Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.

Antibody-Linked Fluorogen-Activating Proteins for Antigen Detection and Cell Ablation.

We demonstrate selective labeling of cell surface proteins using fluorogen-activating proteins (FAPs) conjugated to standard immunoglobulins (IgGs). Conjugation was achieved with a polypeptide reagent comprised of an N-terminal photoactivatable Fc-binding domain and a C-terminal FAP domain. The resulting FAP-antibody conjugates were effective agents for protein detection and cell ablation in cultured mammalian cells and for visualizing cell-cell contacts using a tethered fluorogen assay. Because our approach allows FAP-antibody conjugates to be generated for most currently available IgGs, it should have broad utility for experimental and therapeutic applications.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.

Tethered Fluorogen Assay to Visualize Membrane Apposition in Living Cells.

We describe proof-of-concept for a novel approach for visualizing regions of close apposition between the surfaces of living cells. A membrane-anchored protein with high affinity for a chemical ligand is expressed on the surface of one set of cells, and the cells are co-cultured with a second set of cells that express a membrane-anchored fluorogen-activating protein (FAP). The co-cultured cells are incubated with a bivalent reagent composed of fluorogen linked to the high-affinity ligand, with the concentration of the bivalent reagent chosen to be less than the binding constant for the FAP-fluorogen pair but greater than the binding constant for the ligand-high-affinity protein pair. In these conditions, strong FAP signal is observed only in regions of close proximity between membranes of the two classes of cell, where high local concentration of fluorogen favors binding to the FAP.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.

A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection.

A recently described fluorescence biosensor platform utilizes single-chain Fv (scFvs) that selectively bind and activate fluorogen molecules. In this report we investigated the display of tandem scFv biosensors at the surface of mammalian cells with the aim of advancing current fluorescence detection strategies. We initially screened different peptide linkers to separate each scFv unit, and discovered that tandem proteins joined by either flexible or α-helical linkers properly fold and display at the surface of mammalian cells. Accordingly, we performed a combinatorial scFv-dimer study and identified that fluorescence activation correlated with the cellular location (membrane distal versus proximal) and selections of the different scFvs. Furthermore, in vitro measurements showed that the stability of each scFv monomer unit influenced the folding and cell surface activities of tandem scFvs. Additionally, we investigated the absence or poor signals from some scFv-dimer combinations and discovered that intramolecular and intermolecular scFv chain mispairings led to protein misfolding and/or secretory-pathway-mediated degradation. Furthermore, when tandem scFvs were utilized as fluorescence reporter tags with surface receptors, the biosensor unit and target protein showed independent activities. Thus, the live cell application of tandem scFvs permitted advanced detection of target proteins via fluorescence signal amplification, Förster resonance energy transfer resulting in the increase of Stokes shift and multi-color vesicular traffic of surface receptors.

A single-chain-variable-fragment fluorescence biosensor activates fluorogens from dissimilar chemical families.

Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.

Determining the subcellular location of new proteins from microscope images using local features.

MOTIVATION: Evaluation of previous systems for automated determination of subcellular location from microscope images has been done using datasets in which each location class consisted of multiple images of the same representative protein. Here, we frame a more challenging and useful problem where previously unseen proteins are to be classified. RESULTS: Using CD-tagging, we generated two new image datasets for evaluation of this problem, which contain several different proteins for each location class. Evaluation of previous methods on these new datasets showed that it is much harder to train a classifier that generalizes across different proteins than one that simply recognizes a protein it was trained on. We therefore developed and evaluated additional approaches, incorporating novel modifications of local features techniques. These extended the notion of local features to exploit both the protein image and any reference markers that were imaged in parallel. With these, we obtained a large accuracy improvement in our new datasets over existing methods. Additionally, these features help achieve classification improvements for other previously studied datasets. AVAILABILITY: The datasets are available for download at http://murphylab.web.cmu.edu/data/. The software was written in Python and C++ and is available under an open-source license at http://murphylab.web.cmu.edu/software/. The code is split into a library, which can be easily reused for other data and a small driver script for reproducing all results presented here. A step-by-step tutorial on applying the methods to new datasets is also available at that address. CONTACT: murphy@cmu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform.

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.

Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors.

The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g., EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (ß2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells.

This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2010;00:000-000).

Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.

Cell cycle dependence of protein subcellular location inferred from static, asynchronous images.

Protein subcellular location is one of the most important determinants of protein function during cellular processes. Changes in protein behavior during the cell cycle are expected to be involved in cellular reprogramming during disease and development, and there is therefore a critical need to understand cell-cycle dependent variation in protein localization which may be related to aberrant pathway activity. With this goal, it would be useful to have an automated method that can be applied on a proteomic scale to identify candidate proteins showing cell-cycle dependent variation of location. Fluorescence microscopy, and especially automated, high-throughput microscopy, can provide images for tens of thousands of fluorescently-tagged proteins for this purpose. Previous work on analysis of cell cycle variation has traditionally relied on obtaining time-series images over an entire cell cycle; these methods are not applicable to the single time point images that are much easier to obtain on a large scale. Hence a method that can infer cell cycle-dependence of proteins from asynchronous, static cell images would be preferable. In this work, we demonstrate such a method that can associate protein pattern variation in static images with cell cycle progression. We additionally show that a one-dimensional parameterization of cell cycle progression and protein feature pattern is sufficient to infer association between localization and cell cycle.

Fluorogen-activating single-chain antibodies for imaging cell surface proteins.

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

Detection and assignment of CYP21 mutations using peptide mass signature genotyping.

Congenital adrenal hyperplasia (CAH) is a common inborn error of steroidogenesis. The clinical spectrum of CAH ranges from the severe classical form, which can be fatal in the newborn, to simple virilizing forms or a milder non-classical form which is often not diagnosed until puberty. Recessive mutations in the autosomal gene encoding 21-hydroxylase (CYP21) are responsible for approximately 95% of CAH cases. Since CYP21 genotype is generally predictive of the presence and severity of the disorder, accurate CYP21 genotyping is of clear medical significance. Determining the CYP21 genotype of an individual, using standard methods, is difficult due to the presence of a nearly identical pseudogene (CYP21P) in close proximity to the functional gene. To address the need for a comprehensive test for mutations in the CYP21 gene, we developed a multiplexed peptide mass signature genotyping (PMSG) assay and applied the assay to 151 DNA samples. CAH patients had been previously characterized for the 10 most common mutations. The PMSG assay detected all common mutations; in addition it identified six known rare mutations and also discovered four new mutations (two frameshifts in the first half of the gene, P42fs and S171fs, and two point mutations, H365Y and R479L). This assay has the potential to provide high-throughput, cost-effective analysis of the CYP21 gene to detect known mutations and identify novel variants in samples obtained from patients with CAH, individuals suspected to have CAH, and heterozygous carriers.

Detection and assignment of mutations and minihaplotypes in human DNA using peptide mass signature genotyping (PMSG): application to the human RDS/peripherin gene.

Peptide mass-signature genotyping (PMSG) is a scanning genotyping method that identifies mutations and polymorphisms by translating the sequence of interest in more than one reading frame and measuring the masses of the resulting peptides by mass spectrometry. PMSG was applied to the RDS/peripherin gene of 16 individuals from a family exhibiting autosomal dominant macular degeneration. The method revealed an A-->T transversion in the 5' splice site of intron 2 that is the likely cause of the disease. It also revealed four different minihaplotypes in exon 3 that represent particular combinations of SNPs at four different locations. This study demonstrates the utility of PMSG for identifying and characterizing point mutations and local minihaplotypes that are not readily analyzed by other approaches.

Detection of cystic fibrosis mutations by peptide mass signature genotyping.

BACKGROUND: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis. METHODS: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149-54) were assessed using the PMSG test for the CFTR gene. RESULTS: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. CONCLUSIONS: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.