EGFR trafficking: effect of dimerization, dynamics, and mutation.

Spontaneous dimerization of EGF receptors (EGFR) and dysregulation of EGFR signaling has been associated with the development of different cancers. Under normal physiological conditions and to maintain homeostatic cell growth, once EGFR signaling occurs, it needs to be attenuated. Activated EGFRs are rapidly internalized, sorted through early endosomes, and ultimately degraded in lysosomes by a process generally known as receptor down-regulation. Through alterations to EGFR trafficking, tumors develop resistance to current treatment strategies, thus highlighting the necessity for combination treatment strategies that target EGFR trafficking. This review covers EGFR structure, trafficking, and altered surface expression of EGFR receptors in cancer, with a focus on how therapy targeting EGFR trafficking may aid tyrosine kinase inhibitor treatment of cancer.

Lyophilized liposomal formulation of a peptidomimetic-Dox conjugate for HER2 positive breast and lung cancer.

Nanocarrier-mediated administration of chemotherapeutic drugs can increase the therapeutic index of drugs by reducing off-target site toxicity. Ligand-targeted drug delivery can be utilized to deliver chemotherapeutic drugs to cancer cells selectively and specifically. Here we report the evaluation of a lyophilized formulation of a liposome containing a peptidomimetic-doxorubicin conjugate for targeted delivery of doxorubicin to HER2-positive cancer cells. The lyophilized liposomal formulation exhibited improved release of peptidomimetic-doxorubicin conjugate at pH 6.5 compared to 7.4 and improved cellular uptake in cancer cells at pH 6.5. In vivo studies indicated that pH-sensitive formulation exhibited site-specific formulation delivery and improved anticancer efficacy than free doxorubicin. The findings suggested that combining a lyophilized pH-sensitive liposomal formulation containing trehalose as lyoprotectant with a targeting ligand coupled cytotoxic agent is a potential method for cancer chemotherapy while maintaining long-term stability at 4 °C of the liposome formulation.

Assessment of Antitumor and Antiproliferative Efficacy and Detection of Protein-Protein Interactions in Cancer Cells from 3D Tumor Spheroids.

When compared to two-dimensional (2D) cell cultures, 3D spheroids have been considered suitable in vitro models for drug discovery research and other studies of drug activity. Based on different 3D cell culture procedures, we describe procedures we have used to obtain 3D tumor spheroids by both the hanging-drop and ultra-low-attachment plate methods and to analyze the antiproliferative and antitumor efficacy of different chemotherapeutic agents, including a peptidomimetic. We have applied this method to breast and lung cancer cell lines such as BT-474, MCF-7, A549, and Calu-3. We also describe a proximity ligation assay of the cells from the spheroid model to detect protein-protein interactions of EGFR and HER2. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of 3D spheroids using the hanging-drop method Basic Protocol 2: Growth of spheroids using ultra-low-attachment plates Support Protocol 1: Cell viability assay of tumor spheroids Support Protocol 2: Antiproliferative and antitumor study in 3D tumor spheroids Support Protocol 3: Proximity ligation assay on cells derived from 3D spheroids.

1,3-diarylpyrazolones as potential anticancer agents for non-small cell lung cancer: Synthesis and antiproliferative activity evaluation.

A series of pyrazolone compounds with different substitution patterns have been synthesized using microwave-assisted methods and evaluated their in vitro antiproliferative activity against human lung adenocarcinoma cell lines (A549 and NCI-H522). Among the tested compounds, the pyrazolone P7 exhibited high antiproliferative activity against both A549 and NCIH522 cancer cell lines while being 10 times less cytotoxic to non-cancerous cells. Moreover, our compounds P7 and P11 exhibited higher antiproliferative activity and selectivity against A549 and NCIH522 cells compared with the clinically approved drugs Afatinib and Gefitinib. The cell cycle analysis showed that the compound P7 and P11 arrests the cell cycle at G0/G1 phase, whereas the compounds P13 and P14 involved in G2/M phase arrest. The results from antiproliferative activity screening, cell cycle analysis, and kinase profiling indicate that the suitably substituted 1,3-diarylpyrazolones exhibit high antiproliferative activity against non-small cell lung cancer cells.

A pH-sensitive liposome formulation of a peptidomimetic-Dox conjugate for targeting HER2 + cancer.

Cancer treatment faces the challenge of selective delivery of the cytotoxic drug to the desired site of action to minimize undesired side effects. The liposomal formulation containing targeting ligand conjugated cytotoxic drug can be an effective approach to specifically deliver chemotherapeutic drugs to cancer cells that overexpress a particular cell surface receptor. This research focuses on the in vitro and in vivo studies of a peptidomimetic ligand attached doxorubicin for the HER2 positive lung and breast cancer cells transported by a pH-dependent liposomal formulation system for the enhancement of targeted anticancer treatment. The selected pH-sensitive liposome formulation showed effective pH-dependent delivery of peptidomimetic-doxorubicin conjugate at lower pH conditions mimicking tumor microenvironment (pH-6.5) compared to normal physiological conditions (pH 7.4), leading to the improvement of cell uptake. In vivo results revealed the site-specific delivery of the formulation and enhanced antitumor activity with reduced toxicity compared to the free doxorubicin (Free Dox). The results suggested that the targeting ligand conjugated cytotoxic drug with the pH-sensitive liposomal formulation is a promising approach to chemotherapy.

Quinazoline-tethered hydrazone: A versatile scaffold toward dual anti-TB and EGFR inhibition activities in NSCLC.

Globally, lung cancer and tuberculosis are considered to be very serious and complex diseases. Evidence suggests that chronic infection with tuberculosis (TB) can often lead to lung tumors; therefore, developing drugs that target both diseases is of great clinical significance. In our study, we designed and synthesized a suite of 14 new quinazolinones (5a-n) and performed biological investigations of these compounds in Mycobacterium tuberculosis (MTB) and cancer cell lines. In addition, we conducted a molecular modeling study to determine the mechanism of action of these compounds at the molecular level. Compounds that showed anticancer activity in the preliminary screening were further evaluated in three cancer cell lines (A549, Calu-3, and BT-474 cells) and characterized in an epidermal growth factor receptor (EGFR) binding assay. Cytotoxicity in noncancerous lung fibroblast cells was also evaluated to obtain safety data. Our theoretical and experimental studies indicated that our compounds showed a mechanism of action similar to that of erlotinib by inhibiting the EGFR tyrosine kinase. In turn, the antituberculosis activity of these compounds would be produced by the inhibition of enoyl-ACP-reductase. From our findings, we were able to identify two potential lead compounds (5i and 5l) with dual activity and elevated safety toward noncancerous lung fibroblast cells. In addition, our data identified three compounds with excellent anti-TB activities (compounds 5i, 5l, and 5n).

Molecular Targeting of Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor (VEGFR).

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) are two extensively studied membrane-bound receptor tyrosine kinase proteins that are frequently overexpressed in many cancers. As a result, these receptor families constitute attractive targets for imaging and therapeutic applications in the detection and treatment of cancer. This review explores the dynamic structure and structure-function relationships of these two growth factor receptors and their significance as it relates to theranostics of cancer, followed by some of the common inhibition modalities frequently employed to target EGFR and VEGFR, such as tyrosine kinase inhibitors (TKIs), antibodies, nanobodies, and peptides. A summary of the recent advances in molecular imaging techniques, including positron emission tomography (PET), single-photon emission computerized tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging (OI), and in particular, near-IR fluorescence imaging using tetrapyrrolic-based fluorophores, concludes this review.

Click Conjugation of Boron Dipyrromethene (BODIPY) Fluorophores to EGFR-Targeting Linear and Cyclic Peptides.

Through a simple 1,3-cycloaddition reaction, three BODIPY-peptide conjugates that target the extracellular domain of the epidermal growth factor receptor (EGFR) were prepared and their ability for binding to EGFR was investigated. The peptide ligands K(N3)LARLLT and its cyclic analog cyclo(K(N3)larllt, previously shown to have high affinity for binding to the extracellular domain of EGFR, were conjugated to alkynyl-functionalized BODIPY dyes 1 and 2 via a copper-catalyzed click reaction. This reaction produced conjugates 3, 4, and 5 in high yields (70-82%). In vitro studies using human carcinoma HEp2 cells that overexpress EGFR demonstrated high cellular uptake, particularly for the cyclic peptide conjugate 5, and low cytotoxicity in light (~1 J·cm-2) and darkness. Surface plasmon resonance (SPR) results show binding affinity of the three BODIPY-peptide conjugates for EGFR, particularly for 5 bearing the cyclic peptide. Competitive binding studies using three cell lines with different expressions of EGFR show that 5 binds specifically to EGFR-overexpressing colon cancer cells. Among the three conjugates, 5 bearing the cyclic peptide exhibited the highest affinity for binding to the EGFR protein.

Peptide-functionalized liposomes as therapeutic and diagnostic tools for cancer treatment.

Clinically efficacious medication in anticancer therapy has been successfully designed with liposome-based nanomedicine. The liposomal formulation in cancer drug delivery can be facilitated with a functionalized peptide that mediates the specific drug delivery opportunities with increased drug penetrability, specific accumulation in the targeted site, and enhanced therapeutic efficacy. This review aims to focus on recent advances in peptide-functionalized liposomal formulation techniques in cancer diagnosis and treatment regarding recently published literature. It also will highlight different aspects of novel liposomal formulation techniques that incorporate surface functionalization with peptides for better anticancer effect and current challenges in peptide-functionalized liposomal drug formulation.

Nucleic Acid Delivery with α-Tocopherol-Polyethyleneimine-Polyethylene Glycol Nanocarrier System.

PURPOSE: Nucleic acid-based therapies are a promising therapeutic tool. The major obstacle in their clinical translation is their efficient delivery to the desired tissue. We developed a novel nanosized delivery system composed of conjugates of α-tocopherol, polyethyleneimine, and polyethylene glycol (TPP) to deliver nucleic acids. METHODS: We synthesized a panel of TPP molecules using different molecular weights of PEG and PEI and analyzed with various analytical approaches. The optimized version of TPP (TPP111 - the 1:1:1 molecular ratio) was self-assembled in water to produce nanostructures and then evaluated in diversified in vitro and in vivo studies. RESULTS: Through a panel of synthesized molecules, TPP111 conjugate components self-assembled in water, forming globular shaped nanostructures of ~90 nm, with high nucleic acid entrapment efficiency. The polymer had low cytotoxicity in vitro and protected nucleic acids from nucleases. Using a luciferase-expressing plasmid, TPP111-plasmid nano-complexes were rapidly up-taken by cancer cells in vitro and induced strong transfection, comparable to PEI. Colocalization of the nano-complexes and endosomes/lysosomes suggested an endosome-mediated uptake. Using a subcutaneous tumor model, intravenously injected nano-complexes preferentially accumulated to the tumor area over 24 h. CONCLUSION: These results indicate that we successfully synthesized the TPP111 nanocarrier system, which can deliver nucleic acids in vitro and in vivo and merits further evaluation.

In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC.

Studies related to lung cancer have shown a link between human epidermal growth factor receptor-2 (HER2) expression and poor prognosis in patients with non-small cell lung cancer (NSCLC). HER2 overexpression has been observed in 3-38% of NSCLC, while strong HER2 protein overexpression is found in 2.5% of NSCLC. However, HER2 dimerization is important in lung cancer, including EGFR mutated NSCLC. Since HER2 dimerization leads to cell proliferation, targeting the dimerization of HER2 will have a significant impact on cancer therapies. A peptidomimetic has been designed that can be used as a therapeutic agent for a subset of NSCLC patients overexpressing HER2 or possessing HER2 as well as EGFR mutation. A cyclic peptidomimetic (18) has been designed to inhibit protein-protein interactions of HER2 with its dimerization partners EGFR and HER3. Compound 18 exhibited antiproliferative activity in HER2-positive NSCLC cell lines at nanomolar concentrations. Western blot analysis showed that 18 inhibited phosphorylation of HER2 and Akt in vitro and in vivo. Stability studies of 18 at various temperature and pH (pH 1 and pH 7.6), and in the presence of liver microsomes indicated that 18 was stable against thermal and chemical degradation. Pharmacokinetic parameters were evaluated in nude mice by administrating single doses of 4 mg/kg and 6 mg/kg of 18 via IV. The anticancer activity of 18 was evaluated using an experimental metastasis lung cancer model in mice. Compound 18 suppressed the tumor growth in mice when compared to control. A proximity ligation assay further proved that 18 inhibits HER2:HER3 and EGFR: HER2 dimerization. Overall, these results suggest that 18 can be a potential treatment for HER2-dimerization related NSCLC.

(-)-Oleocanthal as a Dual c-MET-COX2 Inhibitor for the Control of Lung Cancer.

Lung cancer (LC) represents the topmost mortality-causing cancer in the U.S. LC patients have overall poor survival rate with limited available treatment options. Dysregulation of the mesenchymal epithelial transition factor (c-MET) and cyclooxygenase 2 (COX2) initiates aggressive LC profile in a subset of patients. The Mediterranean extra-virgin olive oil (EVOO)-rich diet already documented to reduce multiple malignancies incidence. (-)-Oleocanthal (OC) is a naturally occurring phenolic secoiridoid exclusively occurring in EVOO and showed documented anti-breast and other cancer activities via targeting c-MET. This study shows the novel ability of OC to suppress LC progression and metastasis through dual targeting of c-MET and COX-2. Western blot analysis and COX enzymatic assay showed significant reduction in the total and activated c-MET levels and inhibition of COX1/2 activity in the lung adenocarcinoma cells A549 and NCI-H322M, in vitro. In addition, OC treatment caused a dose-dependent inhibition of the HGF-induced LC cells migration. Daily oral treatment with 10 mg/kg OC for 8 weeks significantly suppressed the LC A549-Luc progression and prevented metastasis to brain and other organs in a nude mouse tail vein injection model. Further, microarray data of OC-treated lung tumors showed a distinct gene signature that confirmed the dual targeting of c-MET and COX2. Thus, the EVOO-based OC is an effective lead with translational potential for use as a prospective nutraceutical to control LC progression and metastasis.

Pseurotin A as a novel suppressor of hormone dependent breast cancer progression and recurrence by inhibiting PCSK9 secretion and interaction with LDL receptor.

Hypercholesterolemia has been documented to drive hormone-dependent breast cancer (BC) progression and resistance to hormonal therapy. Proprotein convertase subtilisin/kexin type-9 (PCSK9) regulates cholesterol metabolism through binding to LDL receptor (LDLR) and targeting the receptor for lysosomal degradation. Inhibition of PCSK9 is an established strategy to treat hypercholesterolemia. Pseurotin A (PS) is a unique spiro-heterocyclic γ-lactam alkaloid isolated from the fungus Aspergillus fumigatus. Preliminary studies indicated that PS lowered PCSK9 secretion in cultured HepG2 hepatocellular carcinoma cells, with an IC50 value of 1.20 µM. Docking studies suggested the ability of PS to bind at the PCSK9 narrow interface pocket that accommodates LDLR. Surface plasmon resonance (SPR) showed PS ability to inhibit the PCSK9-LDLR interaction at a concentration range of 10-150 µM. PS showed in vitro dose-dependent reduction of PCSK9, along with increased LDLR levels in hormone-dependent BT-474 and T47D breast cancer (BC) cell lines. In vivo, daily oral 10 mg/kg PS suppressed the progression of the hormone-dependent BT-474 BC cells in orthotopic nude mouse xenograft model. Immunohistochemistry (IHC) investigation of BT-474 breast tumor tissue proved the PS ability to reduce PCSK9 expression. PS also effectively suppressed BT-474 BC cells locoregional recurrence after primary tumor surgical excision. Western blot analysis showed decreased PCSK9 expression in liver tissues of PS-treated mice compared to vehicle-treated control group. PS treatment significantly reduced PCSK9 expression and normalized LDLR levels in collected primary and recurrent breast tumors at the study end. PS-treated mice showed reduced plasma cholesterol and 17ß-estradiol levels. Inhibition of tumor recurrence was associated with significant reductions in plasma level of the human BC recurrence marker CA 15-3 in treated mice at the study end. Histopathological examination of various PS-treated mice organs indicated lack of metastatic tumor cells and any pathological changes. The results of this study provide the first evidence for the suppression of the hormone-dependent breast tumor progression and recurrence by targeting the PCSK9-LDLR axis. PS is a novel first-in-class PCSK9-targeting lead appropriate for the use to control hormone-dependent BC progression and recurrence.

Targeting EGFR Overexpression at the Surface of Colorectal Cancer Cells by Exploiting Amidated BODIPY-Peptide Conjugates.

Three BODIPY-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) at the extracellular domain were synthesized, and their specificity for binding to EGFR was investigated. Peptide sequences containing seven amino acids, GLARLLT (2) and KLARLLT (4), and 13 amino acids, GYHWYGYTPQNVI (3), were conjugated to carboxyl BODIPY dye (1) by amide bond formation in up to 73% yields. The BODIPY-peptide conjugates and their "parent" peptides were determined to bind to EGFR experimentally using SPR analysis and were further investigated using computational methods (AutoDock). Results of SPR, competitive binding and docking studies propose that conjugate 6 including the GYHWYGYTPQNVI sequence binds to EGFR more effectively than conjugates 5 and 7, bearing the smaller peptide sequences. Findings in human carcinoma HEp2 cells overexpressing EGFR showed nontoxic behavior in the presence of activated light (1.5 J cm-2 ) and in the absence of light for all BODIPYs. Furthermore, conjugate 6 showed about five-fold higher accumulation within HEp2 cells compared with conjugates 5 and 7, localizing preferentially in the cell ER and lysosomes. Our findings suggest that BODIPY-peptide conjugate 6 is a promising contrast agent for detection of colorectal cancer and potentially other EGFR-overexpressing cancers.

Genotype Driven Therapy for Non-Small Cell Lung Cancer: Resistance, Pan Inhibitors and Immunotherapy.

Eighty-five percent of patients with lung cancer present with Non-small Cell Lung Cancer (NSCLC). Targeted therapy approaches are promising treatments for lung cancer. However, despite the development of targeted therapies using Tyrosine Kinase Inhibitors (TKI) as well as monoclonal antibodies, the five-year relative survival rate for lung cancer patients is still only 18%, and patients inevitably become resistant to therapy. Mutations in Kirsten Ras Sarcoma viral homolog (KRAS) and epidermal growth factor receptor (EGFR) are the two most common genetic events in lung adenocarcinoma; they account for 25% and 20% of cases, respectively. Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor tyrosine kinase, and ALK rearrangements are responsible for 3-7% of NSCLC, predominantly of the adenocarcinoma subtype, and occur in a mutually exclusive manner with KRAS and EGFR mutations. Among drug-resistant NSCLC patients, nearly half exhibit the T790M mutation in exon 20 of EGFR. This review focuses on some basic aspects of molecules involved in NSCLC, the development of resistance to treatments in NSCLC, and advances in lung cancer therapy in the past ten years. Some recent developments such as PD-1-PD-L1 checkpoint-based immunotherapy for NSCLC are also covered.

Synthesis, Characterization, and Evaluation of Near-IR Boron Dipyrromethene Bioconjugates for Labeling of Adenocarcinomas by Selectively Targeting the Epidermal Growth Factor Receptor.

A series of five boron dipyrromethene (BODIPY) bioconjugates containing an epidermal growth factor receptor (EGFR)-targeted pegylated LARLLT peptide and/or a glucose or biotin ethylene diamine group were synthesized, and the binding capability of the new conjugates to the extracellular domain of EGFR was investigated using molecular modeling, surface plasmon resonance, fluorescence microscopy, competitive binding assays, and animal studies. The BODIPY conjugates with a LARLLT peptide were found to bind specifically to EGFR, whereas those lacking the peptide bound weakly and nonspecifically. All BODIPY conjugates showed low cytotoxicity (IC50 > 94 µM) in HT-29 cells, both in the dark and upon light activation (1.5 J/cm2). Studies of nude mice bearing subcutaneous human HT-29 xenografts revealed that only BODIPY conjugates bearing the LARLLT peptide showed tumor localization 24 h after intravenous administration. The results of our studies demonstrate that BODIPY bioconjugates bearing the EGFR-targeting peptide 3PEG-LARLLT show promise as near-IR fluorescent imaging agents for colon cancers overexpressing EGFR.

An N6-methyladenosine at the transited codon 273 of p53 pre-mRNA promotes the expression of R273H mutant protein and drug resistance of cancer cells.

Mutant p53 proteins that promote cancer cell invasive growth, metastasis and drug resistance emerge as therapeutic targets. Previously, we reported that suppression of ceramide glycosylation restored wild-type p53 protein and tumor suppressing function in cancer cells heterozygously carrying p53 R273H, a hot-spot missense mutation; however, the mechanisms underlying the control of mutant protein expression remain elusive. Herein, we report that an N6-methyladenosine (m6A) at the point-mutated codon 273 (G > A) of p53 pre-mRNA determines the mutant protein expression. Methylation of the transited adenosine was catalyzed by methyltransferase like 3 (METTL3), and this m6A-RNA promoted a preferential pre-mRNA splicing; consequently, the produced p53 R273H mutant protein resulted in acquired multidrug resistance in colon cancer cells. Furthermore, glycosphingolipids (particularly globotriaosylceramide) generated from serial ceramide glycosylation were seen to activate cSrc and ß-catenin signaling so as to upregulate METTL3 expression, in turn promoting expression of p53 R273H mutant protein, with consequent drug resistance. Conversely, either silencing METTL3 expression by using small interfering RNA (siRNA) or inhibiting RNA methylation with neplanocin A suppressed m6A formation in p53 pre-mRNA, and substantially increased the level of phosphorylated p53 protein (Ser15) and its function in cells heterozygously carrying the R273H mutation, thereby re-sensitizing these cells to anticancer drugs. Concordantly, suppression of ceramide glycosylation repressed METTL3 expression and m6A formation in p53 pre-mRNA, thus sensitizing cells carrying R273H to anticancer drugs. This study uncovers a novel function of pre-mRNA m6A as a determinant of mutant protein expression in cancer cells heterozygously carrying the TP53 R273H mutation. Suppressing both RNA methylation and ceramide glycosylation might constitute an efficacious and specific approach for targeting TP53 missense mutations coding for a G > A transition, thereby improving cancer treatments.

Design of cyclic and d-amino acids containing peptidomimetics for inhibition of protein-protein interactions of HER2-HER3.

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2-positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2-mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18) exhibited antiproliferative activity in HER2-overexpressing lung cancer cell lines with IC50 values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d-amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18, compound 32, a cyclic, d-amino acid-containing peptidomimetic, was found to have an IC50 value in the nanomolar range in HER2-overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d-amino acid analogs of 18, compound 32, binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.

Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a ß-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide.

A peptidomimetic with a chiral switch is an inhibitor of epidermal growth factor receptor heterodimerization.

Among different types of EGFR dimers, EGFR-HER2 and HER2-HER3 are well known in different types of cancers. Targeting dimerization of EGFR will have a significant impact on cancer therapies. A symmetric peptidomimetic was designed to inhibit the protein-protein interaction of EGFR. The peptidomimetic (Cyclo(1,10)PpR (R) Anapa-FDDF-(R)-Anapa)R, compound 18) was shown to exhibit antiproliferative activity with an IC50 of 194 nM in HER2-expressing breast cancer cell lines and 18 nM in lung cancer cell lines. The peptidomimetic has a Pro-Pro sequence in the structure to stabilize the ß-turn and a ß-amino acid, amino napthyl propionic acid. To investigate the effect of the chirality of ß-amino acid on the structure of the peptide and its antiproliferative activity, diastereoisomers of compound 18 were designed and synthesized. Structure-activity relationships of these compounds indicated that there is a chiral switch at ß-amino acid in the designed compound. The peptidomimetic with R configuration at ß-amino acid and with a L-Pro-D-Pro sequence was the most active compound (18). Using enzyme complement fragmentation assay and proximity ligation assay, we show that compound 18 inhibits HER2:HER3 and EGFR:HER2 dimerization. Surface plasmon resonance studies suggested that compound 18 binds to the HER2 extracellular domain and in particular to domain IV. The anticancer activity of compound 18 was evaluated using a xenograft model of breast cancer in mice; compound 18 suppressed the tumor growth in mice compared to control. Compound 18 was also shown to have a synergistic effect with erlotinib on EGFR mutated lung cancer cell lines.