Tumor-On-A-Chip Models for Predicting In Vivo Nanoparticle Behavior.

Nanosized drug formulations are broadly explored for the improvement of cancer therapy. Prediction of in vivo nanoparticle (NP) behavior, however, is challenging, given the complexity of the tumor and its microenvironment. Microfluidic tumor-on-a-chip models are gaining popularity for the in vitro testing of nanoparticle targeting under conditions that simulate the 3D tumor (microenvironment). In this review, following a description of the tumor microenvironment (TME), the state of the art regarding tumor-on-a-chip models for investigating nanoparticle delivery to solid tumors is summarized. The models are classified based on the degree of compartmentalization (single/multi-compartment) and cell composition (tumor only/tumor microenvironment). The physiological relevance of the models is critically evaluated. Overall, microfluidic tumor-on-a-chip models greatly improve the simulation of the TME in comparison to 2D tissue cultures and static 3D spheroid models and contribute to the understanding of nanoparticle behavior. Interestingly, two interrelated aspects have received little attention so far which are the presence and potential impact of a protein corona as well as nanoparticle uptake through phagocytosing cells. A better understanding of their relevance for the predictive capacity of tumor-on-a-chip systems and development of best practices will be a next step for the further refinement of advanced in vitro tumor models.

Deciphering Structural Determinants Distinguishing Active from Inactive Cell-Penetrating Peptides for Cytosolic mRNA Delivery.

The formation of noncovalent complexes by mixing of positively charged polymers with negatively charged oligonucleotides (ONs) is a widely explored concept in nanomedicine to achieve cellular delivery of ONs. Uptake of ON complexes occurs through endocytosis, which then requires release of ON from endosomes. As one type of polymer, cell-penetrating peptides (CPPs) are being used which are peptides of about 8-30 amino acids in length. However, only a few CPPs yield effective cytosolic ON delivery and activity. Several strategies have been devised to increase cellular uptake and enhance endosomal release, among which an increase of osmotic pressure through the so-called proton sponge effect, disruption of membrane integrity through membrane activity, and disulfide-mediated polymerization. Here, we address the relevance of these concepts for mRNA delivery by incorporating structural features into the human lactoferrin-derived CPP, which shows uptake but not delivery. The incorporation of histidines was explored to address osmotic pressure and structural motifs of the delivery-active CPP PepFect14 (PF14) to address membrane disturbance, and finally, the impact of polymerization was explored. Whereas oligomerization increased the stability of polyplexes against heparin-induced decomplexation, neither this approach nor the incorporation of histidine residues to promote a proton-sponge effect yielded activity. Also, the replacement of arginine residues with lysine or ornithine residues, as in PF14, was without effect, even though all polyplexes showed cellular uptake. Ultimately, sufficient activity could only be achieved by transferring amphipathic sequence motifs from PF14 into the hLF context with some benefit of oligomerization demonstrating overarching principles of delivery for CPPs, lipid nanoparticles, and other types of delivery polymers.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

The potential of RNA-based therapy for kidney diseases.

Inherited kidney diseases (IKDs) are a large group of disorders affecting different nephron segments, many of which progress towards kidney failure due to the absence of curative therapies. With the current advances in genetic testing, the understanding of the molecular basis and pathophysiology of these disorders is increasing and reveals new potential therapeutic targets. RNA has revolutionized the world of molecular therapy and RNA-based therapeutics have started to emerge in the kidney field. To apply these therapies for inherited kidney disorders, several aspects require attention. First, the mRNA must be combined with a delivery vehicle that protects the oligonucleotides from degradation in the blood stream. Several types of delivery vehicles have been investigated, including lipid-based, peptide-based, and polymer-based ones. Currently, lipid nanoparticles are the most frequently used formulation for systemic siRNA and mRNA delivery. Second, while the glomerulus and tubules can be reached by charge- and/or size-selectivity, delivery vehicles can also be equipped with antibodies, antibody fragments, targeting peptides, carbohydrates or small molecules to actively target receptors on the proximal tubule epithelial cells, podocytes, mesangial cells or the glomerular endothelium. Furthermore, local injection strategies can circumvent the sequestration of RNA formulations in the liver and physical triggers can also enhance kidney-specific uptake. In this review, we provide an overview of current and potential future RNA-based therapies and targeting strategies that are in development for kidney diseases, with particular interest in inherited kidney disorders.

Constrained peptides mimic a viral suppressor of RNA silencing.

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA.

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.

Light-Responsive Elastin-Like Peptide-Based Targeted Nanoparticles for Enhanced Spheroid Penetration.

We describe here a near infrared light-responsive elastin-like peptide (ELP)-based targeted nanoparticle (NP) that can rapidly switch its size from 120 to 25 nm upon photo-irradiation. Interestingly, the targeting function, which is crucial for effective cargo delivery, is preserved after transformation. The NPs are assembled from (targeted) diblock ELP micelles encapsulating photosensitizer TT1-monoblock ELP conjugates. Methionine residues in this monoblock are photo-oxidized by singlet oxygen generated from TT1, turning the ELPs hydrophilic and thus trigger NP dissociation. Phenylalanine residues from the diblocks then interact with TT1 via π-π stacking, inducing the re-formation of smaller NPs. Due to their small size and targeting function, the NPs penetrate deeper in spheroids and kill cancer cells more efficiently compared to the larger ones. This work could contribute to the design of "smart" nanomedicines with deeper penetration capacity for effective anticancer therapies.

Delivery of antisense oligonucleotides for splice-correction of androgen receptor pre-mRNA in castration-resistant prostate cancer models using cell-penetrating peptides.

BACKGROUND: Cell-penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre-mRNA, thereby preventing the generation of the splice variant AR-V7 mRNA. AON-mediated knockdown of AR-V7 resulted in inhibition of androgen-independent cell proliferation. In this study, we evaluated the CPP-mediated delivery of this AON into castration-resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). METHODS: Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter-Glo cell viability assay. The effectivity of CPP-mediated delivery of the splice-correcting AON-intronic splicing enhancer (ISE) targeting the ISE in the castration-resistant prostate cancer (CRPC)-derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR-V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR-V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. RESULTS: The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine-rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier-associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 µM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON-ISE directed against AR pre-mRNA achieved significant downregulation of AR-V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen-independent cell proliferation of DuCaP and VCaP cells. CONCLUSIONS: This proof-of-principle study constitutes the basis for further development of CPP-mediated delivery of AONs for targeted therapy in prostate cancer.

The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.

Antisense oligonucleotides (ASOs) are a promising class of therapeutics that are starting to emerge in the clinic. Determination of intracellular concentrations required for biologic effects and identification of effective delivery vehicles are crucial for understanding the mode of action and required dosing. Here, we investigated which nuclear oligonucleotide concentration is needed for a therapeutic effect for a triplet repeat-targeting ASO in a muscle cell model of myotonic dystrophy type 1 (DM1). For cellular delivery, ASOs were complexed into nanoparticles using the cationic cell-penetrating peptides nona-arginine and PepFect14 (PF14). Although both peptides facilitated uptake, only PF14 led to a dose-dependent correction of disease-typical abnormal splicing. In line with this observation, time-lapse confocal microscopy demonstrated that only PF14 mediated translocation of the ASOs to the nucleus, which is the main site of action. Through fluorescence lifetime imaging, we could distinguish intact oligonucleotide from free fluorophore, showing that PF14 also shielded the ASOs from degradation. Finally, we employed a combination of live-cell fluorescence correlation spectroscopy and immunofluorescence microscopy and demonstrated that intranuclear blocking-type oligonucleotide concentrations in the upper nanomolar range were required to dissolve nuclear muscleblind-like protein 1 foci, a hallmark of DM1. Our findings have important implications for the clinical use of ASOs in DM1 and provide a basis for further research on other types of ASOs.-Van der Bent, M. L., Paulino da Silva Filho, O., Willemse, M., Hällbrink, M., Wansink, D. G., Brock, R. The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.

The impact of circulation in a heart-lung machine on function and survival characteristics of red blood cells.

Extracorporeal circulation is accompanied by changes in red blood cell morphology and structural integrity that affect cell function and survival, and thereby may contribute to the various side effects of heart-lung machine-assisted surgery. Our main objectives were to determine the effect of circulation of red blood cells in a stand-alone extracorporeal circuit on several parameters that are known to be affected by, as well as contribute to red blood cell aging. As a source of RBCs, we employed blood bank storage units of different ages. In order to assess the relevance of our in vitro observations for the characterization of extracorporal circulation technology, we compared these changes in those of patients undergoing extracorporeal circulation-assisted cardiac surgery. Our results show that circulation in a heart-lung machine is accompanied by changes in red blood cell volume, an increase in osmotic fragility, changes in deformability and aggregation behavior, and alterations in the exposure of phosphatidylserine and in microvesicle generation. RBCs from 1-week-old concentrates showed the highest similarities with the in vivo situation. These changes in key characteristics of the red blood cell aging process likely increase the susceptibility of red blood cells to the various mechanical, osmotic, and immunological stress conditions encountered during and after surgery in the patient's circulation, and thereby contribute to the side effects of surgery. Thus, aging-related parameters in red blood cell structure and function provide a foundation for the validation and improvement of extracorporeal circulation technology.

Super-resolution Imaging of Structure, Molecular Composition, and Stability of Single Oligonucleotide Polyplexes.

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements.

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100-1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 µm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

Penetration in 3D tumor spheroids and explants: Adding a further dimension to the structure-activity relationship of cell-penetrating peptides.

Drug delivery into tumors and metastases is a major challenge in the eradication of cancers such as epithelial ovarian carcinoma. Cationic cell-penetrating peptides (CPPs) are a promising group of delivery vehicles to mediate cellular entry of molecules that otherwise poorly enter cells. However, little is known about their penetration behavior in tissues. Here, we investigated penetration of cationic CPPs in 3D ovarian cancer spheroids and patient-derived 3D tumor explants. Penetration kinetics and distribution after long-term incubation were imaged by confocal microscopy. In addition, spheroids and tumor explants were dissociated and cell-associated fluorescence determined by flow cytometry. CPPs with high uptake activity showed enhanced sequestration in the periphery of the spheroid, whereas less active CPPs were able to penetrate deeper into the tissue. CPPs consisting of d-amino acids were advantageous over l-amino acid CPPs as they showed less but long lasting cellular uptake activity, which benefitted penetration and retention over time. In primary tumor cultures, in contrast to nonaarginine, the amphipathic CPP penetratin was strongly sequestered by cell debris and matrix components pointing towards arginine-rich CPPs as a preferred choice. Overall, the data show that testing in 3D models leads to a different choice of the preferred peptide in comparison to a standard 2D cell culture.

An opportunistic route to success: Towards a change of paradigm to fully exploit the potential of cell-penetrating peptides.

About 25years ago it was demonstrated that certain peptides possess the ability to cross the plasma membrane. This led to the development of cell-penetrating peptides (CPPs) as vectors to mediate the cellular entry of (macro-)molecules that do not show cell entry by themselves. Nonetheless, in spite of an early bloom of promising pre-clinical studies, not a single CPP-based drug has been approved, yet. It is a paradigm in CPP research that the peptides are taken up by virtually all cells. In exploratory research and early preclinical development, this assumption guides the choice of the therapeutic target. However, while this indiscriminatory uptake may be the case for tissue culture experiments, in an organism this is clearly not the case. Biodistribution analyses demonstrate that CPPs only target a very limited number of cells and many tissues are hardly reached at all. Here, we review biodistribution analyses of CPPs and CPP-based drug delivery systems. Based on this analysis we propose a paradigm change towards a more opportunistic approach in CPP research. The application of CPPs should focus on those pathophysiologies for which the relevant target cells have been shown to be reached in vivo.

A Conjugate of an Anti-Epidermal Growth Factor Receptor (EGFR) VHH and a Cell-Penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation.

Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors, and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell-penetrating peptide (CPP), corresponding to amino acids 38-59 of human lactoferrin, and the recombinant llama single-domain antibody (VHH) 7D12, which binds the human epidermal growth factor receptor (EGFR), was generated by sortase A mediated transpeptidation. The conjugate blocks EGF-mediated EGFR activation with higher efficacy than that of both modalities alone; a phenomenon that is caused by both effective receptor blockade and internalization. Thus, the VHH-CPP conjugate shows a combination of activities that implement a highly powerful new design principle to block receptor activation by its clearance from the cell surface.

Frapbot: An open-source application for FRAP data.

We introduce Frapbot, a free-of-charge open source software web application written in R, which provides manual and automated analyses of fluorescence recovery after photobleaching (FRAP) datasets. For automated operation, starting from data tables containing columns of time-dependent intensity values for various regions of interests within the images, a pattern recognition algorithm recognizes the relevant columns and identifies the presence or absence of prebleach values and the time point of photobleaching. Raw data, residuals, normalization, and boxplots indicating the distribution of half times of recovery (t1/2 ) of all uploaded files are visualized instantly in a batch-wise manner using a variety of user-definable fitting options. The fitted results are provided as .zip file, which contains .csv formatted output tables. Alternatively, the user can manually control any of the options described earlier. © 2017 International Society for Advancement of Cytometry.

Mimicking Tumors: Toward More Predictive In Vitro Models for Peptide- and Protein-Conjugated Drugs.

Macromolecular drug candidates and nanoparticles are typically tested in 2D cancer cell culture models, which are often directly followed by in vivo animal studies. The majority of these drug candidates, however, fail in vivo. In contrast to classical small-molecule drugs, multiple barriers exist for these larger molecules that two-dimensional approaches do not recapitulate. In order to provide better mechanistic insights into the parameters controlling success and failure and due to changing ethical perspectives on animal studies, there is a growing need for in vitro models with higher physiological relevance. This need is reflected by an increased interest in 3D tumor models, which during the past decade have evolved from relatively simple tumor cell aggregates to more complex models that incorporate additional tumor characteristics as well as patient-derived material. This review will address tissue culture models that implement critical features of the physiological tumor context such as 3D structure, extracellular matrix, interstitial flow, vascular extravasation, and the use of patient material. We will focus on specific examples, relating to peptide-and protein-conjugated drugs and other nanoparticles, and discuss the added value and limitations of the respective approaches.

Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design.

Cell-penetrating peptides (CPPs) enhance the cellular uptake of membrane-impermeable molecules. Most CPPs are highly cationic, potentially increasing the risk of toxic side effects and leading to accumulation in organs such as the liver. As a consequence, there is an unmet need for less cationic CPPs. However, design principles for effective CPPs are still missing. Here, we demonstrate a design principle based on a classification of peptides according to accumulated side-chain polarity and hydrophobicity. We show that in comparison to randomly selected peptides, CPPs cover a distinct parameter space. We designed peptides of only six to nine amino acids with a maximum of three positive charges covering this property space. All peptides were tested for cellular uptake and subcellular distribution. Following an initial round of screening we enriched the collection with short and hydrophobic peptides and introduced d-amino acid substitutions and lactam bridges which increased cell uptake, in particular for long-term incubation. Using a GFP complementation assay, for the most active peptides we demonstrate cytosolic delivery of a biologically active cargo peptide.

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion.

Sevuparin is a novel drug candidate in phase II development as a treatment for vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS-RBCs) to stimulated cultured endothelial cells in vitro. Importantly, sevuparin prevents vaso-occlusion and normalizes blood flow in an in vivo mouse model of SCD vaso-occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P- and L-selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso-occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti-adhesive efficacy similar to the low molecular weight heparin tinzaparin both in vitro and in vivo. These results suggest that the anti-adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso-occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

A microarray-based approach to evaluate the functional significance of protein-binding motifs.

Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs. To this end, peptides corresponding to protein interaction motifs were spotted as a microarray. First, peptides were titrated with a pan-specific binder and the apparent K(d) value of this binder for each peptide was determined. For phosphotyrosine-containing peptides, an anti-phosphotyrosine antibody was employed. Then, in the presence of the pan-specific binder, arrays were competitively titrated with cell lysate and competition constants were determined. Using the Cheng-Prusoff equation, binding constants for the pan-specific binder and inhibition constants for the lysates were converted into affinity constants for the lysate. We experimentally validate this method using a phosphotyrosine-binding SH2 domain as a further reference. Furthermore, strong binders correlated with binding motifs engaging in numerous interactions as predicted by Scansite. This method provides a highly parallel and robust approach to identify peptides corresponding to interaction motifs with strong binding capacity for proteins in the cell lysate.