Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces.

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. STATEMENT OF SIGNIFICANCE: We describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts - two short peptide "tags" and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.

Homogeneous Immunoassay Using a Tri-Part Split-Luciferase for Rapid Quantification of Anti-TNF Therapeutic Antibodies.

Anti-TNF therapeutics bind and sequester tumor necrosis factor (TNF) to prevent downstream signaling and are clinically important in the treatment of several autoimmune diseases. Effective treatment with these drugs requires frequent therapeutic drug monitoring (TDM). Current analytical methods, including reporter gene assay (RGA), enzyme-linked immunosorbent assay (ELISA), and mobility shift assay (MSA), can be technically rigorous, slow, and expensive. These qualities prevent the implementation of point-of-care testing and ultimately limit the frequency and utility of monitoring. An assay simple enough to be performed in the clinic would enable increased TDM frequency, more accurate dosing, and improved patient outcomes. Toward this end, we developed a homogeneous immunoassay based on a tri-part split-luciferase system for "add-and-read" detection of anti-TNF therapeutics. In our platform, two small fragments of the split-luciferase, called ß9 and ß10, are each fused to a different interacting protein. The binding of each of these proteins to anti-TNF antibodies forces the split-luciferase components into proximity where they reform the active luciferase. We identified the fusion proteins, ß9-protein A (ß9-A) and ß10-TNF, as promising binding pairs. We systematically adjusted assay conditions to optimize the signal/background (S/B) ratio, limit of detection (LOD), and percent recovery. The assay has a large dynamic range (0.5-32 µg/mL) and is sensitive enough to monitor both subtherapeutic and supratherapeutic serum concentrations of anti-TNF antibodies, as demonstrated in clinical samples.

Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery.

Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine - specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding - high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.

Split-enzyme fragment as a single affinity tag that enables protein expression, purification, and functional assays.

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.

CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide.

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays.

Protein-fragment complementation is a valuable tool for monitoring protein interactions. In complementation assays, the reporter fragments are directly fused to the interacting proteins, eliminating the possibility of monitoring native interactions. In principle, complementation could be achieved by placing the reporter fragments on antibodies which bind to the proteins of interest, enabling the monitoring of endogenous protein interactions or detection of a single protein in a homogeneous immunoassay. Previous reports have demonstrated proof-of-concept of this approach; however, current complementation systems have not met the practical requirements as suitable fusion partners for antibodies while providing the sensitivity needed for immunoassays. To surmount these challenges, we created a first-in-class, tri-part split luciferase consisting of two 11-residue peptides that are used as the antibody appendages. As an initial proof-of-concept, we used antibody-peptide fusions and found them to be capable of quantifying pg/mL concentrations of soluble or cell-bound HER2, proving this unique complementation system overcomes previous limitations and transforms this approach from merely possible to practical and useful. As shown herein, this dual-peptide system provides a rapid, simple, and sensitive "add-and-read" homogeneous immunoassay platform that can be broadly adapted as an alternative to traditional immunoassays, and in the future should enable complementation to be expanded to monitoring endogenous protein interactions.

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 µM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 ß-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and ß-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

A chimeric p53 evades mutant p53 transdominant inhibition in cancer cells.

Because of the dominant negative effect of mutant p53, there has been limited success with wild-type (wt) p53 cancer gene therapy. Therefore, an alternative oligomerization domain for p53 was investigated to enhance the utility of p53 for gene therapy. The tetramerization domain of p53 was substituted with the coiled-coil (CC) domain from Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains proper nuclear localization in breast cancer cells detected via fluorescence microscopy and shows a similar expression profile of p53 target genes as wt-p53. Additionally, similar tumor suppressor activities of p53-CC and wt-p53 were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD), and colony-forming assays. Furthermore, p53-CC was found to cause apoptosis in four different cancer cell lines, regardless of endogenous p53 status. Interestingly, the transcriptional activity of p53-CC was higher than wt-p53 in 3 different reporter gene assays. We hypothesized that the higher transcriptional activity of p53-CC over wt-p53 was due to the sequestration of wt-p53 by endogenous mutant p53 found in cancer cells. Co-immunoprecipitation revealed that wt-p53 does indeed interact with endogenous mutant p53 via its tetramerization domain, while p53-CC escapes this interaction. Therefore, we investigated the impact of the presence of a transdominant mutant p53 on tumor suppressor activities of wt-p53 and p53-CC. Overexpression of a potent mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53, p53-CC retains the same level of tumor suppressor activity. Finally, viral transduction of wt-p53 and p53-CC into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53.

Changing the subcellular location of the oncoprotein Bcr-Abl using rationally designed capture motifs.

PURPOSE: Bcr-Abl, the causative agent of chronic myelogenous leukemia (CML), localizes in the cytoplasm where its oncogenic signaling leads to proliferation of cells. If forced into the nucleus Bcr-Abl causes apoptosis. To achieve nuclear translocation, binding domains for capture of Bcr-Abl were generated and attached to proteins with signals destined for the nucleus. These resulting proteins would be capable of binding and translocating endogenous Bcr-Abl to the nucleus. METHODS: Bcr-Abl was targeted at 3 distinct domains for capture: by construction of high affinity intracellular antibody domains (iDabs) to regions of Bcr-Abl known to promote cytoplasmic retention, via its coiled coil domain (CC), and through a naturally occurring protein-protein interaction domain (RIN1). These binding domains were then tested for their ability to escort Bcr-Abl into the nucleus using a "protein switch" or attachment of 4 nuclear localization signals (NLSs). RESULTS: Although RIN1, ABI7-iDab, and CCmut3 constructs all produced similar colocalization with Bcr-Abl, only 4NLS-CCmut3 produced efficient nuclear translocation of Bcr-Abl. CONCLUSIONS: We demonstrate that a small binding domain can be used to control the subcellular localization of Bcr-Abl, which may have implications for CML therapy. Our ultimate future goal is to change the location of critical proteins to alter their function.

Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

Disruption of Bcr-Abl coiled coil oligomerization by design.

Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

Controlling subcellular delivery to optimize therapeutic effect.

This article focuses on drug targeting to specific cellular organelles for therapeutic purposes. Drugs can be delivered to all major organelles of the cell (cytosol, endosome/lysosome, nucleus, nucleolus, mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes and proteasomes) where they exert specific effects in those particular subcellular compartments. Delivery can be achieved by chemical (e.g., polymeric) or biological (e.g., signal sequences) means. Unidirectional targeting to individual organelles has proven to be immensely successful for drug therapy. Newer technologies that accommodate multiple signals (e.g., protein switch and virus-like delivery systems) mimic nature and allow for a more sophisticated approach to drug delivery. Harnessing different methods of targeting multiple organelles in a cell will lead to better drug delivery and improvements in disease therapy.

The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain.

Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein.

Controlling subcellular localization to alter function: Sending oncogenic Bcr-Abl to the nucleus causes apoptosis.

Altering the subcellular localization of signal transducing proteins is a novel approach for therapeutic intervention. Mislocalization of tumor suppressors, oncogenes, or factors involved in apoptosis results in aberrant functioning of these proteins, leading to disease. In the case of chronic myelogenous leukemia (CML), cytoplasmic Bcr-Abl causes oncogenesis/proliferation. On the other hand, nuclear entrapment of endogenous Bcr-Abl (in K562 human leukemia cells) causes apoptosis. The goal of this study was to determine whether ectopically expressed Bcr-Abl could cause apoptosis of K562 cells when specifically directed to the nucleus via strong nuclear localization signals (NLSs). A single NLS from SV40 large T-antigen or four NLSs were subcloned to Bcr-Abl (1NLS-Bcr-Abl or 4NLS-Bcr-Abl). When transfected into K562 cells, only 4NLS-Bcr-Abl translocated to the nucleus. Bcr-Abl alone was found to localize in the cell cytoplasm, colocalizing with actin due to its actin binding domain. 1NLS-Bcr-Abl also localized with actin. Apoptosis induced by 4NLS-Bcr-Abl was evaluated 24h post-transfection by morphologic determination, DNA staining, and caspase-3 assay. This is the first demonstration that altering the location of ectopically expressed Bcr-Abl can kill leukemia cells. Multiple NLSs are required to overcome Bcr-Abl binding to actin, thus driving it into the nucleus and causing apoptosis.