Biomolecular condensates: new opportunities for drug discovery and RNA therapeutics

Numerous examples now exist where biomolecular condensates are mechanistically linked to disease.Small-molecule drugs used in the clinic today can interact with biomolecular condensates, whether intended or not, possibly affecting their pharmacology.Recent clinical success has demonstrated the utility of RNA therapeutics to help patients and propelled interest in this field.Endogenous RNA is a key constituent in both physiological and pathological condensates, and a potential link between prospective RNA therapeutics and condensates is emerging.Understanding the interaction of both small molecules and RNA therapeutics with biomolecular condensates may improve the drug discovery process. Biomolecular condensates organize cellular functions in the absence of membranes. These membraneless organelles can form through liquid–liquid phase separation coalescing RNA and proteins into well-defined, yet dynamic, structures distinct from the surrounding cellular milieu. Numerous physiological and disease-causing processes link to biomolecular condensates, which could impact drug discovery in several ways. First, disruption of pathological condensates seeded by mutated proteins or RNAs may provide new opportunities to treat disease. Second, condensates may be leveraged to tackle difficult-to-drug targets lacking binding pockets whose function depends on phase separation. Third, condensate-resident small molecules and RNA therapeutics may display unexpected pharmacology. We discuss the potential impact of phase separation on drug discovery and RNA therapeutics, leveraging concrete examples, towards novel clinical opportunities. Biomolecular condensates organize cellular functions in the absence of membranes. These membraneless organelles can form through liquid–liquid phase separation coalescing RNA and proteins into well-defined, yet dynamic, structures distinct from the surrounding cellular milieu. Numerous physiological and disease-causing processes link to biomolecular condensates, which could impact drug discovery in several ways. First, disruption of pathological condensates seeded by mutated proteins or RNAs may provide new opportunities to treat disease. Second, condensates may be leveraged to tackle difficult-to-drug targets lacking binding pockets whose function depends on phase separation. Third, condensate-resident small molecules and RNA therapeutics may display unexpected pharmacology. We discuss the potential impact of phase separation on drug discovery and RNA therapeutics, leveraging concrete examples, towards novel clinical opportunities. Membraneless organelles, or biomolecular condensates, support compartmentalization and spatiotemporal regulation of biochemical activities inside cells [1.Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357: eaaf4382Crossref PubMed Scopus (1287) Google Scholar, 2.Lyon A.S. et al.A framework for understanding the functions of biomolecular condensates across scales.Nat. Rev. Mol. Cell Biol. 2021; 22: 215-235Crossref PubMed Scopus (121) Google Scholar, 3.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1891) Google Scholar]. Biomolecular condensates (hereafter condensates), a term coined to represent the coalescence of biomolecules (see Glossary) like RNA and proteins [3.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1891) Google Scholar], are functional, nonstoichiometric, and dynamic assemblies that, like membrane-bound organelles, create subcellular environments but lack enclosing membranes or import/export machinery. Dynamism and function are key discriminators between condensates and aggregates (Box 1). While alternative models exist [4.Darzacq X. Tjian R. Weak multivalent biomolecular interactions: a strength versus numbers tug of war with implications for phase partitioning.RNA. 2022; 28: 48-51Crossref PubMed Scopus (0) Google Scholar,5.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (216) Google Scholar], theoretical modeling and experiments in purified settings and cells support the notion that liquid–liquid phase separation, the same phenomenon forming oil droplets in water, contributes to condensate formation and regulation.Box 1Condensates versus aggregates, and the cellular contextThe original definition of biomolecular condensates is all-inclusive, and was coined to represent the intracellular coalescence of biomolecules in a manner that does not involve a membrane [3.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1891) Google Scholar]. Phase separation is defined as demixing of two (or more) phases, while a phase transition is the change in molecular organization within a system changing its material properties like liquid-to-solid. Both phenomena may be at play in the context of cellular condensates.Biomolecular condensates and aggregates can be readily differentiated [9.Alberti S. Hyman A.A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.Nat. Rev. Mol. Cell Biol. 2021; 22: 196-213Crossref PubMed Scopus (126) Google Scholar]. Here, we define aggregates as non-native, nonfunctional assemblies, that are irreversible at the cellular timescale without involving degradation pathways. Aggregates often involve misfolded proteins (and possibly RNA) that assume a non-native conformation. Importantly, in the case of unstructured proteins, misfolding may lead to a more ordered and less-dynamic structure, causing abnormal interactions [125.Iadanza M.G. et al.A new era for understanding amyloid structures and disease.Nat. Rev. Mol. Cell Biol. 2018; 19: 755-773Crossref PubMed Scopus (347) Google Scholar]. For further discussion about differences and shared features between condensates and aggregates, we direct the reader to a recent review [9.Alberti S. Hyman A.A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.Nat. Rev. Mol. Cell Biol. 2021; 22: 196-213Crossref PubMed Scopus (126) Google Scholar].Certain elements of phase separation in cells can be explained by spontaneous, thermodynamically driven events. However, models describing the formation and function of condensates are necessarily reductionistic and cannot fully incorporate the cellular complexity. First, numerous biomolecules can be forced to undergo phase separation in purified settings. However, this may not occur under physiological conditions [126.Alberti S. et al.Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176: 419-434Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar]. The reader may reference publications discussing best practices for condensate reconstitution in purified settings and the study of phase separation in live cells [2.Lyon A.S. et al.A framework for understanding the functions of biomolecular condensates across scales.Nat. Rev. Mol. Cell Biol. 2021; 22: 215-235Crossref PubMed Scopus (121) Google Scholar,11.Roden C. Gladfelter A.S. RNA contributions to the form and function of biomolecular condensates.Nat. Rev. Mol. Cell Biol. 2021; 22: 183-195Crossref PubMed Scopus (119) Google Scholar,126.Alberti S. et al.Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176: 419-434Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar]. Second, ATP-driven processes can modulate condensates inside cells. These include post-translational modification of proteins [34.Su X. et al.Phase separation of signaling molecules promotes T cell receptor signal transduction.Science. 2016; 352: 595-599Crossref PubMed Scopus (511) Google Scholar,87.Gruijs da Silva L.A. et al.Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation.EMBO J. 2022; 41e108443Crossref PubMed Scopus (7) Google Scholar,90.Qamar S. et al.FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions.Cell. 2018; 173: 720-734.e15Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar,127.Rai A.K. et al.Kinase-controlled phase transition of membraneless organelles in mitosis.Nature. 2018; 559: 211-216Crossref PubMed Scopus (166) Google Scholar] and RNA [91.Lee J.H. et al.Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.Mol. Cell. 2021; 81: 3368-3385.e9Abstract Full Text Full Text PDF PubMed Google Scholar,92.Ries R.J. et al.m6A enhances the phase separation potential of mRNA.Nature. 2019; 571: 424-428Crossref PubMed Scopus (246) Google Scholar], chaperones [128.Yoo H. et al.Chaperones directly and efficiently disperse stress-triggered biomolecular condensates.Mol. Cell. 2022; 82: 741-755.e11Abstract Full Text Full Text PDF PubMed Google Scholar], and helicases [129.Hondele M. et al.DEAD-box ATPases are global regulators of phase-separated organelles.Nature. 2019; 573: 144-148Crossref PubMed Scopus (140) Google Scholar]. Moreover, there is significant interplay between membrane-bound organelles and condensates, as membranes create environments that may promote or inhibit phase separation [75.Maharana S. et al.RNA buffers the phase separation behavior of prion-like RNA binding proteins.Science. 2018; 360: 918-921Crossref PubMed Scopus (460) Google Scholar]. Despite their complexity, the nonequilibrium nature of cellular condensates and the contribution of the heterogeneous environment in which they exist may be described with mathematical models [8.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (177) Google Scholar,130.Jacobs W.M. Self-assembly of biomolecular condensates with shared components.Phys. Rev. Lett. 2021; 126258101Crossref Scopus (5) Google Scholar]. This allows for a quantitative understanding of condensate formation and their composition in cells, and supports interesting condensate properties, like the nonequilibrium flux of biomolecules in and out of condensates as biochemical reactions take place [8.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (177) Google Scholar].Finally, phase separation is not the only phenomenon that can generate functional and dynamic macromolecular assemblies inside cells. For instance, loading of biomolecules to multivalent but static cellular structures or active concentration of biomolecules in defined subcellular volumes by molecular motors may lead to assemblies in many ways analogous to condensates formed by phase separation [2.Lyon A.S. et al.A framework for understanding the functions of biomolecular condensates across scales.Nat. Rev. Mol. Cell Biol. 2021; 22: 215-235Crossref PubMed Scopus (121) Google Scholar]. The original definition of biomolecular condensates is all-inclusive, and was coined to represent the intracellular coalescence of biomolecules in a manner that does not involve a membrane [3.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1891) Google Scholar]. Phase separation is defined as demixing of two (or more) phases, while a phase transition is the change in molecular organization within a system changing its material properties like liquid-to-solid. Both phenomena may be at play in the context of cellular condensates. Biomolecular condensates and aggregates can be readily differentiated [9.Alberti S. Hyman A.A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.Nat. Rev. Mol. Cell Biol. 2021; 22: 196-213Crossref PubMed Scopus (126) Google Scholar]. Here, we define aggregates as non-native, nonfunctional assemblies, that are irreversible at the cellular timescale without involving degradation pathways. Aggregates often involve misfolded proteins (and possibly RNA) that assume a non-native conformation. Importantly, in the case of unstructured proteins, misfolding may lead to a more ordered and less-dynamic structure, causing abnormal interactions [125.Iadanza M.G. et al.A new era for understanding amyloid structures and disease.Nat. Rev. Mol. Cell Biol. 2018; 19: 755-773Crossref PubMed Scopus (347) Google Scholar]. For further discussion about differences and shared features between condensates and aggregates, we direct the reader to a recent review [9.Alberti S. Hyman A.A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.Nat. Rev. Mol. Cell Biol. 2021; 22: 196-213Crossref PubMed Scopus (126) Google Scholar]. Certain elements of phase separation in cells can be explained by spontaneous, thermodynamically driven events. However, models describing the formation and function of condensates are necessarily reductionistic and cannot fully incorporate the cellular complexity. First, numerous biomolecules can be forced to undergo phase separation in purified settings. However, this may not occur under physiological conditions [126.Alberti S. et al.Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176: 419-434Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar]. The reader may reference publications discussing best practices for condensate reconstitution in purified settings and the study of phase separation in live cells [2.Lyon A.S. et al.A framework for understanding the functions of biomolecular condensates across scales.Nat. Rev. Mol. Cell Biol. 2021; 22: 215-235Crossref PubMed Scopus (121) Google Scholar,11.Roden C. Gladfelter A.S. RNA contributions to the form and function of biomolecular condensates.Nat. Rev. Mol. Cell Biol. 2021; 22: 183-195Crossref PubMed Scopus (119) Google Scholar,126.Alberti S. et al.Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176: 419-434Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar]. Second, ATP-driven processes can modulate condensates inside cells. These include post-translational modification of proteins [34.Su X. et al.Phase separation of signaling molecules promotes T cell receptor signal transduction.Science. 2016; 352: 595-599Crossref PubMed Scopus (511) Google Scholar,87.Gruijs da Silva L.A. et al.Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation.EMBO J. 2022; 41e108443Crossref PubMed Scopus (7) Google Scholar,90.Qamar S. et al.FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions.Cell. 2018; 173: 720-734.e15Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar,127.Rai A.K. et al.Kinase-controlled phase transition of membraneless organelles in mitosis.Nature. 2018; 559: 211-216Crossref PubMed Scopus (166) Google Scholar] and RNA [91.Lee J.H. et al.Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.Mol. Cell. 2021; 81: 3368-3385.e9Abstract Full Text Full Text PDF PubMed Google Scholar,92.Ries R.J. et al.m6A enhances the phase separation potential of mRNA.Nature. 2019; 571: 424-428Crossref PubMed Scopus (246) Google Scholar], chaperones [128.Yoo H. et al.Chaperones directly and efficiently disperse stress-triggered biomolecular condensates.Mol. Cell. 2022; 82: 741-755.e11Abstract Full Text Full Text PDF PubMed Google Scholar], and helicases [129.Hondele M. et al.DEAD-box ATPases are global regulators of phase-separated organelles.Nature. 2019; 573: 144-148Crossref PubMed Scopus (140) Google Scholar]. Moreover, there is significant interplay between membrane-bound organelles and condensates, as membranes create environments that may promote or inhibit phase separation [75.Maharana S. et al.RNA buffers the phase separation behavior of prion-like RNA binding proteins.Science. 2018; 360: 918-921Crossref PubMed Scopus (460) Google Scholar]. Despite their complexity, the nonequilibrium nature of cellular condensates and the contribution of the heterogeneous environment in which they exist may be described with mathematical models [8.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (177) Google Scholar,130.Jacobs W.M. Self-assembly of biomolecular condensates with shared components.Phys. Rev. Lett. 2021; 126258101Crossref Scopus (5) Google Scholar]. This allows for a quantitative understanding of condensate formation and their composition in cells, and supports interesting condensate properties, like the nonequilibrium flux of biomolecules in and out of condensates as biochemical reactions take place [8.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (177) Google Scholar]. Finally, phase separation is not the only phenomenon that can generate functional and dynamic macromolecular assemblies inside cells. For instance, loading of biomolecules to multivalent but static cellular structures or active concentration of biomolecules in defined subcellular volumes by molecular motors may lead to assemblies in many ways analogous to condensates formed by phase separation [2.Lyon A.S. et al.A framework for understanding the functions of biomolecular condensates across scales.Nat. Rev. Mol. Cell Biol. 2021; 22: 215-235Crossref PubMed Scopus (121) Google Scholar]. Recent developments in the field of intracellular phase separation have illuminated a link between condensates, disease, and the action of drugs currently used to treat patients [6.Banani S.F. et al.Genetic variation associated with condensate dysregulation in disease.Dev. Cell. 2022; 57: 1776-1788.e8Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar,7.Kilgore H.R. Young R.A. Learning the chemical grammar of biomolecular condensates.Nat. Chem. Biol. 2022; (Published online June 27, 2022)https://doi.org/10.1038/s41589-022-01046-yCrossref PubMed Scopus (0) Google Scholar]. Here, we highlight emerging trends in the field of condensates likely to influence drug discovery, particularly RNA-based therapeutics. We review the mechanistic impact that dysregulated phase separation has on disease and how this may be leveraged for new therapeutic approaches. We also discuss how condensates may aid modulation of difficult-to-drug targets through novel modalities, along with affecting the pharmacology of existing therapies. We aim to provide readers with forward-looking insights into a drug discovery era that integrates the evolving fields of biomolecular condensates and RNA therapeutics to deliver novel opportunities for patients. Intracellular phase separation is explained by borrowing concepts from polymer science, where phase boundaries effectively substitute lipid membranes while permitting diffusion between two phases (Figure 1A ) [1.Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357: eaaf4382Crossref PubMed Scopus (1287) Google Scholar,3.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1891) Google Scholar,8.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (177) Google Scholar]. Phase separation is driven by the energy reduction caused by demixing (by satisfying attractive or repulsive intermolecular forces) that opposes entropy-driven mixing of all components. In its simplest form, this leads to the formation of a two-phase system where the condensate phase assumes different material properties depending on constituents’ concentration, interaction strength, valency, and system parameters like temperature and pH. Phase separation does not have to result in liquid, freely-fusing assemblies; condensates can present a continuum of material properties (Figure 1B) [2.Lyon A.S. et al.A framework for understanding the functions of biomolecular condensates across scales.Nat. Rev. Mol. Cell Biol. 2021; 22: 215-235Crossref PubMed Scopus (121) Google Scholar,3.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1891) Google Scholar,9.Alberti S. Hyman A.A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.Nat. Rev. Mol. Cell Biol. 2021; 22: 196-213Crossref PubMed Scopus (126) Google Scholar]. In cells, attractive forces leading to phase separation include RNA–RNA, protein–RNA, DNA–DNA, protein–DNA, and protein–protein interactions, involving structured domains (e.g., reader modules) and intrinsically disordered regions (IDRs) [10.Sabari B.R. et al.Biomolecular condensates in the nucleus.Trends Biochem. Sci. 2020; 45: 961-977Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,11.Roden C. Gladfelter A.S. RNA contributions to the form and function of biomolecular condensates.Nat. Rev. Mol. Cell Biol. 2021; 22: 183-195Crossref PubMed Scopus (119) Google Scholar] (Figure 1C). 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