SARS-CoV-2 Spike Protein Unlikely to Bind to Integrins via the Arg-Gly-Asp (RGD) Motif of the Receptor Binding Domain: Evidence From Structural Analysis and Microscale Accelerated Molecular Dynamics.

The Receptor Binding Domain (RBD) of SARS-CoV-2 virus harbors a sequence of Arg-Gly-Asp tripeptide named RGD motif, which has also been identified in extracellular matrix proteins that bind integrins as well as other disintegrins and viruses. Accordingly, integrins have been proposed as host receptors for SARS-CoV-2. However, given that the microenvironment of the RGD motif imposes a structural hindrance to the protein-protein association, the validity of this hypothesis is still uncertain. Here, we used normal mode analysis, accelerated molecular dynamics microscale simulation, and protein-protein docking to investigate the putative role of RGD motif of SARS-CoV-2 RBD for interacting with integrins. We found, that neither RGD motif nor its microenvironment showed any significant conformational shift in the RBD structure. Highly populated clusters of RBD showed no capability to interact with the RGD binding site in integrins. The free energy landscape revealed that the RGD conformation within RBD could not acquire an optimal geometry to allow the interaction with integrins. In light of these results, and in the event where integrins are confirmed to be host receptors for SARS-CoV-2, we suggest a possible involvement of other residues to stabilize the interaction.

DisintegrinDB: The first integrated database resource of disintegrins from snake venoms.

Nowadays, a large number of databases have been developed gathering different types of therapeutic peptides including antimicrobial, antiviral and scorpion toxins peptides facilitating the searching for these molecules and their structural characteristics and pharmacology. Disintegrins, a family of small non-enzymatic and cysteine-rich proteins found in the snake venom may have a potential role in terms of novel therapeutic leads for cancer treatment. Despite their therapeutic effect, no database dedicated to disintegrins is available yet. Indeed, accessible information related to disintegrins are either scattered or fragmented in different databases from which it becomes extremely difficult to collect all the properties related to a particular disintegrin without exploring numerous databases available through distinct websites. Here, we propose DisintegrinDB as a first unique resource centralizing data related to disintegrins from snake venom. DisintegrinDB aims to facilitate the search on a given disintegrin and centralizes all the information on these peptides, helping researchers to retrieve all relevant related information.

The First Snake Venom KTS/Disintegrins-Integrin Interactions Using Bioinformatics Approaches.

Snake venom contains a number of active molecules that have been shown to possess high anti-tumor activities; disintegrins are an excellent example among these. Their ability to interact and bind with integrins suggests that they could be very valuable molecules for the development of new cancer therapeutic approaches. However, in the absence of a clear Lysine-Threonine-Serine (KTS) Disintegrins Integrin interaction model, the exact compound features behind it are still unknown. In this study, we investigated the structural characteristics of three KTS-disintegrins and the interaction mechanisms with the α1ß1 integrin receptor using in silico bioinformatics approaches. Normal mode analysis showed that the flexibility of the KTSR motif and the C-terminal region play a key role and influence the KTS-Disintegrin-integrin interaction. Protein-protein docking also suggested that the interaction involving the KTSR motif is highly dependent on the residue following K21, S23 and R24. These findings contribute to a better understanding of the KTS-Disintegrin-Integrin structural differences and their interactions with α1ß1 receptors, which could improve the selection process of the best active molecules for antitumor therapies.

RK, the first scorpion peptide with dual disintegrin activity on α1ß1 and αvß3 integrins.

Scorpion peptides are well known for their pharmaceutical potential on different targets. These include mainly the ion channels which were found to be highly expressed in many diseases, including cancer, auto-immune pathologies and Alzheimer. So far, however, the disintegrin activity had only been characterized for snake venom molecules. Herein, we present the first short peptide, purified from the venom of Buthus occitanus tunetanus, (termed RK) able to inhibit the cell adhesion of Glioblastoma, Melanoma and Rat pheochromocytoma to different extracellular matrix (ECM) receptors. Anti-integrin antibody assay suggests that RK interacts with both α1ß1 and αvß3 with a more pronounced effect for the former. The examination of the primary structure of RK suggests the involvement of two motifs: KSS, analogue to KTS which was characterized for α1ß1 Snake venom disintegrins, and ECD, analogue to RGD which was found to be active on αvß3. To assess their roles in the disintegrin activity of RK, we conducted a computational analysis. The molecular docking study shows that RK involves mainly two segments to interact with the α1ß1 integrin, but the peptide does not implicate the KSS motif in the interaction. The molecular modeling study, suggests the key contribution of the ECD segment in the interaction with αvß3 integrin.

RK1, the first very short peptide from Buthus occitanus tunetanus inhibits tumor cell migration, proliferation and angiogenesis.

Scorpion toxins have been the subject of many studies which explore their pharmacological potential toward diverse molecular targets, known to monitor key mechanisms in cancer such as proliferation, migration and angiogenesis. The few peptides from scorpion venom that have an anti-tumor effect are generally cytotoxic. Herein, we present the first description of a short 14 amino acid peptide (called RK1), purified from the venom of Buthus occitanus tunetanus, with the particular capabilities, among different other scorpion peptides, to inhibit cell proliferation, migration and angiogenesis of U87 (Glioblastoma) and IGR39 (Melanoma). Moreover, RK1 is a first peptide derived from scorpion venom exhibiting a potential anti-tumoral activity with no manifest toxicity. Our results suggest that, in terms of its primary structure, RK1 is unique compared to a variety of known peptides purified from scorpion venoms. In addition, RK1 is the first natural peptide able to abolish completely the proliferation of cancer cells. The Chicken chorioallantoic membrane model revealed that RK1 strongly inhibits ex-vivo vascular growth. RK1 could open new perspective for the pharmaceutical application of short scorpion venom peptides in anticancer activity and may represent the first member of a new group of scorpion peptides.

A New Protein Extract Inhibitor from Hypobranchial Purple Gland of Hexaplex trunculus, a Mediterranean Mollusk, Impairs the Motility of Human Glioblastoma U87 and the HeLa Cell Line of Cervical Carcinoma Cells.

The aim of this study is to evaluate the effect of hypobranchial gland protein extracts (HGPEs) of Hexaplex trunculus on the viability, cell adhesion, and migration of human U87 glioblastoma cells and the HeLa cell line obtained from epithelial cervical carcinoma cells. Analysis of the HGPE on polyacrylamide gel (12%) shows a variety of proteins whose molecular weights vary between 12 and 1OO kDa. Chromatographic analysis shows 16 peaks obtained at various retention times. Cytotoxic effect was observed after 24 hours of incubation at the concentrations 20, 40, and 60 µg/ml in a dose-dependent manner. Concentrations giving 50% inhibition (IC50) are 22 µg/ml for U87 and 15 µg/ml for HeLa cells. Our results show inhibition of U87 and HeLa cancer cell adhesion at concentrations of 10 and 20 µg/ml, respectively. High-pressure liquid chromatography fractions did not show antiadhesive effect on both cancer cell lines. The presence of HGPEs completely blocked the migration of the two cancer cell lines at 10 µg/ml. This inhibition is dose-dependent. IC50 is about 2.5 µg/ml for both cancer cells. The HGPE of Hexaplex trunculus may have the potential to serve as a model for future anticancer drug development with probably a synergistic activity of the proteins of this extract.

Hyalomma dromedarii (Acari: Ixodidae) Salivary Gland Extract Inhibits Angiogenesis and Exhibits In Vitro Antitumor Effects.

Hard ticks (Acari: Ixodidae) are blood-sucking ectoparasites characterized by the extended period of their attachment to their host. To access their bloodmeal, ticks secrete saliva containing a range of molecules that target the host's inflammation, immune system, and hemostatic components. Some of these molecules reportedly possess antiangiogenic and antitumor properties. The present study describes our investigation, the first of its kind, of the antiangiogenic and antitumoral effects of the Hyalomma dromedarii Koch, 1844 (Acari: Ixodidae), salivary gland extract (SGE), which inhibited the adhesion and migration of Human Umbilical Vein Endothelial Cells (HUVECs) in a dose-dependent manner, as well as angiogenesis in the Chick Chorioallantoic Membrane model. Interestingly, H. dromedarii SGE exerted an antiproliferative effect on U87 glioblastoma cells and inhibited their adhesion and migration to fibrinogen. These results open up new possibilities for characterizing and developing new molecules involved in the key steps of tumor progression.

Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli.

Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).

TRAIL receptor gene editing unveils TRAIL-R1 as a master player of apoptosis induced by TRAIL and ER stress.

TRAIL induces selective tumor cell death through TRAIL-R1 and TRAIL-R2. Despite the fact that these receptors share high structural homologies, induction of apoptosis upon ER stress, cell autonomous motility and invasion have solely been described to occur through TRAIL-R2. Using the TALEN gene-editing approach, we show that TRAIL-R1 can also induce apoptosis during unresolved unfolded protein response (UPR). Likewise, TRAIL-R1 was found to co-immunoprecipitate with FADD and caspase-8 during ER stress. Its deficiency conferred resistance to apoptosis induced by thaspigargin, tunicamycin or brefeldin A. Our data also demonstrate that tumor cell motility and invasion-induced by TRAIL-R2 is not cell autonomous but induced in a TRAIL-dependant manner. TRAIL-R1, on the other hand, is unable to trigger cell migration owing to its inability to induce an increase in calcium flux. Importantly, all the isogenic cell lines generated in this study revealed that apoptosis induced TRAIL is preferentially induced by TRAIL-R1. Taken together, our results provide novel insights into the physiological functions of TRAIL-R1 and TRAIL-R2 and suggest that targeting TRAIL-R1 for anticancer therapy is likely to be more appropriate owing to its lack of pro-motile signaling capability.

CC5 and CC8, two homologous disintegrins from Cerastes cerastes venom, inhibit in vitro and ex vivo angiogenesis.

Angiogenesis constitutes a fundamental step in tumor progression. Thus, targeting tumour angiogenesis has been identified to be promising in cancer treatment. In this work, CC5 and CC8, two highly homologous disintegrins isolated from the venom Cerastes cerastes viper from the south of Tunisia, were assessed for their anti-angiogenic effect by testing their ability to interfere with viability, adhesion, migration and angiogenesis of Human Microvascular Endothelial Cells, HMEC-1 and HBMEC. We found that CC5 and CC8 displayed pro-apoptotic potential in HMEC-1 cells. Anoïkis like induced by these two disintegrins was evidenced by cell detachment, down regulation of FAK/AKT/PI3K axis and caspase activation. In addition, both CC5 and CC8 exhibited in vitro anti-adhesive, anti-migratory and anti-proliferative effects on endothelial cells HBMEC. These effects appeared to require RGD and/or WGD loops disintegrin. CC5 and CC8 also inhibited tube-formation on matrigel and displayed potent anti-angiogenic activities as assessed ex vivo, using both the embryo chick chorioallantoic membrane model (CAM) and rat aortic ring assay. Altogether our results demonstrate that CC5 and CC8, are potent inhibitors of angiogenesis, by disrupting αvß3 and α5ß1 binding. The use of CC5 and/or CC8 could provide a beneficial tool to inhibit abnormal angiogenesis and to induce cancer regression.