Protein phase separation: A novel therapy for cancer?

3.1. Epigenetics

Mounting evidence has suggested that epigenetic modifications that regulate transcriptional activity are dysregulated in cancer (Dawson & Kouzarides, 2012). Aberrant nucleosome remodelling, DNA or RNA methylation, histone modifications, and the expression of long non‐coding RNAs are crucial epigenetic mechanisms associated with the occurrence, progression and metastasis of cancers (Park & Han, 2019). Recently, phase separation has been shown to be involved in the process of epigenetic modifications, which may exert an important influence on the initiation and development of cancers, including nucleosome reshape, chromosome condensation, m⁶A modification and long non‐coding RNAs ‐based paraspeckles regulation (Fox, Nakagawa, Hirose, & Bond, 2018; Larson et al., 2017; Plys et al., 2019; Ries et al., 2019; Sanulli et al., 2019; Wang et al., 2019). Thus, it is necessary to reveal the close relationships between phase separation and epigenetics, and it will help us better discover new mechanisms that regulate epigenetics and may lead to unexpected discoveries of new targets for cancer.

In the epigenetic regulation of DNA, researchers have observed that HP1α, a heterochromatin protein 1 (HP1) paralog that mediates the compression of underlying chromatin and leads to gene silencing, has been identified to be capable of forming phase‐separation condensates (Larson et al., 2017; Sanulli et al., 2019). The phase‐separated HP1α condensates can achieve heterochromatin‐mediated gene silencing through the isolation of compacted chromatin, which also enables the recruitment of repressive factors. Furthermore, histone modifications, H3K9me2 and H3K9me3, have been proven to regulate chromosome compartmentalization by promoting HP1α/SUV39H‐induced phase separation (Wang et al., 2019). Recent studies have also reported that linker histone H1, internucleosome linker length, histone acetylation and multi‐bromodomain proteins regulate the phase separation of chromatin to provide the functional diversity of the genome (Gibson et al., 2019). Additionally, chromobox 2 (CBX2), a subunit of polycomb repressive complex 1 (PRC1), drives phase separation to form PcG condensates, which can concentrate nucleosomes and DNA to silence genes (Plys et al., 2019; Tatavosian et al., 2019). To date, the relationship between phase separation and chromatin compaction has been increasingly demonstrated (Narlikar, 2020).

In the epigenetic regulation of RNA, m⁶A is the most prevalent RNA methylation and its associated proteins are frequently up‐regulated in various human cancers (Lan et al., 2019). Recent work has shown that m⁶A promotes the formation of phase‐separated YTHDF–m⁶A–mRNA complexes in cells (Gao et al., 2019; Ries et al., 2019). The higher is the degree of RNA methylation, the stronger the phase separation will be, concomitant with the decline of mRNA translation efficiency. Moreover, long non‐coding RNAs molecules are the newest understood players in gene regulation, and the paraspeckles formed around the long non‐coding RNAs scaffold protein are the workplace for long non‐coding RNAs to regulate gene expression (Yamazaki et al., 2018). Recent discoveries have revealed that the subdomains of NEAT1_2 long non‐coding RNAs preferentially bind NONO/SFPQ proteins, which oligomerize and recruit additional proteins via phase separation to complete paraspeckles assembly (Fox et al., 2018).

3.2. Transcription

Aberrant gene transcription initiates the uncontrolled growth and proliferation of cancer cells (Bradner, Hnisz, & Young, 2017). Many participants are involved in the process of transcription, including transcription factors, cofactors, coactivators, RNA polymerases II, super‐enhancers, and transcription elongation factor, and have been recognized as the focus of cancer target research. For example, many transcription factors are consistently overexpressed or translocated to form fusion genes in cancers and account for approximately 20% of all oncogenes identified so far, such as MYC, YAP/TAZ, KLF4, NRF2, EWS–FLI and MLL rearrangement (Dang, 2012; Janes, 2011; Muller & Vousden, 2013). Additionally, dysregulation of the RNA polymerase transcription apparatus plays an essential role in the malignant transformation of cancer (Bywater, Pearson, McArthur, & Hannan, 2013). Intriguingly, recent studies have shown that phase separation plays an important role in transcription progress (Boija et al., 2018; Lu et al., 2018; Sabari et al., 2018). Understanding the mechanisms of cancer‐related transcriptional condensates, their behaviours and structures may lead to novel therapeutic insights.

Researchers have reported that the activation domains (ADs) of transcription factors OCT4 and GCN4, which generally consist of low‐complexity domain, promote the formation of phase‐separated condensates with mediators (MED1 and MED15) to activate gene expression (Boija et al., 2018). Furthermore, the transcriptional factors MYC, p53, NANOG, SOX2, RARα, and GATA2 are concentrated in MED1–IDR droplets (Boija et al., 2018). Subsequently, there is growing evidence that phase separation plays an important role in transcriptional regulation. YAP and TAZ, pervasively activated transcription factors in cancers, promote specific gene expression through phase separation (Cai et al., 2019; Franklin & Guan, 2020; Lu et al., 2020). Moreover, heat shock transcription factor 1 (HSF1), which is closely associated with malignant evolution of cancers, forms phase‐separated nuclear stress bodies that can sequester HSF1 and thus down‐regulate its function (Gaglia et al., 2020). As transcription proceeds, positive transcription elongation factor b (P‐TEFb) facilitates the hyperphosphorylation of the C‐terminal domain (CTD) of human RNA polymerase II (RNA Pol II) by inducing phase separation (Boehning et al., 2018; Guo et al., 2019; Lu et al., 2018). Thus, the phase‐separated condensates concentrate the substrates and kinases for highly efficient reactions, leading to the hyperphosphorylation of the C‐terminal domain, robust RNA processing and transcriptional elongation. Furthermore, low‐complexity domain–low‐complexity domain interactions of transcription factors promote the formation of a local high concentration of homotypic low‐complexity domain hubs or heterotypic low‐complexity domain hubs to stabilize DNA binding, recruit RNA Pol II, and activate transcription (Chong et al., 2018). Thus, it is worth noting that the selection of the LC domains of FET proteins (such as EWS) as the fusion partner of various DNA binding domains of transcriptional factors (such as FLI1) may easily lead to the BAF complex and RNA Pol II recruitment through phase transition in tumour‐specific enhancers and finally drives oncogenic gene expression programmes in Ewing's sarcoma (Boulay et al., 2017; Kwon et al., 2013).

Additionally, super‐enhancers, large clusters of enhancers that control gene transcription, were demonstrated to exhibit properties of phase‐separated condensates due to multivalent interactions and will be disrupted by chemicals that perturb condensates (Hnisz, Shrinivas, Young, Chakraborty, & Sharp, 2017). Specifically, bromodomain‐containing 4 (BRD4) and mediator complex subunit 1 (MED1), two crucial components of super‐enhancers, mediate the formation of phase‐separated condensates at the sites of super‐enhancers‐driven transcription (Sabari et al., 2018), which can concentrate and compartmentalize the transcription apparatuses at super‐enhancers‐regulated genes and recruit super‐enhancers components to activate transcription. Another study has suggested that enhancer RNA‐dependent ribonucleoprotein complex exhibits the properties of liquid‐like condensates in human breast cancer cells, improving the rapidity and extent of response to transcriptional programmes (Nair et al., 2019).

3.3. Translation

mRNA translation, which includes four steps:‐ initiation, elongation, termination,and ribosome recycling plays a fundamental role in many aspects of cell metabolism. Many oncogenes (MYC, RAS and PIK3CA) and tumour suppressors (PTEN and TP53) can affect the translation machinery, making aberrant translation a common characteristic of tumour cells (Bhat et al., 2015; Chalhoub & Baker, 2009; Prior, Lewis, & Mattos, 2012). Because RNA is an important component of phase separation, studies have reported that the multiple steps in the translation process are controlled by phase separation, including ribosome biogenesis, translation termination, mRNA stability, and deadenylation (Mitrea et al., 2018; Sachdev et al., 2019; Tsang et al., 2019; Xiao et al., 2019). Additionally, RNA/protein bodies phase separation plays critical role in translational regulation (Sachdev et al., 2019). Thus, the research for links between phase separation and translation may lead to a better understanding of the pathology of cancer.

Nucleophosmin 1 undergoes phase separation through both heterotypic interactions with multivalent R‐motif proteins or rRNA and homotypic interaction between its polyampholytic intrinsically disordered regions (Mitrea et al., 2016; Mitrea et al., 2018). Nnucleophosmin 1 can sequest these components away from the nuclear environment, enabling the regulation of ribosome biogenesis and other processes within the confines of the nucleolar matrix. Additionally, translation termination factor Sup35 in yeast has been shown to initiate or terminate translation activities in response to stress changes by phase separation to form gels (Franzmann et al., 2018). The phase separation of Sup35 causes the liquid compartments to harden into gels subsequently and sequesters the termination factor, while making the gels dissolve to cause translation to restart. Fragile X mental retardation protein and cell cycle‐associated protein 1 are functionally involved in mRNA stability and translational repression. Overexpression of fragile X mental retardation protein in breast cancers enhances lung metastasis, while cell cycle‐associated protein 1 expression is significantly up‐regulated in hepatocellular carcinoma, which are closely related to cancer cell cycle and cell proliferation (Luca et al., 2013; Zhang, You, et al., 2018). The latest research has shown that the C‐terminal disordered regions of fragile X mental retardation protein and cell cycle‐associated protein 1 can repress translation by deadenylating mRNA via phase separation (Kim et al., 2019).

Additionally, many cytoplasmic condensates, including stress granules, P granules and processing bodies, are involved in translational regulation. Regarding the well‐studied membraneless organelles, stress granules, many of their important components can undergo phase separation, such as RNA helicase DDX3X (Saito et al., 2019), RNA‐binding protein hnRNPA1 (Molliex et al., 2015), TIA‐1 (Rayman, Karl, & Kandel, 2018), and poly(A)‐binding protein (PAB1) (Riback et al., 2017), which can regulate the formation versus dissolution and biological functions of stress granules. Additionally, RNA helicase LAF‐1 in P granules that can phase separate into condensates in vitro is essential to promote the assembly of P granules (Elbaum‐Garfinkle et al., 2015). Likewise, the PB component Pat1 promotes PB assembly by enhancing the phase separation of DEAD‐box protein Dhh1 and RNA to modulate the decay or storage of mRNAs (Sachdev et al., 2019). Additionally, similar to cytoplasmic RNP granules, nuclear bodies are also reported to be assembled by proteins and RNA molecules selectively. Studies have shown that RNA‐binding motif protein 14 (RBM14) in nuclear paraspeckles, which functions as a regulator of gene transcription and pre‐mRNA splicing, can undergo phase separation to form hydrogels with amyloid‐like properties to regulate multiple aspects of RNA metabolism (Hennig et al., 2015; Xiao et al., 2019).

3.4. Signal transduction

In cancer cells, genetic and epigenetic alterations typically map to signalling pathways that control cell growth and proliferation, cell motility, cell death and fate, which can fuel cancer progression (Sever & Brugge, 2015). According to the location, intracellular cancer‐related signal transduction can be divided into the following three categories:‐ signal transduction on the cell membrane, in local areas of the cell and in the shuttle region (O'Connor, 2015; Schubbert, Shannon, & Bollag, 2007; Simon & Rout, 2014). Thus, phase separation plays a role in many signal transduction pathways and further understanding of its role in signal transduction will undoubtedly provide new ideas for cancer therapy.

3.5. Protein degradation

Protein degradation plays a pivotal role in maintaining protein homeostasis, especially ubiquitin‐mediated degradation and autophagy‐dependent degradation (Hoeller & Dikic, 2009; Levy, Towers, & Thorburn, 2017). A multitude of E3 ligases and their specific substrates recognition in the ubiquitin‐mediated protein degradation pathway make them as significant therapeutic targets for cancer (Hoeller & Dikic, 2009). Autophagy, a process that degrades long‐lived cellular proteins and organelles, is also important in many diseases, including cancer (Levy et al., 2017). Phase separation makes it possible to further understand these two important physiological processes and identifying key proteins associated with phase separation in degradation may play a crucial role in cancer treatment.

Speckle‐type BTB/POZ protein (SPOP), a substrate adaptor of the cullin3‐RING ubiquitin ligase, is one of the first proteins specifically associated with cancer that undergoes phase separation. Recent work has shown that SPOP undergoes phase separation with substrates and co‐localizes with them in membraneless organelles within cells (Bouchard et al., 2018). The disorder of phase separation will interfere with substrate recruitment to ligases, leading to the accumulation of proto‐oncogenic proteins. Ubiquilin‐2 mediates the proteasomal targeting of proteins for degradation by binding to their polyubiquitin chains and subunits of the proteasome. Once the phase separation of ubiquilin‐2 is disrupted by polyUb‐tagged substrates, ubiquilin‐2‐mediated ubiquitinated substrates will be transported from stress granules or other membraneless organelles to protein quality control systems, such as the proteasome (Dao et al., 2018). Additionally, a comprehensive analysis of the substrates of 20S proteasome has revealed that they are highly disordered (Myers et al., 2018), suggesting a close association with phase‐separated condensates.

Recently, pre‐autophagosomal structure was identified as a phase‐separated condensate of autophagy‐related proteins (Fujioka et al., 2020; Jin & Cui, 2020). The autophagy‐related 1 complex undergoes phase separation to form condensates in vitro, which can be inhibited by point mutations and impair pre‐autophagosomal structure formation. Studies have shown that autophagy defects lead to the accumulation of p62 aggregates, facilitating tumourigenesis (Moscat, Karin, & Diaz‐Meco, 2016). Recent work has reported that p62‐formed condensates in vivo have liquid‐like properties and the phase separation is induced by the polyubiquitin chain to drive autophagic cargo concentration and segregation (Sun, Wu, Zheng, Li, & Yu, 2018). Additionally, FIP200 and DAXX could both promote p62 phase condensation (Turco et al., 2019; Yang, Kato, et al., 2019). Moreover, PAB1‐binding protein 1 (PBP1) binds to the mechanistic target of rapamycin complex 1 (TORC1) specifically through an additional methionine‐rich, low‐complexity domain that can cause phase separation and form reversible fibrils (Kato et al., 2019; Yang, Willis, et al., 2019). Mutants that weaken phase separation show decreased ability to inhibit TORC1 and induce autophagy. Additionally, mTORC1 modulates the size and biophysical properties of PGL granules that are assembled by phase separation, determining their susceptibility to autophagic degradation (Zhang, Wang, Du, & Zhang, 2018).

4.1. Regulating scaffold proteins in phase separation

The scaffold proteins are crucial factors affecting phase‐separation formation. In addition to containing the low‐complexity domains, the concentration of the proteins involved in phase separation is extremely important to drive phase separation (Alberti & Dormann, 2019). The condensates formed by phase‐separated proteins are essentially collections of molecules with a high concentration in a certain space, indicating that the alteration of concentration will influence the formation and scale of phase separation, thus affecting their normal function (Sabari et al., 2018; Yang, Willis, et al., 2019). However, as summarized in Table 1, many phase‐separated proteins have been reported to be aberrantly high or low expressed in cancers, affecting their ability to normally and orderly undergo phase separation followed by influencing the corresponding biological processes leading to tumourigenesis. Hence, normalizing the content of proteins may be an important and effective direction for phase‐separation regulation. Fortunately, there are already some proven strategies to regulate protein content, as described below. In terms of the down‐regulation of target proteins, proteolysis targeting chimera (PROTAC) technology provides a powerful means to accurately degrade target proteins by linking them to E3 ligases while autophagy‐targeting chimera (AUTAC) technology facilitates the selective degradation of target proteins through the autophagy‐dependent pathway (Takahashi et al., 2019; Zou, Ma, & Wang, 2019). To increase protein levels, blocking their degradation is a feasible method and has been well studied, such as the use of proteasome inhibitors and lysosomal inhibitors (Mauthe et al., 2018; Nunes & Annunziata, 2017). However, the precise regulation of these methods for phase separation remains to be further studied. In addition to these above methods of regulating the survival time of whole proteins, modulating the aggregation of proteins in specific areas within cells may be a novel therapeutic insight. For example, some phase separation occurs in membranous organelles, such as nuclei and Golgi bodies (Plys et al., 2019; Rebane et al., 2019; Sabari et al., 2018). Thus controlling the transport of scaffold proteins between the cytoplasm and organelles is beneficial to regulate phase separation.

Many recent studies have shown that post‐translational modification is recognized as a pivotal regulator of the assembly and performance of biomolecular condensates, by regulating the weak multivalent interaction that triggers phase separation, often acting as a switch when protein concentrations near thresholds. For example, the phosphorylation of low‐complexity domain in fragile X mental retardation protein can enhance phase separation and translation inhibition, and the translation can be resumed by inhibiting the phosphorylation of fragile X mental retardation protein by casein kinase II (CK2) or promoting the dephosphorylation by protein phosphatase 2A (PP2A) (Bartley et al., 2016; Kim et al., 2019; Tsang et al., 2019). In contrast to phosphorylation, methylation of fragile X mental retardation protein decreases phase‐separation propensity, which can be regulated by protein arginine methyltransferase 1 (PRMT1) (Blackwell, Zhang, & Ceman, 2010). In autophagy‐related proteins, phosphorylation inhibits phase separation and then impairs pre‐autophagosomal structure formation, a process that is catalysed by TORC1 under nutrient‐rich conditions (Fujioka et al., 2020). Another autophagy‐related protein, p62, can undergo phase separation when the K63 and K48 polyubiquitin chains bind to it, and the scale of phase separation depends on the valence of polyubiquitin chains (Sun et al., 2018). Moreover, the phosphorylation of p62 by CK2 can promote the formation of p62 condensates (Sun et al., 2018). Consequently, controlling the activity of enzymes associated with post‐translational modification, such as phosphatases, is a possible way to regulate phase separation and warrants further study experiments to prove its feasibility.

4.2. Interfering with phase separation‐associated client molecules

Client molecules, such as RNA and molecular chaperones, are also crucial for the development of phase separation. For example, recent discoveries have revealed that, in many cases, RNA appears to act as a seed to facilitate phase separation and is involved in most of the fluid‐like condensates formed in cells via phase separation (Banerjee, Milin, Moosa, Onuchic, & Deniz, 2017; Fox et al., 2018; Kim et al., 2019; Tsang et al., 2019). In particular, secondary structures allow mRNAs to self‐associate and determine whether an mRNA is recruited to or excluded from liquid compartments (Langdon et al., 2018). Another study has suggested that RNA can fluidize condensates formed through the phase separation of LAF‐1 by decreasing the viscosity and increasing internal molecular dynamics (Elbaum‐Garfinkle et al., 2015). Interfering with RNA to affect phase separation seems a potential regulatory strategy, and the cells themselves already have some of these regulatory elements. As important players in RNA metabolism from transcription to degradation, RNA helicases have been increasingly reported to be essential regulators for phase‐separated RNA–protein complexes, particularly the DEAD‐box RNA helicase family (Linder & Jankowsky, 2011). For example, DDX3X mediates the maturation and decomposition of stress granules, while Dhh1 promotes processing bodies assembly by binding to Pat1 (Linder & Jankowsky, 2011; Sachdev et al., 2019; Saito et al., 2019). These findings suggest that the indirect intervention of RNA by regulating RNA helicases may be beneficial to regulate phase separation.

Additionally, the molecules bound to proteins involved in phase separation are also important regulatory components, such as molecular chaperones and ligands. Molecular chaperones can regulate the structures of proteins to prevent aggregation and facilitate efficient folding (Hartl, Bracher, & Hayer‐Hartl, 2011). Recent work has shown that they are involved in the negative regulation to form aberrant, solid‐like stress granules (Mateju et al., 2017). Molecular chaperone disorders usually induce toxic misfolded protein aggregates, which are more common in neurodegenerative diseases (Bobori, Theocharopoulou, & Vlamos, 2017; Qamar et al., 2018), but their link to cancer‐related phase separation is only beginning to be understood. Moreover, studies have shown that the steroid hormone 17β‐estradiol (E2) facilitates the interaction between oestrogen receptor (ER) and MED1 by binding the ligand‐binding domain (LBD) of oestrogen receptor, leading to the formation of ER–MED1 condensates and promoting ER‐mediated transcription (Boija et al., 2018). Another study has validated that E2 can rapidly induce transcriptional activation but by facilitating the assembly of another phase‐separated complex enhancer RNA‐dependent ribonucleoprotein (Nair et al., 2019). From the above examples, more critical interactions are likely awaiting discovery in various phase‐separation processes, and the design of protein–protein interaction inhibitors for these highly specific interactions may be an effective strategy for targeted regulation of phase separation.

4.3. Controlling environmental conditions for phase separation

The energy supply of ATP, which can physically rearrange and reorganize the molecular components to maintain the functional state, is essential in phase separation (Brangwynne, Mitchison, & Hyman, 2011). In the case of the RNA helicases and molecular chaperones mentioned above, the important prerequisite for their functioning is ATP consumption (Hartl et al., 2011; Linder & Jankowsky, 2011). Thus, the regulation of the energy supply in phase separation can provide auxiliary assistance to control phase separation, such as regulating related proteins in glycolysis, the tricarboxylic acid cycle, respiratory chain, and ATPase activity (Nilsson & Tarnopolsky, 2019). However, more practice is needed to verify how to achieve the ATP regulation of phase separation.

Additionally, phase separation, which is highly sensitive to disturbances in the cellular environment, is also influenced by pH, temperature, and the salt concentration. A study has validated that a tiny change in pH due to external stress can regulate the conversion of condensates formed by the phase separation of Sup35 into gels while increasing the pH triggers dissolution of the gels, concomitant with translation restart (Franzmann et al., 2018). This suggests that it is attainable to influence phase separation by controlling pH and it may be achieved by regulating sodium hydrogen exchanger, which plays a major role in regulating intracellular pH (Charruyer & Ghadially, 2018). Similar to pH, temperature is also an important environmental condition in regulating phase separation that occurs only in a specific temperature range (Shin & Brangwynne, 2017). The formation of phase‐separated condensates can be controlled by changing the local temperature of the cell. Because photothermal therapy has been studied in cancer, which combines mild hyperthermia with the precise thermal ablation of cancer cells (Liu et al., 2017; Moy & Tunnell, 2017), the temperature regulation of phase separation seems biologically feasible. However, further research is needed to support its implementation.

4.4. Modulating the external media required for phase separation

Noteworthily, external media such as the membrane surface have been increasingly reported to be involved in phase separation. Studies have shown that when T‐cell receptor is activated, its downstream signalling molecules spontaneously undergo phase separation on the membrane, thus promoting signal transmission (Su et al., 2016). Phase separation also regulates the dwell time of SOS proteins on the membrane to affect the activation of downstream Ras proteins (Huang et al., 2019). Additionally, the phase‐separated pre‐autophagosomal structure condensates are tethered to the vacuolar membrane via specific protein–protein interactions to promote the initiation of autophagy (Fujioka et al., 2020). Another example is the formation of tight junctions between cells. By forming adherens junctions, the cell membrane recruits zonula proteins to nascent adhesion sites in large quantities to cross the phase‐separation threshold (Beutel et al., 2019). The above studies indicate that influencing the interactions between proteins and membrane or interfering with membrane formation, thus affecting phase separation and modulating the scale of important biological processes, are promising tasks worth exploring (Zerial & McBride, 2001). To achieve this, identification of the interactions among the membrane, membrane proteins and phase‐separated proteins is required. A tagging system, pupylation‐based interaction tagging (PUP‐IT) (Liu et al., 2018), may help to identify these interactions, from which targeted inhibitors can be developed to regulate phase separation.

5.1. Phase‐separation modulators targeting disordered regions

A hallmark of scaffold proteins are their disordered regions, which is the key molecular driving force underlying the assembly of phase‐separation condensates through mediating multivalent protein–protein (and protein–RNA) interactions (Shin & Brangwynne, 2017). Although no drug that directly targets disordered protein is approved clinically, studies have shown that specifically binding to disordered regions may be achievable. For example, researchers have identified a small‐molecule compound, EPI‐001, that could selectively interact with the disordered region of the androgen receptor (De Mol et al., 2016). Furthermore, entropy seems to be a key factor of selectivity for the modulator targeting disordered region, and specific interactions binding to disordered regions can be optimized through a combination of entropically and enthalpically favourable interactions (Heller, Sormanni, & Vendruscolo, 2015). The entropy of the disordered protein can be expanded after binding small molecules to finally influence the behaviour of proteins (Heller et al., 2015; Heller, Bonomi, & Vendruscolo, 2018). Such small molecules usually lack well‐defined binding sites and generate a fuzzy complex with disordered protein (Fuxreiter, 2018; Miskei et al., 2017). A recently reported research has shown that an acridine derivative, AIM4, has high anti‐TDP‐43 aggregation effects and further demonstrated that AIM4 binds to residues in disordered region of TDP‐43 to inhibit its phase separation (Girdhar et al., 2020), providing a valuable reference to develop future compounds targeting disordered region.

Additionally, by binding to the allosteric sites of proteins, allosteric regulators can cause changes in protein conformation and may affect the phase‐separation phenomenon. Recent research has demonstrated that allosteric sites present within disordered regions facilitate the signalling interactions for various biological mechanisms, which also supports that allosteric regulators may interfere with phase separation (Rehman, Rahman, Arshad, & Chen, 2019). Of course, further theoretical research and discovery are extremely necessary to design small‐molecule compounds targeting disordered regions, the vital research direction of phase‐separation drugs in the future.

5.2. Phase‐separation regulators targeting structured region

Post‐translational modification of scaffold proteins can control the condensates assembly by regulating the weak multivalent interaction that triggers phase separation. Thus the enzymes (such as PARP and HDAC) in these post‐translational modification process can also be the ideal target to regulate phase separation. For example, PAR level acts as a dynamic trigger for biomolecular condensate formation, which is associated with cancer, viral infections and neurodegenerative diseases (Leung, 2020). PARP inhibitors have been clinically approved for cancer and may result from interfering with PAR‐mediated condensates. Additionally, studies have shown that PARP inhibitors can reduce the neurotoxicity of neurodegenerative diseases that are tightly associated with mis‐regulated phase separation, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease, indicating that it perhaps exerts a therapeutic effect through a similar phase‐separation regulation pathway (Kam et al., 2018; McGurk et al., 2018). In addition, some of the scaffold proteins are kinases, such as BRD4 and P‐TEFb, and have inhibitors in development, which bind to structured region of these proteins. Although there have been no in‐depth studies to show whether they interfere with protein phase separations, these inhibitors are closely related to the dysfunction of proteins. Because phase separation is a crucial way for these proteins to function, it is conceivable that these inhibitors may affect the formation and dynamic changes of phase‐separated condensates by regulating its conformation or post‐translational modification. However, until now, no direct and exact experimental evidence is available to confirm these suppositions. Therefore, the effect of current inhibitors on protein phase separation and role of current therapies on cellular condensates deserve more attention.

5.3. High‐throughput screening of phase‐separation modulators

Phase separation can establish visual models in vitro as well as in vivo and combined with the rapid development of high‐content imaging technology, perform the large‐scale high‐throughput screening of phase‐separation modulators is convenient. Anthony A. Hyman, who was the first to describe phase separation, has developed the systems that are cell based and protein based for the drug screening of phase separation in treating neurodegenerative diseases. Results available in bioRxiv have shown that lipoamide and lipoic acid dramatically reduce the aggregation of stress granule proteins, further recovering neuronal and organismal FUS‐associated defects (https://www.biorxiv.org/content/10.1101/721001v2). Moreover, a new technique, called phase‐separated condensate‐aided enrichment of biomolecular interactions in test tubes (CEBIT), has been developed to observe the effects of compounds on the interaction between proteins (Zhou et al., 2020). Specifically, this technique fuses one interacting partner with a scaffold that can form phase‐separation condensates and labels the two proteins with different fluorescent proteins, GFP and mCherry, so that the interaction between the two proteins can be evaluated by enrichment of the mCherry‐labelled recruiting protein in the GFP‐labelled phase‐separation condensates. This in vitro model can also be used to screen for inhibitors of client molecules recruitment in phase separation. Thus, in vitro and in vivo high‐throughput screening is a possible route for phase‐separation modulators development.

In general, the design and development of phase separation‐related drugs are still in their infancy. Certainly, there are many technological challenges regarding the identification of drugs based on phase separation as discussed by Asher Mullard (2019) in Nature Reviews Drug Discovery. For example, because it is not guaranteed that compounds that work well in vivo condensates models can translate into in vivo models, advanced imaging techniques are needed to observe the phase separation in vivo. Additionally, the means to assess the material properties of phase condensates inside a cell, such as liquid like or gel like, will be cutting‐edge technology. Other tools that can control or initiate condensate formation inside the cell will help researchers determine the exact function of phase separation and provide a way to assess the effect of small molecules on phase‐separation dynamics in cells. Additionally, Derek Lowe, a scientist at the Novartis Institutes for BioMedical Research, adds: “Not only would you have to track your compounds in the cell, you'd also have to track them into locations that are so small you need advanced imaging techniques just to convince yourself they exist”. More efforts are needed to further explore this field.

6.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Cidlowski, et al., 2019; Alexander, Fabbro, et al., 2019; Alexander, Kelly, et al., 2019).