaling proteins. Science 363, 1093–1097 (2019).
Shrinivas, K. et al. Enhancer features that drive formation of transcriptional condensates. Mol. Cell 75, 549–561 (2019).
Choi, J.-M., Holehouse, A. S. & Pappu, R. V. Physical principles underlying the complex biology of intracellular phase transitions. Annu. Rev. Biophys. 49, 107–133 (2020).
McSwiggen, D. T., Mir, M., Darzacq, X. & Tjian, R. Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences. Genes Dev. 33, 1619–1634 (2019).
Peng, A. & Weber, S. C. Evidence for and against liquid–liquid phase separation in the nucleus. Noncoding RNA 5, 50 (2019).
Boija, A. et al. Transcription factors activate genes through the phase-separation capacity of their activation domains. Cell 175, 1842–1855 (2018).
Guo, Y. E. et al. Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572, 543–548 (2019). This work establishes that post-translational modifications of the transcription apparatus can regulate its partitioning into different condensates involved in RNA synthesis and splicing.
Henninger, J. E. et al. RNA-mediated feedback control of transcriptional condensates. Cell 184, 207–225 (2021).
Lu, S. et al. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein. Nat. Commun. 12, 502 (2021).
Yu, X. et al. The STING phase-separator suppresses innate immune signalling. Nat. Cell Biol. 23, 330–340 (2021).
Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).
Dao, T. P. et al. Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages. Preprint at bioRxiv https://doi.org/10.1101/2021.11.12.467822 (2021).
Cho, W.-K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412–415 (2018).
Cho, W.-K. et al. RNA polymerase II cluster dynamics predict mRNA output in living cells. eLife 5, e13617 (2016).
Klein, I. A. et al. Partitioning of cancer therapeutics in nuclear condensates. Science 368, 1386–1392 (2020). This study showed that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics, suggesting that further understanding of condensate chemical grammar may facilitate advances in disease therapy.
Schuster, B. S. et al. Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior. Proc. Natl Acad. Sci. USA 117, 11421–11431 (2020).
Alshareedah, I., Moosa, M. M., Pham, M., Potoyan, D. A. & Banerjee, P. R. Programmable viscoelasticity in protein–RNA condensates with disordered sticker–spacer polypeptides. Nat. Commun. 12, 6620 (2021).
Roden, C. & Gladfelter, A. S. RNA contributions to the form and function of biomolecular condensates. Nat. Rev. Mol. Cell Biol. 22, 183–195 (2021).
Savastano, A., Ibáñez de Opakua, A., Rankovic, A. & Zweckstetter, M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates. Nat. Commun. 11, 6041 (2020).
Guillén-Boixet, J. et al. RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation. Cell 181, 346–361 (2020).
Sanders, D. W. et al. Competing protein–RNA interaction networks control multiphase intracellular organization. Cell 181, 306–324 (2020).
Boeynaems, S. et al. Spontaneous driving forces give rise to protein−RNA condensates with coexisting phases and complex material properties. Proc. Natl Acad. Sci. USA 116, 7889–7898 (2019).
Holehouse, A. S., Ginell, G. M., Griffith, D. & Böke, E. Clustering of aromatic residues in prion-like domains can tune the formation, state, and organization of biomolecular condensates. Biochemistry 60, 3566–3581 (2021).
Kar, M. et al. Glycine-rich peptides from FUS have an intrinsic ability to self-assemble into fibers and networked fibrils. Biochemistry 60, 3213–3222 (2021).
Rubinstein, M. & Semenov, A. N. Thermoreversible gelation in solutions of associating polymers. 2. Linear dynamics. Macromolecules 31, 1386–1397 (1998).
Tanaka, F. & Ishida, M. Microphase formation in mixtures of associating polymers. Macromolecules 30, 1836–1844 (1997).
Leibler, L. Theory of microphase separation in block copolymers. Macromolecules 13, 1602–1617 (1980).
Murthy, A. C. et al. Molecular interactions underlying liquid−liquid phase separation of the FUS low-complexity domain. Nat. Struct. Mol. Biol. 26, 637–648 (2019).
Qamar, S. et al. FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation–π interactions. Cell 173, 720–734 (2018).
Kim, T. H. et al. Interaction hot spots for phase separation revealed by NMR studies of a CAPRIN1 condensed phase. Proc. Natl Acad. Sci. USA 118, e2104897118 (2021).
Kim, T. H. et al. Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation. Science 365, 825–829 (2019).
Wyman, J. & Gill, S. J. Ligand-linked phase changes in a biological system: applications to sickle cell hemoglobin. Proc. Natl Acad. Sci. USA 77, 5239–5242 (1980).
Ruff, K. M., Dar, F. & Pappu, R. V. Polyphasic linkage and the impact of ligand binding on the regulation of biomolecular condensates. Biophys. Rev. 2, 021302 (2021).
Ruff, K. M., Dar, F. & Pappu, R. V. Ligand effects on phase separation of multivalent macromolecules. Proc. Natl Acad. Sci. USA 118, e2017184118 (2021).
Burslem, G. M. & Crews, C. M. Proteolysis-targeting chimeras as therapeutics and tools for biological discovery. Cell 181, 102–114 (2020).
Gerry, C. J. & Schreiber, S. L. Unifying principles of bifunctional, proximity-inducing small molecules. Nat. Chem. Biol. 16, 369–378 (2020).
Forman-Kay, J. D., Ditlev, J. A., Nosella, M. L. & Lee, H. O. What are the distinguishing features and size requirements of biomolecular condensates and their implications for RNA-containing condensates? RNA 28, 36–47 (2021).
Andersen, J. S. et al. Nucleolar proteome dynamics. Nature 433, 77–83 (2005).
Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).
Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).
Langdon, E. M. & Gladfelter, A. S. A new lens for RNA localization: liquid–liquid phase separation. Annu. Rev. Microbiol. 72, 255–271 (2018).
Woodruff, J. B., Hyman, A. A. & Boke, E. Organization and function of non-dynamic biomolecular condensates. Trends Biochem. Sci. 43, 81–94 (2018).
Lafontaine, D. L. J., Riback, J. A., Bascetin, R. & Brangwynne, C. P. The nucleolus as a multiphase liquid condensate. Nat. Rev. Mol. Cell Biol. 22, 165–182 (2021).
Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25, 833–840 (2018).
Nojima, T. et al. RNA polymerase II phosphorylated on CTD serine 5 interacts with the spliceosome during co-transcriptional splicing. Mol. Cell 72, 369–379 (2018).
Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A phase separation model for transcriptional control. Cell 169, 13–23 (2017).
Vibet, S. et al. Differential subcellular distribution of mitoxantrone in relation to chemosensitization in two human breast cancer cell lines. Drug Metab. Dispos. 35, 822–828 (2007).
Smith, P. J., Sykes, H. R., Fox, M. E. & Furlong, I. J. Subcellular distribution of the anticancer drug mitoxantrone in human and drug-resistant murine cells analyzed by flow cytometry and confocal microscopy and its relationship to the induction of DNA damage. Cancer Res. 52, 4000–4008 (1992).
Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).
Bradner, J. E., Hnisz, D. & Young, R. A. Transcriptional addiction in cancer. Cell 168, 629–643 (2017).
Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).
Christensen, C. L. et al. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor. Cancer Cell 26, 909–922 (2014).
Kwiatkowski, N. et al. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 511, 616–620 (2014).
Fanning, S. W. et al. Estrogen receptor α somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation. eLife 5, e12792 (2016).
Nagalingam, A. et al. Med1 plays a critical role in the development of tamoxifen resistance. Carcinogenesis 33, 918–930 (2012).
Pommier, Y., Leo, E., Zhang, H. & Marchand, C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 17, 421–433 (2010).
Bielskutė, S. et al. Low amounts of heavy water increase the phase separation propensity of a fragment of the androgen receptor activation domain. Protein Sci. 30, 1427–1437 (2021).
Petronilho, E. C. et al. Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands. Chem. Sci. 12, 7334–7349 (2021).
Babinchak, M. W. et al. Small molecules as potent biphasic modulators of protein liquid–liquid phase separation. Nat. Commun. 11, 5574 (2020).
Krainer, G. et al. Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions. Nat. Commun. 12, 1085 (2021).
Fang, M. Y. et al. Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD. Neuron 103, 802–819 (2019).
Alshareedah, I., Kaur, T. & Banerjee, P. R. Methods for characterizing the material properties of biomolecular condensates. In Methods in Enzymology, Vol. 646 (ed. Keating, C. D.), Ch. 6, 143–183 (Academic Press, 2021).
Wang, S. et al. Targeting liquid–liquid phase separation of SARS-CoV-2 nucleocapsid protein promotes innate antiviral immunity by elevating MAVS activity. Nat. Cell Biol. 23, 718–732 (2021).
Hadjadj, J. et al. Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369, 718–724 (2020).
Li, Y. et al. SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes. Proc. Natl Acad. Sci. USA 118, e2022643118 (2021).
Wu, J. et al. SARS-CoV-2 ORF9b inhibits RIG-I–MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Rep. 34, 108761 (2021).
Zotta, A., Hooftman, A. & O’Neill, L. A. J. SARS-CoV-2 targets MAVS for immune evasion. Nat. Cell Biol. 23, 682–683 (2021).
Risso-Ballester, J. et al. A condensate-hardening drug blocks RSV replication in vivo. Nature 595, 596–599 (2021).
We thank A. Dall’agnese for providing the images presented in Fig. 3a and A. Boija and K. Overholt for helpful discussions. Funding: H.R.K. is supported by a fellowship from the Damon Runyon Cancer Research Foundation (grant number 2458-22). R.A.Y. is supported by NIH grants R01 GM123511 and R01 MH104610, NCI grant CA155258, NSF grant PHY2044895, the 3D Genome Consortium of St. Jude Children’s Research Hospital, and funds from Novo Nordisk.
R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics and Dewpoint Therapeutics. H.R.K. is a consultant of Dewpoint Therapeutics.
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Kilgore, H.R., Young, R.A. Learning the chemical grammar of biomolecular condensates. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01046-y