Small molecules in regulating protein phase separation

Abstract

Biomolecular condensates formed by phase separation are involved in many cellular processes. Dysfunctional or abnormal condensates are closely associated with neurodegenerative diseases, cancer and other diseases. Small molecules can effectively regulate protein phase separation by modulating the formation, dissociation, size and material properties of condensates. Discovery of small molecules to regulate protein phase separation provides chemical probes for deciphering the underlying mechanism and potential novel treatments for condensate-related diseases. Here we review the advances of small molecule regulation of phase separation. The discovery, chemical structures of recently found small molecule phase separation regulators and how they modulate biological condensates are summarized and discussed. Possible strategies to accelerate the discovery of more liquid-liquid phase separation (LLPS)-regulating small molecules are proposed.

Introduction

Liquid-liquid phase separation (LLPS) is a key physiological process in organizing biomolecules to form essential membrane-less organelles (also known as condensates). Condensates are involved in many biological processes including stress response, transcriptional regulation, and signal transduction [ 1– 3] . According to available LLPS databases, more than 70 condensates have been defined in living systems and there are over 40,000 LLPS-associated proteins [ 4, 5] .

Biomolecules that undergo LLPS in cells can be divided into two categories, scaffolds and clients [6]. Scaffolds are necessary for condensate formation, and scaffold molecules can usually undergo phase separation in vitro. However, clients are not necessary for the formation of condensates and are recruited into condensates through their interactions with scaffolds. Scaffolds are biological macromolecules, such as proteins or nucleic acids, that contain multiple interaction domains or intrinsically disordered regions (IDRs) that mediate multivalent interactions [7].

LLPS is closely associated with diseases, such as cancer, infectious diseases, and most notably neurodegenerative diseases [ 8– 10] . Patients with neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer′s disease and Parkinson′s disease, carry pathological inclusions that are condenses of pathological states. Key proteins in pathologic aggregates, such as FUS, TDP-43, α-synuclein, and Tau, undergo liquid-liquid phase separation and liquid-to-solid phase transition [11]. Thus, targeting LLPS has been considered as a promising therapeutic approach [12].

In this review, we summarize and discuss the advances of small molecule regulation of phase separation. We begin with the first commonly used phase separation regulator 1,6-hexanediol (1,6-HD) and describe its characteristics and limitations. We then review a list of recently discovered small molecules that regulate phase separation. We next discuss possible strategies to promote the discovery of more LLPS-regulating small molecules.

Overview of Small Molecule Regulation of Biomolecular Condensates

Since Brangwynne et al. [13] found in 2009 that the Caenorhabditis elegans P granules exhibit liquid-like behaviors, and Li et al. [14] reconstructed condensates in vitro in 2012, the biological functions of condensates attract great interest. In 2019, Alberti et al. [15] summarized the strategy to study the biological function of condensates, that is, whether the disruption of condensates will disrupt function. Experimentally, the condensates can be disrupted, usually by constructing LLPS-deficient protein mutants, or by using phase separation inhibitors. 1,6-Hexadiol is the most popular small molecule regulator for LLPS, as it was found to efficiently disrupt intracellular and in vitro condensates [16].

Regulating disease-related condensates provides a novel way to treat neurodegenerative diseases or cancer. Since 2019, several efforts have been made to screen small molecule drugs that can regulate condensates. These studies have identified some new phase separation regulators through cell-based or protein-based phenotypic screening. In vitro studies have also shown that the effects of small molecules on condensates include enhancement, dissociation, dual effect, inducing condensate formation, and modification of material properties ( Figure 1). Although the molecular grammar of phase separation has been much studied, the chemical grammar of interactions between small molecules and condensates remains unclear. At present, new small molecules are mainly obtained by screening, and the selectivity and molecular mechanism of small molecules on condensates need to be further clarified.

Figure 1 .

Effect of small molecules on condensates

The effects of small molecules on condensates can be divided into enhancing, dissolving, enhancing or dissolving (dual effect), inducing condensate formation, and modifying condensate properties. Small molecules such as metal ions and TMAO enhance phase separation [ 17, 18] . 1,6-HD is a typical condensate destroyer. ATP and bis-ANS enhance LLPS at low concentrations and inhibit LLPS at high concentrations [ 19, 20] . Huezole can selectively induce tubulin condensates [21]. There are also small molecules that significantly modify the material properties of condensates, such as Ro-3306 [22].

The Most Used Phase Separation Regulator — 1,6-Hexadiol

1,6-Hexanediol (1,6-HD) is a representative small molecule that can disrupt intracellular or in vitro condensates within minutes at a concentration of 5%‒15%. 1,6-HD can dissolve many condensates formed by scaffold proteins in vitro, such as FUS, hnRNPA1 and TDP-43, as well as a variety of membrane-less organelles in cells, such as P granules, stress granules, P bodies, Cajal bodies, splicing speckles and HP1α foci [ 16, 23– 25] . However, 1,6-HD cannot dissolve solid-like structures, such as FUS fibers in vitro and membranous organelles, solid-like protein aggregates, and cytoskeletons in cells [16].

In 2002, Ribbeck et al. [26] found that treatment of cells with trans-cyclohexane-1,2-diol or hexane-1,2-diol resulted in non-selective opening of the nuclear pore complexes (NPCs), while treatment with less hydrophobic 1,2,3-hexanetriol did not. It was proposed that aliphatic alcohols may disrupt hydrophobic interactions between FG residues repeats of nucleoporins. In 2003, Shulga et al. [27] studied the effects of a series of alcohols and found that methanol, ethanol, isopropyl alcohol, n-butanol, cyclohexanol and 1,6-hexanediol all promoted the opening of NPCs to varying degrees, and the hydrophobicity (log P) and the half maximal effective concentration (EC ₅₀) of these alcohols were roughly proportional. These findings support the idea that hydrophobic aliphatic alcohols disrupt weak hydrophobic interactions between nucleoporins. In 2016, Lin et al. [28] compared the effects of 1,6-HD, 1,5-HD, 1,5-PD (1,5-pentanediol) and 1,4-BD (1,4-butanediol) on the prion-like domain of FUS in vitro, and found that 1,6-HD, with the strongest hydrophobicity, had the strongest ability to disrupt LLPS. The above evidence shows that various alcohols can affect phase separation by disrupting weak hydrophobic interaction, and the more hydrophobic alcohol have stronger ability to disrupt LLPS.

1,6-HD is often used to distinguish between liquid-like condensate and solid-like condensate and to study the biological function of LLPS in cells. Sabari et al. [29] used 1,6-HD to destroy the MED1 transcription condensates that form at super enhancers. They found that the abundance of BRD4, MED1 and RNA Pol II at super enhancers and downstream gene regions decreased significantly after 1,6-HD treatment, which indicates that the disruption of the integrity of transcription condensates will affect transcription. 1,6-HD can directly and promptly destroy the condensates. Ulianov et al. [30] investigated the role of LLPS in the three-dimensional genome of living cells and found that 1,6-HD-sensitive LLPS is not the primary force supporting genome organization, but contributes to the fine-tuning of the 3D genome. In general, 1,6-HD has the following advantages in LLPS regulation: 1) 1,6-HD is an aliphatic alcohol with strong hydrophobicity, which can effectively disrupt weak hydrophobic interactions between proteins and nucleic acids. 2) It can be used for in vitro experiments and treatment of living cells. 3) The phenotype changes rapidly. 4) The disrupting effect can be reversed by washing out. 5) It is inexpensive and easy to use. For these reasons, 1,6-HD has been widely used in most LLPS-related studies.

However, 1,6-HD has drawbacks that limit its use. First, 1,6-HD is not a universal inhibitor of LLPS. The chemical properties of 1,6-HD determine that it can only disrupt hydrophobic interactions rather than electrostatic interactions. Condensates driven by electrostatic interactions are known immune to 1,6-HD, such as Tau condensates, as well as VRN1-DNA condensates in vitro [ 31, 32] . Nor can 1,6-HD dissolve the core of mammalian stress granules purified in vitro [33]. The replication compartments (RCs) formed by cells infected with herpes simplex virus type 1 (HSV1) are liquid-like condensates but are stable in the presence of 1,6-HD [34]. Second, high concentration of 1,6-HD needs to be used. The commonly used concentration is a mass to volume ratio of 10%, corresponding to a molar concentration of about 850 mM. At such high concentrations, 1,6-HD is likely to disrupt many non-specific interactions. Third, the effect of 1,6-HD is neither specific nor selective. When used to treat cells, 1,6-HD dissolves various condensates at the same time and disrupts a wide range of protein-protein interactions within cells. For multi-component condensates, 1,6-HD cannot affect only specific proteins in the condensates. Fourth, 1,6-HD shows significant cytotoxicity at working concentrations. 1,6-HD results in decreased cell survival and significant changes in cell morphology, possibly due to high osmotic pressure and loss of cell membrane integrity [ 1, 16] . Finally, the molecular mechanism of 1,6-HD remains elusive. Although there is some evidence that its hydrophobicity is associated with the ability to disrupt LLPS, more explanation at the molecular level is needed.

Discovery of Small Molecules That Regulate Phase Separation

Current research indicates the central role of condensates in biological processes and pathology. Targeting condensates has been recognized as a promising disease therapy. Therefore, both scientific research and therapeutic needs are driving the discovery of new LLPS-regulating molecules.

The discovery of new LLPS-regulating molecules first began in the field of neurodegenerative diseases. Table 1 shows emerging small-molecule regulators for phase separation. In 2019, Fang et al. [35] identified a series of small molecules with planar structures, such as Mitoxantrone, through high-throughput screening. These planar molecules disassembled stress granules (SGs) in human iPSC-derived motor neurons and prevented the recruitment of Amyotrophic lateral sclerosis (ALS)-related proteins into SGs. In another work, Wheeler et al. [36] screened a small library of FDA-approved drugs and found that lipoamide and lipoic acid disassembled SGs and regulated FUS phase separation in vitro, and recovered motor defects in Drosophila melanogaster model of ALS. This work suggests that the regulation of disease-associated condensates may be a novel approach of drug design. In 2020, Girdhar et al. [37] found that an acridine derivative, AIM4, which was previously reported to inhibit aggregation of the ALS-related protein TDP-43, also disrupted LLPS. This suggests that small molecules that inhibit aggregation and regulate phase separation may have similar structural features. The formation of RNA foci by phase separation is a common pathological feature of nucleotide repeat expansion disorders. The expansion of the hexanucleotide GGGGCC in the C9orf72 gene is the most common mutation associated with ALS and frontotemporal dementia (FTD) [ 38, 39] . Jain et al. [40] found that ammonium acetate and doxorubicin could inhibit RNA condensates in vitro and RNA foci in cells. In another study, Simone et al. [41] found that small molecules targeting the secondary structure of RNA were able to inhibit RNA foci in C9orf72 patient neurons. The LLPS of Tau, a microtubule-associated protein, likely contributes to the nucleation of microtubules and the formation of microtubule bundles [42]. The liquid-to-solid transition of Tau condensates is thought to be involved in Alzheimer′s disease [ 43– 46] . Myricetin, a natural antioxidant flavonoid, has been discovered to inhibit LLPS of full-length Tau in vitro, and significantly inhibit pathological phosphorylation and abnormal aggregation of Tau in neuronal cells [47]. In a recent work, Ramesh et al. [48] attempted to rationally design small molecules based on the chemical grammar of LLPS. By integrating a variety of chemical groups that offer diverse interactions, small molecule modulators based on cyclic dipeptide (CDP) was rationally designed to effectively inhibit Tau LLPS and amyloid aggregation in vitro.

Table 1 Emerging small molecule drug candidates that target condensates

Small molecule	Discovery method	Target condensate	Associate diseases	Effect	Reference
Mitoxantrone	Cell-based screen	Stress granule	ALS, FTD	Dissolution of SGs, inhibition of TDP-43 accumulation	[35]
Lipoamide, lipoic acid	Cell-based and protein-based screen	Stress granule	ALS	Reduction of SG formation	[36]
AIM4	Previously found to inhibit TDP-43 amyloid aggregation in vitro	TDP-43 2C-A315T condensate	ALS	Inhibition of the in vitro condensates of TDP-43 2C-A315T	[ 37, 49]
DB1246, DB1247, DB1273	Identification of C9orf72 repeat RNA G-quadruplexes binders by FRET-based screen	RNA foci in cell	ALS, FTD	Reduction of RNA foci in C9orf72 patient neurons	[41]
Myricetin	In vitro test	Tau condensate	AD	Inhibition of the in vitro LLPS of full-length Tau	[47]
SMM 1c	Rationally designed cyclic dipeptide-based small molecule	Zn-mediated Tau condensate	AD	Inhibition and dissolution of Zn-mediated Tau condensates in vitro	[48]
Myricetin	Previously found to prevent the oligomerization of α-Syn	α-Syn condensate	PD	Delaying the liquid-to-solid phase transition of α-Syn condensates in vitro	[ 50, 51]
Curcumin	Previously found to inhibit α-Syn aggregation	α-Syn condensate	PD	Inhibition of α-Syn amyloid aggregation under LLPS in vitro	[ 52, 53]
Nelfinavir, mesylate, nilotinib, LDK378	In vitro screen	SARS-CoV -2 N protein condensate	COVID-19	Influence on the number, size, and shape of N protein condensates in vitro	[54]
CVL218, PJ34	Previously found to bind to N protein	SARS-CoV -2 N protein condensate	COVID-19	Increasing the size of N protein condensates	[55]
GCG	Cell-based screen	SARS-CoV -2 N protein condensate	COVID-19	Inhibition of N protein condensates in cells	[56]
Cyclopamine	RSV inhibitor	RSV inclusion body	Respiratory illness caused by RSV	Hardening RSV inclusion bodies in vivo	[ 57, 58]
Elvitegravir	Cell-based screen	SRC-1 condensate	Cancer	Inhibition of SRC-1 condensates	[59]
ET516	Cell-based screen	AR condensate	Prostate cancer	Inhibition of AR condensates	[ 60, 61]
GSK-J4	In vitro screen	Core regulatory circuitry condensate	Metastatic and chemo-resistant osteosarcoma	Inhibition of core regulatory circuitry condensates	[62]
BAY 249716, BAY 1892005	In vitro screen to identify compounds that interact with mutant p53	p53 condensate	Cancer	Dissolution of p53 structural mutant condensates and condensation of p53 DNA binding mutant	[63]
SHP099	SHP2 allosteric inhibitor	SHP2 condensate	Noonan syndrome and juvenile myelomonocytic leukemias	Inhibition of SHP2 condensates	[ 64, 65]

Abbreviations: FRET, fluorescence resonance energy transfer; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019.

Preventing the liquid-to-solid phase transition of disease-associated proteins is another potential therapeutic strategy for neurodegenerative diseases. In a systematic study by Jonchhe et al. [66], the effects of different ions and small molecules on the Tau liquid-to-solid phase transition were studied. They found that hydrophobic alkyl chains and aromatic rings delayed the liquid-to-solid phase transition in the Tau condensate, and charged groups facilitate the liquid-to-solid transition in a manner similar to the Hofmeister effect. The aggregation of α-synuclein (α-Syn) is a hallmark of Parkinson′s disease (PD). Recent evidence suggests that α-Syn condensates become amyloid aggregates through a liquid-to-solid phase transition [ 67, 68] . Myricetin was discovered to delay the liquid-to-solid phase transition while not affecting the formation of α-Syn condensates [50]. Another small natural molecule, curcumin, has been shown to regulate α-Syn amyloid aggregation during protein phase separation [52].

The N protein LLPS plays an important role in the assembly of SARS-CoV-2 [56]. Therefore, targeting N protein condensation is considered as a potential treatment for COVID-19. Iserman et al. [69] tested 1,6-HD, lipoic acid, and kanamycin, each of which could inhibit LLPS of N protein with RNA in vitro. Kanamycin affects protein-RNA interactions by binding to nucleic acids [70]. In 2021, Jack et al. [54] identified that nelfinavir mesylate, nilotinib, and LDK378 can affect the number, size, and shape of N protein condensates through screening of FDA-approved drug library in vitro. All of them inhibit the proliferation of SARS-CoV-2 in host cells. In another work, Zhao et al. [55] found that poly ADP-ribose polymerase (PARP) inhibitors CVL218 and PJ34 can increase the size of N protein condensates. The combination of CVL218 and remdesivir enhances the effects of individual drugs. It is hypothesized that the increased size of the condensates may reduce the local protein density and facilitate the entry of other drugs into the condensates. In addition, (-)-gallocatechin gallate (GCG), a natural product, can disrupt the LLPS of N protein and inhibit the replication of SARS-CoV-2 [56]. Hardening the virus-replicating condensates appears to be an effective strategy. Risso-Ballester et al. [57] reported that the steroidal alkaloid cyclopamine inhibits human respiratory syncytial virus (RSV) replication by hardening virus-induced condensates. This strategy of hardening condensates has the potential to block the function of specific condensates, thus promising applications in many cases.

There is compelling evidence that condensates are associated with cancer [ 71, 72] . Targeting condensates has become a new cancer treatment strategy. Yes-associated protein (YAP) is a transcriptional coactivator and abnormal YAP activation is prevalent in a variety of cancers [73]. Zhu et al. [59] found that SRC-1 is an important coactivator in YAP/TEAD transcriptional condensates. The anti-HIV drug elvitegravir (EVG) can directly bind to SRC-1 and inhibit SRC-1 LLPS, thus suppressing YAP transcriptional activity and tumor cell growth. Androgen receptor (AR) plays a key role in the development of prostate cancer. AR mutation makes it resistant to AR antagonists used in clinic [ 74, 75] . Another work by the same research group has shown that the inhibitory effect of AR antagonist on the transcription condensates of drug-resistant mutated AR becomes weaker or becomes a promoter, which may be the cause of resistance [60]. Through a LLPS-based phenotypic screen, it was found that small molecule ET516 can disrupt wild-type AR and mutant AR condensates, suppress AR transcriptional activity, and significantly inhibit the proliferation of prostate cancer cells. In another work targeting super enhancers to treat osteosarcoma, the H3K27 demethylase inhibitor, GSK-J4 was found to specifically attenuate core regulatory circuitry (CRC) condensates and inhibit proliferation of various osteosarcoma cell lines [62]. p53, as a cancer suppressor, is often downregulated or mutated in tumors. p53 structural mutants form aggregates, and p53 aggregation may transit between condensate states. Lemos et al. [63] found that two aminothiazoles, BAY 249716 and BAY 1892005 could regulate the condensation of p53 mutants in cancer cells. In addition, the molecular mechanism of several anticancer drugs may also be related to condensates. Recent studies have found that oxaliplatin, the most common anticancer drug, may cause liquid-liquid demixing of nucleoli and lead to cell cycle arrest [76].

Abnormal condensation of the non-receptor protein tyrosine phosphatase SHP2 has been found to associate with Noonan syndrome and juvenile myelomonocytic leukemias. Zhu et al. [64] found that the LLPS ability of SHP2 is related to its conformation, and the small molecule allosteric inhibitor SHP099 can regulate the conformation of SHP2 and thus affect its LLPS potential.

Small Molecular Additives, Cellular Metabolites, or Molecular Tools That Regulate Phase Separation

For drugs that regulate phase separation, strict clinical concentrations need to be followed, as well as consideration of molecular toxicity and side effects. There are many small molecules that can regulate phase separation, such as 1,6-HD, regardless of limitations such as working concentration or cytotoxicity. Monovalent salt ions weaken electrostatic interactions through electrostatic shielding effect, and indeed NaCl and KCl are often used to disrupt condensates in vitro or in cells [ 32, 77] . However, at high salt concentrations, hydrophobic interactions are enhanced due to salting-out effects. At this point, some proteins, such as FUS, undergo reentrant phase separation driven by enhanced hydrophobic interactions [78]. Metal ions tend to bind to proteins or nucleic acids and have also been reported to affect phase separation. Manganese ions have been reported to induce α-Syn to form solid-like condensates and to promote α-Syn liquid-to-solid phase transition [17]. The metal cofactor Zn ²⁺ inhibits Cu/Zn superoxide dismutase (SOD1) LLPS through conformational transition [79]. Zinc ions can promote LLPS of full-length human Tau in vitro [80]. Denaturants, such as urea and guanidinium, disrupt the tertiary structure of proteins and weaken hydrophobic interactions between solutes, thus inhibiting phase separation. In contrast, trimethylamine N-oxide (TMAO) stabilizes the three-dimensional structure of proteins and has been reported to promote phase separation of the disordered region of TDP-43 [18]. In addition, D ₂O may be an enhancer of LLPS. It has been reported that small amounts of heavy water increase the LLPS tendency of a fragment of the androgen receptor activation domain [81]. These findings highlight the need to fully consider solution conditions in biophysical experiments such as nuclear magnetic resonance (NMR) studies.

Metabolites in cells can also regulate phase separation. Adenosine triphosphate (ATP) is the energy fuel of cells. And ATP has been reported to have a two-stage effect on phase separation. The phase separation is enhanced at low ATP concentration and inhibited at high ATP concentration [ 19, 82– 84] . In addition, other molecules such as bis-ANS have similar dual effects, which are related to bivalence [20]. The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is a crucial regulator of intracellular signaling. In fact, cAMP itself promotes the phase separation of PKA regulatory subunit, RIα [85]. Glucose is another abundant endogenous substance in cells. It has been reported to promote calmodulin condensation [61].

Theoretically, small molecules can regulate not only the dissociation, size, and composition of condensates, but also the formation and material properties of condensates. Ado et al. [21] found that small molecules with self-assembly potential can induce specific proteins to form condensates. They identified a non-peptidic small molecule called huezole that selectively enriches tubulin to form condensates at 20 μM in vitro and in cell. This means that it might be possible to use small molecules to induce condensates with specific components that could exogenously control cellular processes. Wang et al. [22] showed that the material properties of condensates affect their biological functions, and that small molecules can influence the activity of condensates by modulating the material properties. They identified small molecules Ro-3306 and vanillic acid that increase the fusion propensity of transcription factor TFEB condensates. The modification of the condensate’s material properties upregulates the transcriptional activity of TFEB, thereby regulating the autophagy-lysosome pathway.

Concluding Remarks and Future Perspectives

As a basic principle of organizing biomolecules, liquid-liquid phase separation has been widely accepted. The roles of biomolecular condensates in cellular processes have been much studied and the association with a variety of diseases has been established. In comparison, discovering small molecules that can disrupt specific LLPS just emerges.

Most of the recently discovered LLPS-regulating small molecules have been validated to bind to the target. The small molecule AIM4, which inhibits the aggregation and phase separation of TDP-43, binds to the prion-like domain of TDP-43 [37]. Small molecules that specifically bind to C9orf72 repeating RNA G-quadruplex reduce RNA foci in the neurons of patients [41]. A rationally designed cyclic dipeptide molecule has strong binding ability with both Tau and Zn and can inhibit zinc-mediated Tau phase separation [48]. SARS-CoV -2 N protein binding small molecules CVL218 and PJ34 affect the size and kinetic properties of N protein condensates [55]. GCG directly binds to N protein and disrupts N protein LLPS [56]. Elvitegrair directly binds to SRC-1, disturbing SRC-1 LLPS to constrain YAP-dependent cancer cell growth [59]. The small molecule ET516 that disrupts AR condensates directly binds to the disordered N-terminal domain [60]. GSK-J4 disrupts CRC condensate by directly binding to HOXB8-IDR [62]. Small molecules BAY 249716 and BAY 1892005 interact with p53 and regulate phase behavior of p53 mutants [63].

So far, 1,6-HD remains the only widely used small molecule in LLPS studies. Because of the limitations and toxicity of 1,6-HD, the discovery of small molecules with new structure or better function can provide more powerful tools for research. Considering that intracellular and in vitro condensates that are usually micron scale are an easily observable phenotype, high throughput screening can be performed by high-content imaging techniques, thus identification of small molecules that affect condensates is feasible. Small molecules can also be designed to directly target proteins or nucleic acids. Although many LLPS proteins often have disordered regions that make it difficult to find ligands, there are successful examples of small molecules that bind to disordered proteins such as p53 and c-Myc [ 86, 87] . A new idea is that small molecules that can regulate phase separation do not require a strong binding to the target [88]. Lipoamide, for example, affects the material properties of FUS condensates, but does not directly bind to proteins with high affinity [36].

In fact, most currently known LLPS-regulating small molecules are obtained by phenotypic screening. It remains challenging to identify targets and molecular mechanisms of the novel LLPS-regulating molecules discovered by phenotypic screening. Some cell model-based screening efforts lack in vitro validation to determine whether the small molecules regulate condensates directly. In addition, when screening for the purpose of treating diseases, the druggability and kinetic parameters of molecules have to be taken into account. So, researchers often screen compounds that are FDA-approved drugs or have good druggability, which only covers a small chemical space. Figure 2 shows the chemical structure of some emerging LLPS-regulating small molecules. Most of the small molecules discovered so far have certain common characteristics, such as strong hydrophobicity, high molecular weight, more specifically, these molecules usually have multiple aromatic rings, or have long alkyl chains, or have multiple functional groups that can provide a variety of chemical interactions. In addition, whether the small molecules regulating the condensates are selective remains elusive. Rational drug design may be a good strategy if the LLPS mechanism of the target is well studied. For example, allosteric inhibitors of SHP2 specifically bind to it to regulate the conformation and phase separation of SHP2. For disordered proteins, specific binding ligands can also be obtained through computer-aided drug design strategies or high throughput screening.

Figure 2 .

Chemical structure of representative condensate-regulating small molecules

In summary, biomolecular condensates have received extensive attention as targets for drug discovery. Small molecules that regulate condensates have shown potential to treat related diseases. Further elucidation of the physical and chemical mechanism of the interaction between small molecules and condensates will promote the development of novel therapies.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported in part by the grant from the National Natural Science Foundation of China (No. 22237002).