"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Affimers and nanobodies as molecular probes and their applications in imaging.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay.

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4-5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.

Selective Affimers Recognise the BCL-2 Family Proteins BCL-xL and MCL-1 through Noncanonical Structural Motifs*.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-xL and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons.

A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

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.

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk.

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.

Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action.

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Control of conformation in α-helix mimicking aromatic oligoamide foldamers through interactions between adjacent side-chains.

The design, synthesis and structural characterization of non-natural oligomers that adopt well-defined conformations, so called foldamers, is a key objective in developing biomimetic 3D functional architectures. For the aromatic oligoamide foldamer family, use of interactions between side-chains to control conformation is underexplored. The current manuscript addresses this objective through the design, synthesis and conformational analyses of model dimers derived from 3-O-alkylated para-aminobenzoic acid monomers. The O-alkyl groups on these foldamers are capable of adopting syn- or anti-conformers through rotation around the Ar-CO/NH axes. In the syn-conformation this allows the foldamer to act as a topographical mimic of the α-helix whereby the O-alkyl groups mimic the spatial orientation of the i and i + 4 side-chains from the α-helix. Using molecular modelling and 2D NMR analyses, this work illustrates that covalent links and hydrogen-bonding interactions between side-chains can bias the conformation in favour of the α-helix mimicking syn-conformer, offering insight that may be more widely applied to control secondary structure in foldamers.

FGFR3 mutation increases bladder tumourigenesis by suppressing acute inflammation.

Recent studies of muscle-invasive bladder cancer show that FGFR3 mutations are generally found in a luminal papillary tumour subtype that is characterised by better survival than other molecular subtypes. To better understand the role of FGFR3 in invasive bladder cancer, we examined the process of tumour development induced by the tobacco carcinogen OH-BBN in genetically engineered models that express mutationally activated FGFR3 S249C or FGFR3 K644E in the urothelium. Both occurrence and progression of OH-BBN-driven tumours were increased in the presence of an S249C mutation compared to wild-type control mice. Interestingly, at an early tumour initiation stage, the acute inflammatory response in OH-BBN-treated bladders was suppressed in the presence of an S249C mutation. However, at later stages of tumour progression, increased inflammation was observed in S249C tumours, long after the carcinogen administration had ceased. Early-phase neutrophil depletion using an anti-Ly6G monoclonal antibody resulted in an increased neutrophil-to-lymphocyte ratio at later stages of pathogenesis, indicative of enhanced tumour pathogenesis, which supports the hypothesis that suppression of acute inflammation could play a causative role. Statistical analyses of correlation showed that while initial bladder phenotypes in morphology and inflammation were FGFR3-dependent, increased levels of inflammation were associated with tumour progression at the later stage. This study provides a novel insight into the tumour-promoting effect of FGFR3 mutations via regulation of inflammation at the pre-tumour stage in the bladder. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular function. VEGF-A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, proteolysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and de-ubiquitination has important consequences for the endothelial cell response and vascular physiology.

Site-Specific Labeling of Affimers for DNA-PAINT Microscopy.

Optical super-resolution techniques allow fluorescence imaging below the classical diffraction limit of light. From a technology standpoint, recent methods are approaching molecular-scale spatial resolution. However, this remarkable achievement is not easily translated to imaging of cellular components, since current labeling approaches are limited by either large label sizes (antibodies) or the sparse availability of small and efficient binders (nanobodies, aptamers, genetically-encoded tags). In this work, we combined recently developed Affimer reagents with site-specific DNA modification for high-efficiency labeling and imaging using DNA-PAINT. We assayed our approach using an actin Affimer. The small DNA-conjugated affinity binders could provide a solution for efficient multitarget super-resolution imaging in the future.

Antibody Mimetics for the Detection of Small Organic Compounds Using a Quartz Crystal Microbalance.

Conventional immunoassays rely on antibodies that provide high affinity, specificity, and selectivity against a target analyte. However, the use of antibodies for the detection of small-sized, nonimmunogenic targets, such as pharmaceuticals and environmental contaminants, presents a number of challenges. Recent advances in protein engineering have led to the emergence of antibody mimetics that offer the high affinity and specificity associated with antibodies, but with reduced batch-to-batch variability, high stability, and in vitro selection to ensure rapid discovery of binders against a wide range of targets. In this work we explore the potential of Affimers, a recent example of antibody mimetics, as suitable bioreceptors for the detection of small organic target compounds, here methylene blue. Target immobilization for Affimer characterization was achieved using long-chained alkanethiol linkers coupled with oligoethylene glycol (LCAT-OEG). Using quartz crystal microbalance with dissipation monitoring (QCM-D), we determine the affinity constant, KD, of the methylene blue Affimer to be comparable to that of antibodies. Further, we demonstrate the high selectivity of Affimers for its target in complex matrixes, here a limnetic sample. Finally, we demonstrate an Affimer-based competition assay, illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, nonimmunogenic compounds.

Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk.

An enhanced thrombotic environment and premature atherosclerosis are key factors for the increased cardiovascular risk in diabetes. The occlusive vascular thrombus, formed secondary to interactions between platelets and coagulation proteins, is composed of a skeleton of fibrin fibres with cellular elements embedded in this network. Diabetes is characterised by quantitative and qualitative changes in coagulation proteins, which collectively increase resistance to fibrinolysis, consequently augmenting thrombosis risk. Current long-term therapies to prevent arterial occlusion in diabetes are focussed on anti-platelet agents, a strategy that fails to address the contribution of coagulation proteins to the enhanced thrombotic milieu. Moreover, antiplatelet treatment is associated with bleeding complications, particularly with newer agents and more aggressive combination therapies, questioning the safety of this approach. Therefore, to safely control thrombosis risk in diabetes, an alternative approach is required with the fibrin network representing a credible therapeutic target. In the current review, we address diabetes-specific mechanistic pathways responsible for hypofibrinolysis including the role of clot structure, defects in the fibrinolytic system and increased incorporation of anti-fibrinolytic proteins into the clot. Future anti-thrombotic therapeutic options are discussed with special emphasis on the potential advantages of modulating incorporation of the anti-fibrinolytic proteins into fibrin networks. This latter approach carries theoretical advantages, including specificity for diabetes, ability to target a particular protein with a possible favourable risk of bleeding. The development of alternative treatment strategies to better control residual thrombosis risk in diabetes will help to reduce vascular events, which remain the main cause of mortality in this condition.

Inhibition of complement C3 and fibrinogen interaction: a potential novel therapeutic target to reduce cardiovascular disease in diabetes.

BACKGROUND: Enhanced complement C3 incorporation into the fibrin network in diabetes is one mechanism for impaired fibrinolysis and increased thrombosis risk in this condition. Our aim was to develop new strategies to modulate fibrinolysis in diabetes by interfering with fibrin-C3 interaction. METHODS: To modulate interaction between fibrinogen and C3 we used a novel technique by screening fibrinogen with a phage display library of 3 billion random, conformational 9AA peptides (termed adhirons). The effect of high affinity fibrinogen binding adhirons, released by the addition of excess C3, on fibrin clot lysis and structure was assessed in turbidimetric assays. Fibrinogen-C3 interactions were further studied by peptide microarray techniques and modelled with the website PepSite2. FINDINGS: Ten high affinity fibrinogen binding adhirons, released by C3, were available for turbidimetric analysis. One adhiron (A6) was found to have a sequence homology with C3 and studied further. In the absence of C3, adhiron A6 failed to modulate fibrin clot lysis time (mean 644 s [SE 13] and 620 [14] without and with adhiron A6, respectively). However, adhiron A6 abolished C3-induced prolongation of clot lysis, reducing mean lysis time from 728 s (SE 25) to 632 (24) (p=0·01). The peptide microarray screening of C3 identified two peptide motifs within the ß chain of fibrinogen (residues 424-433, 435-445) that bound to C3. PepSite2 predicted that adhiron A6 binds to similar areas on the ß chain of fibrinogen. INTERPRETATION: Using a novel phage display system, we discovered an adhiron that shared sequence homology with C3 and abolished C3-induced prolongation of fibrin clot lysis by interfering with C3-fibrinogen interaction within the ß chain. This technique offers a unique method to identify new therapeutic targets for the reduction of diabetes-specific thrombosis risk. FUNDING: Sir Jules Thorn Charitable Trust.

A place for precision medicine in bladder cancer: targeting the FGFRs.

Bladder tumors show diverse molecular features and clinical outcome. Muscle-invasive bladder cancer has poor prognosis and novel approaches to systemic therapy are urgently required. Non-muscle-invasive bladder cancer has good prognosis, but high recurrence rate and the requirement for life-long disease monitoring places a major burden on patients and healthcare providers. Studies of tumor tissues from both disease groups have identified frequent alterations of FGFRs, including mutations of FGFR3 and dysregulated expression of FGFR1 and FGFR3 that suggest that these may be valid therapeutic targets. We summarize current understanding of the molecular alterations affecting these receptors in bladder tumors, preclinical studies validating them as therapeutic targets, available FGFR-targeted agents and results from early clinical trials in bladder cancer patients.

Vascular endothelial growth factors: multitasking functionality in metabolism, health and disease.

Vascular endothelial growth factors (VEGFs) bind to VEGF receptor tyrosine kinases (VEGFRs). The VEGF and VEGFR gene products regulate diverse regulatory pathways in mammalian development, health and disease. The interaction between a particular VEGF and its cognate VEGFR activates multiple signal transduction pathways which regulate different cellular responses including metabolism, gene expression, proliferation, migration, and survival. The family of VEGF isoforms regulate vascular physiology and promote tissue homeostasis. VEGF dysfunction is implicated in major chronic disease states including atherosclerosis, diabetes, and cancer. More recent studies implicate a strong link between response to VEGF and regulation of vascular metabolism. Understanding how this family of multitasking cytokines regulates cell and animal function has implications for treating many different diseases.

Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions.

Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.

Therapeutic drug monitoring of immunotherapies with novel Affimer-NanoBiT sensor construct.

Concentration-therapeutic efficacy relationships have been observed for several therapeutic monoclonal antibodies (TmAb), where low circulating levels can result in ineffective treatment and high concentrations can cause adverse reactions. Rapid therapeutic drug monitoring (TDM) of TmAb drugs would provide the opportunity to adjust an individual patient's dosing regimen to improve treatment results. However, TDM for immunotherapies is currently limited to centralised testing methods with long sample-collection to result timeframes. Here, we show four point-of-care (PoC) TmAb biosensors by combining anti-idiotypic Affimer proteins and NanoBiT split luciferase technology at a molecular level to provide a platform for rapid quantification (<10 minutes) for four clinically relevant TmAb (rituximab, adalimumab, ipilimumab and trastuzumab). The rituximab sensor performed best with 4 pM limit of detection (LoD) and a quantifiable range between 8 pM-2 nM with neglectable matrix effects in serum up to 1%. After dilution of serum samples, the resulting quantifiable range for all four sensors falls within the clinically relevant range and compares favourably with the sensitivity and/or time-to-result of current ELISA standards. Further development of these sensors into a PoC test may improve treatment outcome and quality of life for patients receiving immunotherapy.