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The mammalian two-hybrid system as a powerful tool for high-throughput drug screening
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The mammalian two-hybrid system as a powerful tool for high-throughput drug screening Q5
Daniela Patrício and Margarida Fardilha Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine – iBiMED, University of Aveiro, 3810-193 Aveiro, Portugal
Protein–protein interactions (PPIs) are the backbone of signaling pathways, responsible for the basis of cell communication and, when deregulated, several diseases. Consequently, identifying and modulating PPIs can unravel the pathophysiological mechanisms of diseases. The two-hybrid system, particularly the mammalian two-hybrid system (MTH), is an efficient technique to validate PPIs ex vivo. Combining MTH with high-throughput screening has a huge advantage in biomedical research. In this review, we describe methodologies developed from MTH and the role of these adaptations in PPI discovery. We also highlight the powerful contribution of MTH to the identification of disease-related PPIs and its use in the development of potential new drug screens.
Introduction Q2 PPIs are one of the most important mechanisms used by mammalian cells to regulate several cellular and molecular activities [1]. Both modification and regulation of protein function involve transient PPIs [2], which provide a fast cellular mechanism to respond to extracellular stimuli [3]. PPI characterization is essential to understand diverse cellular functions [2] and unravel the pathophysiological mechanism of diseases, and PPIs have emerged as promising drug targets [4–6]. The need to understand signaling pathways and cellular interactions has increased the use of high-throughput (HTP) interaction methodologies, which can be classified as binary or co-complex. While binary methods provide a direct pair-wise comparison of proteins, including techniques such as yeast twohybrid (YTH) and its variants, co-complex methods tag one specific bait protein and test its interaction with a group of prey proteins, such as when happens when co-immunoprecipitation is combined with mass spectrometry [7–9]. YTH was developed to overcome the need to biochemically purify and test PPIs. The introduction of YTH [10] revolutionized the proteomics field, and it has become the most used technique for PPI investigations [11]. YTH uses reconstitution of the GAL4 transcription factor from Saccharomyces cerevisiae to identify PPIs, Corresponding author: Fardilha, M. ([email protected]) 1359-6446/ã 2020 Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.drudis.2020.01.022
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and has been used to identify numerous binary PPIs and for interactome studies on a whole-organism scale. The first organism interactome identified by YTH was that of S. cerevisiae [12]. Since 2000, the interactomes of several further species have been identified, from yeast to Drosophila melanogaster. Moreover, it is estimated that 50% of interactions described in PubMed were identified by YTH screenings [10,13,14]. In YTH screenings, falsepositives and false-negatives are often recorded, as well as autoactivation of the reporter gene (RG), even when the interaction does not occur [15]. Thus, MTH was developed to overcome some of these drawbacks and to allow better extrapolation, based on environment mimics [16]. The first method to identify PPIs in mammalian cells was described in 1991 by Dang et al., and since then, several MTH applications have been developed, especially focused on the recognition of deregulated signaling pathways in diseases. Thus, here we review the MTH assay and its contribution to PPI modulation and the search for new potential drugs.
The MTH system: principle and concepts MTH is one variant of YTH. Similar to YTH, the principle of MTH relies in the fusion of the cDNAs of interest with both the DNA-binding domain (DNA-BD) and the DNA-activation domain (DNA-AD), named bait and prey plasmid, respectively [17]. These plasmids are then co-introduced into the appropriate host cells along with the reporter plasmid [10,18] (Fig. 1).
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FIGURE 1
Conventional mammalian two-hybrid assay. (a) Cloning of the proteins ORF (gene X and Y) of interest and transfection of bait, prey, and reporter plasmids with the DNA-binding domain (DNA-BD) and DNA-activation domain (DNA-AD), respectively. The plasmids are transfected together to evaluate the protein–protein interactions (PPI between protein X and Y; control vectors are also transfected to further analyze the interaction. (b) Interaction between protein X and Y. (i) Reporter gene is expressed when the recognition of the upstream activation site (UAS) through the BD in protein X occurs and there is an interaction with protein Y; the interactions ensures that BD and AD are close and recruits RNA polymerase I to start the reporter transcription. (ii) When Protein X and Y do not come in close proximity, transcription of the report gene does not occur.
The first study in mammalian cells was performed by Dang and colleagues in 1991 on Chinese hamster ovary cells [16]. This system was based on GAL4-BD from YTH and VP16-AD (from Herpes virus), with chloramphenicol acetyltransferase (CAT) as the RG, to analyze the complex formed between leucine zipper transcription factors. Later, it was confirmed that the interaction of proteins could be studied in other mammalian cells, such as HeLa cells [15]. In 1997, Lou et al. reported the expression of two chimeric proteins from Simian virus 40 T antigens and their interactions with mouse p53 antitumor protein by expression of the CAT gene under the control of five consensus GAL4-binding sites [15]. This methodology has gained favor because it allows PPI identification and modulation in an environment that mimics the complex cellular context in which the interactions naturally occur. Furthermore, it allows the study of drugs and small molecules that might modulate an interaction of interest [18].
Advantages of MTH for PPI identification MTH is straightforward because all plasmids, including the reporter plasmid, are transfected, in contrast to the intrinsic RG, which is located in the nucleus. This allows the detection of nuclear and non-nuclear proteins; however, in conventional YTH, hybrid proteins might not be expressed stably in yeast or might be moved inefficiently into the nucleus to induce the RG expression [19]. The use of positive controls with subtracts such as secreted alkaline phosphatase (SEAP), CAT, and luciferase, avoids false-positives and negatives [20]. In terms of the target proteins, the native physiological context might require cofactors and regulatory proteins for post-translational modifications, such as phosphorylation, or 2
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assist the target proteins with any conformational alterations that are required for them to interact. Despite such advantages, MTH still has practical constraints and is more difficult to implement in the laboratory compared with YTH [18].
Adaptations to conventional MTH Adaptations to conventional MTH have been developed to overcome the need to identify PPIs specific to particular cellular regions (Table 1).
Mammalian PPI trap Mammalian PPI trap (MAPPIT) is a two-hybrid assay that exploits the unique properties of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway, and is based on type I cytokine signal transduction. The ligand-induced activation and reorganization leads to cross-phosphorylation and activation of receptor-associated cytosolic JAKs. JAK activation results in the phosphorylation of conserved tyrosine motifs in the cytosolic tail of the receptor or signaling molecules, such as STATs. Phosphorylated STATs migrate to the nucleus, where they act as transcription factors. In MAPPIT, the bait is fused to the C terminus of the leptin receptor (LR) with a mutation on three tyrosine residues, and the prey is linked to six functional STAT3 recruitment sites of a gp130 chain. When the bait and prey proteins interact, JAK is able to phosphorylate the three tyrosines on the LR and a ligand stimulus leads to STAT3 activation, inducing the transcription of the STAT3-responsive luciferase reporter [21,22] (Fig. 2a). Advantages of MAPPIT include a near-optimal physiological context and the separation of interactor and effector zones. Given
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TABLE 1
Q3 Advantages and limitations of the conventional MTH assay and its adaptationsa Advantages
Limitations
Refs
MTH
Rapid and quantitative; PPIs mimic better in vivo situation Transcription factor activated by complementary cytokine receptor; ligand-dependent interaction allows fast discrimination of false positives and preliminary mapping of interaction site on bait protein Allows identification of inhibitors of target PPI, because prey protein is fused with inhibitory molecule of JAK/STAT signal Highly sensitive; able to detect dynamic changes; effective enough to be used in identification of transmembrane and cytosolic proteins Uses TerT as a yield functional transcriptional regulator; cell high-throughput arrays Parallel analysis of thousands of proteins for interacting partners (cDNA Library screen); quantitative; cost-effective; cell high-throughput arrays Allows mapping of phosphorylated residues on RTKs; suitable for weak or dynamic interactions and for identification of new pharmaceuticals Ideal for highly sensitive detection of PPIs; provides semiquantitative results
Less suitable for cDNA library screenings
[15,16,18]
Incompatible with full-length transmembrane proteins; requires PPIs to occur in cytoplasm submembrane region; cannot be used to study proteins involved in STAT signaling pathway
[21,37,46]
Cannot be used to study proteins involved in STAT signaling pathway
[24]
Signal readout is indirect; cannot be used to study proteins involved in STAT signaling pathway
[25,55]
–
[28]
–
[2]
Works only with membrane-associated baits; not suitable for real-time PPI analysis
[31,47,48]
–
[32]
MAPPIT
Reverse MAPPIT
KISS
trMTH CAPPIA
MaMTH
iMTH a
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MTH adaptation
Some variants might not present limitations because of the lack of studies using that technique.
PPIs occur in the context of an activated receptor, MAPPIT is appropriate to use to analysis PPIs involved in signal transduction pathways [21,23].
Reverse MAPPIT Reverse MAPPIT is similar to MAPPIT. Members of the type I cytokine receptor family are activated, altering the conformation of the receptor complex, which leads to juxtaposition and consequent activation of JAKs. However, compared with MAPPIT, this methodology uses a bait fused to a functional receptor chimera capable of activate STAT, and an inhibitory domain is fused to the prey. This results in JAK-STAT suppression by the action of a functional leptin chimeric bait receptor. The disturbance of the bait–prey interaction, either by a competing protein or an organic molecule, restores the signal, leading to a positive readout [24] (Fig. 2b). In addition, in reverse MAPPIT, the interference in a PPI by a competitor molecule results in dissociation of an inhibitor from an active receptor system, allowing the STAT complex to form and transcribe the RG. Thus, it can avoid problems associated with the membrane characteristics and permeability of lower eukaryotes or prokaryotes. However, an intrinsic limitation involves the localization of the bait and inhibitory prey proteins to the submembrane compartment [24].
technique can also detect dynamic changes and is effective enough for transmembrane and cytosolic protein identification. The indirect readout represents the major limitation, because it can stop spatial and temporal analysis of PPIs [27] (Fig. 2c). All the methodologies described thus far rely on endogenous STAT3, which represents a common limitation because it might be unsuitable to study PPIs involved in STAT3 signaling [27].
Tetracycline-repressor based MTH Tetracycline repressor-based MTH (trMTH) is based on the tetracycline repressor (TetR)–tetracycline operator (TetO) interaction (TetR-TetO). TetR-TetO is used in mammalian protein expression systems to achieve highly sensitive regulation of gene expression. The replacement of the dimerization domain (DD) of TetR with interacting bait and prey molecules yields a functional transcriptional regulator. The small molecule doxycycline can disrupt the TetR dimer and turn off the transcription activator function and the expression downstream of the RG. This system has a built-in negative selection tool via the doxycycline disruption assay. It is an ideal platform for cell-based HTP screening for therapeutic small-molecule and/or peptide PPI disruptors [28,29].
Kinase Substrate Sensor
Cell array PPI assays
In Kinase Substrate Sensor (KISS), the bait is coupled to the kinase domain of Tyrosine Kinase 2 (TYK2) and the prey is fused to a gp130 cytokine receptor chain. The bait–prey interaction leads to the phosphorylation of the gp130 anchor via TYK2, activating STAT3 and the transcription of STAT3-dependent reporter system. This technique enables the identification of in situ PPIs in mammalian cells and their modulation under physiological or pharmacological conditions [25,26]. This highly sensitive
Cell array PPI assay (CAPPIA) combines the study of the PPIs in a cDNA library with the MTH assay in adherent mammalian cells. An autofluorescence-based and GAL4-driven reporter plasmid (GAL4-pZsGreen) was developed as a RG. Interactions can be easily and directly detected through a DNA array scanner or under a HTP microscope [30]. CAPPIA allows the quantitative detection of specific PPIs in various mammalian cells and the identification of PPI modulators. In addition, several prey proteins can be tested www.drugdiscoverytoday.com
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FIGURE 2
Outline of cytokine receptor-based interaction traps. (a) Mammalian protein–protein interaction trap (MAPPIT): the different components and mechanisms of this cytokine receptor-based interaction trap are represented. Bait (X) and prey (Y) are fused to complementary fragments of a type I cytokine receptor. Ligandinduced activation and reorganization leads to cross-phosphorylation and activation of receptor-associated cytosolic Janus kinase (JAK). JAK activation leads to signal transducer and activator of transcription 2 (STAT2) activation and, consequently, RNA polymerase I recruitment. (b) Reverse MAPPIT: in the reverse mode, the bait is tethered to a cytokine receptor that has intact STAT recruitment sites and the prey is coupled to an inhibitory domain (ID), such as an inhibitory phosphatase of JAK. Upon disruption of bait–prey binding, the ID dissociates and JAK is able to phosphorylate STAT docking sites and STATs, leading to reporter gene activation. (c) Kinase substrate sensor (KISS): the bait is fused to the kinase-containing portion of tyrosine kinase 2 (TYK2) and the prey protein is coupled to Q1 the gp130 anchor, similar to MAPPIT. The bait–prey interaction leads to STAT3 phosphorylation, resulting in reporter gene expression. Adapted from Refs [21,24,25].
against a bait of interest, rendering CAPPIA a cost-effective PPI-HTP assay [2,30]. This technique has been mainly used to identify hormone-dependent PPIs, particularly steroid hormone receptors, such as the androgen receptor (AR), given that the AR is a ligand-dependent transcription factor involved in several physiological processes, in humans. CAPPIA is a technique that allows PPI identification and monitoring in different physiological conditions, the screening of ligand-dependent PPIs, and the identification of the dose response of the compounds tested [2].
Mammalian membrane two-hybrid assay Recently, a method based on the membrane split-ubiquitin assay was developed [31]. The mammalian membrane two-hybrid assay (MaMTH) can be conducted in most cell lines and is suitable for HTP screenings. It requires an integral membrane, because the bait protein is fused with a C terminus of ubiquitin and an artificial transcription factor (TF), and the prey protein is fused with the N terminus of the ubiquitin. When the N and C termini of the ubiquitin come into close proximity because of the bait–prey interaction, pseudo-ubiquitin is formed and proteolytic cleavage occurs, releasing the TF, which then activates the RG expression in 4
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the nucleus [31] (Fig. 3). MaMTH enables researchers to map phosphorylated residues on receptor tyrosine kinases (RTKs) that mediate PPIs with adaptor proteins; to monitor dynamic PPIs, and to use the increased amount of adaptor-protein recruitment to identify new pharmaceuticals [31].
Inducible MTH The most recent MTH adaptation, inducible MTH (iMTH), presents higher sensitivity compared with MTH. It uses a DNABD stronger than yeast GAL4-BD. To boost the activity of the prey protein–AD, a complex of multiple copies of the AD is fused to the Q6 prey, to which a small-molecule dimerizer (rapalog) binds, increasing the sensitivity up to 100-fold compared with classic MTH approaches [32].
The role of MTH in PPI discovery, validation, and modulation Over the past few decades, MTH has been used mainly to validate YTH screenings [15]. In 2010, Naz and Dhandapani used MTH to detect false positives after an YTH screen. Human ZP3 was cloned into YTH bait vectors to find reactive proteins in a human testis
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FIGURE 3
Membrane mammalian two-hybrid (MaMTH). The bait protein is an integral membrane fused with a C terminus of ubiquitin and an artificial transcription factor (TF). The prey protein is fused with the N terminus of ubiquitin. When the N and C termini of ubiquitin come in close proximity through the interaction of the bait and prey, pseudo-ubiquitin formation occurs, resulting in proteolytic cleavage and the release of the TF, which then activates expression of the reporter gene.
cDNA library. Six clones were identified and further confirmed by MTH [33]. Later, Tham et al. performed YTH to identify potential interactors of DENV-2 in Aedes aegypti, responsible for dengue transition. In total, 36 midgut proteins where identified as possible interactors, and three proteins (NS1, prM, and E proteins) were confirmed as interactors with DENV-2, by MTH [34]. Later, MTH was used to identify PPIs involved in virus–cell communication. Jiang and Luo showed that adaptive and compensatory mutations occurring in different nonstructural (NS) proteins promote Hepatitis C virus (HCV) production in cell culture. PPIs between NS proteins involved in HCV cell penetration were confirmed by MTH, confirming that adaptative mutations in NS protein occur and alter the association of the core
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with lipid droplets, allowing HCV cell assembly [35]. Hu et al. performed a YTH screen to identify proteins from a human fetal brain cDNA library interacting with NS2 of Influenza A virus (IAV). CHD3 had a positive readout and the interaction was further confirmed by MTH. The authors showed that NS2 of IAV has a nuclear export signal (NES1) motif that is crucial for the NS2–CHD3 interaction, required for IAV penetration [36]. Bovijn et al. studied the impact of the upper activation of toll-like receptor (TLRs) on pathogen recognition, highlighting pathogenassociated molecular patterns [37]. Oligomerization of TLRs trigger multiple signaling pathways, activating inflammatory and immune responses [38]. The authors used a random mutagenesis and MAPPIT assay to identify myeloid differentiation primary response gene 88 (MyD88) adapter-like protein (Mal) mutations that disrupt Mal/TLR4 and/or MyD88/TLR4 interactions. Results showed four potential binding sites and the AB-loop in the Mal TIR domain, contributing to the formation of TLR4/Mal/MyD88 complex [37]. Mal dimerization leads to the formation of binding platforms to which MyD88 or TLR4 bind [37], consequently triggering inflammatory and immune responses. In 2019, Kumar et al. reported a novel interaction between HIV-1 Nef, a mediator of HIV replication, and Ca2+/calmodulindependent protein kinase II (CAMKIId). Nef activates calcium signaling as a positive feedback mechanism to regulate HIV replication in human cells. MTH was performed to confirm the Nef–CAMKIId interaction and screen for peptide inhibitor activity. The findings suggested Nef as a CAMKIId activator through elevation of Ca2+ concentration, which inhibited ASK-1 and p38-mediated apoptosis of infected cells [39]. The interaction between MMD2 (an E3 ubiquitin ligase) and p53 was used to validate the reverse MAPPIT assay, prove its sensitivity, and evaluate the modulation effect of Nutlin-3, a known inhibitor of this interaction, confirming the ability of this approach to identify PPI disruptors [24]. The MDM2–p53 interaction was disrupted with an IC50 value of 0.4 mM [24], which approximates the IC50 value previously reported for Nutlin-3 in Biacore experiments with purified proteins (0.1 mM) [40]. In 2012, Liu et al. studied the role of fucoxanthin in drug resistance through the attenuation of rifampin-induced CYP3A4 and MDR1 gene expression. The pregnane X receptor (PXR)-mediated pathway mediates CYP3A4 and MDR1 expression in HepG2 hepatoma cells, and the interaction between PXR and SRC1 (which acts as a main co-activator). Whereas fucoxanthin negatively affects the PXR–SRC1 interaction, rifampin strongly increases it. However, Q7 when cells were exposed to fucoxanthin and rifampin, the rifampin-induced PXR–SRC1 interaction was attenuated, suggesting that fucoxanthin is a PXR antagonist. This could be a potential way to prevent transcriptional activation of PXR-regulated genes, given that fucoxanthin is used to prevent drug resistance in patients receiving chronic therapy with PXR agonists [41]. Leverson et al. identified a small molecule that inhibits MCL-1, inducing clear on-target cellular activity in cancer cells. Molecules derived from indole-2-carboxylic acids, such as A-1210477, induce intrinsic apoptosis. A-1210477 binds MCL-1 with subnanomolar affinity and can disrupt endogenous MCL-1–BH3-only protein (NOXA and MULE) interactions. Based on MTH and a cell imaging platform that tracks BCL-2 family interactions in real time, it was shown that A-1210477 selectively disrupts MCL-1–NOXA in cancer cells [42]. www.drugdiscoverytoday.com
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Recently, Arao et al. showed the contribution of the estrogen receptor alpha (ERa) F domain to species-specific ER modulator 4-hydroxy-tamoxifen (4OHT)-dependent ERa ligand-binding domain (LBD) homodimerization using MTH. In the nuclear receptor (NR) field, MTH has been used to assess the ligand-dependent coactivator or corepressor recruitment activity of NR LBDs. The effect of 4OHT on mouse and human ERa LBD revealed that RG expression was higher in cells expressing the mouse ERa LBD. The authors showed that MTH enables the evaluation of LBD dimerization activity of certain substances that do not activate the AF-2 transactivation function of LBD (such as 4OHT or SERMs) [43]. Thus, MTH has proved to be useful for identifying and studying PPIs, highlighting its versatility. It can be applied to different cell lines, enabling tissue-specific PPI study. On a pharmacological and therapeutic level, MTH has already been applied to study different diseases, increasing the knowledge of the deregulated PPIs involved.
MTH and its role in HTP screening Most PPI studies with MTH were done on an individual gene scale, but there is an increasing need to increase putative interactors with a bait protein. In 2001, Suzuki et al. performed the first MTH-HTP screening for PPIs [44]. In this study, a mouse full-length enriched cDNA library was constructed using a PCR-based process. A MTHHTP assay was run to test 3500 bait proteins and 3400 prey proteins in a total of 1.2 107 possible interactions. Overall, 145 PPIs were identified in the assay, further analyzed using a bioinformatic approach, which was crucial to understand the role and function of uncharacterized proteins, and to unravel the associated molecular mechanisms [44]. Later, Ravasi et al. confirmed that it was possible to perform an extensive MTH-based assay. PPI maps were established using pairwise analyses of 1988 human and 1727 mouse transcription factors. A cDNA library of human and mouse transcription factors was constructed out of all DNA-binding transcription factors able to express a full-length protein, resulting in 1222 human and 1112 mouse cDNA clones. MTH screening revealed 762 and 877 interactions, respectively [45]. In 2008, following the development of CAPPIA, Fiebitz et al. studied a large number of PPIs simultaneously. This technique allowed the screening of 160 different bait–prey combinations, associated with the human AR. The study was performed in the presence of 10 nM of the synthetic androgenic ligand R1881. A specific interaction between the AR-LBD and the AR-N-terminal domain (AR-NTD) was identified and a dose-dependent induction of receptor expression in the presence of R1881 was observed. The effect of two antagonists was also evaluated (medroxyprogesterone acetate and hydroxyflutamide), showing an inhibitory effect on the AR-LBD–AR-NTD interaction. Thus, CAPPIA was shown to be one of the most economical HTP detection assays for PPIs in mammalian cells [2]. Q8 The use of MTH for PPI HTP screening was first reported in 2012 by Lievens and colleagues [18]. Later, Rolland et al. performed a human proteome assay using four techniques, including MAPPIT. All binary pairs identified in seven public databases were tested and validated using the different techniques. Half of the interactome was systematically screened, resulting in double the number of high-quality binary PPIs available from the literature (14 000 interactions). This research resulted in the human interactome data set covering Space II, reported in 2014 (HI-II-14), showing that known candidate cancer 6
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gene products are highly connected in the human interactome network [46]. Recently, the group responsible for MaMTH development adapted the method to map the genome-wide RTK–phosphatase interactome [47]. After a membrane YTH screen, a huge number of PPIs were identified, most of them for the first time. MaMTH was further used to validate some of the PPIs and nine interactions were confirmed [48].
Impact of MTH on drug discovery The combination of MTH and HTP can be applied to therapeutic research, making it possible to evaluate the effects of drugs and small compounds on specific PPIs, rendering treatments target specific. In cancer research, it has been possible to increase the identification of drugs able to inhibit and/or modulate PPIs involved in tumor suppression, chemotherapeutic resistance, and other mechanisms. Li et al. screened 3000 compounds to identify a disruptor of the MDM2–p53 interaction, given that it was thought that p53 is activated when MDM2 is released from the complex. The authors developed MTH to study MDM2–p53, with a dual-luciferase RG to exclude false positives. Nutlin-3, caylin-1, and three hit compounds (SL-01–03) had an inhibitory effect on MDM2/p53, increasing the levels of active p53, which inhibits tumor proliferation [49]. Bhatia et al. also used HTP drug screening to identify small molecules able to inhibit proteins involved in p53 suppression. MAGE proteins are highly present in human tumors, and are associated with chemotherapeutic drug resistance. KAP-1 is a central molecule involved in p53 regulation. In this study, after screening 14 400 compounds from the Maybridge HitFinder library, seven were identified as possible molecules able to modulate the MAGE–KAP-1 interaction. The authors performed an MTH assay and concluded that Class I MAGE proteins bind with the KAP-1 RBCC binding region, suppressing p53. Additionally, three small molecules (HTS11125, GK02432, and SPB02) were shown to have a direct effect on MAGE–KAP-1, indicating them as promising candidates for its modulation and, thus, as potential anticancer drugs [50]. Constitutive androstane receptor (CAR) inhibition and PXR activation appear to be crucial to improve the effects of chemotherapeutic drugs, given that CAR inhibition attenuates multidrug resistance in aggressive cancers. Cherian et al. performed an HTP screening of 1000 compounds to identify CAR inhibitors. In total, 25 compounds displayed dose-responsive inhibitory effects, but only CINPA1 was selected, because it had a potent inhibitory effect on hCAR1 and no agonistic activity against PXR. MTH was performed to validate the ability of CINPA1 to inhibit CAR–CAR coactivator interactions. Given that CINPA1 proved to be a specific CAR inhibitor, it could help researchers to better understand CAR functions or could even be a suitable molecule to be used along with chemotherapy for a better tumor response [51]. Recently, Yasui et al. identified two suitable inhibitors of the interaction between B cell lymphoma 6 (BCL6) and its corepressor (BCoR). BCL6 and BCoR are responsible for the differentiation and proliferation of lymphocytes. Several compounds were synthetized to find a drug with inhibitory activity, but with increased membrane permeability and high stability in aqueous solution, rendering them suitable for MTH assays. MTH was performed and two of the developed compounds were identified to have good
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Concluding remarks PPI identification is a key to understand signaling pathways responsible for all cellular processes and the molecular
mechanisms behind many diseases. The study of PPIs as drug targets can be challenging and requires adequate systems to select lead compounds, but is a straightforward way to interfere with the disease signal cascade. MTH and its variations are flexible methodologies because they can be adapted to the PPIs of interest. Comparing MTH with YTH is important to highlight their ability to mimic the complex cellular context in which the interactions occur. Although MTH is a versatile strategy to study binary PPIs, it also has a crucial role in drug discovery when allied to HTP screening. From a pharmacological and therapeutic view, MTH allows the extrapolation of results to the roles of human proteins and interactions, and PPI modulation. The successful application of MTH in HTP is an alternative method to discover small-molecule inhibitors and represents a valuable approach for the discovery and development of new antiviral, anticancer, and other drugs. Although most of the work using MTH be applied to cancer and chemotherapy, this methodology presents many opportunities for PPI modulation and library drug screening in other diseases, such as viral infections or male infertility. Q9
Acknowledgments We thank the Portuguese Foundation for Science and Technology (FCT), European Union, QREN, FEDER and COMPETE for funding iBiMED (UID/BIM/04501/2013, POCI-01-0145-FEDER-007628 and UID/BIM/04501/2019), the research project PTDC/BBB-BQB/ 3804/2014 and an individual scholarship from DP (SFRH/BD/ 137487/2018).
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15 Luo, Y. et al. (1997) Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system. Biotechniques 22, 350–352 16 Dang, C.V. et al. (1991) Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization. Mol. Cell Biol. 11, 954–962 17 Stynen, B. et al. (2012) Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian splitluciferase system. Microbiol. Mol. Biol. Rev. 76, 331–382 18 Lievens, S. et al. (2012) The use of mammalian two-hybrid technologies for highthroughput drug screening. Methods 58, 335–342 19 Mendonc¸a, D.B. et al. (2013) Mammalian two-hybrid assays for studies of interaction of p300 with transcription factors. Methods Mol. Biol. 977, 323–338 20 Hoat, T. et al. (2009) Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential. BMC Cell Biol. 10, 69 21 Eyckerman, S. et al. (2002) Design and use of a mammalian protein-protein interaction trap (MAPPIT). Sci. Signal. 2002, 118 22 Lemmens, I. et al. (2015) MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions. Methods Mol. Biol. 1278, 447–455 23 Lievens, S. et al. (2016) Proteome-scale binary interactomics in human cells. Mol. Q11 Cell Proteomics Xx, YYY–ZZZ 24 Eyckerman, S. et al. (2005) Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells. Nat. Methods 2, 427–433 25 Lievens, S. et al. (2014) Kinase substrate sensor (KISS), a mammalian in situ protein interaction sensor. Mol. Cell. Proteomics 13, 3332–3342 26 Masschaele, D. et al. (2018) KISS: A mammalian two-hybrid method for in situ analysis of protein–protein interactions. Methods Mol. Biol. 1794, 269–278 27 Snider, J. et al. (2015) Fundamentals of protein interaction network mapping. Mol. Q12 Syst. Biol. Xx, YYY–ZZZ 28 Thibodeaux, G.N. et al. (2009) A tetracycline repressor-based mammalian twohybrid system to detect protein–protein interactions in vivo. Anal. Biochem. 386, 129–131 29 Moncivais, K. and Zhang, Z.J. (2012) Tetracycline repressor-based mammalian twohybrid systems. Methods Mol. Biol. 812, 259–273
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cellular PPI inhibitory activity. The authors identified these compounds as good candidate drugs for autoimmune diseases and cancer treatment [52]. Although most studies using MTH for drug discovery have been performed on cancer, the association of MTH with HTP screening could also be applied to PPI modulation in other biological contexts. In recent research on male fertility, HTP screening was performed of 3242 ion channel library ligands. In total, 384 compounds showed positive alteration of functional sperm motility and calcium rates of the sperm of subfertile patients. Furthermore, protein phosphatase 1 (PPP1) and its catalytic subunits were studied using YTH for PPI discovery [53,54]. In human testis, all four PPP1 isoforms are expressed [55], but PPP1CC2 has enriched expression in testis and spermatozoa (but low expression in brain, lung, spleen, and thymus) [56–58]. In spermatozoa, PPP1CC2 is present throughout the flagellum, midpiece, and posterior region of the head [54], suggesting a role in motility and acrosome reaction. Studying PPIs already known to be involved in sperm motility or dysfunction, combined with HTP drug screening, could be the next step in the effective identification of targets for the treatment of specific sperm dysfunctions. Thus, the combination of MTH-HTP has significant potential for drug discovery, allowing the development of drugs that can enhance sperm function or can be used as targeted contraceptive molecules [59].
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30 Fiebitz, A. et al. (2008) High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays. BMC Genomics 9, 68 31 Petschnigg, J. et al. (2014) The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nat. Methods 11, 585–592 Q13 32 Riegel, E. et al. (2017) A sensitive, semi-quantitative mammalian two-hybrid assay. Biotechniques 62 Xx, YYY–ZZZ 33 Naz, R.K. and Dhandapani, L. (2010) Identification of human sperm proteins that interact with human zona pellucida3 (ZP3) using yeast two-hybrid system. J. Reprod. Immunol. 84, 24–31 34 Tham, H.-W. et al. (2015) Protein-protein interactions between A. aegypti midgut and dengue virus 2, two-hybrid screens using the midgut cDNA library. J. Infect. Dev. Ctries 9, 1338 35 Jiang, J. and Luo, G. (2012) Cell Culture-Adaptive Mutations Promote Viral ProteinProtein Interactions and Morphogenesis of Infectious Hepatitis C Virus. J. Virol. 86, 8987–8997 36 Hu, Y. et al. (2015) CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2. Cell. Mol. Life Sci. 72, 971–982 37 Bovijn, C. et al. (2013) Identification of binding sites for myeloid differentiation primary response gene 88 (MyD88) and Toll-like receptor 4 in MyD88 adapter-like (Mal). J. Biol. Chem. 288, 12054–12066 38 Kawai, T. and Akira, S. (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 Q14 39 Kumar, P. et al. (2019) HIV-1 Nef physically associate with CAMKIId – ASK-1 complex to inhibit p38MAPK signalling and apoptosis in infected cells. Life Sci. Xx, YYY–ZZZ 40 Vassilev, L.T. et al. (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848 41 Liu, C.-L. et al. (2012) Fucoxanthin attenuates rifampin-induced cytochrome P450 3A4 (CYP3A4) and multiple drug resistance 1 (MDR1) gene expression through pregnane x receptor (PXR)-mediated pathways in human hepatoma HepG2 and colon adenocarcinoma LS174T cells. Mar. Drugs 10, 242–257 42 Leverson, J.D. et al. (2015) Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax). Cell Death Dis. 6, 1590 43 Arao, Y. and Korach, K.S. (2018) Detecting the ligand-binding domain dimerization Q15 activity of estrogen receptor alpha using the mammalian two-hybrid assay. J. Vis. Exp. Xx, YYY–ZZZ
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44 Suzuki, H. et al. (2001) Protein-protein interaction panel using mouse full-length cDNAs. Genome Res. 11, 1758–1765 45 Ravasi, T. et al. (2010) An atlas of combinatorial transcriptional regulation in mouse and man. Cell 140, 744–752 46 Rolland, T. et al. (2014) A proteome-scale map of the human interactome network. Cell 159, 1212–2126 47 Saraon, P. et al. (2017) Detecting membrane protein-protein interactions using the mammalian membrane two-hybrid (MaMTH) assay. Curr. Protoc. Chem. Biol. 9, 38–54 48 Yao, Z. et al. (2017) A global analysis of the receptor tyrosine kinase-protein phosphatase interactome. Mol. Cell. 65, 347–360 49 Li, J. et al. (2011) A Cell-based high-throughput assay for the screening of smallmolecule inhibitors of p53–MDM2 interaction. J. Biomol. Screen. 16, 45045–45046 50 Bhatia, N. et al. (2011) Identification of novel small molecules that inhibit protein– protein interactions between MAGE and KAP-1. Arch Biochem Biophys. 508, 217–221 51 Cherian, M.T. et al. (2015) CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor. Mol. Pharmacol. 87, 878–889 52 Yasui, T. et al. (2017) Discovery of a novel B-cell lymphoma 6 (BCL6)-corepressor interaction inhibitor by utilizing structure-based drug design. Bioorg. Med. Chem. 25, 4876–4886 53 Fardilha, M. et al. (2004) Alternatively spliced protein variants as potential Q16 therapeutic targets for male infertility and contraception. Ann. N. Y. Acad. Sci. Xx, YYY–ZZZ 54 Fardilha, M. et al. (2011) Identification of the human testis protein phosphatase 1 interactome. Biochem. Pharmacol. 82, 1403–1415 55 Shima, H. et al. (1993) Identification of PP1 catalytic subunit isotypes PP1 gamma 1, PP1 delta and PP1 alpha in various rat tissues. Biochem. Biophys. Res. Commun. 192, 1289–1296 56 Takizawa, N. et al. (1994) Tissue distribution of isoforms of type-1 protein phosphatase PP1 in mouse tissues and its diabetic alterations. J. Biochem 116, 411–415 57 da Cruz, e and Silva, E. et al. (1995) Differential expression of protein phosphatase 1 isoforms in mammalian brain. J. Neurosci. 15, 3375–3389 58 Trinkle-Mulcahy, L. et al. (2001) Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J. Cell Sci. 114, 4219–4228 59 Martins da Silva, S.J. et al. (2017) Drug discovery for male subfertility using highthroughput screening: a new approach to an unsolved problem. Hum. Reprod. 32, 974–984
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The mammalian two-hybrid system as a powerful tool for high-throughput drug screening Q5
Daniela Patrício and Margarida Fardilha Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine – iBiMED, University of Aveiro, 3810-193 Aveiro, Portugal
Protein–protein interactions (PPIs) are the backbone of signaling pathways, responsible for the basis of cell communication and, when deregulated, several diseases. Consequently, identifying and modulating PPIs can unravel the pathophysiological mechanisms of diseases. The two-hybrid system, particularly the mammalian two-hybrid system (MTH), is an efficient technique to validate PPIs ex vivo. Combining MTH with high-throughput screening has a huge advantage in biomedical research. In this review, we describe methodologies developed from MTH and the role of these adaptations in PPI discovery. We also highlight the powerful contribution of MTH to the identification of disease-related PPIs and its use in the development of potential new drug screens.
Introduction Q2 PPIs are one of the most important mechanisms used by mammalian cells to regulate several cellular and molecular activities [1]. Both modification and regulation of protein function involve transient PPIs [2], which provide a fast cellular mechanism to respond to extracellular stimuli [3]. PPI characterization is essential to understand diverse cellular functions [2] and unravel the pathophysiological mechanism of diseases, and PPIs have emerged as promising drug targets [4–6]. The need to understand signaling pathways and cellular interactions has increased the use of high-throughput (HTP) interaction methodologies, which can be classified as binary or co-complex. While binary methods provide a direct pair-wise comparison of proteins, including techniques such as yeast twohybrid (YTH) and its variants, co-complex methods tag one specific bait protein and test its interaction with a group of prey proteins, such as when happens when co-immunoprecipitation is combined with mass spectrometry [7–9]. YTH was developed to overcome the need to biochemically purify and test PPIs. The introduction of YTH [10] revolutionized the proteomics field, and it has become the most used technique for PPI investigations [11]. YTH uses reconstitution of the GAL4 transcription factor from Saccharomyces cerevisiae to identify PPIs, Corresponding author: Fardilha, M. ([email protected]) 1359-6446/ã 2020 Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.drudis.2020.01.022
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and has been used to identify numerous binary PPIs and for interactome studies on a whole-organism scale. The first organism interactome identified by YTH was that of S. cerevisiae [12]. Since 2000, the interactomes of several further species have been identified, from yeast to Drosophila melanogaster. Moreover, it is estimated that 50% of interactions described in PubMed were identified by YTH screenings [10,13,14]. In YTH screenings, falsepositives and false-negatives are often recorded, as well as autoactivation of the reporter gene (RG), even when the interaction does not occur [15]. Thus, MTH was developed to overcome some of these drawbacks and to allow better extrapolation, based on environment mimics [16]. The first method to identify PPIs in mammalian cells was described in 1991 by Dang et al., and since then, several MTH applications have been developed, especially focused on the recognition of deregulated signaling pathways in diseases. Thus, here we review the MTH assay and its contribution to PPI modulation and the search for new potential drugs.
The MTH system: principle and concepts MTH is one variant of YTH. Similar to YTH, the principle of MTH relies in the fusion of the cDNAs of interest with both the DNA-binding domain (DNA-BD) and the DNA-activation domain (DNA-AD), named bait and prey plasmid, respectively [17]. These plasmids are then co-introduced into the appropriate host cells along with the reporter plasmid [10,18] (Fig. 1).
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FIGURE 1
Conventional mammalian two-hybrid assay. (a) Cloning of the proteins ORF (gene X and Y) of interest and transfection of bait, prey, and reporter plasmids with the DNA-binding domain (DNA-BD) and DNA-activation domain (DNA-AD), respectively. The plasmids are transfected together to evaluate the protein–protein interactions (PPI between protein X and Y; control vectors are also transfected to further analyze the interaction. (b) Interaction between protein X and Y. (i) Reporter gene is expressed when the recognition of the upstream activation site (UAS) through the BD in protein X occurs and there is an interaction with protein Y; the interactions ensures that BD and AD are close and recruits RNA polymerase I to start the reporter transcription. (ii) When Protein X and Y do not come in close proximity, transcription of the report gene does not occur.
The first study in mammalian cells was performed by Dang and colleagues in 1991 on Chinese hamster ovary cells [16]. This system was based on GAL4-BD from YTH and VP16-AD (from Herpes virus), with chloramphenicol acetyltransferase (CAT) as the RG, to analyze the complex formed between leucine zipper transcription factors. Later, it was confirmed that the interaction of proteins could be studied in other mammalian cells, such as HeLa cells [15]. In 1997, Lou et al. reported the expression of two chimeric proteins from Simian virus 40 T antigens and their interactions with mouse p53 antitumor protein by expression of the CAT gene under the control of five consensus GAL4-binding sites [15]. This methodology has gained favor because it allows PPI identification and modulation in an environment that mimics the complex cellular context in which the interactions naturally occur. Furthermore, it allows the study of drugs and small molecules that might modulate an interaction of interest [18].
Advantages of MTH for PPI identification MTH is straightforward because all plasmids, including the reporter plasmid, are transfected, in contrast to the intrinsic RG, which is located in the nucleus. This allows the detection of nuclear and non-nuclear proteins; however, in conventional YTH, hybrid proteins might not be expressed stably in yeast or might be moved inefficiently into the nucleus to induce the RG expression [19]. The use of positive controls with subtracts such as secreted alkaline phosphatase (SEAP), CAT, and luciferase, avoids false-positives and negatives [20]. In terms of the target proteins, the native physiological context might require cofactors and regulatory proteins for post-translational modifications, such as phosphorylation, or 2
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assist the target proteins with any conformational alterations that are required for them to interact. Despite such advantages, MTH still has practical constraints and is more difficult to implement in the laboratory compared with YTH [18].
Adaptations to conventional MTH Adaptations to conventional MTH have been developed to overcome the need to identify PPIs specific to particular cellular regions (Table 1).
Mammalian PPI trap Mammalian PPI trap (MAPPIT) is a two-hybrid assay that exploits the unique properties of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway, and is based on type I cytokine signal transduction. The ligand-induced activation and reorganization leads to cross-phosphorylation and activation of receptor-associated cytosolic JAKs. JAK activation results in the phosphorylation of conserved tyrosine motifs in the cytosolic tail of the receptor or signaling molecules, such as STATs. Phosphorylated STATs migrate to the nucleus, where they act as transcription factors. In MAPPIT, the bait is fused to the C terminus of the leptin receptor (LR) with a mutation on three tyrosine residues, and the prey is linked to six functional STAT3 recruitment sites of a gp130 chain. When the bait and prey proteins interact, JAK is able to phosphorylate the three tyrosines on the LR and a ligand stimulus leads to STAT3 activation, inducing the transcription of the STAT3-responsive luciferase reporter [21,22] (Fig. 2a). Advantages of MAPPIT include a near-optimal physiological context and the separation of interactor and effector zones. Given
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TABLE 1
Q3 Advantages and limitations of the conventional MTH assay and its adaptationsa Advantages
Limitations
Refs
MTH
Rapid and quantitative; PPIs mimic better in vivo situation Transcription factor activated by complementary cytokine receptor; ligand-dependent interaction allows fast discrimination of false positives and preliminary mapping of interaction site on bait protein Allows identification of inhibitors of target PPI, because prey protein is fused with inhibitory molecule of JAK/STAT signal Highly sensitive; able to detect dynamic changes; effective enough to be used in identification of transmembrane and cytosolic proteins Uses TerT as a yield functional transcriptional regulator; cell high-throughput arrays Parallel analysis of thousands of proteins for interacting partners (cDNA Library screen); quantitative; cost-effective; cell high-throughput arrays Allows mapping of phosphorylated residues on RTKs; suitable for weak or dynamic interactions and for identification of new pharmaceuticals Ideal for highly sensitive detection of PPIs; provides semiquantitative results
Less suitable for cDNA library screenings
[15,16,18]
Incompatible with full-length transmembrane proteins; requires PPIs to occur in cytoplasm submembrane region; cannot be used to study proteins involved in STAT signaling pathway
[21,37,46]
Cannot be used to study proteins involved in STAT signaling pathway
[24]
Signal readout is indirect; cannot be used to study proteins involved in STAT signaling pathway
[25,55]
–
[28]
–
[2]
Works only with membrane-associated baits; not suitable for real-time PPI analysis
[31,47,48]
–
[32]
MAPPIT
Reverse MAPPIT
KISS
trMTH CAPPIA
MaMTH
iMTH a
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MTH adaptation
Some variants might not present limitations because of the lack of studies using that technique.
PPIs occur in the context of an activated receptor, MAPPIT is appropriate to use to analysis PPIs involved in signal transduction pathways [21,23].
Reverse MAPPIT Reverse MAPPIT is similar to MAPPIT. Members of the type I cytokine receptor family are activated, altering the conformation of the receptor complex, which leads to juxtaposition and consequent activation of JAKs. However, compared with MAPPIT, this methodology uses a bait fused to a functional receptor chimera capable of activate STAT, and an inhibitory domain is fused to the prey. This results in JAK-STAT suppression by the action of a functional leptin chimeric bait receptor. The disturbance of the bait–prey interaction, either by a competing protein or an organic molecule, restores the signal, leading to a positive readout [24] (Fig. 2b). In addition, in reverse MAPPIT, the interference in a PPI by a competitor molecule results in dissociation of an inhibitor from an active receptor system, allowing the STAT complex to form and transcribe the RG. Thus, it can avoid problems associated with the membrane characteristics and permeability of lower eukaryotes or prokaryotes. However, an intrinsic limitation involves the localization of the bait and inhibitory prey proteins to the submembrane compartment [24].
technique can also detect dynamic changes and is effective enough for transmembrane and cytosolic protein identification. The indirect readout represents the major limitation, because it can stop spatial and temporal analysis of PPIs [27] (Fig. 2c). All the methodologies described thus far rely on endogenous STAT3, which represents a common limitation because it might be unsuitable to study PPIs involved in STAT3 signaling [27].
Tetracycline-repressor based MTH Tetracycline repressor-based MTH (trMTH) is based on the tetracycline repressor (TetR)–tetracycline operator (TetO) interaction (TetR-TetO). TetR-TetO is used in mammalian protein expression systems to achieve highly sensitive regulation of gene expression. The replacement of the dimerization domain (DD) of TetR with interacting bait and prey molecules yields a functional transcriptional regulator. The small molecule doxycycline can disrupt the TetR dimer and turn off the transcription activator function and the expression downstream of the RG. This system has a built-in negative selection tool via the doxycycline disruption assay. It is an ideal platform for cell-based HTP screening for therapeutic small-molecule and/or peptide PPI disruptors [28,29].
Kinase Substrate Sensor
Cell array PPI assays
In Kinase Substrate Sensor (KISS), the bait is coupled to the kinase domain of Tyrosine Kinase 2 (TYK2) and the prey is fused to a gp130 cytokine receptor chain. The bait–prey interaction leads to the phosphorylation of the gp130 anchor via TYK2, activating STAT3 and the transcription of STAT3-dependent reporter system. This technique enables the identification of in situ PPIs in mammalian cells and their modulation under physiological or pharmacological conditions [25,26]. This highly sensitive
Cell array PPI assay (CAPPIA) combines the study of the PPIs in a cDNA library with the MTH assay in adherent mammalian cells. An autofluorescence-based and GAL4-driven reporter plasmid (GAL4-pZsGreen) was developed as a RG. Interactions can be easily and directly detected through a DNA array scanner or under a HTP microscope [30]. CAPPIA allows the quantitative detection of specific PPIs in various mammalian cells and the identification of PPI modulators. In addition, several prey proteins can be tested www.drugdiscoverytoday.com
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FIGURE 2
Outline of cytokine receptor-based interaction traps. (a) Mammalian protein–protein interaction trap (MAPPIT): the different components and mechanisms of this cytokine receptor-based interaction trap are represented. Bait (X) and prey (Y) are fused to complementary fragments of a type I cytokine receptor. Ligandinduced activation and reorganization leads to cross-phosphorylation and activation of receptor-associated cytosolic Janus kinase (JAK). JAK activation leads to signal transducer and activator of transcription 2 (STAT2) activation and, consequently, RNA polymerase I recruitment. (b) Reverse MAPPIT: in the reverse mode, the bait is tethered to a cytokine receptor that has intact STAT recruitment sites and the prey is coupled to an inhibitory domain (ID), such as an inhibitory phosphatase of JAK. Upon disruption of bait–prey binding, the ID dissociates and JAK is able to phosphorylate STAT docking sites and STATs, leading to reporter gene activation. (c) Kinase substrate sensor (KISS): the bait is fused to the kinase-containing portion of tyrosine kinase 2 (TYK2) and the prey protein is coupled to Q1 the gp130 anchor, similar to MAPPIT. The bait–prey interaction leads to STAT3 phosphorylation, resulting in reporter gene expression. Adapted from Refs [21,24,25].
against a bait of interest, rendering CAPPIA a cost-effective PPI-HTP assay [2,30]. This technique has been mainly used to identify hormone-dependent PPIs, particularly steroid hormone receptors, such as the androgen receptor (AR), given that the AR is a ligand-dependent transcription factor involved in several physiological processes, in humans. CAPPIA is a technique that allows PPI identification and monitoring in different physiological conditions, the screening of ligand-dependent PPIs, and the identification of the dose response of the compounds tested [2].
Mammalian membrane two-hybrid assay Recently, a method based on the membrane split-ubiquitin assay was developed [31]. The mammalian membrane two-hybrid assay (MaMTH) can be conducted in most cell lines and is suitable for HTP screenings. It requires an integral membrane, because the bait protein is fused with a C terminus of ubiquitin and an artificial transcription factor (TF), and the prey protein is fused with the N terminus of the ubiquitin. When the N and C termini of the ubiquitin come into close proximity because of the bait–prey interaction, pseudo-ubiquitin is formed and proteolytic cleavage occurs, releasing the TF, which then activates the RG expression in 4
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the nucleus [31] (Fig. 3). MaMTH enables researchers to map phosphorylated residues on receptor tyrosine kinases (RTKs) that mediate PPIs with adaptor proteins; to monitor dynamic PPIs, and to use the increased amount of adaptor-protein recruitment to identify new pharmaceuticals [31].
Inducible MTH The most recent MTH adaptation, inducible MTH (iMTH), presents higher sensitivity compared with MTH. It uses a DNABD stronger than yeast GAL4-BD. To boost the activity of the prey protein–AD, a complex of multiple copies of the AD is fused to the Q6 prey, to which a small-molecule dimerizer (rapalog) binds, increasing the sensitivity up to 100-fold compared with classic MTH approaches [32].
The role of MTH in PPI discovery, validation, and modulation Over the past few decades, MTH has been used mainly to validate YTH screenings [15]. In 2010, Naz and Dhandapani used MTH to detect false positives after an YTH screen. Human ZP3 was cloned into YTH bait vectors to find reactive proteins in a human testis
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FIGURE 3
Membrane mammalian two-hybrid (MaMTH). The bait protein is an integral membrane fused with a C terminus of ubiquitin and an artificial transcription factor (TF). The prey protein is fused with the N terminus of ubiquitin. When the N and C termini of ubiquitin come in close proximity through the interaction of the bait and prey, pseudo-ubiquitin formation occurs, resulting in proteolytic cleavage and the release of the TF, which then activates expression of the reporter gene.
cDNA library. Six clones were identified and further confirmed by MTH [33]. Later, Tham et al. performed YTH to identify potential interactors of DENV-2 in Aedes aegypti, responsible for dengue transition. In total, 36 midgut proteins where identified as possible interactors, and three proteins (NS1, prM, and E proteins) were confirmed as interactors with DENV-2, by MTH [34]. Later, MTH was used to identify PPIs involved in virus–cell communication. Jiang and Luo showed that adaptive and compensatory mutations occurring in different nonstructural (NS) proteins promote Hepatitis C virus (HCV) production in cell culture. PPIs between NS proteins involved in HCV cell penetration were confirmed by MTH, confirming that adaptative mutations in NS protein occur and alter the association of the core
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with lipid droplets, allowing HCV cell assembly [35]. Hu et al. performed a YTH screen to identify proteins from a human fetal brain cDNA library interacting with NS2 of Influenza A virus (IAV). CHD3 had a positive readout and the interaction was further confirmed by MTH. The authors showed that NS2 of IAV has a nuclear export signal (NES1) motif that is crucial for the NS2–CHD3 interaction, required for IAV penetration [36]. Bovijn et al. studied the impact of the upper activation of toll-like receptor (TLRs) on pathogen recognition, highlighting pathogenassociated molecular patterns [37]. Oligomerization of TLRs trigger multiple signaling pathways, activating inflammatory and immune responses [38]. The authors used a random mutagenesis and MAPPIT assay to identify myeloid differentiation primary response gene 88 (MyD88) adapter-like protein (Mal) mutations that disrupt Mal/TLR4 and/or MyD88/TLR4 interactions. Results showed four potential binding sites and the AB-loop in the Mal TIR domain, contributing to the formation of TLR4/Mal/MyD88 complex [37]. Mal dimerization leads to the formation of binding platforms to which MyD88 or TLR4 bind [37], consequently triggering inflammatory and immune responses. In 2019, Kumar et al. reported a novel interaction between HIV-1 Nef, a mediator of HIV replication, and Ca2+/calmodulindependent protein kinase II (CAMKIId). Nef activates calcium signaling as a positive feedback mechanism to regulate HIV replication in human cells. MTH was performed to confirm the Nef–CAMKIId interaction and screen for peptide inhibitor activity. The findings suggested Nef as a CAMKIId activator through elevation of Ca2+ concentration, which inhibited ASK-1 and p38-mediated apoptosis of infected cells [39]. The interaction between MMD2 (an E3 ubiquitin ligase) and p53 was used to validate the reverse MAPPIT assay, prove its sensitivity, and evaluate the modulation effect of Nutlin-3, a known inhibitor of this interaction, confirming the ability of this approach to identify PPI disruptors [24]. The MDM2–p53 interaction was disrupted with an IC50 value of 0.4 mM [24], which approximates the IC50 value previously reported for Nutlin-3 in Biacore experiments with purified proteins (0.1 mM) [40]. In 2012, Liu et al. studied the role of fucoxanthin in drug resistance through the attenuation of rifampin-induced CYP3A4 and MDR1 gene expression. The pregnane X receptor (PXR)-mediated pathway mediates CYP3A4 and MDR1 expression in HepG2 hepatoma cells, and the interaction between PXR and SRC1 (which acts as a main co-activator). Whereas fucoxanthin negatively affects the PXR–SRC1 interaction, rifampin strongly increases it. However, Q7 when cells were exposed to fucoxanthin and rifampin, the rifampin-induced PXR–SRC1 interaction was attenuated, suggesting that fucoxanthin is a PXR antagonist. This could be a potential way to prevent transcriptional activation of PXR-regulated genes, given that fucoxanthin is used to prevent drug resistance in patients receiving chronic therapy with PXR agonists [41]. Leverson et al. identified a small molecule that inhibits MCL-1, inducing clear on-target cellular activity in cancer cells. Molecules derived from indole-2-carboxylic acids, such as A-1210477, induce intrinsic apoptosis. A-1210477 binds MCL-1 with subnanomolar affinity and can disrupt endogenous MCL-1–BH3-only protein (NOXA and MULE) interactions. Based on MTH and a cell imaging platform that tracks BCL-2 family interactions in real time, it was shown that A-1210477 selectively disrupts MCL-1–NOXA in cancer cells [42]. www.drugdiscoverytoday.com
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Recently, Arao et al. showed the contribution of the estrogen receptor alpha (ERa) F domain to species-specific ER modulator 4-hydroxy-tamoxifen (4OHT)-dependent ERa ligand-binding domain (LBD) homodimerization using MTH. In the nuclear receptor (NR) field, MTH has been used to assess the ligand-dependent coactivator or corepressor recruitment activity of NR LBDs. The effect of 4OHT on mouse and human ERa LBD revealed that RG expression was higher in cells expressing the mouse ERa LBD. The authors showed that MTH enables the evaluation of LBD dimerization activity of certain substances that do not activate the AF-2 transactivation function of LBD (such as 4OHT or SERMs) [43]. Thus, MTH has proved to be useful for identifying and studying PPIs, highlighting its versatility. It can be applied to different cell lines, enabling tissue-specific PPI study. On a pharmacological and therapeutic level, MTH has already been applied to study different diseases, increasing the knowledge of the deregulated PPIs involved.
MTH and its role in HTP screening Most PPI studies with MTH were done on an individual gene scale, but there is an increasing need to increase putative interactors with a bait protein. In 2001, Suzuki et al. performed the first MTH-HTP screening for PPIs [44]. In this study, a mouse full-length enriched cDNA library was constructed using a PCR-based process. A MTHHTP assay was run to test 3500 bait proteins and 3400 prey proteins in a total of 1.2 107 possible interactions. Overall, 145 PPIs were identified in the assay, further analyzed using a bioinformatic approach, which was crucial to understand the role and function of uncharacterized proteins, and to unravel the associated molecular mechanisms [44]. Later, Ravasi et al. confirmed that it was possible to perform an extensive MTH-based assay. PPI maps were established using pairwise analyses of 1988 human and 1727 mouse transcription factors. A cDNA library of human and mouse transcription factors was constructed out of all DNA-binding transcription factors able to express a full-length protein, resulting in 1222 human and 1112 mouse cDNA clones. MTH screening revealed 762 and 877 interactions, respectively [45]. In 2008, following the development of CAPPIA, Fiebitz et al. studied a large number of PPIs simultaneously. This technique allowed the screening of 160 different bait–prey combinations, associated with the human AR. The study was performed in the presence of 10 nM of the synthetic androgenic ligand R1881. A specific interaction between the AR-LBD and the AR-N-terminal domain (AR-NTD) was identified and a dose-dependent induction of receptor expression in the presence of R1881 was observed. The effect of two antagonists was also evaluated (medroxyprogesterone acetate and hydroxyflutamide), showing an inhibitory effect on the AR-LBD–AR-NTD interaction. Thus, CAPPIA was shown to be one of the most economical HTP detection assays for PPIs in mammalian cells [2]. Q8 The use of MTH for PPI HTP screening was first reported in 2012 by Lievens and colleagues [18]. Later, Rolland et al. performed a human proteome assay using four techniques, including MAPPIT. All binary pairs identified in seven public databases were tested and validated using the different techniques. Half of the interactome was systematically screened, resulting in double the number of high-quality binary PPIs available from the literature (14 000 interactions). This research resulted in the human interactome data set covering Space II, reported in 2014 (HI-II-14), showing that known candidate cancer 6
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gene products are highly connected in the human interactome network [46]. Recently, the group responsible for MaMTH development adapted the method to map the genome-wide RTK–phosphatase interactome [47]. After a membrane YTH screen, a huge number of PPIs were identified, most of them for the first time. MaMTH was further used to validate some of the PPIs and nine interactions were confirmed [48].
Impact of MTH on drug discovery The combination of MTH and HTP can be applied to therapeutic research, making it possible to evaluate the effects of drugs and small compounds on specific PPIs, rendering treatments target specific. In cancer research, it has been possible to increase the identification of drugs able to inhibit and/or modulate PPIs involved in tumor suppression, chemotherapeutic resistance, and other mechanisms. Li et al. screened 3000 compounds to identify a disruptor of the MDM2–p53 interaction, given that it was thought that p53 is activated when MDM2 is released from the complex. The authors developed MTH to study MDM2–p53, with a dual-luciferase RG to exclude false positives. Nutlin-3, caylin-1, and three hit compounds (SL-01–03) had an inhibitory effect on MDM2/p53, increasing the levels of active p53, which inhibits tumor proliferation [49]. Bhatia et al. also used HTP drug screening to identify small molecules able to inhibit proteins involved in p53 suppression. MAGE proteins are highly present in human tumors, and are associated with chemotherapeutic drug resistance. KAP-1 is a central molecule involved in p53 regulation. In this study, after screening 14 400 compounds from the Maybridge HitFinder library, seven were identified as possible molecules able to modulate the MAGE–KAP-1 interaction. The authors performed an MTH assay and concluded that Class I MAGE proteins bind with the KAP-1 RBCC binding region, suppressing p53. Additionally, three small molecules (HTS11125, GK02432, and SPB02) were shown to have a direct effect on MAGE–KAP-1, indicating them as promising candidates for its modulation and, thus, as potential anticancer drugs [50]. Constitutive androstane receptor (CAR) inhibition and PXR activation appear to be crucial to improve the effects of chemotherapeutic drugs, given that CAR inhibition attenuates multidrug resistance in aggressive cancers. Cherian et al. performed an HTP screening of 1000 compounds to identify CAR inhibitors. In total, 25 compounds displayed dose-responsive inhibitory effects, but only CINPA1 was selected, because it had a potent inhibitory effect on hCAR1 and no agonistic activity against PXR. MTH was performed to validate the ability of CINPA1 to inhibit CAR–CAR coactivator interactions. Given that CINPA1 proved to be a specific CAR inhibitor, it could help researchers to better understand CAR functions or could even be a suitable molecule to be used along with chemotherapy for a better tumor response [51]. Recently, Yasui et al. identified two suitable inhibitors of the interaction between B cell lymphoma 6 (BCL6) and its corepressor (BCoR). BCL6 and BCoR are responsible for the differentiation and proliferation of lymphocytes. Several compounds were synthetized to find a drug with inhibitory activity, but with increased membrane permeability and high stability in aqueous solution, rendering them suitable for MTH assays. MTH was performed and two of the developed compounds were identified to have good
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Concluding remarks PPI identification is a key to understand signaling pathways responsible for all cellular processes and the molecular
mechanisms behind many diseases. The study of PPIs as drug targets can be challenging and requires adequate systems to select lead compounds, but is a straightforward way to interfere with the disease signal cascade. MTH and its variations are flexible methodologies because they can be adapted to the PPIs of interest. Comparing MTH with YTH is important to highlight their ability to mimic the complex cellular context in which the interactions occur. Although MTH is a versatile strategy to study binary PPIs, it also has a crucial role in drug discovery when allied to HTP screening. From a pharmacological and therapeutic view, MTH allows the extrapolation of results to the roles of human proteins and interactions, and PPI modulation. The successful application of MTH in HTP is an alternative method to discover small-molecule inhibitors and represents a valuable approach for the discovery and development of new antiviral, anticancer, and other drugs. Although most of the work using MTH be applied to cancer and chemotherapy, this methodology presents many opportunities for PPI modulation and library drug screening in other diseases, such as viral infections or male infertility. Q9
Acknowledgments We thank the Portuguese Foundation for Science and Technology (FCT), European Union, QREN, FEDER and COMPETE for funding iBiMED (UID/BIM/04501/2013, POCI-01-0145-FEDER-007628 and UID/BIM/04501/2019), the research project PTDC/BBB-BQB/ 3804/2014 and an individual scholarship from DP (SFRH/BD/ 137487/2018).
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cellular PPI inhibitory activity. The authors identified these compounds as good candidate drugs for autoimmune diseases and cancer treatment [52]. Although most studies using MTH for drug discovery have been performed on cancer, the association of MTH with HTP screening could also be applied to PPI modulation in other biological contexts. In recent research on male fertility, HTP screening was performed of 3242 ion channel library ligands. In total, 384 compounds showed positive alteration of functional sperm motility and calcium rates of the sperm of subfertile patients. Furthermore, protein phosphatase 1 (PPP1) and its catalytic subunits were studied using YTH for PPI discovery [53,54]. In human testis, all four PPP1 isoforms are expressed [55], but PPP1CC2 has enriched expression in testis and spermatozoa (but low expression in brain, lung, spleen, and thymus) [56–58]. In spermatozoa, PPP1CC2 is present throughout the flagellum, midpiece, and posterior region of the head [54], suggesting a role in motility and acrosome reaction. Studying PPIs already known to be involved in sperm motility or dysfunction, combined with HTP drug screening, could be the next step in the effective identification of targets for the treatment of specific sperm dysfunctions. Thus, the combination of MTH-HTP has significant potential for drug discovery, allowing the development of drugs that can enhance sperm function or can be used as targeted contraceptive molecules [59].
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