Changes in ganglioside content affect the binding of Clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells

Epsilon-toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent-resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside G(M1) dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to G(M1) or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, G(M3), a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane-anchored sialic acid of G(M3) within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells.     

ErbB-targeted therapeutic approaches in human cancer

The overexpression and aberrant function of the epidermal growth factor receptor (EGFR, erbB1, HER1) and its ligands and coreceptors in a wide spectrum of epithelial cancers have provided a rationale for targeting this signaling network with novel treatment approaches. Several antireceptor therapeutic strategies have been pursued, but two stand ahead in their clinical development. One approach has been the generation of small molecules that compete with adenosine triphosphate (ATP) for binding to the receptor's kinase pocket, thus blocking receptor activation and the transduction of postreceptor signals. The second approach utilizes humanized monoclonal antibodies generated against the receptor's ligand-binding extracellular domain. These antibodies block binding of receptor-activating ligands and, in some cases, can induce receptor endocytosis and downregulation. Clinical studies already suggest that both of these approaches, either alone or in combination with standard anticancer therapies, are well tolerated and can induce clinical responses and tumor stabilization in a variety of common carcinomas.     

Rational bases for the development of EGFR inhibitors for cancer treatment

Growth factor receptors and their ligands not only regulate normal cell processes but have been also identified as key regulators of human cancer formation. The epidermal growth factor receptor (EGFR/ErbB1/HER1) belongs to the ErbB/HER-family of tyrosine kinase receptors (RTKs). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Several evidences suggest that cooperation of multiple ErbB receptors and ligands is required for the induction of cell transformation. In this respect, EGFR, upon activation, sustains a complex and redundant network of signal transduction pathways with the contribution of other trans-membrane receptors. EGFR has been found to be expressed and altered in a variety of malignancies and clearly it plays a significant role in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis and metastatic spread. Moreover, amplification of the EGFR gene and mutations in the EGFR tyrosine kinase domain have been recently reported in human carcinomas. As a result, investigators have developed approaches to inhibit the effects of EGFR activation, with the aim of blocking tumor growth and invasion. A number of agents targeting EGFR, including specific antibodies directed against its ligand-binding domain and small molecules inhibiting its tyrosine kinase activity are either in clinical trials or are already approved for clinical treatment. This article reviews the EGFR role in carcinogenesis and tumor progression as rational bases for the development of specific therapeutic inhibitors.     

Role of N‐glycosylation in EGFR ectodomain ligand binding

The epidermal growth factor receptor (EGFR) is a tyrosine kinase protein, overexpressed in several cancers. The extracellular domain of EGFR is known to be heavily glycosylated. Growth factor (mostly epidermal growth factor or EGF) binding activates EGFR. This occurs by inducing the transition from the autoinhibited tethered conformation to an extended conformation of the monomeric form of EGFR and by stabilizing the flexible preformed dimer. Activated EGFR adopts a back‐to‐back dimeric conformation after binding of another homologous receptor to its extracellular domain as the dimeric partner. Several antibodies inhibit EGFR by targeting the growth factor binding site or the dimeric interfaces. Glycosylation has been shown to be important for modulating the stability and function of EGFR. Here, atomistic MD simulations show that N‐glycosylation of the EGFR extracellular domain plays critical roles in the binding of growth factors, monoclonal antibodies, and the dimeric partners to the monomeric EGFR extracellular domain. N‐glycosylation results in the formation of several noncovalent interactions between the glycans and EGFR extracellular domain near the EGF binding site. This stabilizes the growth factor binding site, resulting in stronger interactions (electrostatic) between the growth factor and EGFR. N‐glycosylation also helps maintain the dimeric interface and plays distinct roles in binding of antibodies to spatially separated epitopes of the EGFR extracellular domain. Analysis of SNP data suggests the possibility of altered glycosylation with functional consequences. Proteins 2017; 85:1529–1549. © 2017 Wiley Periodicals, Inc.     

A role for epidermal growth factor receptor in idiopathic pulmonary fibrosis onset

In idiopathic pulmonary fibrosis (IPF) patients the presence of missense polymorphisms (SNP) in members of the epidermal growth factor receptor (EGFR) family or their genetic association could influence the binding affinity of natural ligands, modifying the expression and the behavior of the correlated genes. EGFR family members are particularly involved in the epithelial injury and fibrotic process in IPF. Genetic variations in HER family of receptors may alter the possible therapeutic efficacy of EGFR inhibitors. This study aimed to analyze the relationships between IPF and specific EGF receptor family functional polymorphisms. We tested the presence of common EGFR, HER2 and HER3 non-synonymous SNPs in the peripheral blood of 20 Italian IPF patients and their association with the disease. Our data indicated that the HER2 variant allele frequency was significantly lower in patients than in controls, with an odds ratio of 0.31 (95% CI 0.080, 0.98). Our finding suggests that HER2 variant could be a protective factor against IPF onset.     

Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K_d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K_d, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K_d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.     

Fibronectin type III domain based monobody with high avidity

Significant efforts have been made to improve the target-binding strength of numerous affinity molecules that are widely used in biomedical research and clinical applications. While many antibody-like fragments have been successfully optimized through affinity maturation, the process is time-consuming and restricted by the stability, solubility, and expression level of the protein sequences. To generate a fibronectin type III domain (FN3) based monobody that binds to the tumor-related biomarkers with exceptional high strength and stability, we developed a multivalent strategy by fusing an alphavbeta3-binding FN3 monobody with a short COMP pentamerization domain through a linker that facilitates appropriate display of multivalent FN3 domains. The fusion protein was highly expressed in the soluble fraction of Escherichia coli and efficiently self-assembled into a pentameric molecule that could be readily purified. Compared to the monomeric form, the pentameric monobody bound to alphavbeta3 integrin much more tightly with significantly slower off-rate, while still maintaining its excellent specificity toward alphavbeta3 when tested by using purified integrins and integrin-expressing cell lines. The multivalent strategy we describe here could be applied to engineer other FN3 monobodies to acquire significantly improved targeting-binding strengths.     

Targeting Adenoviral Vectors by Using the Extracellular Domain of the Coxsackie-Adenovirus Receptor: Improved Potency via Trimerization

Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.     

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.     

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5?h in human serum compared to the half-life of 2?h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.     

Evaluation of a maleimido derivative of CHX-A' DTPA for site-specific labeling of affibody molecules

Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.     

Structural insights and biomedical potential of IgNAR scaffolds from sharks

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.     

Quantitative Analysis of Ligand-EGFR Interactions: A Platform for Screening Targeting Molecules

Epidermal growth factor receptor (EGFR) is often constitutively stimulated in many cancers owing to the binding of ligands such as epidermal growth factor (EGF). Therefore, it is necessary to investigate the interaction between EGFR and its targeting biomolecules. The main aim of this study was to estimate the binding affinity and adhesion force of two targeting molecules, anti-EGFR monoclonal antibody (mAb LA1) and the peptide GE11 (YHWYGYTPQNVI), with respect to EGFR and to compare these values with those obtained for the ligand, EGF. Surface plasmon resonance (SPR) was used to determine the equilibrium dissociation constant (K_D) for evaluating the binding affinity. Atomic force microscopy (AFM) was performed to estimate the adhesion force. In the case of EGFR, the K_D of EGF, GE11, and mAb LA1 were 1.77 × 10⁻⁷, 4.59 × 10⁻⁴ and 2.07 × 10⁻⁹, respectively, indicating that the binding affinity of mAb LA1 to EGFR was higher than that of EGF, while the binding affinity of GE11 to EGFR was the lowest among the three molecules. The adhesion force between EGFR and mAb LA1 was 210.99 pN, which is higher than that observed for EGF (209.41 pN), while the adhesion force between GE11 and EGFR was the lowest (59.51 pN). These results suggest that mAb LA1 binds to EGFR with higher binding affinity than EGF and GE11. Moreover, the adhesion force between mAb LA1 and EGFR was greater than that observed for EGF and GE11. The SPR and AFM experiments confirmed the interaction between the receptor and targeting molecules. The results of this study might aid the screening of ligands for receptor targeting and drug delivery.     

Structural insights and biomedical potential of IgNAR scaffolds from sharks

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.     

Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ?-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope ((??m)Tc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.     

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target

The binding process of A9 peptide toward HER2‐DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2‐DIVMP, multiple ligand‐based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.     

Development of Targeting Ligands for HER2 Receptor: Simplified Receptor Model Employed for Selecting Highly Specific Molecules

The interaction between Herceptin (Fab)-derived dimeric peptide and HER2-DIVMP, a synthetic peptide mimicking the subdomain IV of the receptor HER2, was monitored by fluorescence spectroscopy method. The obtained results confirmed that the adopted mimetic receptor fragment represents a reliable model system to monitor the ability of selected ligands to bind the receptor target, avoiding the involvement of cells or cellular extracts in a preliminary binding assay. Moreover, HER2-DIVMP has the characteristics of being easily integrated in rapid, efficient and inexpensive screening method for optimizing small molecules as targeting ligands for the receptor-mediated anticancer therapy.     

Mechanisms of resistance to HER family targeting antibodies

The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.     

A SPR strategy for high-throughput ligand screenings based on synthetic peptides mimicking a selected subdomain of the target protein: A proof of concept on HER2 receptor

The discovery of pharmaceutical agents is a complex, lengthy and costly process, critically depending on the availability of rapid and efficient screening methods. In particular, when targets are large, multidomain proteins, their complexity may affect unfavorably technical feasibility, costs and unambiguity of binding test interpretation.A possible strategy to overcome these problems relies on molecular design of receptor fragments that are: sensible targets for ligand screenings, conformationally stable also as standalone domains, easily synthesized and immobilized on chip for Biacore experiments.An additional desirable feature for new ligands is the ability of selectively targeting alternative conformational states typical of many proteins.To test the feasibility of such approach on a case with potential applicative interest, we developed a surface plasmon resonance (SPR)-based screening method for drug candidates toward HER2, a Tyr-kinase receptor targeted in anticancer therapies. HER2 was mimicked by HER2-DIVMP, a modified fragment of it immobilized onto the sensor surface specifically modeling HER2 domain IV in its bounded form, designed by structural comparison of HER2 alone and in complex with Herceptin, a monoclonal therapeutic anti-HER2 antibody.This design and its implementation in SPR devices was validated by investigating Herceptin- HER2-DIVMP affinity, measuring its dissociation constant (K_D = 19.2 nM). An efficient synthetic procedure to prepare the HER2-DIVMP peptide was also developed. The HER2-DIVMP conformational stability suggested by experimental and computational results, makes it also a valuable candidate as a mold to design new molecules selectively targeting domain IV of HER2.     

Design and Optimization of Peptide Ligands to Target Breast Cancer-Positive HER2 by Grafting and Truncation of MIG6 Peptide

The human epidermal growth factor receptor 2 (EGFR2 or HER2) has been established as a therapeutic target for HER2-positive breast cancer. Although a number of small-molecule agents have been developed to target HER2, many adverse drug reactions (ADRs) such as side effects and drug resistance are frequently observed in the chemotherapeutics. Previously, peptides derived from MIG6 protein, a natural negative regulator of EGFR and HER2, have been shown to destabilize EGFR dimerization (Zhang et al. 2007). Here, we grafted a MIG6 fragment (³³⁶KSLPSYLNGVMPPTQSFAPDPKYVSS³⁶¹) in crystal interaction site from EGFR to HER2, truncated the fragment to obtain a short segment (³⁴⁶MPPTQSFA³⁵³) with reserved binding capability (K _d = 121.6 ± 14.5 μM) to HER2 kinase domain, and optimized the segment to improve its affinity for the domain. Consequently, three peptides (MLPNQSFA, MFPNQSFA and MFPYQSFA) were successfully designed to exhibit moderate or high potency (K _d = 78.0 ± 9.8, 28.7 ± 3.9 and 65.1 ± 7.2 μM, respectively) towards HER2 kinase domain, which are expected to destabilize HER2 dimerization and then suppress the kinase activation in breast cancer.