Search Results (103)

Cutaneous melanoma is an extremely dangerous tumor disease with poor prognosis at advanced stages. Accounting for a small percentage of all skin tumors, malignant melanoma leads the mortality rate in this group of cancers. Clearly, the search for new drugs and therapeutic approaches for the treatment of cutaneous melanoma is a highly pressing issue in modern medicine. In this study, novel recombinant proteins with anti-melanoma activity, called RGD-apoptins, were produced in an E. coli expression system, and their properties were evaluated in human cell models. These chimeric proteins consist of two parts, each tumor-specific. One part of the chimeric molecule is the RGD peptide, which binds to αVβ3 integrins widely expressed on the surface of malignant melanocytes. The other part is the viral protein apoptin, known to induce programmed cell death in tumor cells but not in normal cells. This molecular design aims to enhance the specificity of potential therapeutic agent toward malignant melanoma cells while reducing cytolytic effects on healthy tissue. In a resazurin assay, RGD-apoptins decreased the viability of MeWo human melanoma cells and did not affect the viability of HaCaT human keratinocyte cell line and primary skin fibroblasts. Using an annexin V assay, we confirmed that malignant melanocytes death occurs via apoptosis. Transcriptomic analysis allowed us to dynamically evaluate the spectrum of differentially expressed genes 24 and 48 h after treating melanoma cells with recombinant RGD-apoptin. Full article

Integrin-mediated binding is important for the metastatic dissemination of different types of cancer cells. Snake venom disintegrins obtustatin and echistatin are potent, irreversible, and selective inhibitors of α1β1 and αvβ3 integrins, respectively. Obtustatin is one of the shortest disintegrins yet described, containing 41 amino acids. It has a similar pattern of cysteines to the other disintegrin echistatin but with a KTS motif rather than a classic RGD in its active site. A surface acoustic wave biosensor was applied to prove the molecular recognition of disintegrins by their substrates. The human erythrocyte ghost cells were immobilized at the sensors to allow for the detection of kinetic binding constants of disintegrins compared to the surface of giant unilamellar vesicles (GUVs). Obtustatin binds to the erythrocyte ghost membrane with affinity in the mid-nanomolar range (2.32 × 10^–7 M), and echistatin in the low micromolar range, which indicates specific molecular recognition for both disintegrins, but the higher response for obtustatin. The data directly confirm that disintegrins bind to the erythrocyte ghost membrane, thereby supporting the previously overlooked presence of integrins in red blood cell membranes. Full article

The SARS-CoV-2 spike (S1) protein facilitates viral entry through binding to angiotensin-converting enzyme 2 (ACE2), but it also contains an Arg–Gly–Asp (RGD) motif that may enable interactions with RGD-binding integrins on ACE2-negative cells. Here, we provide quantitative evidence for this alternative binding pathway using a live-cell, label-free resonant waveguide grating (RWG) biosensor. RWG technology allowed us to monitor real-time adhesion kinetics of live cells to RGD-displaying substrates, as well as cell adhesion to S1-coated surfaces. To characterize the strength of the integrin–S1 interaction, we determined the dissociation constant using two complementary approaches. First, we performed a live-cell competitive binding assay on RGD-displaying surfaces, where varying concentrations of soluble S1 were added to cell suspensions. Second, we recorded the adhesion kinetics of cells on S1-coated surfaces and fitted the data using a kinetic model based on coupled ordinary differential equations. By comparing the results from both methods, we estimate that approximately 33% of the S1 molecules immobilized on the Nb₂O₅ biosensor surface are capable of initiating integrin-mediated adhesion. These findings support the existence of an alternative integrin-dependent entry route for SARS-CoV-2 and highlight the effectiveness of label-free RWG biosensing for quantitatively probing virus–host interactions under physiologically relevant conditions without the need of the isolation of the interaction partners from the cells. Full article

Background/Objectives: Tetrastatin, the globular non collagenous (NC1) domain of the α4 chain of collagen IV, was previously demonstrated to inhibit melanoma progression. We identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin and demonstrated its anti-angiogenic activity mediated through αvβ3 and α5β1 binding. As QS-13 peptide was not fully soluble in aqueous solution, we designed new peptides with better water solubility. The present work aimed to investigate the interactions of ten QS-13-derived peptides, exhibiting improved hydro-solubility, with αvβ3 and α5β1 integrins. Methods: Using bioinformatics tools such as GROMACS, VMD, and the Autodock4 suite, we investigated the ability of the substituted peptides to bind αvβ3 and α5β1 integrins in silico. Results: We demonstrated in silico that all substituted peptides were able to bind both integrins at the RGD-binding site and determined their theoretical binding energy. Conclusions: The new soluble peptides should be able to compete with natural integrin ligands such as fibronectin, but also FGF1, FGF2, IGF1, and IGF2. Taken together, these findings suggest that the QS-13-derived peptides are reliable anti-angiogenic and anti-tumor agents. Full article

The opportunistic bacteria Serratia proteamaculans are able to penetrate human cells. It was previously shown that the bacterial surface protein OmpX promotes bacterial adhesion. In addition, infection with bacteria that synthesize the OmpX protein enhances the expression of EGFR and β1 integrin involved in the invasion of S. proteamaculans. Therefore, this work was aimed at determining the mechanism of interaction of S. proteamaculans with receptors of eukaryotic cells. Both integrin-linked kinase (ILK) and EGFR tyrosine kinase have been shown to be involved in the invasion of these bacteria. During infection, EGFR is first phosphorylated at Tyr845, which is carried out by c-Src kinase transmitting a signal from nearby receptors. The S. proteamaculans invasion depends on c-Src and focal adhesion kinase (FAK), which can both transmit a signal between β1 integrin and EGFR and participate in cytoskeletal rearrangements. These bacteria have been shown to interact with integrin not through the RGD binding site, and integrin binding to the RGD peptide enhances adhesion, invasion, and expression of α5 and β1 integrin subunits in response to infection. On the other hand, bacterial adhesion and increased expression of integrins during infection are caused by OmpX. Thus, OmpX interacts with integrins, and the participation of the α5 and β1 integrin subunits in the S. proteamaculans invasion allows us to assume that the receptor of OmpX is α5β1 integrin. Full article

We showed that multiple inflammatory cytokines (e.g., CCL5, CXCL12, CX3CL1, CD40L, and FGF2) bind to the allosteric site (site 2) of integrins, distinct from the classical RGD-binding site (site 1), and allosterically activate integrins. A major inflammatory lipid mediator 25-hydroxycholesterol is known to bind to site 2 and allosterically activates integrins and induces inflammatory signals (e.g., IL-6 and TNF secretion). Thus, site 2 is involved in inflammatory signaling. Neuregulin-1 (NRG1) is known to suppresses the progression of inflammatory diseases, fibrosis, and insulin resistance. But, the mechanism of anti-inflammatory action of NRG1 is unclear. We previously showed that NRG1 binds to the classical RGD-binding site (site 1). Mutating the 3 Lys residues that are involved in site 1 binding (NRG1 3KE mutant) is defective in binding to site 1 and in ErbB3-mediated mitogenic signals. Docking simulation predicted that NRG1 binds to site 2. We hypothesized that NRG1 acts as an antagonist of site 2 and blocks allosteric activation by multiple cytokines. Here, we describe that NRG1 binds to site 2 but does not activate soluble αvβ3 or αIIbβ3 in 1 mM Ca²⁺, unlike inflammatory cytokines. Instead, NRG1 suppressed integrin activation by several inflammatory cytokines, suggesting that NRG1 acts as a competitive inhibitor of site 2. Wild-type NRG1 is not suitable for long-term treatment due to its mitogenicity. We showed that the non-mitogenic NRG1 3KE mutant still bound to site 2 and inhibited allosteric activation of soluble and cell-surface integrins, suggesting that NRG1 3KE may have potential as a therapeutic. Full article

Background/Objectives: Integrin α_Vβ₃ plays a crucial role in tumor angiogenesis and cancer progression, making it a key target for radiolabeled probes used in imaging and therapy. A previously developed probe, [¹²⁵I]bcRGD, exhibited high selectivity for α_Vβ₃ but limited tumor accumulation due to rapid blood clearance. This study aimed to address this issue through two strategies: (1) conjugating albumin-binding molecules to enhance systemic circulation and (2) dimerizing RGD peptides to improve binding affinity via multivalency effects. Methods: Three [¹²⁵I]bcRGD derivatives were synthesized: [¹²⁵I]bcRGD_pal (with palmitic acid), [¹²⁵I]bcRGD_iba (with 4-(p-iodophenyl)butyric acid), and [¹²⁵I]bcRGD_dimer (a dimeric bicyclic RGD peptide). Their physicochemical properties, α_Vβ₃-selectivity, albumin-binding capacity, and biodistribution were assessed in vitro and in vivo using tumor-bearing mice. Tumor models included α_Vβ₃-high U-87 MG and α_Vβ₃-low A549 xenografts. Results: [¹²⁵I]bcRGD_pal and [¹²⁵I]bcRGD_iba exhibited prolonged blood retention (30-fold and 55-fold vs. [¹²⁵I]bcRGD, respectively) and increased tumor accumulation (3.9% ID/g and 3.6% ID/g at 2 h, respectively). Despite improved systemic circulation, tumor-to-blood ratios remained low (<1), indicating limited tumor retention. [¹²⁵I]bcRGD_dimer achieved significantly greater tumor accumulation (4.2% ID/g at 2 h) and favorable tumor-to-blood (22) and tumor-to-muscle (14) ratios, with a 5.4-fold higher uptake in U-87 MG tumors compared to A549 tumors. Conclusions: Dimerization was more effective than albumin binding in enhancing bcRGD’s tumor-targeting potential. The dimeric probe demonstrated improved tumor accumulation, favorable pharmacokinetics, and preserved integrin selectivity. These findings provide a foundation for further structural optimization of bicyclic RGD peptides for integrin α_Vβ₃-targeted imaging and therapy applications. Full article

Integrins, an important superfamily of cell adhesion receptors, play an essential role in cancer progression, metastasis, and angiogenesis, establishing them as prime targets for both diagnostic and therapeutic applications. Despite their significant potential, integrin-targeted therapies have faced substantial challenges in clinical trials, including variable efficacy and unmet high expectations. Nevertheless, the consistent expression of integrins on tumor and stromal cells underscores their ongoing relevance and potential. Traditional RGD-based imaging and therapeutic agents have faced limitations, such as inconsistent target expression and rapid systemic clearance, which have reduced their effectiveness. To overcome these challenges, recent research has focused on advancing RGD-based strategies and exploring innovative solutions. This review offers a thorough analysis of the latest developments in the RGD–integrin field, with a particular focus on addressing previous limitations. It delves into new dual-targeting approaches and cutting-edge RGD-based agents designed to improve both tumor diagnosis and therapeutic outcomes. By examining these advancements, this review illuminates new pathways for enhancing the specificity and efficacy of integrin-targeted therapies, paving the way for more effective cancer diagnosis and treatment strategies. Full article

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca²⁺ ions and inhibited by an excess of Zn²⁺. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics. Full article

Chemotherapy remains the primary therapeutic approach in treating cancer. The tumor microenvironment (TME) is the complex network surrounding tumor cells, comprising various cell types, such as immune cells, fibroblasts, and endothelial cells, as well as ECM components, blood vessels, and signaling molecules. The often stiff and dense network of the TME interacts dynamically with tumor cells, influencing cancer growth, immune response, metastasis, and resistance to therapy. The effectiveness of the treatment of solid tumors is frequently reduced due to the poor penetration of the drug, which leads to attaining concentrations below the therapeutic levels at the site. Cell-penetrating peptides (CPPs) present a promising approach that improves the internalization of therapeutic agents. CPPs, which are short amino acid sequences, exhibit a high ability to pass cell membranes, enabling them to deliver drugs efficiently with minimal toxicity. Specifically, the iRGD peptide, a member of CPPs, is notable for its capacity to deeply penetrate tumor tissues by binding simultaneously integrins ανβ3/ανβ5 and neuropilin receptors. Indeed, ανβ3/ανβ5 integrins are characteristically expressed by tumor cells, which allows the iRGD peptide to home onto tumor cells. Notably, the respective dual-receptor targeting mechanism considerably increases the permeability of blood vessels in tumors, enabling an efficient delivery of co-administered drugs or nanoparticles into the tumor mass. Therefore, the iRGD peptide facilitates deeper drug penetration and improves the efficacy of co-administered therapies. Distinctively, we will focus on the iRGD mechanism of action, drug delivery systems and their application, and deliberate future perspectives in developing iRGD-conjugated therapeutics. In summary, this review discusses the potential of iRGD in overcoming barriers to drug delivery in cancer to maximize treatment efficiency while minimizing side effects. Full article

The efficacy of glioblastoma treatment is closely associated with complete tumor resection. However, conventional surgical techniques often result in incomplete removal, leading to poor prognosis. A major challenge is the accurate delineation of tumor margins from healthy tissues. Imaging-guided surgery, particularly using fluorescent probes, is a promising solution for intraoperative guidance. The recently developed ‘always-on’ types of targeted fluorescence probes generate signals irrespective of their presence in tumor cells or in blood circulation, hampering their effectiveness. Here, we propose a novel activatable fluorescence imaging probe, Q-cRGD, that targets glioma cells via the specific binding of the cyclic Arg-Gly Asp-containing pentapeptide (cRGD) to integrins. The Q-cRGD probe was synthesized by conjugating a near-infrared (NIR) dye to a tryptophan quencher via a disulfide linkage, including a cRGD-targeting ligand. This activatable probe remained inactive until the redox-responsive cleavage of the disulfide linkage occurred within the target cell. The zwitterionic nature of NIR dyes minimizes nonspecific interactions with serum proteins, thereby enhancing the tumor-to-background signal ratio (TBR). An in vivo fluorescence imaging study demonstrated a TBR value of 2.65 within 3 h of the intravenous injection of Q-cRGD, confirming its potential utility in imaging-guided brain cancer surgery. Full article

The key factor that enables pathogenic bacteria to establish successful infections lies largely in their ability to escape the host’s immune response and adhere to host surfaces. Vitronectin (Vn) is a multidomain glycoprotein ubiquitously present in blood and the extracellular matrix of several tissues, where it plays important roles as a regulator of membrane attack complex (MAC) formation and as a mediator of cell adhesion. Vn has emerged as an intriguing target for several microorganisms. Vn binding by bacterial receptors confers protection from lysis resulting from MAC deposition. Furthermore, through its Arg-Gly-Asp (RGD) motif, Vn can bind several host cell integrins. Therefore, Vn recruited to the bacterial cell functions as a molecular bridge between bacteria and host surfaces, where it triggers several host signaling events that could promote bacterial internalization. Each bacterium uses different receptors that recognize specific Vn domains. In this review, we update the current knowledge of Vn receptors of major bacterial pathogens, emphasizing the role they may play in the host upon Vn binding. Focusing on the structural properties of bacterial proteins, we provide details on the residues involved in their interaction with Vn. Furthermore, we discuss the possible involvement of Vn adsorption on biomaterials in promoting bacterial adhesion on abiotic surfaces and infection. Full article

Integrin α_IIbβ₃ mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the α_IIb and β₃ subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to α_IIbβ₃ integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the α_IIb and β₃ subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the β₃ subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin. Full article

HER2-positive breast cancer, characterised by overexpressed HER2 levels, is associated with aggressive tumour behaviour and poor prognosis. Trastuzumab is a standard treatment; however, approximately 50% of patients develop resistance within one year. This study investigates the role of ITGβ3 in promoting stemness and resistance in HER2-positive breast cancer cell lines (HCC1954 and SKBR3). The findings demonstrate that chronic exposure to trastuzumab upregulates stem cell markers (SOX2, OCT4, KLF4, NANOG, SALL4, ALDH, BMI1, Nestin, Musashi 1, TIM3, CXCR4). Given the documented role of RGD-binding integrins in drug resistance and stemness, we specifically investigated their impact on resistant cells. Overexpression of ITGβ3 enhances the expression of these stem cell markers, while silencing ITGβ3 reduces their expression, suggesting a major role for ITGβ3 in maintaining stemness and resistance. Further analysis reveals that ITGβ3 activates the Notch signalling pathway, known for regulating stem cell maintenance. The combination of trastuzumab and cilengitide, an integrin inhibitor, significantly decreases the expression of stem cell markers in resistant cells, indicating a potential therapeutic strategy to overcome resistance. These results identify the importance of ITGβ3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy. Full article

Numerous human adenovirus (AdV) types are endowed with arginine–glycine–aspartic acid (RGD) sequences that enable them to recognize vitronectin-binding (αv) integrins. These RGD-binding cell receptors mediate AdV entry into host cells, a crucial early step in virus infection. Integrin interactions with adenoviruses not only initiate receptor-mediated endocytosis but also facilitate AdV capsid disassembly, a prerequisite for membrane penetration by AdV protein VI. This review discusses fundamental aspects of AdV–host interactions mediated by integrins. Recent efforts to re-engineer AdV vectors and non-viral nanoparticles to target αv integrins for bioimaging and the eradication of cancer cells will also be discussed. Full article