High-Throughput profiling of SH2 domains using Affimer reagents: unravelling protein interaction networks

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Heseltine, Gregory J. Billenness, Heather L Martin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3959018/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Despite SH2 domains, being pivotal in protein interactions linked to various diseases like cancer, we lack specific research tools for intracellular assays. Understanding SH2-mediated interactions and creating effective inhibitors requires tools which target individual protein domains. Affimer reagents exhibit promise, yet their potential against the extensive SH2 domain family remains largely unexplored. Our study aimed to bridge this gap by identifying Affimer reagents that selectively bind to 22 out of 41 SH2 domains. These reagents enabled a medium-throughput screening approach resembling siRNA studies, shedding light on their functionality. Notably, select Affimers demonstrated the ability to curtail the nuclear translocation of pERK, with Grb2 being a prominent target. Further analyses revealed that these Grb2-specific Affimer reagents displayed competitive inhibition with impressive metrics: IC50s ranging from 270.9 nM to 1.22 µM, together with low nanomolar binding affinities. Moreover, they exhibited the ability to pull down endogenous Grb2 from cell lysates, illustrating their efficacy in binding the Grb2 SH2 domain. This comprehensive assessment underscores the potential of Affimer reagents as domain-specific inhibitors. Their viability for medium/high-throughput phenotypic screening presents a promising avenue via which to identify and characterize potential drug targets within the SH2 domain family. Biological sciences/Biological techniques Biological sciences/Biological techniques/High throughput screening Biological sciences/Biological techniques/Imaging Biological sciences/Cell biology/Cell signalling Biological sciences/Cell biology/Cellular imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Src Homology 2 (SH2) domains are phosphotyrosine (pTyr)-binding modules found in over 120 human proteins [ 1 ]. Approximately 100 amino acids in length, the SH2 structure consists of a central anti-parallel β-sheet flanked on both sides by an α-helix [ 2 ]. These form two binding sites; a conserved pTyr binding pocket and a variable pocket that binds residues C-terminal to the pTyr. In total, a four to seven amino acid motif is bound by the domain [ 3 ]. SH2s are usually found in conjunction with either catalytic domains or other binding domains, such as SH3s [ 4 ]. SH2 domains constitute the largest class of pTyr-binding modules and are found in a wide variety of proteins including kinases, adaptor proteins, transcription factors and phosphatases [ 5 , 6 ]. Through their binding of phosphorylated targets, they mediate protein-protein interactions (PPIs) and are involved in numerous intracellular signalling pathways. Many of these SH2-regulated interactions play key roles in processes that become dysregulated in disease, such as cell cycling, proliferation and apoptosis [ 7 , 8 ]. SH2 domains are therefore potential therapeutic targets for the treatment of several disorders including cancer, and study of their function could lead to a better understanding of numerous cancer signalling pathways [ 9 , 10 ]. Though recognised as promising disease targets, there is still a lack of research reagents available for SH2 domains [ 11 ] and the scarcity of SH2 inhibitors that are effective in intracellular assays has hampered study of SH2-mediated mechanisms [ 2 , 12 ]. The highly conserved sequence and structure of SH2 domains raises significant challenges in generating specific binding reagents or inhibitors [ 2 , 13 ]. As a result, the function of many SH2s has not yet been explored [ 14 ] and it is widely acknowledged that protein-specific SH2 inhibitors would be highly valuable research tools that would enable the discovery of novel biology and new pharmaceutical targets in various disorders [ 1 , 15 ]. The development of these could also lead to the detection of residues that may be exploitable for protein-specific drug design [ 11 ]. Analysis of intracellular protein function can be achieved through techniques such as gene knockout or RNA interference. However, these methods are impractical for studying domain-specific interactions as they result in the deletion of the entire protein [ 12 ]. In order to observe the cellular functions of SH2 domains, binding molecules acting at the protein level are needed [ 14 ]. In recent years, the development of scaffold based binding proteins (SBPs) has aided targeted disruption of PPIs [ 15 – 17 ]. SBPs have many advantageous features; ability to be expressed intracellularly, high solubility, high stability and lack of disulphide bonds, and include, amongst others, Designed Ankyrin Repeat Proteins (DARPins) and monobodies as well as the Affimers [ 18 – 20 ] utilised in this study. These easily producible proteins have been used for a range of biological and therapeutic applications to date [ 21 ]. Monobodies targeting the SH2 domain of Abl have been shown to bind Bcr-Abl allosterically and inhibit its function resulting in apoptosis of chronic myeloid leukaemia cells [ 22 , 23 ]. Our group have previously identified Affimers that bind the SH2 domains of the Grb family of proteins and the individual SH2 domains of PI3K [ 19 ]. These studies demonstrate the potential for SBPs in modulation of SH2 mediated signalling events. However, to date, there has not been available a toolbox of SH2 modulating reagents to allow the roles of individual SH2 containing proteins, and even individual domains, to be delineated in desired cellular phenotypes. Here we have developed a toolbox of Affimer reagents that bind 38 SH2 domains representing approximately a third of the known SH2-domain containing proteins. The specificity of the toolbox is comparable to those seen with ScFv screens [ 24 ] and we have proved that the toolbox functions intracellularly by the identification of Grb2 as a major SH2 protein in the MAPK pathway using a phenotypic screen looking at phosphorylation and subsequent nuclear translocation of ERK. These specific Grb2-binding Affimers were shown to have nanomolar affinities and IC 50 values demonstrating the utility of the toolbox. RESULTS High-throughput screen of SH2 domains to identify Affimer binders Building on our previously published work identifying Affimers that specifically bound the SH2 domains of the Grb family of proteins, p85 and p55 [ 19 ] we sought to generate a toolbox of reagents to dissect the roles of SH2 domain/proteins intracellularly and test the application of such reagents in a medium throughput screen. To achieve this an additional 32 SH2 domains encompassing the main molecular functions of SH2 domain containing proteins (excluding those associated with RAS regulation and ubiquitination) [ 25 ] were selected. These 32 SH2 domains were then expressed and purified in a small-scale high throughput manner utilising 3mL cultures and the KingFisher Flex yielding between 16–173 µg of protein. These proteins were then used for isolation of Affimers from our phage library using a competitive approach [ 18 ]. Three panning rounds were sufficient to yield substantial amplification for 18 of the targets whilst a fourth panning round increased this to 27 targets giving an 84% success rate at this stage. No amplification was achieved for Ptpn11-N, Src2, Syk-C, Stat2 and Yes, possibly owing to poor protein quality/quantity or low levels of biotinylation. Target binding for 24–48 clones for the 27 successful targets was assessed by phage ELISA and successful binders sent for sequencing to identify unique clones, yielding 621 unique clones. The range of clones per target varied from 1–48 (Table 1 ). Table 1 Summary of phage display screening and isolation of Affimer clones to BAP-tagged SH2 domains. Final hit rates and unique clones isolated for each successfully screened SH2 domain. Targets for which positive hit criteria was lowered to ≥ 3x that of the negative control (compared to the standard criteria of ≥ 10x that of the negative control) are highlighted in italics. For SH2 domains screened twice, both hit rates are shown in column 2. SH2 target Phage ELISA hit rate (%) Number sequenced Unique clones Abl1 60 48 6 Abl2 100 48 42 Bmx 98 48 22 Crk 83 48 21 Fyn 90 48 20 Grb2 92 48 30 Grb7 73 / 15 61 48 Grb10 42 / 13 34 12 Grb14 92 / 10 52 8 Lck 91 87 35 Lyn 8 8 4 Nck1 81 48 4 Nck2 15 8 7 p85α-C 23 48 9 p85α-N 92 48 24 p85β-C 54 48 8 p85β-N 98 48 8 p55γ-C 81 48 13 p55γ-N 100 48 35 PLCγ1-T 27 / 4 21 13 PLCγ1-N 88 10 2 PLCγ2-T 8 6 3 PLCγ2-N 48 / 4 33 11 She 66 48 8 Ship1 52 / 50 54 26 Ship2 25 / 25 36 27 Src1 17 8 4 Stat1 2 48 14 Stat3 92 48 47 Stat4 31 48 48 Stat5a 29 7 2 Stat5b 2 6 5 Stat6 4 1 1 Syk-N 69 42 18 Tec 4 7 6 Tns1 38 48 4 Vav1 81 48 26 As SH2 domains share high structural homology [ 25 ], a microarray approach was used to determine the specificity of these binders for their target domain in a rapid fashion. BAP-tagged SH2 domains were printed onto streptavidin coated slides, five spots for each SH2 domain, 10 buffer spots per array and 14 arrays per slide. Three SH2 domains, Nck1, Stat2 and Tec, were excluded from the microarray as the detection antibody bound to these proteins in optimisation experiments. The six SH2 domains we had previously targeted [ 19 ] were also included on the microarrays, giving a total of 35 SH2 domains. A maximum of five clones per SH2 domain were then tested for specificity (clones were selected based on their sequence and signal in the phage ELISA). HA-tagged Affimers (5 µg/ml) were applied to microarrays and detected with an anti-HA antibody (1 µg/ml). Of the 162 Affimers tested, 54 showed no binding at the cutoff of 50x the signal from buffer only spots (Fig. 1 ). This was in contrast to the phage ELISAs used in Affimer identification, however this appeared to be target dependent with four targets that showed no binding (She, Tns1, p85α-C and p85β-C) that was possibly the result of protein denaturation [ 26 – 28 ]. Re-testing these non-binding Affimers in an ELISA format only identified 14 that bound their targets, however these included all Affimers targeting p85α-C suggesting this target had indeed become denatured on the microarray. From the remaining 108 Affimers that showed binding in the microarray, 51 showed specificity for their respective target (Fig. 1 ). Affimer reagents were deemed specific if off-target interactions were ≤ 10% of the signal shown for the intended target, in accordance with previous work on SH2 domain-binding antibody fragments [ 11 , 24 ]. In total, specific Affimers were found for 22 SH2 domains giving a specific hit rate of 63% (Table 2 ). Table 2 Target-specific Affimer clones as determined by SH2 protein microarray. Table summarising specific Affimer clones for SH2 targets as identified by protein microarray. Clones were deemed specific if off-target interactions showed a signal < 10% of that of the intended target. Target​ Number of specific clones​ Abl1​ 1​ Abl2​ 5​ Bmx​ 4​ Crk​ 3​ Fyn​ 4​ Grb2​ 5​ Grb10​ 1​ Grb14​ 2​ Lyn​ 3​ P55γ-C​ 5​ P85α-N​ 2​ P85β-N​ 1​ P55γ-N​ 1​ PLCγ1-N​ 1​ PLCγ2-T​ 1​ PLCγ2-N​ 1​ Ship1​ 1​ Ship2​ 1​ Stat3​ 3​ Stat4​ 3​ Stat6​ 1​ Syk-N​ 2​ Intracellular pathway screening Having identified a toolbox of SH2-binding Affimers with good target specificity, we investigated their application towards understanding the roles of SH2 domain containing proteins in cell signalling. For this we used an assay from our previous work on the modulation of Ras with Affimers [ 29 ], and examined the nuclear translocation of phosphorylated extracellular signal-regulated kinase (pERK) with high content imaging as a measure of EGFR signalling. By testing our toolbox of SH2-binding Affimers with this assay we hoped to identify those SH2 domain containing proteins with roles in this pathway as a proof of principle for the utility of the toolbox, in particular we anticipated that we should be able to identify Grb2 owing to its well characterised role with this pathway [ 30 – 32 ]. To achieve this 119 of the 162 Affimers used in the microarray were subcloned into the mammalian expression vector pCMV6-tGFP. Our previous pERK nuclear translocation assay was adapted to a screening format of four 96 well-plates with each plate featuring a maximum of 30 Affimers to 62 negative controls (a non-targeting Affimer) and four positive controls of a Ras-inhibiting Affimer K6. HEK293 cells were reverse-transfected with these constructs and pERK nuclear translocation assessed 48 hr later. The assay quality of the screen was assessed by robust Z’ factor analysis which yielded a value of 0.52 indicating the screen had a difference of 12 standard deviations between the positive and negative controls showing that it was likely to pick up a number of hits. The screen was repeated in triplicate and 18 hits identified as those Affimers with robust Z scores of less than − 3, identifying SH2 domains involved in positive EGFR signalling (Fig. 2 a). Intriguingly 3 Affimers (Lck A7, Lyn A2, p85αN C6) increased pERK nuclear translocation as indicated by robust Z scores of greater than + 3. The 18 hits were then validated by individual assessment of pERK translocation and all were confirmed as hits (Fig. 2 b) including 12 Affimers targeting Grb2. It was unsurprising that Grb2-binding Affimers were the major hits from this screen as this is the predominant SH2 domain containing protein in the EGFR signalling pathway [ 30 – 32 ], but this confirms the utility of this screening technique as it highlighted a known major player in this pathway. PI3K hits (p85αC A1 and F4, p85βN A3 and p55ɣC B5) were also seen and this confirms our previous work [ 19 ] as there is a degree of cross talk between the Akt pathway activated by PI3K upon EGF stimulation and the ERK pathway [ 33 ]. The two remaining hits Plcy2N A8 and Lyn A4 have all been linked to MAPK pathway signalling in other cell types with other stimulating agents, the majority of which show heterogeneity with the EGF receptor (ErbB1) [ 34 – 36 ]. These signalling events are not the major pathway for ERK phosphorylation and this is reflected in the relatively small reductions in pERK compared to those seen with Grb2 (Fig. 2 a). Thus, we have demonstrated the utility of using binding proteins in a high-throughput screen and that the SH2 Affimer toolbox functions in vitro to identify SH2 domains with both major and minor roles in MAPK signalling. Characterisation of Grb2 Affimers Having identified Grb2 Affimers has the major hit from the pERK screen the four Affimers that had shown specificity for Grb2 (as measured by microarray) were then characterised further. Initially their competitive inhibitory capabilities were quantified by fluorescence polarisation, as measured by the displacement of a fluorescently labelled SH2 phosphopeptide from the Grb2 SH2 domain, giving IC 50 values ranging from 270 nM (Affimer Grb2 F1) to 1.22 µM (Affimer Grb2 D6) (Fig. 3 a). The two Affimers (A4 and F1) showing nanomolar IC 50 values were taken forward for more detailed characterisation. Surface plasmon resonance showed the affinity of these Affimers to full length Grb2 to be low nanomolar (A4 K D = 11.8 ± 6.9 nM; F1 K D = 34.8 ± 16.9 nM) comparable with the affinity of Grb2 for its intracellular targets [ 37 ], demonstrating these Affimers are able to compete in vitro for binding of the SH2 domain of Grb2. In vitro binding was confirmed by the ability of these Affimers to immunoprecipitate Grb2 from HEK293 lysates (Fig. 3 b). Next, we explored if these Affimers demonstrated a dose-response in terms of inhibition of pERK nuclear translocation by correlating GFP intensity with pERK nuclear intensity, as GFP intensity increased, i.e dose of Affimer, pERK nuclear intensity decreased and for both A4 and F1 the correlation was significant (Pearson correlation p = 0.0003 A4, p = 0.0004 F1; Fig. 3 c and d). This demonstrates that the SH2 toolbox contains high affinity, specific Affimer binders that can block SH2 function in normal cells in a dose-dependent manner. To determine the wider relevance of these results we determined if they were applicable to cancer cells. The effects of these two Grb2 Affimers were explored in two cancer cell lines, U-2-OS and HeLa. Both A4 and F1 were able to immunoprecipate Grb2 from both HeLa and U-2-OS lysates (Fig. 4 a and b). However, only F1 was able to inhibit pERK nuclear translocation in both cell lines (One-way ANOVA with Dunnett’s post hoc test p < 0.0001 for U-2 OS and p = 0.0237 for HeLa; Fig. 4 c-f). These data are consistent with A4 being a marginal hit in the screen (z = -3.27 ± 0.66) DISCUSSION Isolating specific and potent SH2 inhibitors has proven a significant challenge in the past, to the extent that SH2 domains were deemed ‘undruggable’ targets [ 9 , 38 ]. However, the development of scaffold based binding reagents, SBPs, has allowed the specific targeting of interaction domains previously abandoned as disease targets. In this work, we have created a toolbox of SH2-binding Affimers to aid in the exploration of the roles of SH2 domain-containing proteins in cellular signalling and function and to determine the ability to use this type of reagents set in high-throughput phenotypic screens. To achieve this, we successfully isolated protein-specific Affimer binders to 22 SH2 domains. This success was not only due to the stringent phage display process used, but also the incorporation of an N-terminal BAP tag on the SH2 antigens. This allowed site-directed in vivo biotinylation of the target protein for phage display screening, thus removing the need for chemical biotinylation, which has previously resulted in the coupling of a biotin molecule to free lysines in the SH2 domain binding site. This method also allows the presentation of the target protein in its native conformation, an advantage when isolating Affimers that will function in cell-based assays. The hit rate of 80% achieved is higher than previously reported hit rates from SH2 domain screening [ 24 ]. The specific hit rate of 63% is comparable to previous screens using ScFvs [ 5 , 24 ] and both these studies only targeted 20 SH2 domains. An Abl SH2-binding monobody isolated by Wojcik et al. [ 23 ] showed cross-reactivity to three other SH2s in a protein microarray used at a tenth of the concentration of the Affimers in this study, indicating that the specificity of SH2-binding Affimers is favourable when compared with similar non-antibody reagents raised against SH2s. Additionally, this monobody could not distinguish between Abl1 and Abl2 unlike some of the Abl binding Affimers identified in this study. Whilst the previous screens identified SH2 binders, no assessment of their function in vitro in live cells was undertaken as these screens identified ScFvs that bound SH2 domains in lysates or fixed cells [ 5 , 24 ]. This was important for the utility of the SH2 binding Affimer toolbox. Shp2 SH2 binding monobodies have been shown to be functional in inhibiting ERK phosphorylation in HCC1171 lung cancer cells [ 17 ] and our previous work [ 19 , 29 ] show SBPs function intracellularly. This intracellular functionality was utilised to screen the SH2 Affimer toolbox in a pERK translocation assay. Popular methods for investigating MAPK signalling include western blotting and SRE luciferase assays, which can be slow and labour intensive [ 39 ] so high-content imaging was used together with GFP-tagged SH2-binding Affimer constructs yielding a simple, time efficient and sensitive assay. This approach is easily modified to screen different endpoints, for example modulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by using AKT phosphorylation as the measurable endpoint [ 40 ], or any other phenotypic change that can be imaged or measured. The identification of 18 Affimers for 6 SH2 domains that reduced EGF-induced pERK translocation indicating they inhibited MAPK signalling demonstrates a hit rate of 22%. These included 12 Affimers isolated against the Grb2 SH2 domain which is not surprising giventhe canonical role of Grb2 in Ras-activated MAPK signalling [ 30 – 32 ]. Of the remaining six Affimers four were isolated against the C-terminal SH2 domains of PI3K subunits p55ɣ (B5), p85α (A1 and F4) and p85β (A3) in conjunction with our previous work showing Affimers binding the N-terminal SH2 of p85 increase AKT phosphorylation (pAKT) levels [ 19 ]. Stimulation of the PI3K pathway leads to phosphorylation and activation of its downstream effector AKT. Activated AKT has been shown to inhibit ERK phosphorylation via its interaction with RAF [ 33 ]. Both the N and C-terminal subunits are involved in inhibition of the catalytic p110 subunit [ 41 , 42 ]. This suggests a mechanism where Affimer binding of the PI3K regulatory subunit’s SH2 domain leads to increased AKT activity and subsequent inhibition of ERK phosphorylation. Characterisation of the specific Grb2 Affimers identified as inhibiting pERK translocation showed IC 50 values ranging from 0.27–1.22 µM, as measured by fluorescence polarisation, which is in line with the IC 50 values for Grb2 SH2-binding phosphopeptides [ 43 ]. The most potent Affimer, F1, had an IC 50 comparable to bicyclic peptide inhibitors of the Grb2 SH2 domain [ 44 ]. This demonstrates that the inhibitive ability of the Grb2 SH2 Affimers is equal to, or surpasses, previously developed Grb2 SH2 inhibitors. The nanomolar affinities of the Grb2 Affimers for Grb2 is in line with that of the antibody fragments that bind the Grb2 SH2 with K D s in the nanomolar range [ 24 ], the SHP2 SH2 domain binding monobodies [ 17 ], as well as phosphopeptide binders [ 43 ]. Higher picomolar affinities have been achieved with Grb2 SH2 small molecule inhibitors that mimic the phosphorylated tyrosine residue in Grb2 SH2 binding partners [ 45 ]. In spite of their high affinity for Grb2 SH2, the small scFv antibody fragments lacked the ability to immunoprecipitate endogenous Grb2 from clarified lysate, in contrast to the Grb2-SH2 Affimers tested in this study that were able to pull out detectable levels of endogenous Grb2 from cell lysates from multiple cell lines. These results show the ability of the Affimers to bind low levels of the target, in the context of the whole protein rather than just the isolated SH2 domain. This has positive implications for their use in functional cell-based assays as we have shown with successful inhibition of EGF-stimulated MAPK signalling as measured by pERK translocation. The Grb2 SH2 domain binds its natural substrates via selective recognition of the binding motif pY-X-N-X [ 10 ], a motif that is mimicked by the phosphopeptide binders [ 43 ] and the small molecule inhibitors [ 45 ]. The majority of the Grb2 SH2 Affimers share this binding motif (10/16) including Affimer F1. Interestingly Affimer A4 contains an alternative aromatic residue, tryptophan, and Affimer F5 does not contain this sequence, so this motif alone does not confer Affimer specificity for the Grb2 SH2 domain. Relating results to the specificity motifs in the variable regions of the strongest hit, Affimer F1, reveals the sequence of Y-V-N-V, as in previous work using phosphorylated peptide libraries the sequence pY-V-N-V to have the highest affinity for the Grb2 SH2 [ 46 , 47 ]. The level of MAPK inhibition seen in this study was closely correlated with similarity to this sequence. The variable regions of A6 and H1, which failed to significantly reduce pERK, show little similarity to this sequence. This provides strong evidence that the effects seen in this assay are due to the binding of the Grb2 SH2 domain, rather than some unknown off-target effects. Importantly, Affimer reagents can utilize this motif to bind the Grb2 SH2 domain with high specificity without the need for the highly polar phosphorylated tyrosine residue, which can cause promiscuous binding [ 46 – 48 ]. In conclusion this study has demonstrated that Affimers can be isolated that bind SH2 domains in a protein-specific manner with high affinity. The specificity of Affimers for their target SH2 over highly homologous SH2 domains of other proteins and their ability to bind endogenous Grb2 is favourable when compared with previously isolated binding reagents [ 23 , 49 ]. Furthermore, Grb2 SH2-binding Affimers show the ability to inhibit target function. This, in conjunction with the ability of Affimers to fold correctly and bind targets in the cytoplasm, indicates that the SH2-targeting Affimer toolbox or an Affimer toolbox to other protein domains, will be useful for functional cell-based assays to determine the role of different protein domains in biology and disease. This may show the way for future development of proteome domain screening tools for functionally dissecting pathways and identifying key domains on proteins for targeted therapeutics. METHODS SH2 domain production SH2 domains were produced as previously described [ 19 ]. Briefly sequences encoded in kanamycin-resistant pET28 SacBAPvectors were purchased from the Pawson Lab (Samuel Lunenfeld Research Institute, Canada) and a biotin acceptor peptide (BAP) sequence was cloned into the vectors to give an N-terminal BAP-Histag-SH2 domain sequence. For production in Rosetta TM 2 (DE3) cells (Novagen, Merk Millipore), overnight starter cultures were grown at 37°C, 230 rpm in TB medium supplemented with kanamycin (50 µg/ml), chloramphenicol (34 µg/ml), and 1% glucose. These were used to inoculate 3 ml cultures of TB kanamycin that were grown at 37°C, 230 rpm until OD600 reached ca. 1.5 and temperature was reduced to 18°C for 1 h before addition of 0.5 mM IPTG and cultures were grown overnight at 18°C, 230 rpm. His-tagged SH2 proteins were purified from clarified culture lysates on a KingFisher™ Flex robotic platform (ThermoFisher) using His Mag Sepharose Ni beads (GE Healthcare), washed (50 mM NaH 2 PO4; 500 mM NaCl; 20 mM imidazole; pH 7.4) and eluted in 130 µl elution buffer (50 mM NaH 2 PO4; 500 mM NaCl; 300 mM imidazole; 10% glycerol; pH 7.4). The elution buffer also contained 1 mM TCEP. Samples were flash frozen in liquid nitrogen and stored in aliquots at -80°C Phage display and phage ELISA Phage display was completed over four panning rounds, as described previously [ 19 ]. Streptavidin-coated wells were used for the first panning round, followed by Streptavidin-coated magnetic beads (Dynabeads®; Life Technologies) and NeutrAvidin-coated wells in the final panning round. For competitive pans, an additional incubation of target-bound phage with 2.5 µg of non-biotinylated target was performed for 24 hours at room temp before elution. Phage ELISAs were conducted as described previously [ 19 ], and positive clones sent for sequencing. Affimer Production Affimer sequences were cloned into pET11a using the NheI and NotI sites. SH2-binding Affimers were produced in BL21 STAR™ (DE3) E. coli (C601003, Life Technologies, Invitrogen) and affinity purified using Ni-NTA resin as previously described [ 19 ]. For HA-tagged Affimers, Affimer sequences were subcloned into kanamycin-resistant pET-lectra vectors with C-terminal HA, 8xHis-tag sequences and produced in BL21 Star™ (DE3) E.coli cells in 24 well plates. Briefly, 200 µl starter cultures were grown at 37°C, 1050 rpm in a 96-well plate for 6–8 h in LB broth kanamycin (50 µg/ml) + 1% glucose. Cultures were used to inoculate 3ml of LB broth kanamycin in round bottom 24-well plates and grown at 37°C, 1050 rpm until OD600 reached ca. 0.8. Protein expression was induced with 0.5 mM IPTG and cultures were left overnight at 22°C, 1050 rpm. Affimer proteins were purified from clarified lysates using His Mag Sepharose™ Ni beads on a KingFisher Flex™ robotic platform, as for SH2-domains with the exclusion of TCEP from the elution buffer. Samples were flash frozen in liquid nitrogen and stored in aliquots at -80°C. Microarray Protein microarrays were conducted using HA-tagged Affimer reagents and BAP-tagged SH2 domain proteins. SH2 domain protein samples were diluted to 70 µM in PBS containing 20% glycerol and 10 µl samples added to wells in a 384-well microarray plate (Genetix). Proteins were spotted onto the surface of streptavidin-coated 3D-functionalized glass slides (PolyAn), using an ArrayJet Marathon™ non-contact printer. The system buffer contained 47% glycerol, 0.06% Triton™ X-100 (Sigma-Aldrich),0.04% ProClin™ 200 (Sigma-Aldrich) in ddH2O. Each protein spot consisted of 100 ρl solution, with a typical spot size of 200 µm. Proteins were left to dry onto the surface overnight, in a controlled environment of 18–19°C and 50–55% humidity (using the ArrayJet JetMosphere™ system). Slides were scanned at 532 nm using a GenePix® 4300A scanner (Molecular Devices) to visualise and analyse the printed protein spots for any drying artefacts. Slides were incubated with Blocking Buffer 1 (0.1 M Tris-HCl; 50 mM ethanolamine; 0.05% Tween-20, pH 9.0; 140 µl/well) for 15 min at room temperature. Wells were washed x3 with PBST and blocked additionally with Blocking Buffer 2 (2X Casein Blocking Buffer (Sigma-Aldrich); 0.1 M Tris-HCl, pH 8.5; 140 µl/well) for 30 min at room temperature. Arrays were then incubated with 5 µg/ml Affimer in Blocking Buffer 2 (70 µl/well) for 1 h at room temperature, followed by 3x PBST washes. Bound Affimer was detected using an anti-HA-tag AlexaFluorTM 647 conjugated antibody (1:1,000; Thermo Fisher 26183-A647 diluted in Blocking Buffer 2 (70 µl/well), for 1 h at room temperature in the dark. Negative control miniarrays were included on each slide; these controls were incubated with Blocking Buffer 2 and HA-tag antibody only. Slides were washed 3 times with PBST, once with PBS and finally with ddH2O before centrifugation at 200 x g for 5 min to dry. Slides were scanned at 635 nm using a GenePix® 4300A scanner to detect bound HA-tag antibody. Images were analysed using image analysis software GenePix® Pro 7, which automatically detected spots and identified proteins according to the print layout. The local background signal surrounding each spot was also read to enable background correction for each spot. Each miniarray was analysed separately, with the mean fluorescence at 635 nm after subtraction of background fluorescence (F635 – B635) calculated for each SH2 target from the five replicate spots. For analysis of Affimer binding specificities, the F365 – B635 calculated for each SH2 protein spot against that Affimer clone was averaged over three 50 experimental repeats. The Affimer was considered to be a positive hit if the signal for the intended target was ≥ 50x that of the signal for the buffer-only control spot. Cross-reactions to other targets were deemed significant if the signal totalled ≥ 10% of the intended target signal. Purified protein ELISA Purified protein ELISA were performed to test binding of HA-tagged Affimer proteins to their BAP-tagged SH2 target. Wells of Nunc-Immuno™ Maxisorp™ F96 plates were incubated with 50 µl of 5 µg/ml streptavidin (Molecular Probes® Life Technologies) in PBS at 4°C overnight. Plates were blocked with Blocking Buffer overnight at 37°C, washed with PBST, and 50 µl of 10 µg/ml SH2 protein in Blocking Buffer added per well. For streptavidin only controls, 50 µl of Blocking Buffer only was added. SH2s were incubated in the wells for 2 h at room temperature, followed by 1 x wash with PBST and incubation with 50 µl of 10 µg/ml Affimer protein in Blocking Buffer, for 1 h at room temperature. Each Affimer was tested against both SH2- containing and streptavidin-only wells. Wells were washed with PBST and incubated with 50 µl HA-tag antibody (1:20,000 ,Abcam, ab119703) in Blocking Buffer, for 1 h at room temperature. After 1 x wash with PBST, wells were incubated with 50 µl anti-mouse-HRP antibody (1:10,000; Abcam, ab6789) in Blocking Buffer for 1 h at room temperature. Plates were washed x 6 with PBST and HRP was detected using SeramunBlau® fast TMB (Seramun Diagnostica GmbH). Absorbance at 620 nm was read after 3 min and 10 min, before the reaction was stopped with 1 M H 2 SO 4 and the absorbance read again at 450 nm. Cell culture U-2 OS, HEK293 and HeLa cell lines (ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum and 100U/mL penicillin-streptomycin at 37 o C in 5% CO 2 . The identity of all cell lines was verified by STR and all cell lines were mycoplasma negative. Plasmid transfections Affimer DNA was subcloned from pBSTG into pCMV6-tGFP (Origene) using the Affimer-GFP forward and reverse primers. For reverse transfection with 50ng of Affimer DNA using Lipofectamine 2000 (100nl; Invitrogen; HEK293 and U-2 OS cells) or 100ng of Affimer DNA using X-Treme Gene 9 (300nl; Roche; HeLa cells) in 20 uL Opti-MEM were incubated in 96 well Viewpoint plates (PerkinElmer) for 20 mins. 80 uL of cell suspensions were then added (1x10 4 cells/well for HEK293 and U-2 OS cells, and 5 x 10 3 cells/well for HeLa cells). pERK Translocation Assay pERK nuclear translocation was assessed as previously described [ 29 ]. Briefly, cells transiently transfected with GFP-tagged Affimer were starved for 1h in serum-free media and stimulated with 25 ng/ml EGF for 5 mins. Cells were rinsed in DPBS and fixed in 4% paraformaldehyde (PFA) for 15 min. Cells were then permeabilised with ice-cold methanol for 10 min at -20 o C and rinsed with PBS before blocking in 1% milk for 10 mins prior to incubation with anti-pERK antibody (1:100; Cell Signalling Technology 4370) in 1% milk for 1 h at room temperature. Cells were washed 3 times in PBS and incubated with Alexa-Flour 568 (1:1000; Molecular Probes, Invitrogen) and Hoechst 33342 (1:1000; Molecular Probes, Invitrogen) in 1% milk for 1 h at room temperature. Cells were washed 3 times in PBS and stored at 4 o C until imaging. Plates were imaged using ImageXpress® PICO automated cell imaging system (Molecular Devices) and analysed using MetaXpress® High-Content Image Acquisition and Analysis software (Molecular Devices). Fluorescence Anisotropy Fluorescence anisotropy (FA) assays were performed on Grb2-SH2 Affimers. All Affimer and Grb2 SH2 samples were dialysed into 50 mM Tris, 100mM NaCl, pH7.4 prior to use. Assays were set up in 96 well plates and analysed using a Tecan Spark™ 10M microplate reader. 20 µM Affimer solutions were set up in triplicate and sequentially diluted by a factor of 2 across 12 wells. A fluorescein isothiocyanate-labelled phosphopeptide (FYp; FITC-GABA-S-pY-V-N-V-Q) was added to these wells to a final concentration of 20 nM. Grb2 SH2 protein was added to wells to a final concentration of 0.25 µM and the anisotropy measured in each well. Polarisation values for each Affimer concentration were plotted using a logarithmic scale (log10) for the concentration values, and the resultant sigmoidal curve fitted using the logistic function on Origin 9.1 software. From this fit, half maximal inhibitory values (IC50) values were calculated automatically by Origin. Surface Plasmon Resonance Full-length Grb2 protein was expressed from a pET28a vector using the same method as SH2 domain expression. The protein also contained an N-terminal His tag and no BAP tag. All proteins used in Surface Plasmon Resonance (SPR) were further purified using S.E.C, which also functioned as a method to separate the Grb2 monomer from the dimer. Only monomeric fractions were used in SPR. Grb2 was diluted to 5 µg/ml in 10 mM Sodium Acetate, pH 5.6 and immobilised onto Amine-coupling chips (sensor chip CM5, GE Healthcare). Affimer concentrations of 6.25 nM – 400 nM in 10 mM Sodium Acetate, pH 5.6 were flowed over the immobilised Grb2 at a flow rate of 80 µl/min for 1–3 min in succession and binding was measured. A 1M NaCl wash was used for chip regeneration between measurements. Binding curves were fitted using BIAevaluation 3.2 software and K D values calculated from these. An activated flow cell containing no Grb2 that had been capped using ethanolamine was used as the blank. Protein extraction, immunoprecipitation and immunoblotting Protein extraction, immunoprecipitation and immunoblotting were as previously described [ 50 ]. Briefly, cells were washed with ice-cold PBS and lysed in Mammalian Lysis Buffer (50 mM Tris; 150 mM NaCl; 1% (v/v) Nonidet P-40 (Sigma); pH 7.4) supplemented with HALT protease inhibitor cocktail and phosphatase inhibitor 2 (SigmaAldrich), for 30 min on ice, followed by centrifugation at 10,000 x g for 10 min at 4°C. Protein concentrations were measured by BCA assay, as per manufacturer’s instructions (ThermoFisher). For immunoprecipitation mammalian cells lysates, clarified lysates of His-tagged Affimer proteins produced in BL21 StarTM (DE3) E coli ., His-Tag Dynabeads (ThermoFisher) and the Kingfisher Flex (ThermoFisher) were utilised. Dynabeads were incubated with 80 µl clarified lysate in 1x blocking buffer (SigmaAldrich) in wash buffer (100 mM Sodium-phosphate, pH 8.0, 600 mM NaCl, 0.02% Tween-20) for 10 min, and rinsed with wash buffer. Beads were then incubated with 500 µg mammalian cell lysate for 90 mins at room temperature. Following three washes, proteins were eluted by incubation in His elution buffer (300 mM Imidazole, 50 mM Sodium phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20) for 10 min. Immunoprecipitants were heated in 4xSDS-PAGE Sample Buffer (8% (w/v) SDS; 0.2 M Tris-HCl (pH 7); 20% glycerol; 1% bromophenol blue; 20% β-mercaptoethanol) and run on a 15% SDS-PAGE gels before transfer to nitrocellulose membrane using the BioRad Transblot Turbo. Membranes were then blocked in 5% milk in TBS-T before overnight incubation at 4°C with rabbit Grb2 (1:5,000, Abcam ab32037), or rabbit anti-6xHisTag-HRP (1:10,000 for 1hr at room temperature, Abcam, ab1187). Membranes were rinsed three times with TBS-T before 1 hr incubation at room temperature with goat-anti-rabbit HRP (Abcam, ab97051) if required, followed by three more TBS-T rinses, and development using Immunoblot Forte Western HRP (Millipore), according to the manufacturer’s instructions. Blots were imaged using an Amersham™ Imager 600 (GE Healthcare, Chicago, IL). Statistical analysis Statistical analyses were carried out in GraphPad Prism 8.00 software (GraphPad Software, La Jolla, CA), with robust Z scores calculated in Microsoft Excel (Redmond, WA) as per the formulae in Birmingham et al. (2009) [ 51 ]. Statistical assumptions of equal variance for one-way ANOVA were tested with Brown-Forsythe tests. Fluorescent anisotropy data was plotted in Origin 9.1 software (OriginLab Corporation, Northampton, MA) and curves fitted with the logistical function. Declarations Acknowledgments – This work was supported by BB/J014443/1, BB/M011151/1, and MR/P019188/1. We gratefully acknowledge Dr Iain Manfield and funding from the MRC Mid-range equipment call for purchase of the Biacore 1K+ (MC_PC_MR/X013227/1). Author Contributions – DCT, MJ and MJM conceived the experimental plan. SJH, GJB, HLM, AAT, CT, EF, GR and NG conducted experimental work. All authors performed data analysis and critically reviewed and approved the manuscript. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Declaration of Interests MJ is an employee of Avacta Life Sciences and holds shares in the company, the Affimer technology is licensed to Avacta Life Sciences by the University of Leeds. The royalties from the license are managed by ULIP at the University of Leeds and disseminated to the inventors. References Kraskouskaya, D., et al., Progress towards the development of SH2 domain inhibitors. Chemical Society Reviews, 2013. 42(8): p. 3337–3370. Campbell, S.J. and R.M. Jackson, Diversity in the SH2 domain family phosphotyrosyl peptide binding site. Protein Engineering, 2003. 16(3): p. 217–227. Liu, B.A., et al., The Human and Mouse Complement of SH2 Domain Proteins—Establishing the Boundaries of Phosphotyrosine Signaling. 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Usta, D., et al., A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells. Mol Cancer Ther, 2020. 19(8): p. 1736–1750. Manning, B.D. and A. Toker, AKT/PKB Signaling: Navigating the Network. Cell, 2017. 169(3): p. 381–405. Zhang, X., et al., Structure of lipid kinase p110β/p85β elucidates an unusual SH2-domain-mediated inhibitory mechanism. Mol Cell, 2011. 41(5): p. 567–78. Hofmann, B.T. and M. Jücker, Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain. Cell Signal, 2012. 24(10): p. 1950–4. Gram, H., et al., Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display. European Journal of Biochemistry, 1997. 246(3): p. 633–7. Quartararo, J.S., P. Wu, and J.A. Kritzer, Peptide bicycles that inhibit the Grb2 SH2 domain. Chembiochem, 2012. 13(10): p. 1490–6. Furet, P., et al., Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 1. Bioorganic & Medicinal Chemistry Letters, 2000. 10(20): p. 2337–2341. Hart, C.P., et al., Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries. Cell Signal, 1999. 11(6): p. 453–64. Kessels, H.W., A.C. Ward, and T.N. Schumacher, Specificity and affinity motifs for Grb2 SH2-ligand interactions. Proc Natl Acad Sci U S A, 2002. 99(13): p. 8524–9. Müller, K., et al., Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting. J Biol Chem, 1996. 271(28): p. 16500–5. Imhof, D., et al., Sequence specificity of SHP-1 and SHP-2 Src homology 2 domains. Critical roles of residues beyond the pY + 3 position. Journal of Biological Chemistry, 2006 281(29): p. 20271–20282. Martin, H.L., et al., Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition. Cell Rep, 2023. 42(10): p. 113184. Birmingham, A. et al. Statistical methods for analysis of high-throughput RNA interference screens . Nat Methods 6, 569–575, (2009). Additional Declarations Competing interest reported. MJ is an employee of Avacta Life Sciences and holds shares in the company, the Affimer technology is licensed to Avacta Life Sciences by the University of Leeds. The royalties from the license are managed by ULIP at the University of Leeds and disseminated to the inventors DCT and MJM. All other authors do not have a competing interest. Supplementary Files SupplementaryData.docx Cite Share Download PDF Status: Published Journal Publication published 16 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 21 Mar, 2024 Reviews received at journal 15 Mar, 2024 Reviewers agreed at journal 07 Mar, 2024 Reviewers agreed at journal 04 Mar, 2024 Reviewers invited by journal 01 Mar, 2024 Editor assigned by journal 01 Mar, 2024 Editor invited by journal 01 Mar, 2024 Submission checks completed at journal 01 Mar, 2024 First submitted to journal 15 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3959018","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":276353222,"identity":"62259c06-f1a3-49a9-92f7-bd0a63350c0a","order_by":0,"name":"Sophie J. 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The heatmap displays the mean background corrected fluorescent signal at 635 nm for each Affimer against each SH2 domain, normalised to the buffer only control, as shown in the scale bar. N=3 independent experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3959018/v1/b2d4e9f39c65afbcef2b7389.png"},{"id":52036850,"identity":"05d01756-6902-455e-882f-813f2946792c","added_by":"auto","created_at":"2024-03-05 17:13:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of SH2-binding Affimers in the pERK translocation assay.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e HEK293 cells reverse-transfected with either SH2-binding Affimers or control Affimers (green triangles; Alanine - negative control; K6 - positive control) plasmids were starved and stimulated with EGF for 5 minutes. After staining and acquisition, images were analysed for pERK nuclear intensity in GFP positive cells about the fluorescence threshold of 400. To compare ERK phosphorylation levels between all wells on each plate the robust z-score was used, calculated as the (pERK signal of the individual well - median pERK signal of the whole plate)/ median absolute deviation of the pERK signal of the whole plate. A well returning a mean absolute robust z-score of \u0026gt; 3 was considered a hit. Cells highlighted in red reduced pERK nuclear translocation whilst those highlighted in blue increased pERK nuclear. \u003cstrong\u003eb)\u003c/strong\u003e SH2-binding Affimers that reduced pERK nuclear intensity were validated by repeated experiments and the pERK signal normalised to the Alanine Affimer control. Data is expressed as mean ± SEM, n=3 independent experiments. One-Way ANOVA with Dunnett’s post-hoc test compared to Alanine Affimer *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005, **\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3959018/v1/2ad646687c9ced054a336cde.png"},{"id":52035988,"identity":"3a33fc3e-9a27-4645-b017-521cb595f37f","added_by":"auto","created_at":"2024-03-05 17:05:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1344778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrb2 SH2-binding Affimers show competitive inhibition of the Grb2 SH2 and immunoprecipitation of endogenous Grb2 from cell lysate. a) \u003c/strong\u003eFluorescence polarisation (FP) was used as a measure of binding between the Grb2 SH2 and a FITC-labelled phosphopeptide ligand (FYp). A Grb2 SH2-FYp control binding curve was also read on each plate ([pY] = 20nM). Serial dilutions of Affimer were set up in triplicate and the FP measured in each well ([FYp] = 20nM; [Grb2 SH2] = 0.25µM). \u003cstrong\u003eb) \u003c/strong\u003eImmunoprecipitation of endogenous Grb2 from HEK293 cell lysates by Grb2 SH2- binding Affimers A4 and F1. (dotted lines indicate the removal of non-relevant lanes from membrane, see Supplementary Figure 1 for full membranes) \u003cstrong\u003ec)\u003c/strong\u003e Correlation of GFP intensity versus pERK nuclear intensity demonstrating the dose-dependent effects of Grb2 SH2- binding Affimers A4 and F1. \u003cstrong\u003ed)\u003c/strong\u003e Representative images of Grb2 SH2- binding Affimers A4 and F1 in the pERK nuclear translocation assay are shown. Con. – Control Affimer. Data shown is mean ± SEM. n = 3 independent experiments, IC\u003csub\u003e50\u003c/sub\u003es calculated using OriginPro 9.1.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3959018/v1/319868c32198d29c6ae814f6.png"},{"id":52036851,"identity":"73b942fd-1d59-48d5-a8d9-4f9e570fe7d9","added_by":"auto","created_at":"2024-03-05 17:13:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":850874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrb2 SH2-binding Affimers immunoprecipitate endogenous Grb2 from cancer cell lysates and inhibit pERK nuclear translocation. \u003c/strong\u003eImmunoprecipitation of endogenous Grb2 from U-2 OS (\u003cstrong\u003ea)\u003c/strong\u003e) and HeLa (\u003cstrong\u003eb)\u003c/strong\u003e) cell lysates by Grb2 SH2-binding Affimers A4 and F1. (dotted lines indicate the removal of non-relevant lanes from membrane, see Supplementary Figure 1 for full membranes) Grb2 SH2- binding Affimer F1 reduces pERK nuclear translocation when compared to the non-targeting alanine Affimer in both U-2 OS (\u003cstrong\u003ec)\u003c/strong\u003e) and HeLa (\u003cstrong\u003ed)\u003c/strong\u003e) cells. Representative images of Grb2 SH2- binding Affimers A4 and F1 in the pERK nuclear translocation assay are shown in \u003cstrong\u003ee)\u003c/strong\u003e and \u003cstrong\u003ef)\u003c/strong\u003e. Green – Grb-2 binding Affimers, Red – pERK, Blue – Hoechst. Con. – Control Affimer, Ala – Alanine Affimer. Data shown is mean ± SEM. n = 3 independent experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3959018/v1/12244e8bab390029e9f9a58c.png"},{"id":69275001,"identity":"034c4511-91d4-4ffa-8382-b65a9f14de4d","added_by":"auto","created_at":"2024-11-18 16:43:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3453275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3959018/v1/6e28d3b1-1b51-47d9-8c87-3110634352e2.pdf"},{"id":52035991,"identity":"9930d3a8-e4f0-4a2d-b5d6-a51130d482bd","added_by":"auto","created_at":"2024-03-05 17:05:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":357458,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-3959018/v1/0978985dcb25c0a67f816146.docx"}],"financialInterests":"Competing interest reported. MJ is an employee of Avacta Life Sciences and holds shares in the company, the Affimer technology is licensed to Avacta Life Sciences by the University of Leeds. The royalties from the license are managed by ULIP at the University of Leeds and disseminated to the inventors DCT and MJM. All other authors do not have a competing interest.","formattedTitle":"High-Throughput profiling of SH2 domains using Affimer reagents: unravelling protein interaction networks","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSrc Homology 2 (SH2) domains are phosphotyrosine (pTyr)-binding modules found in over 120 human proteins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 100 amino acids in length, the SH2 structure consists of a central anti-parallel β-sheet flanked on both sides by an α-helix [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These form two binding sites; a conserved pTyr binding pocket and a variable pocket that binds residues C-terminal to the pTyr. In total, a four to seven amino acid motif is bound by the domain [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. SH2s are usually found in conjunction with either catalytic domains or other binding domains, such as SH3s [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. SH2 domains constitute the largest class of pTyr-binding modules and are found in a wide variety of proteins including kinases, adaptor proteins, transcription factors and phosphatases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Through their binding of phosphorylated targets, they mediate protein-protein interactions (PPIs) and are involved in numerous intracellular signalling pathways. Many of these SH2-regulated interactions play key roles in processes that become dysregulated in disease, such as cell cycling, proliferation and apoptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. SH2 domains are therefore potential therapeutic targets for the treatment of several disorders including cancer, and study of their function could lead to a better understanding of numerous cancer signalling pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThough recognised as promising disease targets, there is still a lack of research reagents available for SH2 domains [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and the scarcity of SH2 inhibitors that are effective in intracellular assays has hampered study of SH2-mediated mechanisms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The highly conserved sequence and structure of SH2 domains raises significant challenges in generating specific binding reagents or inhibitors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As a result, the function of many SH2s has not yet been explored [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and it is widely acknowledged that protein-specific SH2 inhibitors would be highly valuable research tools that would enable the discovery of novel biology and new pharmaceutical targets in various disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The development of these could also lead to the detection of residues that may be exploitable for protein-specific drug design [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of intracellular protein function can be achieved through techniques such as gene knockout or RNA interference. However, these methods are impractical for studying domain-specific interactions as they result in the deletion of the entire protein [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In order to observe the cellular functions of SH2 domains, binding molecules acting at the protein level are needed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In recent years, the development of scaffold based binding proteins (SBPs) has aided targeted disruption of PPIs [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. SBPs have many advantageous features; ability to be expressed intracellularly, high solubility, high stability and lack of disulphide bonds, and include, amongst others, Designed Ankyrin Repeat Proteins (DARPins) and monobodies as well as the Affimers [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] utilised in this study. These easily producible proteins have been used for a range of biological and therapeutic applications to date [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Monobodies targeting the SH2 domain of Abl have been shown to bind Bcr-Abl allosterically and inhibit its function resulting in apoptosis of chronic myeloid leukaemia cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our group have previously identified Affimers that bind the SH2 domains of the Grb family of proteins and the individual SH2 domains of PI3K [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These studies demonstrate the potential for SBPs in modulation of SH2 mediated signalling events. However, to date, there has not been available a toolbox of SH2 modulating reagents to allow the roles of individual SH2 containing proteins, and even individual domains, to be delineated in desired cellular phenotypes.\u003c/p\u003e \u003cp\u003eHere we have developed a toolbox of Affimer reagents that bind 38 SH2 domains representing approximately a third of the known SH2-domain containing proteins. The specificity of the toolbox is comparable to those seen with ScFv screens [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and we have proved that the toolbox functions intracellularly by the identification of Grb2 as a major SH2 protein in the MAPK pathway using a phenotypic screen looking at phosphorylation and subsequent nuclear translocation of ERK. These specific Grb2-binding Affimers were shown to have nanomolar affinities and IC\u003csub\u003e50\u003c/sub\u003e values demonstrating the utility of the toolbox.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHigh-throughput screen of SH2 domains to identify Affimer binders\u003c/h2\u003e \u003cp\u003eBuilding on our previously published work identifying Affimers that specifically bound the SH2 domains of the Grb family of proteins, p85 and p55 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] we sought to generate a toolbox of reagents to dissect the roles of SH2 domain/proteins intracellularly and test the application of such reagents in a medium throughput screen. To achieve this an additional 32 SH2 domains encompassing the main molecular functions of SH2 domain containing proteins (excluding those associated with RAS regulation and ubiquitination) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] were selected. These 32 SH2 domains were then expressed and purified in a small-scale high throughput manner utilising 3mL cultures and the KingFisher Flex yielding between 16\u0026ndash;173 \u0026micro;g of protein. These proteins were then used for isolation of Affimers from our phage library using a competitive approach [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Three panning rounds were sufficient to yield substantial amplification for 18 of the targets whilst a fourth panning round increased this to 27 targets giving an 84% success rate at this stage. No amplification was achieved for Ptpn11-N, Src2, Syk-C, Stat2 and Yes, possibly owing to poor protein quality/quantity or low levels of biotinylation. Target binding for 24\u0026ndash;48 clones for the 27 successful targets was assessed by phage ELISA and successful binders sent for sequencing to identify unique clones, yielding 621 unique clones. The range of clones per target varied from 1\u0026ndash;48 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of phage display screening and isolation of Affimer clones to BAP-tagged SH2 domains.\u003c/b\u003e Final hit rates and unique clones isolated for each successfully screened SH2 domain. Targets for which positive hit criteria was lowered to \u0026ge;\u0026thinsp;3x that of the negative control (compared to the standard criteria of \u0026ge;\u0026thinsp;10x that of the negative control) are highlighted in italics. For SH2 domains screened twice, both hit rates are shown in column 2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSH2 target\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhage ELISA hit rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber sequenced\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnique clones\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAbl1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAbl2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBmx\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCrk\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFyn\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrb2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrb7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73 / 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrb10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42 / 13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrb14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92 / 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLck\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLyn\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNck1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNck2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep85α-C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep85α-N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep85β-C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep85β-N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep55γ-C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep55γ-N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePLCγ1-T\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 / 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePLCγ1-N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePLCγ2-T\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePLCγ2-N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48 / 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShip1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52 / 50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShip2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 / 25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSrc1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStat1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e48\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e14\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStat3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStat4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStat5a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e29\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStat5b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStat6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSyk-N\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTec\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTns1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e38\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e48\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVav1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs SH2 domains share high structural homology [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], a microarray approach was used to determine the specificity of these binders for their target domain in a rapid fashion. BAP-tagged SH2 domains were printed onto streptavidin coated slides, five spots for each SH2 domain, 10 buffer spots per array and 14 arrays per slide. Three SH2 domains, Nck1, Stat2 and Tec, were excluded from the microarray as the detection antibody bound to these proteins in optimisation experiments. The six SH2 domains we had previously targeted [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were also included on the microarrays, giving a total of 35 SH2 domains. A maximum of five clones per SH2 domain were then tested for specificity (clones were selected based on their sequence and signal in the phage ELISA). HA-tagged Affimers (5 \u0026micro;g/ml) were applied to microarrays and detected with an anti-HA antibody (1 \u0026micro;g/ml). Of the 162 Affimers tested, 54 showed no binding at the cutoff of 50x the signal from buffer only spots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis was in contrast to the phage ELISAs used in Affimer identification, however this appeared to be target dependent with four targets that showed no binding (She, Tns1, p85α-C and p85β-C) that was possibly the result of protein denaturation [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Re-testing these non-binding Affimers in an ELISA format only identified 14 that bound their targets, however these included all Affimers targeting p85α-C suggesting this target had indeed become denatured on the microarray. From the remaining 108 Affimers that showed binding in the microarray, 51 showed specificity for their respective target (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Affimer reagents were deemed specific if off-target interactions were \u0026le;\u0026thinsp;10% of the signal shown for the intended target, in accordance with previous work on SH2 domain-binding antibody fragments [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In total, specific Affimers were found for 22 SH2 domains giving a specific hit rate of 63% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eTarget-specific Affimer clones as determined by SH2 protein microarray.\u003c/b\u003e Table summarising specific Affimer clones for SH2 targets as identified by protein microarray. Clones were deemed specific if off-target interactions showed a signal\u0026thinsp;\u0026lt;\u0026thinsp;10% of that of the intended target.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget​\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of specific clones​\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbl1​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbl2​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBmx​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrk​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFyn​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrb2​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrb10​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrb14​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLyn​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP55γ-C​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP85α-N​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP85β-N​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP55γ-N​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLCγ1-N​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLCγ2-T​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLCγ2-N​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShip1​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShip2​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStat3​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStat4​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStat6​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyk-N​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2​\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular pathway screening\u003c/h2\u003e \u003cp\u003eHaving identified a toolbox of SH2-binding Affimers with good target specificity, we investigated their application towards understanding the roles of SH2 domain containing proteins in cell signalling. For this we used an assay from our previous work on the modulation of Ras with Affimers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and examined the nuclear translocation of phosphorylated extracellular signal-regulated kinase (pERK) with high content imaging as a measure of EGFR signalling. By testing our toolbox of SH2-binding Affimers with this assay we hoped to identify those SH2 domain containing proteins with roles in this pathway as a proof of principle for the utility of the toolbox, in particular we anticipated that we should be able to identify Grb2 owing to its well characterised role with this pathway [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To achieve this 119 of the 162 Affimers used in the microarray were subcloned into the mammalian expression vector pCMV6-tGFP. Our previous pERK nuclear translocation assay was adapted to a screening format of four 96 well-plates with each plate featuring a maximum of 30 Affimers to 62 negative controls (a non-targeting Affimer) and four positive controls of a Ras-inhibiting Affimer K6. HEK293 cells were reverse-transfected with these constructs and pERK nuclear translocation assessed 48 hr later. The assay quality of the screen was assessed by robust Z\u0026rsquo; factor analysis which yielded a value of 0.52 indicating the screen had a difference of 12 standard deviations between the positive and negative controls showing that it was likely to pick up a number of hits. The screen was repeated in triplicate and 18 hits identified as those Affimers with robust Z scores of less than \u0026minus;\u0026thinsp;3, identifying SH2 domains involved in positive EGFR signalling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Intriguingly 3 Affimers (Lck A7, Lyn A2, p85αN C6) increased pERK nuclear translocation as indicated by robust Z scores of greater than +\u0026thinsp;3. The 18 hits were then validated by individual assessment of pERK translocation and all were confirmed as hits (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) including 12 Affimers targeting Grb2. It was unsurprising that Grb2-binding Affimers were the major hits from this screen as this is the predominant SH2 domain containing protein in the EGFR signalling pathway [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], but this confirms the utility of this screening technique as it highlighted a known major player in this pathway. PI3K hits (p85αC A1 and F4, p85βN A3 and p55ɣC B5) were also seen and this confirms our previous work [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] as there is a degree of cross talk between the Akt pathway activated by PI3K upon EGF stimulation and the ERK pathway [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The two remaining hits Plcy2N A8 and Lyn A4 have all been linked to MAPK pathway signalling in other cell types with other stimulating agents, the majority of which show heterogeneity with the EGF receptor (ErbB1) [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These signalling events are not the major pathway for ERK phosphorylation and this is reflected in the relatively small reductions in pERK compared to those seen with Grb2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Thus, we have demonstrated the utility of using binding proteins in a high-throughput screen and that the SH2 Affimer toolbox functions \u003cem\u003ein vitro\u003c/em\u003e to identify SH2 domains with both major and minor roles in MAPK signalling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCharacterisation of Grb2 Affimers\u003c/h2\u003e \u003cp\u003eHaving identified Grb2 Affimers has the major hit from the pERK screen the four Affimers that had shown specificity for Grb2 (as measured by microarray) were then characterised further. Initially their competitive inhibitory capabilities were quantified by fluorescence polarisation, as measured by the displacement of a fluorescently labelled SH2 phosphopeptide from the Grb2 SH2 domain, giving IC\u003csub\u003e50\u003c/sub\u003e values ranging from 270 nM (Affimer Grb2 F1) to 1.22 \u0026micro;M (Affimer Grb2 D6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The two Affimers (A4 and F1) showing nanomolar IC\u003csub\u003e50\u003c/sub\u003e values were taken forward for more detailed characterisation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSurface plasmon resonance showed the affinity of these Affimers to full length Grb2 to be low nanomolar (A4 K\u003csub\u003eD\u003c/sub\u003e = 11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 nM; F1 K\u003csub\u003eD\u003c/sub\u003e = 34.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.9 nM) comparable with the affinity of Grb2 for its intracellular targets [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], demonstrating these Affimers are able to compete \u003cem\u003ein vitro\u003c/em\u003e for binding of the SH2 domain of Grb2. \u003cem\u003eIn vitro\u003c/em\u003e binding was confirmed by the ability of these Affimers to immunoprecipitate Grb2 from HEK293 lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Next, we explored if these Affimers demonstrated a dose-response in terms of inhibition of pERK nuclear translocation by correlating GFP intensity with pERK nuclear intensity, as GFP intensity increased, i.e dose of Affimer, pERK nuclear intensity decreased and for both A4 and F1 the correlation was significant (Pearson correlation p\u0026thinsp;=\u0026thinsp;0.0003 A4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004 F1; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d). This demonstrates that the SH2 toolbox contains high affinity, specific Affimer binders that can block SH2 function in normal cells in a dose-dependent manner. To determine the wider relevance of these results we determined if they were applicable to cancer cells. The effects of these two Grb2 Affimers were explored in two cancer cell lines, U-2-OS and HeLa. Both A4 and F1 were able to immunoprecipate Grb2 from both HeLa and U-2-OS lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). However, only F1 was able to inhibit pERK nuclear translocation in both cell lines (One-way ANOVA with Dunnett\u0026rsquo;s post hoc test p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for U-2 OS and p\u0026thinsp;=\u0026thinsp;0.0237 for HeLa; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-f). These data are consistent with A4 being a marginal hit in the screen (z = -3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIsolating specific and potent SH2 inhibitors has proven a significant challenge in the past, to the extent that SH2 domains were deemed \u0026lsquo;undruggable\u0026rsquo; targets [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, the development of scaffold based binding reagents, SBPs, has allowed the specific targeting of interaction domains previously abandoned as disease targets. In this work, we have created a toolbox of SH2-binding Affimers to aid in the exploration of the roles of SH2 domain-containing proteins in cellular signalling and function and to determine the ability to use this type of reagents set in high-throughput phenotypic screens. To achieve this, we successfully isolated protein-specific Affimer binders to 22 SH2 domains. This success was not only due to the stringent phage display process used, but also the incorporation of an N-terminal BAP tag on the SH2 antigens. This allowed site-directed \u003cem\u003ein vivo\u003c/em\u003e biotinylation of the target protein for phage display screening, thus removing the need for chemical biotinylation, which has previously resulted in the coupling of a biotin molecule to free lysines in the SH2 domain binding site. This method also allows the presentation of the target protein in its native conformation, an advantage when isolating Affimers that will function in cell-based assays. The hit rate of 80% achieved is higher than previously reported hit rates from SH2 domain screening [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The specific hit rate of 63% is comparable to previous screens using ScFvs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and both these studies only targeted 20 SH2 domains. An Abl SH2-binding monobody isolated by Wojcik et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] showed cross-reactivity to three other SH2s in a protein microarray used at a tenth of the concentration of the Affimers in this study, indicating that the specificity of SH2-binding Affimers is favourable when compared with similar non-antibody reagents raised against SH2s. Additionally, this monobody could not distinguish between Abl1 and Abl2 unlike some of the Abl binding Affimers identified in this study.\u003c/p\u003e \u003cp\u003eWhilst the previous screens identified SH2 binders, no assessment of their function \u003cem\u003ein vitro\u003c/em\u003e in live cells was undertaken as these screens identified ScFvs that bound SH2 domains in lysates or fixed cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This was important for the utility of the SH2 binding Affimer toolbox. Shp2 SH2 binding monobodies have been shown to be functional in inhibiting ERK phosphorylation in HCC1171 lung cancer cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and our previous work [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] show SBPs function intracellularly. This intracellular functionality was utilised to screen the SH2 Affimer toolbox in a pERK translocation assay. Popular methods for investigating MAPK signalling include western blotting and SRE luciferase assays, which can be slow and labour intensive [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] so high-content imaging was used together with GFP-tagged SH2-binding Affimer constructs yielding a simple, time efficient and sensitive assay. This approach is easily modified to screen different endpoints, for example modulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by using AKT phosphorylation as the measurable endpoint [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], or any other phenotypic change that can be imaged or measured. The identification of 18 Affimers for 6 SH2 domains that reduced EGF-induced pERK translocation indicating they inhibited MAPK signalling demonstrates a hit rate of 22%. These included 12 Affimers isolated against the Grb2 SH2 domain which is not surprising giventhe canonical role of Grb2 in Ras-activated MAPK signalling [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Of the remaining six Affimers four were isolated against the C-terminal SH2 domains of PI3K subunits p55ɣ (B5), p85α (A1 and F4) and p85β (A3) in conjunction with our previous work showing Affimers binding the N-terminal SH2 of p85 increase AKT phosphorylation (pAKT) levels [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Stimulation of the PI3K pathway leads to phosphorylation and activation of its downstream effector AKT. Activated AKT has been shown to inhibit ERK phosphorylation via its interaction with RAF [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Both the N and C-terminal subunits are involved in inhibition of the catalytic p110 subunit [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This suggests a mechanism where Affimer binding of the PI3K regulatory subunit\u0026rsquo;s SH2 domain leads to increased AKT activity and subsequent inhibition of ERK phosphorylation.\u003c/p\u003e \u003cp\u003eCharacterisation of the specific Grb2 Affimers identified as inhibiting pERK translocation showed IC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.27\u0026ndash;1.22 \u0026micro;M, as measured by fluorescence polarisation, which is in line with the IC\u003csub\u003e50\u003c/sub\u003e values for Grb2 SH2-binding phosphopeptides [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The most potent Affimer, F1, had an IC\u003csub\u003e50\u003c/sub\u003e comparable to bicyclic peptide inhibitors of the Grb2 SH2 domain [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This demonstrates that the inhibitive ability of the Grb2 SH2 Affimers is equal to, or surpasses, previously developed Grb2 SH2 inhibitors. The nanomolar affinities of the Grb2 Affimers for Grb2 is in line with that of the antibody fragments that bind the Grb2 SH2 with K\u003csub\u003eD\u003c/sub\u003es in the nanomolar range [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the SHP2 SH2 domain binding monobodies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], as well as phosphopeptide binders [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Higher picomolar affinities have been achieved with Grb2 SH2 small molecule inhibitors that mimic the phosphorylated tyrosine residue in Grb2 SH2 binding partners [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In spite of their high affinity for Grb2 SH2, the small scFv antibody fragments lacked the ability to immunoprecipitate endogenous Grb2 from clarified lysate, in contrast to the Grb2-SH2 Affimers tested in this study that were able to pull out detectable levels of endogenous Grb2 from cell lysates from multiple cell lines. These results show the ability of the Affimers to bind low levels of the target, in the context of the whole protein rather than just the isolated SH2 domain. This has positive implications for their use in functional cell-based assays as we have shown with successful inhibition of EGF-stimulated MAPK signalling as measured by pERK translocation.\u003c/p\u003e \u003cp\u003eThe Grb2 SH2 domain binds its natural substrates via selective recognition of the binding motif pY-X-N-X [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], a motif that is mimicked by the phosphopeptide binders [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and the small molecule inhibitors [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The majority of the Grb2 SH2 Affimers share this binding motif (10/16) including Affimer F1. Interestingly Affimer A4 contains an alternative aromatic residue, tryptophan, and Affimer F5 does not contain this sequence, so this motif alone does not confer Affimer specificity for the Grb2 SH2 domain. Relating results to the specificity motifs in the variable regions of the strongest hit, Affimer F1, reveals the sequence of Y-V-N-V, as in previous work using phosphorylated peptide libraries the sequence pY-V-N-V to have the highest affinity for the Grb2 SH2 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The level of MAPK inhibition seen in this study was closely correlated with similarity to this sequence. The variable regions of A6 and H1, which failed to significantly reduce pERK, show little similarity to this sequence. This provides strong evidence that the effects seen in this assay are due to the binding of the Grb2 SH2 domain, rather than some unknown off-target effects. Importantly, Affimer reagents can utilize this motif to bind the Grb2 SH2 domain with high specificity without the need for the highly polar phosphorylated tyrosine residue, which can cause promiscuous binding [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion this study has demonstrated that Affimers can be isolated that bind SH2 domains in a protein-specific manner with high affinity. The specificity of Affimers for their target SH2 over highly homologous SH2 domains of other proteins and their ability to bind endogenous Grb2 is favourable when compared with previously isolated binding reagents [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Furthermore, Grb2 SH2-binding Affimers show the ability to inhibit target function. This, in conjunction with the ability of Affimers to fold correctly and bind targets in the cytoplasm, indicates that the SH2-targeting Affimer toolbox or an Affimer toolbox to other protein domains, will be useful for functional cell-based assays to determine the role of different protein domains in biology and disease. This may show the way for future development of proteome domain screening tools for functionally dissecting pathways and identifying key domains on proteins for targeted therapeutics.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSH2 domain production\u003c/h2\u003e \u003cp\u003eSH2 domains were produced as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly sequences encoded in kanamycin-resistant pET28 SacBAPvectors were purchased from the Pawson Lab (Samuel Lunenfeld Research Institute, Canada) and a biotin acceptor peptide (BAP) sequence was cloned into the vectors to give an N-terminal BAP-Histag-SH2 domain sequence. For production in Rosetta\u003csup\u003eTM\u003c/sup\u003e2 (DE3) cells (Novagen, Merk Millipore), overnight starter cultures were grown at 37\u0026deg;C, 230 rpm in TB medium supplemented with kanamycin (50 \u0026micro;g/ml), chloramphenicol (34 \u0026micro;g/ml), and 1% glucose. These were used to inoculate 3 ml cultures of TB kanamycin that were grown at 37\u0026deg;C, 230 rpm until OD600 reached ca. 1.5 and temperature was reduced to 18\u0026deg;C for 1 h before addition of 0.5 mM IPTG and cultures were grown overnight at 18\u0026deg;C, 230 rpm. His-tagged SH2 proteins were purified from clarified culture lysates on a KingFisher\u0026trade; Flex robotic platform (ThermoFisher) using His Mag Sepharose Ni beads (GE Healthcare), washed (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO4; 500 mM NaCl; 20 mM imidazole; pH 7.4) and eluted in 130 \u0026micro;l elution buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO4; 500 mM NaCl; 300 mM imidazole; 10% glycerol; pH 7.4). The elution buffer also contained 1 mM TCEP. Samples were flash frozen in liquid nitrogen and stored in aliquots at -80\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhage display and phage ELISA\u003c/h2\u003e \u003cp\u003ePhage display was completed over four panning rounds, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Streptavidin-coated wells were used for the first panning round, followed by Streptavidin-coated magnetic beads (Dynabeads\u0026reg;; Life Technologies) and NeutrAvidin-coated wells in the final panning round. For competitive pans, an additional incubation of target-bound phage with 2.5 \u0026micro;g of non-biotinylated target was performed for 24 hours at room temp before elution. Phage ELISAs were conducted as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and positive clones sent for sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAffimer Production\u003c/h2\u003e \u003cp\u003eAffimer sequences were cloned into pET11a using the NheI and NotI sites. SH2-binding Affimers were produced in BL21 STAR\u0026trade; (DE3) E. coli (C601003, Life Technologies, Invitrogen) and affinity purified using Ni-NTA resin as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For HA-tagged Affimers, Affimer sequences were subcloned into kanamycin-resistant pET-lectra vectors with C-terminal HA, 8xHis-tag sequences and produced in BL21 Star\u0026trade; (DE3) E.coli cells in 24 well plates. Briefly, 200 \u0026micro;l starter cultures were grown at 37\u0026deg;C, 1050 rpm in a 96-well plate for 6\u0026ndash;8 h in LB broth kanamycin (50 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;1% glucose. Cultures were used to inoculate 3ml of LB broth kanamycin in round bottom 24-well plates and grown at 37\u0026deg;C, 1050 rpm until OD600 reached ca. 0.8. Protein expression was induced with 0.5 mM IPTG and cultures were left overnight at 22\u0026deg;C, 1050 rpm. Affimer proteins were purified from clarified lysates using His Mag Sepharose\u0026trade; Ni beads on a KingFisher Flex\u0026trade; robotic platform, as for SH2-domains with the exclusion of TCEP from the elution buffer. Samples were flash frozen in liquid nitrogen and stored in aliquots at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray\u003c/h2\u003e \u003cp\u003eProtein microarrays were conducted using HA-tagged Affimer reagents and BAP-tagged SH2 domain proteins. SH2 domain protein samples were diluted to 70 \u0026micro;M in PBS containing 20% glycerol and 10 \u0026micro;l samples added to wells in a 384-well microarray plate (Genetix). Proteins were spotted onto the surface of streptavidin-coated 3D-functionalized glass slides (PolyAn), using an ArrayJet Marathon\u0026trade; non-contact printer. The system buffer contained 47% glycerol, 0.06% Triton\u0026trade; X-100 (Sigma-Aldrich),0.04% ProClin\u0026trade; 200 (Sigma-Aldrich) in ddH2O. Each protein spot consisted of 100 ρl solution, with a typical spot size of 200 \u0026micro;m. Proteins were left to dry onto the surface overnight, in a controlled environment of 18\u0026ndash;19\u0026deg;C and 50\u0026ndash;55% humidity (using the ArrayJet JetMosphere\u0026trade; system). Slides were scanned at 532 nm using a GenePix\u0026reg; 4300A scanner (Molecular Devices) to visualise and analyse the printed protein spots for any drying artefacts. Slides were incubated with Blocking Buffer 1 (0.1 M Tris-HCl; 50 mM ethanolamine; 0.05% Tween-20, pH 9.0; 140 \u0026micro;l/well) for 15 min at room temperature. Wells were washed x3 with PBST and blocked additionally with Blocking Buffer 2 (2X Casein Blocking Buffer (Sigma-Aldrich); 0.1 M Tris-HCl, pH 8.5; 140 \u0026micro;l/well) for 30 min at room temperature. Arrays were then incubated with 5 \u0026micro;g/ml Affimer in Blocking Buffer 2 (70 \u0026micro;l/well) for 1 h at room temperature, followed by 3x PBST washes. Bound Affimer was detected using an anti-HA-tag AlexaFluorTM 647 conjugated antibody (1:1,000; Thermo Fisher 26183-A647 diluted in Blocking Buffer 2 (70 \u0026micro;l/well), for 1 h at room temperature in the dark. Negative control miniarrays were included on each slide; these controls were incubated with Blocking Buffer 2 and HA-tag antibody only. Slides were washed 3 times with PBST, once with PBS and finally with ddH2O before centrifugation at 200 x g for 5 min to dry. Slides were scanned at 635 nm using a GenePix\u0026reg; 4300A scanner to detect bound HA-tag antibody. Images were analysed using image analysis software GenePix\u0026reg; Pro 7, which automatically detected spots and identified proteins according to the print layout. The local background signal surrounding each spot was also read to enable background correction for each spot. Each miniarray was analysed separately, with the mean fluorescence at 635 nm after subtraction of background fluorescence (F635 \u0026ndash; B635) calculated for each SH2 target from the five replicate spots. For analysis of Affimer binding specificities, the F365 \u0026ndash; B635 calculated for each SH2 protein spot against that Affimer clone was averaged over three 50 experimental repeats. The Affimer was considered to be a positive hit if the signal for the intended target was \u0026ge;\u0026thinsp;50x that of the signal for the buffer-only control spot. Cross-reactions to other targets were deemed significant if the signal totalled\u0026thinsp;\u0026ge;\u0026thinsp;10% of the intended target signal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePurified protein ELISA\u003c/h2\u003e \u003cp\u003ePurified protein ELISA were performed to test binding of HA-tagged Affimer proteins to their BAP-tagged SH2 target. Wells of Nunc-Immuno\u0026trade; Maxisorp\u0026trade; F96 plates were incubated with 50 \u0026micro;l of 5 \u0026micro;g/ml streptavidin (Molecular Probes\u0026reg; Life Technologies) in PBS at 4\u0026deg;C overnight. Plates were blocked with Blocking Buffer overnight at 37\u0026deg;C, washed with PBST, and 50 \u0026micro;l of 10 \u0026micro;g/ml SH2 protein in Blocking Buffer added per well. For streptavidin only controls, 50 \u0026micro;l of Blocking Buffer only was added. SH2s were incubated in the wells for 2 h at room temperature, followed by 1 x wash with PBST and incubation with 50 \u0026micro;l of 10 \u0026micro;g/ml Affimer protein in Blocking Buffer, for 1 h at room temperature. Each Affimer was tested against both SH2- containing and streptavidin-only wells. Wells were washed with PBST and incubated with 50 \u0026micro;l HA-tag antibody (1:20,000 ,Abcam, ab119703) in Blocking Buffer, for 1 h at room temperature. After 1 x wash with PBST, wells were incubated with 50 \u0026micro;l anti-mouse-HRP antibody (1:10,000; Abcam, ab6789) in Blocking Buffer for 1 h at room temperature. Plates were washed x 6 with PBST and HRP was detected using SeramunBlau\u0026reg; fast TMB (Seramun Diagnostica GmbH). Absorbance at 620 nm was read after 3 min and 10 min, before the reaction was stopped with 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and the absorbance read again at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eU-2 OS, HEK293 and HeLa cell lines (ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum and 100U/mL penicillin-streptomycin at 37\u003csup\u003eo\u003c/sup\u003eC in 5% CO\u003csub\u003e2\u003c/sub\u003e. The identity of all cell lines was verified by STR and all cell lines were mycoplasma negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid transfections\u003c/h2\u003e \u003cp\u003eAffimer DNA was subcloned from pBSTG into pCMV6-tGFP (Origene) using the Affimer-GFP forward and reverse primers. For reverse transfection with 50ng of Affimer DNA using Lipofectamine 2000 (100nl; Invitrogen; HEK293 and U-2 OS cells) or 100ng of Affimer DNA using X-Treme Gene 9 (300nl; Roche; HeLa cells) in 20 uL Opti-MEM were incubated in 96 well Viewpoint plates (PerkinElmer) for 20 mins. 80 uL of cell suspensions were then added (1x10\u003csup\u003e4\u003c/sup\u003ecells/well for HEK293 and U-2 OS cells, and 5 x 10\u003csup\u003e3\u003c/sup\u003e cells/well for HeLa cells).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003epERK Translocation Assay\u003c/h2\u003e \u003cp\u003epERK nuclear translocation was assessed as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, cells transiently transfected with GFP-tagged Affimer were starved for 1h in serum-free media and stimulated with 25 ng/ml EGF for 5 mins. Cells were rinsed in DPBS and fixed in 4% paraformaldehyde (PFA) for 15 min. Cells were then permeabilised with ice-cold methanol for 10 min at -20\u003csup\u003eo\u003c/sup\u003eC and rinsed with PBS before blocking in 1% milk for 10 mins prior to incubation with anti-pERK antibody (1:100; Cell Signalling Technology 4370) in 1% milk for 1 h at room temperature. Cells were washed 3 times in PBS and incubated with Alexa-Flour 568 (1:1000; Molecular Probes, Invitrogen) and Hoechst 33342 (1:1000; Molecular Probes, Invitrogen) in 1% milk for 1 h at room temperature. Cells were washed 3 times in PBS and stored at 4\u003csup\u003eo\u003c/sup\u003eC until imaging. Plates were imaged using ImageXpress\u0026reg; PICO automated cell imaging system (Molecular Devices) and analysed using MetaXpress\u0026reg; High-Content Image Acquisition and Analysis software (Molecular Devices).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence Anisotropy\u003c/h2\u003e \u003cp\u003eFluorescence anisotropy (FA) assays were performed on Grb2-SH2 Affimers. All Affimer and Grb2 SH2 samples were dialysed into 50 mM Tris, 100mM NaCl, pH7.4 prior to use. Assays were set up in 96 well plates and analysed using a Tecan Spark\u0026trade; 10M microplate reader. 20 \u0026micro;M Affimer solutions were set up in triplicate and sequentially diluted by a factor of 2 across 12 wells. A fluorescein isothiocyanate-labelled phosphopeptide (FYp; FITC-GABA-S-pY-V-N-V-Q) was added to these wells to a final concentration of 20 nM. Grb2 SH2 protein was added to wells to a final concentration of 0.25 \u0026micro;M and the anisotropy measured in each well. Polarisation values for each Affimer concentration were plotted using a logarithmic scale (log10) for the concentration values, and the resultant sigmoidal curve fitted using the logistic function on Origin 9.1 software. From this fit, half maximal inhibitory values (IC50) values were calculated automatically by Origin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSurface Plasmon Resonance\u003c/h2\u003e \u003cp\u003eFull-length Grb2 protein was expressed from a pET28a vector using the same method as SH2 domain expression. The protein also contained an N-terminal His tag and no BAP tag. All proteins used in Surface Plasmon Resonance (SPR) were further purified using S.E.C, which also functioned as a method to separate the Grb2 monomer from the dimer. Only monomeric fractions were used in SPR. Grb2 was diluted to 5 \u0026micro;g/ml in 10 mM Sodium Acetate, pH 5.6 and immobilised onto Amine-coupling chips (sensor chip CM5, GE Healthcare). Affimer concentrations of 6.25 nM \u0026ndash; 400 nM in 10 mM Sodium Acetate, pH 5.6 were flowed over the immobilised Grb2 at a flow rate of 80 \u0026micro;l/min for 1\u0026ndash;3 min in succession and binding was measured. A 1M NaCl wash was used for chip regeneration between measurements. Binding curves were fitted using BIAevaluation 3.2 software and \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values calculated from these. An activated flow cell containing no Grb2 that had been capped using ethanolamine was used as the blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction, immunoprecipitation and immunoblotting\u003c/h2\u003e \u003cp\u003eProtein extraction, immunoprecipitation and immunoblotting were as previously described [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Briefly, cells were washed with ice-cold PBS and lysed in Mammalian Lysis Buffer (50 mM Tris; 150 mM NaCl; 1% (v/v) Nonidet P-40 (Sigma); pH 7.4) supplemented with HALT protease inhibitor cocktail and phosphatase inhibitor 2 (SigmaAldrich), for 30 min on ice, followed by centrifugation at 10,000 x\u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. Protein concentrations were measured by BCA assay, as per manufacturer\u0026rsquo;s instructions (ThermoFisher).\u003c/p\u003e \u003cp\u003eFor immunoprecipitation mammalian cells lysates, clarified lysates of His-tagged Affimer proteins produced in BL21 StarTM (DE3) \u003cem\u003eE coli\u003c/em\u003e., His-Tag Dynabeads (ThermoFisher) and the Kingfisher Flex (ThermoFisher) were utilised. Dynabeads were incubated with 80 \u0026micro;l clarified lysate in 1x blocking buffer (SigmaAldrich) in wash buffer (100 mM Sodium-phosphate, pH 8.0, 600 mM NaCl, 0.02% Tween-20) for 10 min, and rinsed with wash buffer. Beads were then incubated with 500 \u0026micro;g mammalian cell lysate for 90 mins at room temperature. Following three washes, proteins were eluted by incubation in His elution buffer (300 mM Imidazole, 50 mM Sodium phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20) for 10 min. Immunoprecipitants were heated in 4xSDS-PAGE Sample Buffer (8% (w/v) SDS; 0.2 M Tris-HCl (pH 7); 20% glycerol; 1% bromophenol blue; 20% β-mercaptoethanol) and run on a 15% SDS-PAGE gels before transfer to nitrocellulose membrane using the BioRad Transblot Turbo. Membranes were then blocked in 5% milk in TBS-T before overnight incubation at 4\u0026deg;C with rabbit Grb2 (1:5,000, Abcam ab32037), or rabbit anti-6xHisTag-HRP (1:10,000 for 1hr at room temperature, Abcam, ab1187). Membranes were rinsed three times with TBS-T before 1 hr incubation at room temperature with goat-anti-rabbit HRP (Abcam, ab97051) if required, followed by three more TBS-T rinses, and development using Immunoblot Forte Western HRP (Millipore), according to the manufacturer\u0026rsquo;s instructions. Blots were imaged using an Amersham\u0026trade; Imager 600 (GE Healthcare, Chicago, IL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were carried out in GraphPad Prism 8.00 software (GraphPad Software, La Jolla, CA), with robust Z scores calculated in Microsoft Excel (Redmond, WA) as per the formulae in Birmingham et al. (2009) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Statistical assumptions of equal variance for one-way ANOVA were tested with Brown-Forsythe tests. Fluorescent anisotropy data was plotted in Origin 9.1 software (OriginLab Corporation, Northampton, MA) and curves fitted with the logistical function.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e \u0026ndash; This work was supported by BB/J014443/1, BB/M011151/1, and MR/P019188/1.\u0026nbsp;We gratefully acknowledge Dr Iain Manfield and funding from the MRC Mid-range equipment call for purchase of the Biacore 1K+ (MC_PC_MR/X013227/1). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions \u0026ndash;\u0026nbsp;\u003c/strong\u003eDCT, MJ and MJM conceived the experimental plan. SJH, GJB, HLM, AAT, CT, EF, GR and NG conducted experimental work. All authors performed data analysis and critically reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMJ is an employee of Avacta Life Sciences and holds shares in the company, the Affimer technology is licensed to Avacta Life Sciences by the University of Leeds. The royalties from the license are managed by ULIP at the University of Leeds and disseminated to the inventors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKraskouskaya, D., et al., Progress towards the development of SH2 domain inhibitors. Chemical Society Reviews, 2013. 42(8): p. 3337\u0026ndash;3370.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell, S.J. and R.M. Jackson, Diversity in the SH2 domain family phosphotyrosyl peptide binding site. Protein Engineering, 2003. 16(3): p. 217\u0026ndash;227.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, B.A., et al., The Human and Mouse Complement of SH2 Domain Proteins\u0026mdash;Establishing the Boundaries of Phosphotyrosine Signaling. Molecular Cell, 2006. 22(6): p. 851\u0026ndash;868.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachida, K. and B.J. Mayer, The SH2 domain: versatile signaling module and pharmaceutical target. Biochimica et Biophysica Acta 2005. 1747(1): p. 1\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePershad, K., et al., Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display. Protein Engineering, Design \u0026amp; Selection, 2010. 23(4): p. 279\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePawson, T., G.D. Gish, and P. Nash, SH2 domains, interaction modules and cellular wiring. Trends Cell Biol, 2001. 11(12): p. 504\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVidal, M., V. Gigoux, and C. Garbay, SH2 and SH3 domains as targets for anti-proliferative agents. Critical Reviews in Oncology/Hematology, 2001. 40(1): p. 175\u0026ndash;186.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaksman, G., S. Kumaran, and O. Lubman, SH2 domains: role, structure and implications for molecular medicine. Expert Rev Mol Med, 2004. 6(3): p. 1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorlacchi, P., et al., Targeting SH2 domains in breast cancer. 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Statistical methods for analysis of high-throughput RNA interference screens\u003c/em\u003e. Nat Methods 6, 569\u0026ndash;575, (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3959018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3959018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite SH2 domains, being pivotal in protein interactions linked to various diseases like cancer, we lack specific research tools for intracellular assays. Understanding SH2-mediated interactions and creating effective inhibitors requires tools which target individual protein domains. Affimer reagents exhibit promise, yet their potential against the extensive SH2 domain family remains largely unexplored. Our study aimed to bridge this gap by identifying Affimer reagents that selectively bind to 22 out of 41 SH2 domains. These reagents enabled a medium-throughput screening approach resembling siRNA studies, shedding light on their functionality. Notably, select Affimers demonstrated the ability to curtail the nuclear translocation of pERK, with Grb2 being a prominent target. Further analyses revealed that these Grb2-specific Affimer reagents displayed competitive inhibition with impressive metrics: IC50s ranging from 270.9 nM to 1.22 \u0026micro;M, together with low nanomolar binding affinities. Moreover, they exhibited the ability to pull down endogenous Grb2 from cell lysates, illustrating their efficacy in binding the Grb2 SH2 domain. This comprehensive assessment underscores the potential of Affimer reagents as domain-specific inhibitors. Their viability for medium/high-throughput phenotypic screening presents a promising avenue via which to identify and characterize potential drug targets within the SH2 domain family.\u003c/p\u003e","manuscriptTitle":"High-Throughput profiling of SH2 domains using Affimer reagents: unravelling protein interaction networks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 17:05:14","doi":"10.21203/rs.3.rs-3959018/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-21T06:08:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-15T05:48:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53907e4b-6fef-40b4-b374-16069402630e","date":"2024-03-07T16:18:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"cdec3e4a-74ab-4599-a8f2-6b08da3bab35","date":"2024-03-04T20:48:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-01T21:02:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-01T20:57:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-01T20:02:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-01T20:00:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-15T15:17:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"520f1936-90dd-475d-80a9-a5aaebeb6ad2","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29128302,"name":"Biological sciences/Biological techniques"},{"id":29128303,"name":"Biological sciences/Biological techniques/High throughput screening"},{"id":29128304,"name":"Biological sciences/Biological techniques/Imaging"},{"id":29128305,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":29128306,"name":"Biological sciences/Cell biology/Cellular imaging"}],"tags":[],"updatedAt":"2024-11-18T16:01:45+00:00","versionOfRecord":{"articleIdentity":"rs-3959018","link":"https://doi.org/10.1038/s41598-024-79357-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-16 15:57:27","publishedOnDateReadable":"November 16th, 2024"},"versionCreatedAt":"2024-03-05 17:05:14","video":"","vorDoi":"10.1038/s41598-024-79357-4","vorDoiUrl":"https://doi.org/10.1038/s41598-024-79357-4","workflowStages":[]},"version":"v1","identity":"rs-3959018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3959018","identity":"rs-3959018","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}