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Design and synthesis of EDA peptides. (a) Peptide sequences were derived from the dimerization arm sequence of EGFR. The overall linker length and positioning of the azide and alkyne amino acids were varied. Non-natural amino acids are show in red and blue. (b) Dimerization arm mimics were synthesized by incorporating non-natural amino acids into the peptide sequence using solid phase peptide synthesis (SPPS). Peptides were cyclized on solid support via copper (I)-catalyzed azide-alkyne cycloaddition prior to resin cleavage. (c) Non-natural amino acids used for the triazole cross-link: N-Fmoc-L-propargylglycine (Pg), N-Fmoc-4-azido-L-homoalanine (Aha), N-Fmoc-5-azido-L-norvaline (Anv), N-Fmoc-6-azido-L-norleucine (Anl).
Source publication
The epidermal growth factor receptor (EGFR) is overexpressed in multiple carcinomas and is the focus of a variety of targeted therapies. Here we report the design of peptide-based compounds that mimic the EGFR dimerization arm and inhibit allosteric activation of EGFR. These peptides are modified to contain a triazolyl bridge between the peptide st...
Contexts in source publication
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... As an alternative strategy to cova- lently constrain the dimerization arm, we utilized cycloaddition chemistry to introduce a 1,4- disubstituted [1,2,3]-triazolyl-containing bridge between the terminal residues of the sequence. A panel of EGFR Dimerization Arm (EDA) peptides was designed using the native sequence of human EGFR (residues 269-278, Fig. 2a). The β-strand and turn residues were conserved from (a) Peptide sequences were derived from the dimerization arm sequence of EGFR. The overall linker length and positioning of the azide and alkyne amino acids were varied. Non-natural amino acids are show in red and blue. (b) Dimerization arm mimics were synthesized by incorporating ...
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... this strategy, the dimerization arm was covalently cross-linked while on solid support to link the terminal residue side chains using copper (I)-catalyzed azide-alkyne [3+2] Huisgen cycloaddition chemistry (Fig. 2b) [24,33,34]. The azide-or alkyne-containing amino acids were incorporated into terminal positions of the sequence to minimize modifications within the di- merization arm itself. Since the optimal bridge length was not known, we modified this length by incorporating different azido-amino acid derivatives (Fig. 2c) that were synthesized ...
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... Huisgen cycloaddition chemistry (Fig. 2b) [24,33,34]. The azide-or alkyne-containing amino acids were incorporated into terminal positions of the sequence to minimize modifications within the di- merization arm itself. Since the optimal bridge length was not known, we modified this length by incorporating different azido-amino acid derivatives (Fig. 2c) that were synthesized as previ- ously described [35]. The methylene units of the azido-amino acids were varied from 2 to 4 units (azido-L-homoalanine, azido-norvaline, or azido-norleucine) to alter the overall length of the triazole linker while the alkyne (propargylglycine) remained fixed. Since the linker asym- metrically connects ...
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... by exchanging the posi- tions of the azido-and alkynyl-amino acids. This was performed to evaluate the effects of the triazole position on inhibitory activity. In addition, two peptide controls were designed: one containing the non-modified sequence of the dimerization arm, and the other containing a scrambled sequence of the dimerization arm (Fig. 2a) ...
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... determine whether the addition of a covalent constraint promoted proteolytic stability, deg- radation of the EDA peptides was measured in the presence of purified proteases, serum, and culture media ( Fig. 4 and S2 Fig.). The rate of peptide degradation was first measured using pu- rified proteases (Fig. 4a). EDA peptides were incubated with a cocktail of immobilized trypsin and chymotrypsin over a time course of four hours. The amount of remaining peptide was quantified by LC/MS relative to an internal standard. While the non-modified control peptide ...
Citations
... To assess stability, the peptide library was incubated with freshly prepared cell lysates to measure proteolytic degradation over time as analyzed by mass spectrometry [ Figure 3A]. 17 Stability was assessed over a 6 h time course, whereby the amount of peptide remaining over time was analyzed by ESI-MS using benzoic acid as an internal control. As expected, the nonconstrained parent peptide was readily degraded with less than 20% detected by 2 h and nearly completely degraded at the 4 h time point. ...
Missense mutations along the leucine-rich repeat kinase 2 (LRRK2) protein are a major contributor to Parkinson's Disease (PD), the second most commonly occurring neurodegenerative disorder worldwide. We recently reported the development of allosteric constrained peptide inhibitors that target and downregulate LRRK2 activity through disruption of LRRK2 dimerization. In this study, we designed doubly constrained peptides with the objective of inhibiting C-terminal of Roc (COR)-COR mediated dimerization at the LRRK2 dimer interface. We show that the doubly constrained peptides are cell-permeant, bind wild-type and pathogenic LRRK2, inhibit LRRK2 dimerization and kinase activity, and inhibit LRRK2-mediated neuronal apoptosis, and in contrast to ATP-competitive LRRK2 kinase inhibitors, they do not induce the mislocalization of LRRK2 to skein-like structures in cells. This work highlights the significance of COR-mediated dimerization in LRRK2 activity while also highlighting the use of doubly constrained peptides to stabilize discrete secondary structural folds within a peptide sequence.
... An effective strategy to guide the optimization of peptidomimetics is the theoretical approach [14] to increase the potential of peptidomimetics as therapeutic products. This approach consists of combining different computational strategies [15][16][17][18][19][20][21], ranging from bioinformatics approaches to predicting the conformational plasticity of peptidomimetics, to molecular simulations to predict the binding affinity of peptidomimetics for their targets. ...
Natural peptides are an important class of chemical mediators, essential for most vital processes. What limits the potential of the use of peptides as drugs is their low bioavailability and enzymatic degradation in vivo. To overcome this limitation, the development of new molecules mimicking peptides is of great importance for the development of new biologically active molecules. Therefore, replacing the amide bond in a peptide with a heterocyclic bioisostere, such as the 1,2,3-triazole ring, can be considered an effective solution for the synthesis of biologically relevant peptidomimetics. These 1,2,3-triazoles may have an interesting biological activity, because they behave as rigid link units, which can mimic the electronic properties of amide bonds and show bioisosteric effects. Additionally, triazole can be used as a linker moiety to link peptides to other functional groups.
... An effective strategy to guide the optimization of peptidomimetics is the theoretical approach [14] to increase the potential of peptidomimetics as therapeutic products. This approach consists of combining different computational strategies [15][16][17][18][19][20][21], ranging from bioinformatics approaches to predicting the conformational plasticity of peptidomimetics, to molecular simulations to predict the binding affinity of peptidomimetics for their targets. ...
... Two additional peptidic affinity ligands to EGFR, KCCYSL, and LTVSPWY, were also discovered by phage display and have proven useful for imaging and targeted drug delivery to EGFR-positive tumors [217]. Finally, two EGFR targeting peptides, EDA (sequence: PgYNPTTYQAha) and disruptin (sequence: SVDNPH), were biologically active and inhibit dimerization of EGFR and downstream signaling [5,6,70]. ...
Tumor-homing peptides are widely used for improving tumor selectivity of anticancer drugs and imaging agents. The goal is to increase tumor uptake and reduce accumulation at nontarget sites. Here, we describe current approaches for tumor-homing peptide identification and validation, and provide comprehensive overview of classes of tumor-homing peptides undergoing preclinical and clinical development. We focus on unique mechanistic features and applications of a recently discovered class of tumor-homing peptides, tumor-penetrating C-end Rule (CendR) peptides, that can be used for tissue penetrative targeting of extravascular tumor tissue. Finally, we discuss unanswered questions and future directions in the field of development of peptide-guided smart drugs and imaging agents.
... Other approaches in finding EGFR-binding peptides focused on targeting domain II and discovered the minimal binding motif of the so-called EGFR dimerization arm (QTPYYMNT). Subsequent work showed that this peptide interacts with EGFR and inhibits signaling [29][30][31]. ...
... The sequence of peptide pepEDA is based on published data, which show that this peptide does influence EGFR phosphorylation [29]. In addition, a fluorescein derivative [31], very similar triazolylbridged variants [30], and a dimeric form [54] have been studied. Our results were in agreement with published data regarding the CD spectrum of a very similar linear EDA peptide [30] and the observation that a fluorescently-labeled version was only detected within the cell but not on the surface by laser scanning microscopy [31,55]. ...
... In addition, a fluorescein derivative [31], very similar triazolylbridged variants [30], and a dimeric form [54] have been studied. Our results were in agreement with published data regarding the CD spectrum of a very similar linear EDA peptide [30] and the observation that a fluorescently-labeled version was only detected within the cell but not on the surface by laser scanning microscopy [31,55]. In our wound healing assay, pepEDA produced no effect, which agreed with the previously detected low influence of similar peptides on EGFR signaling. ...
The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.
... This may be particularly exacerbated with the larger peptides required to antagonise the full bZIP domain of cJun. A range of methodologies to alleviate the potential shortcomings of peptide therapeutics have been developed including systematic downsizing [168,169], chemical modifications such as acetylation [170][171][172], incorporation of non-natural amino acids [173,174], or cyclisation of the peptide using linkages such as lactam bridges [175][176][177][178][179][180]. It will also be important to consider that cJun localises to the nucleus [181], so peptides may need additional optimisation to promote cellular and nuclear uptake. ...
The activator protein-1 (AP-1) family of transcription factors modulate a diverse range of cellular signalling pathways into outputs which can be oncogenic or anti-oncogenic. The transcription of relevant genes is controlled by the cellular context, and in particular by the dimeric composition of AP-1. Here, we describe the evidence linking cJun in particular to a range of cancers. This includes correlative studies of protein levels in patient tumour samples and mechanistic understanding of the role of cJun in cancer cell models. This develops an understanding of cJun as a focal point of cancer-altered signalling which has the potential for therapeutic antagonism. Significant work has produced a range of small molecules and peptides which have been summarised here and categorised according to the binding surface they target within the cJun-DNA complex. We highlight the importance of selectively targeting a single AP-1 family member to antagonise known oncogenic function and avoid antagonism of anti-oncogenic function.
... Mizuguchi et al used a synthetic peptide that could mimic the loop of the dimerization arm and revealed that the peptide inhibited the dimerization and autophosphorylation of EGFR in epidermoid carcinoma A431 cells (30). Also in another study, a proteolytically stable and biologically active form of the peptide was designed and has been shown that the peptide can downregulates receptor phosphorylation and dimerization of EGFR and reduces cell growth (31). ...
Background:
Targeted therapy is an important treatment strategy that is widely used for cancer therapy. Epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of Triple-negative breast cancer (TNBC) patients. Although Cetuximab, which targets EGFR, has shown some inhibitory effects on TNBC cells, Cetuximab resistance cases due to ligand-independent activating mutations in the EGFR gene limit its application. Due to various benefits of single chain antibodies (scFvs), the use of these antibodies in cancer targeted therapy is increasing. In this study, a specific anti-EGFR antibody was isolated and evaluated.
Methods:
Panning procedure was used against an immunodominant epitope of EGFR in its dimerization arm using a diverse phage library. Polymerase Chain Reaction (PCR) and fingerprinting were applied to identify the specific clones. The MTT tetrazolium assay was performed to evaluate the inhibitory effects of selected anti- EGFR scFv phage antibody on MDA-MB-468, a TNBC cell line.
Results:
After four round of panning, one dominant pattern was observed in DNA fingerprinting with frequency of 85%. The growth of MDA-MB-468 cells was decreased dose-dependently after treatment with anti-EGFR scFv phage antibody. No significant inhibitory effect of M13KO7 helper phage as negative control on the cell growth of MDA-MB-468 was observed (p> 0.05).
Conclusion:
The selected anti-EGFR scFv with high anti proliferative effect on TNBC cells offers an effective alternative for TNBC targeted therapy. The antibody, which binds to the dimerization arm of EGFR and inhibits EGFR dimerization, could also overcome TNBC cases with Cetuximab resistance due to ligandindependent activating mutations.
... These include all-hydrocarbon-stapled peptides and corresponding peptides with an open cross-link bearing the two olefin side chains (Walensky and Bird 2014;Grossmann et al. 2015). Hanold and coworkers also introduced a non-helical, triazolyl-bridged peptide targeting EGFR dimerization (Hanold et al. 2015). ...
Protein–protein interactions (PPI) are vital in modulating biochemical pathways in many biological processes. Inhibiting PPI is a tremendously important diagnostic and therapeutic strategy in averting pathophysiological cues and disease progression. Targeting PPI as a smart drug discovery tool has been largely overlooked over the years due to their highly dynamic and expansive interfacial areas. However in recent years, researchers have developed new technologies that have the potential to move this approach up the technology development curve and enable the regular discovery of PPI-focused smart drugs. Few drugs are already on the market and some potential drug-like candidates are in clinical trials. In this study we review the application of peptidomimetics as a valuable tool in PPI inhibition in cancer. First, we describe PPI and the general properties of the PPI interface. Next, we discuss the classification of peptidomimetics. Lastly, we focus on the application of peptidomimetics on targeted PPI in cancer pathways.
... Recently, Kennedy et al. utilized this triazole-based β-hairpin stabilization approach to inhibit the dimerization and subsequent allosteric activation of epidermal growth factor receptor (EGFR) that is overexpressed in multiple carcinomas. The authors mimic the β-hairpin dimerization arm of the EGFR in the form of a stable triazole-linked β-hairpin peptide that prevents the receptor dimerization and eventually reduces the cell viability [73]. They demonstrate that the triazole-linked peptides were completely stable against trypsin and chymotrypsin digestion over 4 h, whereas the linear peptide showed a half-life of 1 h. ...
Macrocyclic peptides are a unique class of molecules that display a relatively constrained peptidic backbone as compared to their linear counterparts leading to the defined 3-D orientation of the constituent amino acids (pharmacophore). Although they are attractive candidates for lead discovery owing to the unique conformational features, their peptidic backbone is susceptible to proteolytic cleavage in various biological fluids that compromise their efficacy. In this chapter we review the various classical and contemporary chemical and biological approaches that have been utilized to combat the metabolic instability of macrocyclic peptides. We note that any chemical modification that helps in providing either local or global conformational rigidity to these macrocyclic peptides aids in improving their metabolic stability typically by slowing the cleavage kinetics by the proteases.
... Purification was performed using the same conditions except over a semi-preparatory column with a flow rate of 4 ml/min. FAM-labeled peptides were quantified based on absorbance at 495 nm in 10 mM Tris pH 8 (Bio-Tek Synergy 2) using an extinction coefficient of e = 68,000 M −1 cm −1 as previously described [29]. Biotin-labeled peptides were quantified by measuring ...
Kinases regulate multiple and diverse signaling pathways and misregulation is implicated in a multitude of diseases. Although significant efforts have been put forth to develop kinase-specific inhibitors, specificity remains a challenge. As an alternative to catalytic inhibition, allosteric inhibitors can target areas on the surface of an enzyme, thereby providing additional target diversity. Using cAMP-dependent protein kinase A (PKA) as a model system, we sought to develop a hydrocarbon-stapled peptide targeting the pseudosubstrate domain of the kinase. A library of peptides was designed from a Protein Kinase Inhibitor (PKI), a naturally encoded protein that serves as a pseudosubstrate inhibitor for PKA. The binding properties of these peptide analogs were characterized by fluorescence polarization and surface plasmon resonance, and two compounds were identified with KD values in the 500–600 pM range. In kinase activity assays, both compounds demonstrated inhibition with 25–35 nM IC50 values. They were also found to permeate cells and localize within the cytoplasm and inhibited PKA activity within the cellular environment. To the best of our knowledge, these stapled peptide inhibitors represent some of the highest affinity binders reported to date for hydrocarbon stapled peptides.
