Application of polymersomes in membrane protein study and drug discovery: Progress, strategies, and perspectives

Abstract Membrane proteins (MPs) play key roles in cellular signaling pathways and are responsible for intercellular and intracellular interactions. Dysfunctional MPs are directly related to the pathogenesis of various diseases, and they have been exploited as one of the most sought‐after targets in the pharmaceutical industry. However, working with MPs is difficult given that their amphiphilic nature requires protection from biological membrane or membrane mimetics. Polymersomes are bilayered nano‐vesicles made of self‐assembled block copolymers that have been widely used as cell membrane mimetics for MP reconstitution and in engineering of artificial cells. This review highlights the prevailing trend in the application of polymersomes in MP study and drug discovery. We begin with a review on the techniques for synthesis and characterization of polymersomes as well as methods of MP insertion to form proteopolymersomes. Next, we review the structural and functional analysis of the different types of MPs reconstituted in polymersomes, including membrane transport proteins, MP complexes, and membrane receptors. We then summarize the factors affecting reconstitution efficiency and the quality of reconstituted MPs for structural and functional studies. Additionally, we discuss the potential in using proteopolymersomes as platforms for high‐throughput screening (HTS) in drug discovery to identify modulators of MPs. We conclude by providing future perspectives and recommendations on advancing the study of MPs and drug development using proteopolymersomes.

these, only less than 2% have high-resolution structures consistently found in all databases. 6,10 The dearth of studies that focus on MPs can be contributed by various factors. First, MPs are usually unstable and require a bilayer membrane for them to be folded correctly during protein translation. Second, it is difficult to obtain stable and functional MPs of interest in high yields, as MPs are usually low in numbers and tend to aggregate in the cytoplasm, despite attempts at protein overexpression. 6,11 Importantly, MPs are generally insoluble in aqueous solution due to the incompatibility between the hydrophobic nature of MP surfaces associated with lipid membranes and the hydrophilicity of solvent molecules. The use of amphiphilic agents is thus necessary to extract MPs from the native membranes and maintain them in a stable soluble form. Hence, there is a need to develop synthetic membrane platforms that mimic native biological membrane to provide amphiphilic environments for the MPs and maintain their structural and functional integrity for in vitro protein studies. [12][13][14][15] Conventional methods of MP study include the usage of protein tethered lipid bilayer and supported planar lipid bilayer membranes. 16,17 However, these systems have limitations such as incompatibility between tethered molecules and extra-membranous domains, inaccessibility of region occupied by tethered molecules as well as uncontrollable orientation of inserted MPs and constraints on their biological functions. 16,17 Therefore, cell membrane mimetics with vesicular morphologies known as nano-vesicles have been increasingly used to overcome these limitations. 18 While liposomes are composed of natural nontoxic phospholipids, polymersomes are formed by amphiphilic block copolymers. 19,20 Both types of nano-vesicles are analogous to biological membrane and suitable for MP residence. 17,18,[20][21][22] These nano-vesicles, or small unilamellar vesicles (SUV), have a size of 20-100 nm and have the lowest interfacial area and highest configurational entropy as compared to other morphologies. This makes them more energetically favorable for MP reconstitution. 20 They also have an increased stability over large unilamellar vesicles (LUVs, >100 nm in size) and giant unilamellar vesicles (GUVs, >1 μm in size). 20 Additionally, they contain a concentration gradient, which can play a key role in determining the functions of poreforming channel MPs. 23 While liposomes have been widely used and reviewed for their use in MP reconstitution and the related structural and functional studies, 17 they are limited by low stability. 22 To overcome this limitation, polymersomes have been increasingly adopted for MP studies because of their superior stability. [22][23][24] Liposomes and polymersomes with reconstituted MPs are termed as proteoliposomes and proteopolymersomes, respectively.
Apart from finding a suitable membrane support, it is crucial to ensure that the inserted MPs are folded in the correct orientation and maintain their biological functions, in order to facilitate further characterizations of these MPs. 14,15 Hence, it is imperative to optimize chemical constituents used in the formation of polymersomes or hybrid polymer-lipid systems, [25][26][27][28][29][30] MP production methods, and parameters used in the reconstitution process. 17,31,32 The reconstitution process plays a key role in determining the efficiency of reconstitution, the quality of the inserted MPs, as well as the resolution and capacity of the methods used to study these MPs. 17,31 In this article, we will review the use of polymersomes in MP structural and functional studies, as well as their translational application in highthroughput screening (HTS) for drug discovery (Figure 1). We start by introducing the synthesis and characterization of polymersomes and methods of MP reconstitution to form proteopolymersomes. We then summarize the use of proteopolymersomes in studying both the structures and functions of channel proteins, MP complexes, and membrane receptors. Additionally, we provide a comprehensive list of factors affecting the efficiency of MP insertion and the quality of the inserted MPs. Finally, we discuss the feasibility and current applications of proteo-nano-vesicles in HTS. We conclude by providing future prospects in using polymersomes to engineer artificial cells as well as laying out a roadmap with recommendations for using proteopolymersomes in drug discovery pipeline.

| SYNTHESIS AND CHARACTERIZATION OF POLYMERSOMES
Polymersomes are spherical nanovesicular systems with polymer shells of 5-50 nm in thickness and are formed by the self-assembly of amphiphilic block copolymers. [35][36][37][38] The polymersome membrane provides a physical barrier that isolates the encapsulated materials from external biological environment, while allowing controlled release or exchange of biological molecules due to the presence of a concentration gradient. A major difference between polymersomes and liposomes lies in the chemical versatility to control the thickness of the membranes where liposomes are limited to a membrane thickness of up to 5 nm, while polymersomes can have membrane thickness of up to 50 nm, depending on the type of block copolymers used. 24 This suggests that polymersomes could potentially accommodate larger and higher amounts of MPs than liposomes, although it is important to consider the hydrophobic mismatch that might be present during MP insertion. 24 Due to the higher molecular weight of constituent block polymers and the potential of forming cross-linking structures through UV irradiation, 39,40 polymersomes usually have enhanced mechanical properties, 41,42 higher stability, 43,44 lower dissociation rates, lower permeability, 44 and limited leakage 45 compared to liposomes ( Figure 2a). 20,22 Furthermore, their dense hydrophilic polymer brush-like coronas increases their resistance to degradation and have longer circulation half-lives in vivo. 48

| Diblock copolymers
The most commonly used diblock polymers is poly(butadiene)-b-poly (ethylene oxide) (PBD-PEO)-based. 47,49,51 Their ability to provide more fluidity over other diblock copolymers make them suitable for studying membrane receptors. 52,53 Polystyrene-b-poly(isocyanoalanine[2-thiophen-3-yl-ethyl]amide) (PS-PIAT) diblock copolymers self-assemble into an intrinsically porous bilayer, 54  F I G U R E 1 Polymersomes as platforms for MP study and drug discovery. Polymersomes, which are made up of block copolymers, can mimic biological membranes for reconstitution or incorporation of MPs, including channels, receptors, and protein complexes to form proteopolymersomes (center). Proteopolymersomes can be used to study the structure-function relationship of MPs including the characterization of (a) receptor-ligand binding through the use of surface plasmon resonance (SPR), 33  (PEtOz-PDMS-PEtOz). [60][61][62] To create a polymeric nanocompartment with low permeability, polyisobutylene-polyethylene glycol-polyisobutylene (PIB-PEG-PIB) (BAB) with the PIB unit being impermeable to many molecules, 63 has been used in the formation of polymersomes with the insertion of an Escherichia coli (E. coli) outer MP. 64 Poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) is another type of BAB polymer, which has been used to synthesize polymersomes as nanocarriers for delivery of hydrophilic and hydrophobic drugs. 65 To account for the membrane asymmetry in lipid composition, poly(ethylene oxide)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PEO-PDMS-PMOXA) (ABC) is used. 66,67 ABC polymers can adopt a mixture of hairpin or transmembrane orientations due to steric hindrance and are useful for MP study as they can change their chemical composition to influence the orientation of the inserted integral proteins upon the application of external fields such as electric fields to its membrane leaflets. 68 Recently, an one-pot synthesis method of a new ABC triblock terpolymer, poly(ethylene oxide)-block-poly(2-(3-ethylheptyl)-2-oxazoline)-block-poly (2-ethyl-2-oxazoline) (PEO-PEHOx-PEtOz), using sequential microwaveassisted polymerization has been reported. 69

| Characterization of polymersomes
The hydrodynamic radius, size distribution, and morphology of the formed polymersomes can be characterized by dynamic light scattering (DLS), static light scattering (SLS), optical microscopy, and transmission electron microscopy (TEM). 82 High-throughput scattering methods such as combinatorial small-angle X-ray scattering (SAXS) or wide-angle x-ray scattering (WAXS) can provide information about structural features of colloidal size, including membrane bilayer thickness and internal structure. 83 The small-angle neutron scattering (SANS) technique can study the morphology and thermodynamics of polymer blends and copolymers in polymersomes, as well as the polymersome structure. 84 Optical microscopy can only resolve polymersomes larger than 1 μm in diameter, 85 while higher resolution imaging tools such as TEM, cryo-TEM, and freeze fracture cryo-scanning electron microscopy (FF-Cryo-SEM) are able to give about a 1000-fold increase in resolution and a 100-fold increase in depth of field. 85 In particular, cryo-TEM can avoid the drying process associated artifacts in electron microscopy sample preparation and can provide the information regarding the size, morphology, and bilayer thickness of polymersomes ( Figure 2f). 83 Atomic force microscopy (AFM) can also be used to characterize the mechanical properties of polymersomes. 83

| STRATEGIES FOR MP INSERTION TO FORM PROTEOPOLYMERSOMES
The reconstitution or insertion of MPs in polymersomes has emerged as a powerful tool in studying the structure and functionality of MPs. 86 To retain the structural integrity of MPs and confer their biological functionalities, MPs have to be preserved in amphiphilic environment similar to their native environment such as the use of detergents to prevent denaturation. The protein-detergentmembrane interaction play a key role in MP insertion, which is affected by the different methods of protein production and purification, the type and amount of detergents used, and the different physicochemical properties of polymersomes, including their fluidity and flexibility. MPs can be reconstituted via three major methods: (1) cellbased protein production and detergent mediated reconstitution, 87 (2) cell-free co-translational protein production and direct incorporation, 53 and (3) reconstitution by destabilization of vesicles or supported planar bilayer membranes. [88][89][90][91][92][93] Following reconstitution, purification steps such as dialysis, gel filtration or size exclusion chromatography (SEC), centrifugation, and bio-beads aided procedures should be carried out to remove excess detergents and other reagents to enhance the formation of stable proteopolymersomes.

| Cell-based protein production and detergent mediated reconstitution
The recombinant MPs are first purified from plasmid transformed bacteria cultures, and the purified MPs are solubilized with detergents and emulsified with excess polymers via self-assembly, followed by detergent removal (Figure 3a). 32,94 The addition of detergents allows for ease of MPs solubilization and keeps them in a native environment to facilitate MPs folding and stabilization. Upon protein reconstitution, the detergent molecules need to be removed to aid in the formation of stable vesicles, and residual detergent may also inhibit protein activity. 94   3.2 | Cell-free co-translational protein production and direct incorporation The MP of interest is expressed from a plasmid and directly incorporated into the polymersome (Figure 3b). 116 120 The cell-free method also allows direct access to reaction conditions, where additional agents which aid the reconstitution process such as detergents or protein folding catalysts can be included. 115 The cell-free method overcomes the issues associated with conventional overexpression and reconstitution of MPs into membrane models, such as low protein yields, cytotoxicity, misfolding, and aggregation. 121,122 Upon reconstitution, the proteopolymersome size and morphology can be further fine-tuned through freeze-thaw, extrusion, and sonication methods. 94,114 Polymersomes without MPs, as well as excess cell-free expression reaction reagents, can be removed from proteopolymersomes by methods similar to detergent removal including dialysis, 110 gel filtration or SEC, 86,87 centrifugation, 53 and bio-beads mediated process. 111 A limitation of the direct incorporate approach is that the necessary posttranslational modifications, which are required for the formation of fully functional proteins may not occur, unless known enzymes responsible for these processes are added to the reaction mixture. 123

| Reconstitution by destabilization of vesicles or supported planar bilayer membranes
Membrane destabilization by detergents has been used to reconsti- M1oK1 increased only when protein reconstitution was carried out in the presence of bio-beads. 91 Voltage destabilization is another approach that has been suggested with the insertion of α-hemolysin into supported planar polymer membranes made of PB-PEO diblock copolymers as an example. 92,93 Cell-based protein production followed by detergent-mediated reconstitution has been the predominantly used method in MP inser-

| MEMBRANE TRANSPORT PROTEINS
Membrane transport proteins are MPs that play important roles in maintaining the physiological function of cells. There are two different types of transport (passive and active) across cell membranes. Passive transport requires no energy input as transport follows a concentration gradient and examples include channel proteins. 124 In contrast, active transport requires energy, most commonly from ATP hydrolysis by primary active transporters, which include proton pumps. Active transport is used to carry substances into a cell against the concentration gradient. 125 Liposomes have been used to study membrane transport proteins, in particular channel proteins; however, their highly fluid and leaky nature hinders the retention of molecules, often resulting in inaccurate measurement of these protein functions. 20,125 Polymersomes can overcome these issues with their low passive permeability to low-molecular-weight solute, 44 and have been used widely by researchers to reconstitute and incorporate channel proteins or porins. 99 Apart from studying the functional activity of channel proteins, the activity of protein complexes can also be modeled and studied with proteopolymersomes. These complexes include primary active transporters and MP coupling systems such as NADH:ubiquinone oxidoreductase (Complex I), F 0 F 1 -ATPase, and proton pumps.
We have summarized the various types of channel proteins for passive transport and protein complexes for active transport studied in polymersomes (Table 1).

| OmpF
The outer membrane protein F (OmpF) is a MP that functions as a passive diffusion channel in E. coli and assembles to form a highly stable trimer in membranes. OmpF functions as the main route of outer membrane penetration for many antibiotics, hence studying its structure and function can be of clinical importance in determining bacterial resistance mechanisms and therapeutic advancements. 126 OmpF is the first MP successfully reconstituted with full functionality into PMOXA-PDMS-PMOXA membranes. 25,26,96,97,106 The OmpF reconstitution efficiency is increased with homogenous distribution of MPs and polymers coupled with slow controlled removal of surfactants. 26 The successful passage of antibiotics, such as ampicillin, demonstrates the functional reconstitution of OmpF in polymersomes. 25 implants in living organisms. 99 Other modified OmpF such as OmpF 6His has also been successfully reconstituted in PMOXA-PDMS-PMOXA polymersomes. 98 The structure of the OmpF 6His is determined with circular dichroism (CD) in solution, which indicates that OmpF 6His adopts a β-barrel stable structure in proteopolymersome. Functional reconstitution of OmpF 6His is determined through measuring a significant release of encapsulated acridine orange outside of the proteopolymersomes when the pH was increased from 5 to 7 across the OmpF, which allows for protons to pass through and result in changes in acridine orange. 98

| Aquaporins
Another widely studied class of channel proteins is the aquaporins (AQPs), which are water channels that can mediate bidirectional, transmembrane water flow in the presence of an osmotic gradient. Its dysfunction is associated with multiple human diseases, such as glaucoma, cancer, epilepsy, and obesity. 127 Several AQPs have been reconstituted into PMOXA-PDMS copolymer-based polymersomes via detergent-mediated reconstitution including AQP1-5, which are highly specific for water and AQP3, 7, 9, and 10, which mediate glycerol flux. 95 The functionality of reconstituted AQPs as solute transporters of water or glycerol is studied with stopped flow light scattering kinetics, where a hyperosmotic gradient is first imposed across the membrane of the AQP proteopolymersomes, and then a hypertonic gradient is applied. Outflow from the polymersome results in faster shrinking and increase in light scattering, indicating higher water permeability as a result of functional AQP reconstitution. 95 Aquaporin Z (AQPZ), which can maintain water permeability while retaining uncharged solutes (i.e., glucose, glycerol, salt, and urea), is reconstituted in PMOXA-PDMS-PMOXA polymersomes, where it shows 90 times higher water permeability than polymersomes without AQPZ insertion, as well as high rejection rates of salt, glycerol, and urea ( Figure 4c). 101 18 , micelle-formation tendency is reduced when AQPZ is incorporated. 129 The lens specific Ext). 64 The secondary protein structure of reconstituted FhuA Δ1-159 Ext is determined through CD spectroscopy, which shows β-barrel folding, indicative of correct folding. The functional activity of FhuA Δ1-159 Ext is proven via kinetic analysis of 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) uptake by encapsulated HRP. 64

| Ion channels
Ion channels, such as gramicidin A (gA), 104 ionomycin, 105 and KcsA 59 have also been studied in polymersomes. Ion channel gA, which allows for the transport of protons and monovalent ions, is reconstituted in a series of PMOXA-PDMS-PMOXA polymersomes with membrane thickness ranging from 9.2 to 16.2 nm, where membranes thicker than 12.1 nm did not result in successful reconstitution of gA protein, potentially due to hydrophobic mismatch of the protein to polymersome membrane. 104 The functionality of gA is investigated through encapsulation of pyranine, a pH-sensitive dye in polymersomes, where quenching of fluorescence intensity indicates gA activity due to transport of protons into the polymersomes. Other methods such as monitoring for fluorescence changes of the Asante NaTRIUM Green-2 (ANG-2) dye that is specific for Na + transport and Asante Potassium Green-2 (APG-2) dye that is specific for K + transport have also been used to determine gA functionality (Figure 4d). 104 Ionomycin, which allows for transport of Ca 2+  GUVs via film rehydration, and its transport functionality is studied through analyzing fluorescent increases in the calcium sensitive Asante Calcium Green (ACG) dye due to influx of Ca 2+ ions into the polymersome. 105 In addition, the permeability of ionomycin can be determined with stopped flow apparatus. 105 KcsA, which allows for transport of K + ions, has also been studied in The ability to add pores or synthetic channels to polymersomes could lead to novel membrane composites with unique selectivity and permeability. For instance, α-hemolysin, involved in pore formation, has been inserted into PBD-PEO polymersomes using cell-free cotranslational incorporation approach, which increased permeability to encapsulated calcein dye. 52 In addition to porins, synthetic pores selfassembled from either a dendritic dipeptide or a dendritic ester have also been successfully synthesized into stable helical pores in PEO-PBD polymersomes to enhance polymersomes permeability. 134  used as controls which showed no binding. 53,109,110 In the individual system, the binding of dansyl-labeled dopamine to DRD2 proteopolymersome illustrates a half-maximal effective concentration (EC 50 ) of 30 μM, which is much higher than the known EC 50 of its native ligand dopamine in the nanomolar range. 149 While it is not discussed, this discrepancy can be due to the low amount of protein incorporation at only 25%, the presence of dansyl label leading to steric hindrance, and potential protein misfolding due to cellfree synthesis that resulted in reduced ligand binding capacity. In the The display of eGFP on the surface of polymersomes illustrates the proper insertion of the peptide anchors into the polymeric membranes and co-localization of these peptides and polymersomes is shown through SEC (Figure 6e). 113 The study also shows an inversely propor-

| FACTORS AFFECTING MP STUDIES IN PROTEOPOLYMERSOMES
A good cell membrane mimetic should be morphologically similar to the biological bilayer membrane, equivalent thickness in a liquid crystalline phase, without any change in the membrane fluidity, which may affect the equilibrium distribution of the different MPs. 164 There are several factors that affect the folding, function, and dynamic equilibrium of MPs in proteopolymersomes. We will discuss three key groups of factors below, including membrane composition, MP expression and reconstitution system, and protein states ( Table 2).

| Membrane composition
The asymmetricity of copolymers used in polymersome synthesis could result in different thickness and fluidity of the formed polymersomes, thereby affecting the orientation and functionality of the inserted MPs. A study with aquaporins with His-tag shows that the percentages of nonphysiological orientation as characterized by the exposure of His-tag to the external solution for lipids and ABA triblock copolymer were near 50%, which is in reasonable agreement with random insertion into the membranes. However, for ABC and CBA triblock polymers, there was a preferred physiologic orientation of 72% in the ABC system, and a nonphysiologic orientation of 81% in the CBA system (Figure 7a). 29 In addition, designing triblock copolymers with different molecular weights for the A and C blocks can facilitate efficient protein encapsulation and stabilization. This can be done through having the longer end with a higher molecular weight segregating on the outside of the polymersome due to a larger radius of curvature with a differing volume fraction, and the smaller end segregating on the inside of the polymersome. 165 The conformational  a strong negative mismatch, resulting in symmetric deformations in the upper and the lower leaflets, and could potentially lead to an expulsion of the MP. 167 A membrane formed from amphiphilic block copolymers can withstand larger hydrophobic mismatches of more than 22% in the membrane thickness than lipid-based membranes, which can typically only withstand mismatches of 2%-3%. 167  Nano-vesicles, in particular the SUVs, are more stable, have a smaller curvature than GUVs, and provide a local environment that is more similar to that of biological membranes for MPs. This property makes them more suitable for MP studies, particular for structural studies. 20,43,180 In addition, a key advantage of these nano-vesicles lies in the clearly defined compartments segregated by the copolymer layers, which creates a concentration gradient that allows the transport of solutes and hence the measurement of the activity of poreforming proteins across the membrane. 104,123,181 Hence

| MP expression and reconstitution system
Cell-free protein expression systems have been widely adopted to produce structurally intact mammalian MPs, 185 and to overcome the limitations of the conventional protein production with use of E. coli or yeast. 116,176 Cell-free protein production generates a large amount of properly folded and biologically active proteins to be mixed with sufficient copolymers for optimal reconstitution for extensive MP studies. While cell-free approaches come with many advantages, the required enzymes supplemented in vitro which may be inferior to the quality control systems found in cells, and may contribute to making some misfolded and inactive MPs in the mixture that confounds quantifications of MP properties. 186 Therefore, it is important to optimize the protein expression and reconstitution system to use for MP studies depending on the quantity and stability of the MPs required.  27,43,61,62,90,107 The lipid/polymer-to-protein concentration and ratio also affects MP activity. 188 For instance, a ratio of 1:1 results in the highest NADH-decylubiquinone oxireductase activity ( Figure 7d). 90

| Protein states
The  [190][191][192][193] Hence, the characterization of the interactions between drug candidates and MPs using a cell-free system to directly observe their functional modulation and structural perturbation in a high-throughput setting can greatly facilitate the speed, specificity, and quality of drug discovery. 192 MP inserted nano-vesicles such as proteopolymersomes and proteoliposomes, either freely residing in microplates or immobilized onto a membrane bilayer, serve as excellent cell-free HTS platforms (Figure 8a). 196,197 7.1 | Structural and conformational screening HTS of small molecules can be performed based on either modulating the protein-protein interactions (PPIs) 198,199 (Figure 8c,d). Specifically, a low-throughput proteoliposome-based fluorescent dye permeability assay was modified, optimized, and converted to a robust HTS assay to screen for compounds capable of interfering with p7 channel function ( Figure 8c). 194 To eliminate nonspecific hits, melittin channelforming peptide is used in a counter screen. 194 Similarly, a proteoliposome flux assay using a fluorescent dye was applied in a HTS and the study identified new activators and inhibitors of four different K + channels. GIRK2, TRAAK, Slo1 and hERG, all of which are important MPs to control ion homeostasis and cell signaling ( Figure 8d). 195 In another study, hERG channel is expressed through a cell-free expression system and integrated into a biomimetic lipid bilayer platform. The properly folded and functional hERG channel is used for probing the channel and drug interactions through a fluorescence polarization assay and can be adopted for HTS to discover novel channel blockers. 208 All these studies illustrate the potential of using proteopolymersome for similar functional screening studies.
Once lead compounds are identified, high-resolution studies are required to understand the binding sites of small molecule modulators as well as how they perturb the MP of interest. An example of a highresolution study is using solid-state MAS NMR to investigate the binding and structural change of a small molecule water channel inhibitor (NSC13691) to AQPZ (Figure 9a-c) in proteoliposomes, together with their functional inhibition in a stopped-flow water permeability assay (Figure 9d). 34 Future directions in HTS for drug discovery can include mass production of nano-vesicles 209  particular proteopolymersomes, as HTS platforms in drug discovery. The main obstacles in using proteopolymersomes in the HTS process lie in the lack of robust and scalable production methods to produce large batches of proteopolymersomes with perfect monodispersity and reproducibility. 217,218 Once these obstacles are overcome, we propose that proteopolymersomes are suitable to be used as HTS platforms to discover novel therapeutics targeting the reconstituted protein of interest and provide corresponding recommendations as well as a roadmap for using proteopolymersomes in drug discovery pipeline ( Figure 10).
1. Development of proteopolymersome-based screening platform: Exploiting the use of polymersomes in drug discovery can potentially be adapted to all membrane-bound protein targets through careful selection of suitable reconstitution methods and polymer mixtures to engineer the desired proteopolymersome as a F I G U R E 9 Structure-based elucidation of inhibitor binding site in AqpZ proteoliposome by solid-state MAS NMR. (a) Two dimensional (2D) 15 N-13 C α spectra of AqpZ with inhibitor (NSC13691) (red) and without inhibitor (blue) recorded by solid-state MAS NMR illustrating that AqpZ-drug interaction leads to perturbation in chemical shift of AqpZ structure with significant perturbations being highlighted. (b) Contour plots of 2D planes ( 15 N, 13C α ) corresponding to AqpZ with inhibitor (red) and without inhibitor (blue) extracted from 3D NCACX spectra with the assigned peaks indicating significant chemical shift perturbations of more than 0.2 ppm in both 13Cα and 15 N planes. (c) Mapping of the ApZdrug interaction site onto the crystal structure (1RC2). The residues that have undergone significant chemical shift perturbations have been highlighted in red. (d) The inhibitor NSC13691 blocks AqpZ function as characterized by a stopped-flow water permeability assay in AqpZ proteoliposomes. A reduced water permeability of AqpZ is observed as characterized by a significant decrease in the rate constant of the AqpZ proteoliposomes shrinkage. Source: Figure 9a-d is reproduced with permission from reference 34, Copyright 2018, Springer Nature F I G U R E 1 0 Prospective research directions and a roadmap for using proteopolymersomes in drug discovery. (a) Proteopolymersomes engineering using suitable block copolymers and gene/protein or interest. (b) Bioassay establishment with proteopolymersomes using appropriate detection methods such as FRET measurement for structural change or fluorescent leakage assay for channel functional assay. (c) Validation of bioassay using positive controls that are known to alter protein structure or modulate protein function in proteopolymersomes. (d) Compare the structural and functional data obtained from proteopolymersomes with known biological data from literature to benchmark the performance of the bioassay established with proteopolymersomes to decide whether to proceed with high-throughput screening (HTS) or more optimizations may be required. (e) HTS of suitable chemical libraries using proteopolymersomes to identify hit compounds or potential drug candidates for further testing. (f) Potential drug candidates may undergo structural optimization by medicinal chemistry to obtain analogs with improved pharmacological properties, which will be tested in proteopolymersomes for their functions. (g) Cell-based studies will be conducted to determine the efficacy of the potential drug candidates in modulating cellular functions as well as elucidate their specificity to the protein target. (h) Use of animal models such as mice to validate the efficacy of potential drug candidates in vivo. (i) To elucidate the mechanism of action of the potential drug candidates using proteopolymersomes, including their binding sites, how they perturb the protein of interest as well as whether they act through competitive or allosteric mechanisms. Schematics were created with BioRender.com.
screening platform (Figure 10a). This platform, in principle, allows the incorporation of any MP for which the complementary DNA is available. An initial characterization of the engineered proteopolymersomes should be performed to ensure good quality control. To achieve inserted MPs of better quality, chaperones or enzymes may be added to assist the MP folding. 110 Furthermore, other cellular components such as organized metabolic reactions and gene expression mechanisms may be included in distinct spatial compartments in the proteopolymersomes 18,218 to achieve the engineering of synthetic cells as a screening platform, which would be compatible in their biological functionalities to drug screening using living cells. This is followed by the characterization of the structure and function of the reconstituted MP to validate its physiological folding and functions. The proteopolymersome constructed will need to be coupled to a detection method such as fluorescence or luminescence measurements to establish the screen platform with sensing capability (Figure 10b). The adaptation into high-throughput formats, including accurate dispensing of proteopolymersomes into multiwells, calibrations, and determination of limit of detection should be optimized.
2. Validation of the screening platform and benchmarking against known biological data: The proteopolymersomes based screening platform can be tested with positive controls known to modulate the MP structure (e.g., conformational dynamics) and function (e.g., ligand binding) to ensure that the MPs are responding to all protein-specific stimuli and to determine the compound efficacy or binding affinities to the MPs (Figure 10c). Testing with positive controls will also determine the assay quality Z-factor and coefficient of variation of the screening platform. 219