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Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins., Oncogenic RAS mutants remain difficult to target with small molecules. Here, the authors show that RAS-binding Affimer proteins inhibit RAS signaling while binding diverse regions on the RAS surface, suggesting the potential to use Affimers as tools to identify new binding pockets and pharmacophores.

Glucocorticoid receptor modulators (GRM) are the first-line treatment for many immune diseases, but unwanted side effects restrict chronic dosing. However, targeted delivery of a GRM payload via an immunology antibody-drug conjugate (iADC) may deliver significant efficacy at doses that do not lead to unwanted side effects. We initiated our α-TNF-GRM ADC project focusing on identifying the optimal payload and a linker that afforded stable attachment to both the payload and antibody, resulting in the identification of the synthetically accessible maleimide-Gly-Ala-Ala linker. DAR 4 purified ADCs were shown to be more efficacious in a mouse contact hypersensitivity model than the parent α-TNF antibody. Analysis of P1NP and corticosterone biomarkers showed there was a sufficient therapeutic window between efficacy and unwanted effects. In a chronic mouse arthritis model, α-TNF-GRM ADCs were more efficacious than both the parent α-TNF mAb and an isotype control bearing the same GRM payload.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA–plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents in molecular and cellular biology. For example, they have been used as inhibitors of protein-protein interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.Here, we describe methods that have been employed in isolating highly specific artificial binding proteins against a wide range of target proteins.

Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents in molecular and cellular biology. For example, they have been used as inhibitors of protein-protein interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.Here, we describe methods that have been employed in isolating highly specific artificial binding proteins against a wide range of target proteins.

Polyoctopamine (POct), an amine-functionalised non-conducting polymer, as the transducer layer in an electrochemical biosensor, is presented. This polymer offers versatile covalent coupling either through thiol linker conjugation, carboxyl or aldehyde functional groups without the requirement of pre- or post-surface activation. The colorectal cancer biomarker carcinoembryonic antigen (CEA) was selected as the target analyte, whilst an antibody and a synthetic binding protein, an Affimer, were used as distinct bioreceptors to demonstrate the versatility of polyoctopamine as a transducer polymer layer for oriented immobilisation of the bioreceptors. The electrodeposited polymer layer was characterised using cyclic voltammetry, electrochemical impedance spectroscopy, and on-sensor chemiluminescent blotting. The performance of optimised POct-based biosensors were tested in spiked human serum. Results showed that the electropolymerisation of octopamine on screen printed gold electrode generates a thin polymer film with low resistance. Close proximity of the immobilised bioreceptors to the transducer layer greatly enhanced the sensitivity detection. The sensitivity of the smaller monomeric bioreceptor (Affimer, 12.6 kDa) to detect CEA was comparable to the dimeric antibody (150 kDa) with limit of detection at 11.76 fM which is significantly lower than the basal clinical levels of 25 pM. However, the Affimer-based sensor had a narrower dynamic range compared to the immunosensor (1–100 fM vs. 1 fM – 100 nM, respectively). All electrochemical measurements were done in less than 5 min with small sample volumes (10 μl). Hence, polyoctopamine features a simple fabrication of impedimetric biosensors using amine-functionalisation technique, provides rapid response time with enhanced sensitivity and label-free detection.

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. We have identified two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signalling pathways with distinct isoform and mutant profiles. Affimer K6 is the first biologic to bind in the SI/SII pocket, whilst Affimer K3 is the first non-covalent inhibitor of the SII region, revealing a novel RAS conformer with a large, druggable SII/α3 pocket. This work demonstrates the potential of using biologics with small interface surfaces to select novel druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

Haematologica 106(4), 1616-1623 (2020). doi:10.3324/haematol.2019.239558, Published by Ferrata Storti Foundation, Pavia

The structure of Scytalidium thermophilum catalase in complex with its well known inhibitor 3-amino-1,2,4-triazole revealed that the inhibitor occupies a surface pocket at the end of the lateral channel. This pocket corresponds to the site of NADPH binding in mammalian catalases. Peroxide-independent phenolic substrate oxidation is likely to occur in a similar manner to NADPH oxidation., The catalase from Scytalidium thermophilum is a homotetramer containing a heme d in each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, similar to 32 angstrom from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Cu2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Cu2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Qmutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (similar to 60%WTECAO TPQ), I342F/E573Qhas almost no amine oxidase activity (similar to 4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+ -substrate reduced TPQ(TPQ(AmQ)) CutTPQsemiquinone (TPQ(SQ)) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQ(SQ) or destabilizes Cu+-TPQ(SQ): Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state: Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.

Antibodies have been indispensable tools in molecular biology, biochemistry and medical research. However, a number of issues surrounding validation, specificity and batch variation of commercially available antibodies have prompted research groups to develop novel non-antibody binding reagents. The ability to select highly specific monoclonal non-antibody binding proteins without the need for animals, the ease of production and the ability to site-directly label has enabled a wide variety of applications to be tested, including imaging. In this review, we discuss the success of a number of non-antibody reagents in imaging applications, including the recently reported Affimer.

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic &beta, cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant.

Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an "active" enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme-inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-β-lactamase and its inhibitor protein, such that target binding disrupts the enzyme-inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics.

Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to limit such threats, but plant viral diseases can be extremely challenging to diagnose. An ideal method for plant virus diagnosis would be a device which can be implemented easily in the field. Such devices require a binding reagent that is specific for the virus of interest. We chose to investigate the use of Affimer reagents, artificial binding proteins and a model plant virus Cowpea Mosaic virus (CPMV) empty virus like particles (eVLPs). CPMV-eVLP mimic the morphology of wild-type (WT) CPMV but lack any infectious genomic material and so do not have biocontainment issues. We have produced and purified an Affimer reagent selected for its ability to bind to CPMV-eVLP and have shown that the selected Affimer also specifically binds to WT CPMV. We have produced a 3.4 Å structure of WT CPMV bound to the Affimer using cryo-electron microscopy. Finally, we have shown that this Affimer is capable of reliably detecting the virus in crude extracts of CPMV-infected leaves and can therefore form the basis for the future development of diagnostic tests.

Fibroblast growth factor receptor 3 (FGFR3) is a transmembrane tyrosine kinase protein in the fibroblast growth factor receptor family, which plays a key role in many biological processes. Over-expression and activating mutations in FGFR3 are frequent in non-invasive bladder cancer, highlighting this protein as a potential biomarker for recurrent bladder cancer detection. Affimer reagents isolated against recombinant FGFR3 were assessed for their affinity using double-sandwich ELISA and SPR. Anti-FGFR3-14 and FGFR3-21 Affimer proteins showed strong binding to FGFR3 and were used for fabrication of impedimetric electrochemical biosensors. A decrease in impedance was observed when the sensors were exposed to increasing concentrations of FGFR3. The successful impedimetric biosensors were capable of detecting sub-pM to nM concentrations of recombinant FGFR3 protein in phosphate buffered saline as well as in synthetic urine.

This article presents a simple and highly reliable method for preparing PDMS microfluidic double emulsion devices that employs a single-step oxidative plasma treatment to both pattern the wettability of the microchannels and to bond the chips. As a key component of our strategy we use epoxy glue to define the required hydrophobic zones and then remove this after plasma treatment, but prior to bonding. This novel approach achieves surface modification and device sealing in a single process, which reduces chip preparation times to minutes and eliminates the need for unreliable coating processes. The second key element of our procedure is the maintenance of the patterned surfaces, where we demonstrate that immediate filling of the prepared device with water or the use of solvent-extracted PDMS vastly extends the operational lifetimes of the chips. The reliability of this technique is confirmed by generating water-in-oil-in-water (W/O/W) double emulsion droplets with controlled core/shell structures and volumes, and diameters as small as 55 μm. Its versatility is shown by simply using a different placement of the epoxy glue on the same chip design to create oil-in-water-in-oil (O/W/O) double emulsion droplets. Both W/O/W and O/W/O double emulsion droplets can therefore be created from the same soft-lithography mould. This simple method overcomes one of the key problems limiting the wider use of double emulsions – lack of reliability – while its speed and simplicity will facilitate the high-throughput production of monodisperse double emulsions. It could also be readily extended to produce microfluidic chips with more complex hydrophilic and hydrophobic patterns.

Ras proteins are small GTPases that are mutationally activated in around 30% of all human cancers. Oncogenic mutations in Ras trigger uncontrolled cellular differentiation and division through uninhibited Ras-GTP signaling. Despite major efforts in developing inhibitors, lack of treatments directly targeting Ras in cancer led to the current assumption that Ras is undruggable. A new approach, which involves use of biologics, has shown great potential for development of Ras inhibitors, as demonstrated by recent increase in the number of antibody mimetic reagents targeting Ras, including single domain antibodies, monobodies and DARPins. We have developed modulators of Ras activity using novel artificial binding proteins, termed Affimers. Affimers are small 91-amino-acid scaffold proteins that constrain one or two randomized nine amino acid loop regions for molecular recognition. Affimers isolated against KRas, the most commonly mutated Ras family member, displayed low nanomolar binding affinities, were shown to be effective at inhibiting nucleotide exchange and blocked interaction between Ras and its effector Raf. When expressed in mammalian cells, Affimers bound with endogenous Ras and inhibited Ras-mediated signaling. Site-directed mutagenesis of Affimer variable regions revealed three residues critical for binding and inhibition. X-ray crystal structure of Affimer in complex with KRas demonstrated that these residues bind into a hydrophobic pocket on Ras, previously described with small molecules. Currently, we are determining whether this interaction gives insight into new modes of therapeutic development using molecular docking in attempt to mimic the Affimer residues with small molecules. This project provides a unique opportunity to further our understanding of Ras biology through the development of reagents that modulate Ras activity. In addition, structural insights into mode of inhibition allow the modelling and design of small-molecule compounds, providing novel therapeutic strategies to slow Ras-addicted tumor growth. Citation Format: Katarzyna Haza, Heather Martin, Christian Tiede, Kevin Tipping, Chi Trinh, Holly Foster, Rachel Trowbridge, Richard Foster, Thomas Edwards, Alexander Breeze, Michael McPherson, Darren Tomlinson. Development of small-molecule RAS inhibitors using Affimer reagents [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B24.

Cancer is frequently characterised by dysregulation of the cellular signalling processes that govern proliferation, survival and attachment. Understanding such dysregulation continues to present a challenge given the importance of protein-protein interactions in intracellular processes. Exploring this protein-protein interactome requires novel tools capable of discriminating between highly homologous proteins, individual domains and post-translational modifications. This review examines the potential of scaffold-based binding proteins to fulfil these requirements. It also explores protein-protein interactions in the context of intracellular signalling pathways and cancer, and demonstrates the uses of scaffold proteins as functional moderators, biosensors and imaging reagents. This review also highlights the timeliness and potential to develop international consortia to develop and validate highly specific "proteome" scaffold-based binding protein reagents with the ultimate aim of developing screening tools for studying the interactome.

Significance Autoimmune disease pathogenesis is driven by inflammation, induced partly by IgG autoantibody-containing immune complexes binding to Fc gamma receptors (FcγRs). These receptors are valid therapeutic targets in the treatment of autoimmunity. FcγRIIIa is one of a family of highly homologous receptors for IgG antibodies; previous attempts at therapeutic blockade have resulted in off-target effects involving cells that express the almost identical protein FcγRIIIb. Here we report the identification of functionally specific protein-based inhibitors (Affimer proteins) of FcγRIIIa and the structural/functional basis of their selectivity. As molecular research tools FcγRIIIa-specific Affimer proteins provide the ability to block IgG interaction with a single receptor. Our findings suggest that highly selective protein-based blocking agents that may have therapeutic applications can be readily produced.

Receptor tyrosine kinases (RTKs) are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

Biopanning was used to generate novel artificial binding proteins which are able to control the formation of synthetic cubic nanoparticles of magnetite., Adhirons are robust, well expressing, peptide display scaffold proteins, developed as an effective alternative to traditional antibody binding proteins for highly specific molecular recognition applications. This paper reports for the first time the use of these versatile proteins for material binding, and as tools for controlling material synthesis on the nanoscale. A phage library of Adhirons, each displaying two variable binding loops, was screened to identify specific proteins able to interact with [100] faces of cubic magnetite nanoparticles. The selected variable regions display a strong preference for basic residues such as lysine. Molecular dynamics simulations of amino acid adsorption onto a [100] magnetite surface provides a rationale for these interactions, with the lowest adsorption energy observed with lysine. These proteins direct the shape of the forming nanoparticles towards a cubic morphology in room temperature magnetite precipitation reactions, in stark contrast to the high temperature, harsh reaction conditions currently used to produce cubic nanoparticles. These effects demonstrate the utility of the selected Adhirons as novel magnetite mineralization control agents using ambient aqueous conditions. The approach we outline with artificial protein scaffolds has the potential to develop into a toolkit of novel additives for wider nanomaterial fabrication.

We gratefully acknowledge financial support from the EPSRC, BBSRC and Royal Society. JRH thanks the ARC (FT120100421) for financial support. JEL and AMB would like to thank University College, Oxford for financial support. JEL would also like to thank The Royal Society for a University Research Fellowship. TGG and AMB were supported by a BBSRC studentships. Orientation-selective DEER (Double Electron-Electron Resonance) measurements were conducted on a series of rigid and flexible molecules containing Cu(ii) ions. A system with two rigidly held Cu(ii) ions was afforded by the protein homo-dimer of copper amine oxidase from Arthrobacter globiformis. This system provided experimental DEER data between two Cu(ii) ions with a well-defined distance and relative orientation to assess the accuracy of the methodology. Evaluation of orientation-selective DEER (os DEER) on systems with limited flexibility was probed using a series of porphyrin-based Cu(ii)-nitroxide and Cu(ii)-Cu(ii) model systems of well-defined lengths synthesized for this project. Density functional theory was employed to generate molecular models of the conformers for each porphyrin-based Cu(ii) dimer studied. Excellent agreement was found between DEER traces simulated using these computed conformers and the experimental data. The performance of different parameterised structural models in simulating the experimental DEER data was also investigated. The results of this analysis demonstrate the degree to which the DEER data define the relative orientation of the two Cu(ii) ions and highlight the need to choose a parameterised model that captures the essential features of the flexibility (rotational freedom) of the system being studied. Publisher PDF

This work was funded in part by a Leeds Cancer Research UK (CRUK) Development Fund and The Wellcome Trust (ISSF) (DJH), The Wellcome Trust (089330), BBSRC (BB/K000306/1 and BB/M006557/1) (AW) and The University of Leeds through Biomedical Health Research Centre support for the Leeds BioScreening Technology Group (MJM, DCT). Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions. Postprint

This work was funded by the University of Leeds. Some 30 years after its discovery, phage display remains one of the most widely used methods of in vitro selection. Initially developed to revolutionize the generation of therapeutic antibodies, phage display is now the first choice for screening artificial binding proteins. Artificial binding proteins can be used as reagents to study protein-protein interactions, target posttranslational modifications, and distinguish between homologous proteins. They can also be used as research and affinity reagents, for diagnostic purposes, and as therapeutics. However, the ability to identify isoform-specific reagents remains highly challenging. We describe an adapted phage display protocol using an artificial binding protein (Affimer) for the selection of isoform-selective binding proteins. Postprint

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001, eLife digest Many of the molecules that are essential for life are too small to be visible inside cells. So, scientists use large complex proteins called antibodies that bind to these molecules to detect whether they are present and show where they are in a cell. As well as being useful tools in experiments, these antibodies can be used to help identify and treat diseases. The body produces antibodies in response to an infection. The antibodies used in experiments are purified from animal blood, but this method of producing antibodies has flaws. For example, it can be difficult to make identical batches of antibody that always behave in the same way. So scientists have developed “alternative binding proteins” that can be made in the laboratory. These proteins are much less complicated and can be developed more quickly than antibodies, and can easily be adapted for a variety of uses. An alternative binding protein called an Affimer behaves in a similar way to an antibody by binding tightly to its target molecule, but is much more stable to acidity and high temperature. Tiede et al. have now tested how well the Affimer works in a wide range of different experiments that normally use antibodies to analyse the amount of a particular molecule inside a cell. The results of the tests show that the Affimer behaves in the same way as antibodies, and sometimes works more effectively. Tiede et al. show that an Affimer can help to reveal how a particular molecule works within a cell, to create detailed pictures of molecules in cells and tissues, and to identify a tumour. It can also be used alongside a new technique called ‘super-resolution microscopy’ that allows researchers to watch the activity of individual molecules. Future challenges are to test the Affimer in even more applications and to encourage its wider use by researchers, alongside other alternative binding proteins, as as replacements for some antibodies. This could ultimately lead to the development of faster and more efficient diagnostic, imaging and therapeutic tests. DOI: http://dx.doi.org/10.7554/eLife.24903.002

Segmented flow microfluidic devices offer an attractive means of studying crystallization processes. However, while they are widely employed for protein crystallization, there are few examples of their use for sparingly soluble compounds due to problems with rapid device fouling and irreproducibility over longer run-times. This article presents a microfluidic device which overcomes these issues, as this is constructed around a novel design of "picoinjector" that facilitates direct injection into flowing droplets. Exploiting a Venturi junction to reduce the pressure within the droplet, it is shown that passive injection of solution from a side-capillary can be achieved in the absence of an applied electric field. The operation of this device is demonstrated for calcium carbonate, where highly reproducible results are obtained over long run-times at high supersaturations. This compares with conventional devices that use a Y-junction to achieve solution loading, where in-channel precipitation of calcium carbonate occurs even at low supersaturations. This work not only opens the door to the use of microfluidics to study the crystallization of low solubility compounds, but the simple design of a passive picoinjector will find wide utility in areas including multistep reactions and investigation of reaction dynamics.

Glypican-3 (GPC3) is a promising new marker for hepatocellular carcinoma, but the reported values for serum GPC3 differ markedly between currently available kits. Here we isolated Affimer non-antibody binding proteins against GPC3 by phage display and developed a new sandwich chemiluminescence immunoassay (CLIA) combining an Affimer with a monoclonal antibody (Affimer-MAb CLIA). The proposed CLIA assay demonstrated a wide linear range 0.03–600 ng/mL) with a good linear correlation coefficient (0.9999), a high detection limitation (0.03 ng/mL) and specificity (0–0.002%) for detection of GPC3. The accuracy, hook effect and stability were demonstrated to be satisfactory. The mean level of GPC3 in serum was higher (>8.5 fold, P

Almost all monofunctional haem catalases contain a highly conserved core containing the active site, which is connected to the exterior of the enzyme by three channels. These channels have been identified as potential routes for substrate flow and product release. To further investigate the role of these molecular channels, a series of mutants ofScytalidium thermophilumcatalase were generated. The three-dimensional structures of four catalase variants, N155A, V123A, V123C and V123T, have been determined at resolutions of 2.25, 1.93, 1.9 and 1.7 Å, respectively. The V123C variant contains a new covalent bond between the S atom of Cys123 and the imidazole ring of the essential His82. This variant enzyme has only residual catalase activity and contains haembinstead of the normal haemd. The H82A variant demonstrates low catalase and phenol oxidase activities (0.2 and 20% of those of recombinant wild-type catalase–phenol oxidase, respectively). The N155A and N155H variants exhibit 4.5 and 3% of the wild-type catalase activity and contain haemd, showing that Asn155 is essential for catalysis but is not required for the conversion of haembto haemd. Structural analysis suggests that the cause of the effect of these mutations on catalysis is the disruption of the ability of dioxygen substrates to efficiently access the active site. Additional mutants have been characterized biochemically to further probe the roles of the different channels. Introducing smaller or polar side chains in place of Val123 reduces the catalase activity. The F160V, F161V and F168V mutants show a marked decrease in catalase activity but have a much lower effect on the phenol oxidase activity, despite containing substoichiometric amounts of haem.