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.

Conformationally constrained cyclic grafted peptidomimetics targeting protein-protein interactions.

Sunflower trypsin inhibitor-1 (SFTI-1) structure is used for designing grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro-Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI-DBF adopts a twisted beta-strand structure in solution. The surface plasmon resonance analysis showed that SFTI-DBF binds to CD58 protein. A model for the protein-SFTI-DBF complex was proposed based on docking studies. These studies suggested that SFTI-1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI-1 grafting.

Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein.

The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.

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.

Targeting protein-protein interaction for immunomodulation: A sunflower trypsin inhibitor analog peptidomimetic suppresses RA progression in CIA model.

Protein-protein interactions (PPI) of co-stimulatory molecules CD2-CD58 are important in the early stage of an immune response, and increased expression of these co-stimulatory molecules is observed in the synovial region of joints in rheumatoid arthritis (RA) patients. A CD2 epitope region that binds to CD58 was grafted on to sunflower trypsin inhibitor (SFTI) template structure to inhibit CD2-CD58 PPI. The peptide was incorporated with an organic moiety dibenzofuran (DBF) in its structure. The designed peptidomimetic was studied for its ability to inhibit CD2-CD58 interactions in vitro, and its thermal and enzymatic stability was evaluated. Stability studies indicated that the grafted peptidomimetic was stable against trypsin cleavage. In vivo studies using the collagen-induced arthritis (CIA) model in mice indicated that the peptidomimetic was able to slow down the progress of arthritis, an autoimmune disease in the mice model. These studies suggest that with the grafting of organic functional groups in the stable peptide template SFTI stabilizes the peptide structure, and these peptides can be used as a template to design stable peptides for therapeutic purposes.

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.

An antagonist of growth hormone-releasing hormone protects against LPS-induced increase of bronchoalveolar lavage fluid protein concentration.

Growth Hormone-Releasing Hormone (GHRH) is a neuropeptide regulating the release of Growth Hormone (GH) from the anterior pituitary gland, and acts as a growth factor in a diverse variety of tissues. GHRH antagonists (GHRHAnt) have been developed to counteract those events, and the beneficial effects of those peptides toward homeostasis have been associated with anti-inflammatory activities. Our lab is interested in delineating the mechanisms governing endothelial barrier function. Our goal is to establish new grounds on the development of efficient countermeasures against Acute Respiratory Distress Syndrome (ARDS), which has been associated with thousands of deaths worldwide due to COVID-19. Herein we demonstrate in vivo that GHRHAnt suppresses LPS-induced increase in bronchoalveolar lavage fluid (BALF) protein concentration, thus protecting the lungs against edema and inflammation.

Peptides and peptidomimetics as therapeutic agents for Covid-19.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Covid-19 pandemic has caused high morbidity and mortality rates worldwide. Virus entry into cells can be blocked using several strategies, including inhibition of protein-protein interactions (PPIs) between the viral spike glycoprotein and cellular receptors, as well as blocking of spike protein conformational changes that are required for cleavage/activation and fusogenicity. The spike-mediated viral attachment and entry into cells via fusion of the viral envelope with cellular membranes involve PPIs mediated by short peptide fragments exhibiting particular secondary structures. Thus, peptides that can inhibit these PPIs may be used as potential antiviral agents preventing virus entry and spread. This review is focused on peptides and peptidomimetics as PPI modulators and protease inhibitors against SARS-CoV-2.

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).

Lipidated Peptidomimetic Ligand-Functionalized HER2 Targeted Liposome as Nano-Carrier Designed for Doxorubicin Delivery in Cancer Therapy.

The therapeutic index of chemotherapeutic agents can be improved by the use of nano-carrier-mediated chemotherapeutic delivery. Ligand-targeted drug delivery can be used to achieve selective and specific delivery of chemotherapeutic agents to cancer cells. In this study, we prepared a peptidomimetic conjugate (SA-5)-tagged doxorubicin (Dox) incorporated liposome (LP) formulation (SA-5-Dox-LP) to evaluate the targeted delivery potential of SA-5 in human epidermal growth factor receptor-2 (HER2) overexpressed non-small-cell lung cancer (NSCLC) and breast cancer cell lines. The liposome was prepared using thin lipid film hydration and was characterized for particle size, encapsulation efficiency, cell viability, and targeted cellular uptake. In vivo evaluation of the liposomal formulation was performed in a mice model of NSCLC. The cell viability studies revealed that targeted SA-5-Dox-LP showed better antiproliferative activity than non-targeted Dox liposomes (Dox-LP). HER2-targeted liposome delivery showed selective cellular uptake compared to non-targeted liposomes on cancer cells. In vitro drug release studies indicated that Dox was released slowly from the formulations over 24 h, and there was no difference in Dox release between Dox-LP formulation and SA-5-Dox-LP formulation. In vivo studies in an NSCLC model of mice indicated that SA-5-Dox-LP could reduce the lung tumors significantly compared to vehicle control and Dox. In conclusion, this study demonstrated that the SA-5-Dox-LP liposome has the potential to increase therapeutic efficiency and targeted delivery of Dox in HER2 overexpressing cancer.

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.

A grafted peptidomimetic for EGFR heterodimerization inhibition: Implications in NSCLC models.

Among the lung cancers, approximately 85% are histologically classified as non-small-cell lung cancer (NSCLC), a leading cause of cancer deaths worldwide. Epidermal growth factor receptors (EGFRs) are known to play a crucial role in lung cancer. HER2 overexpression is detected by immunohistochemistry in 2.4%-38% of NSCLC samples. EGFRs have been targeted with three generations of tyrosine kinase inhibitors (TKIs), and drug resistance has become a major issue; HER2 dimerization with EGFR also plays a major role in the development of resistance to TKI therapy. We have designed grafted peptides to bind to the HER2 extracellular domain (ECD) and inhibit protein-protein interactions of EGFR:HER2 and HER2:HER3. A sunflower trypsin inhibitor (SFTI-1) template was used to graft a peptidomimetic compound. Among several grafted peptides, SFTI-G5 exhibited antiproliferative activity in HER2-positive NSCLC cell lines such as Calu-3 cells with an IC50 value of 0.073 µM. SFTI-G5 was shown to bind to ECD of HER2 and inhibit EGFR:HER2 and HER2:HER3 dimerization and inhibit the phosphorylation of HER2 and downstream signaling proteins. As a proof-of-concept, the in vivo activity of SFTI-G5 was evaluated in two NSCLC mouse models. SFTI-G5 was able to inhibit tumor growth in both models. Furthermore, SFTI-G5 was shown to inhibit EGFR dimerization in tissue samples obtained from in vivo models. These grafted peptides can be used as novel dual inhibitors of EGFR dimerization in NSCLC.

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.

Cancer Vaccines, Treatment of the Future: With Emphasis on HER2-Positive Breast Cancer.

Breast cancer is one of the leading causes of death in women. With improvements in early-stage diagnosis and targeted therapies, there has been an improvement in the overall survival rate in breast cancer over the past decade. Despite the development of targeted therapies, tyrosine kinase inhibitors, as well as monoclonal antibodies and their toxin conjugates, all metastatic tumors develop resistance, and nearly one-third of HER2+ breast cancer patients develop resistance to all these therapies. Although antibody therapy has shown promising results in breast cancer patients, passive immunotherapy approaches have limitations and need continuous administration over a long period. Vaccine therapy introduces antigens that act on cancer cells causing prolonged activation of the immune system. In particular, cancer relapse could be avoided due to the presence of a longer period of immunological memory with an effective vaccine that can protect against various tumor antigens. Cancer vaccines are broadly classified as preventive and therapeutic. Preventive vaccines are used to ward off any future infections and therapeutic vaccines are used to treat a person with active disease. In this article, we provided details about the tumor environment, different types of vaccines, their advantages and disadvantages, and the current status of various vaccine candidates with a focus on vaccines for breast cancer. Current data indicate that therapeutic vaccines themselves have limitations in terms of efficacy and are used in combination with other chemotherapeutic or targeting agents. The majority of breast cancer vaccines are undergoing clinical trials and the next decade will see the fruitfulness of breast cancer vaccine therapy.

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.

Modulation of co-stimulatory signal from CD2-CD58 proteins by a grafted peptide.

Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.

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.