Design, synthesis and purification of analogues of animal venom molecules with cancer cell inhibitory action

Le glioblastome multiforme (GBM) demeure le cancer le plus agressif des cancers du cerveau. Malgré les progrès réalisés durant les dernières décennies dans la compréhension de la pathogenèse du GBM, les thérapies disponibles ne sont pas efficaces vis à vis de la malignité de la maladie, d’où la nécessité de trouver de nouveaux traitements plus efficaces. L’exploitation du venin de scorpion tunisien Androctonus australis hector a permis d’isoler deux molécules de petites tailles H8 et AaTs-1 dotées d’activités anticancéreuses sur les cellules U87 du glioblastome. H8 agit sur la migration de ces cellules. L’exploration de la structure de ce peptide a donné 5 séquences possibles que nous avons synthétisées et testé leurs effets sur ces mêmes cellules. Un, parmi les cinq peptides synthétisés, a montré une activité proche du peptide natif mais toujours moins efficace. Ainsi nous avons conclu qu’aucune des molécules synthétisées ne correspond à H8. AaTs-1 est un tetrapeptide qui possède une activité antiproliférative sur les cellules U87 avec une IC50 de l’ordre de 0,5 mM. Afin d’augmenter l’efficacité de ce tetrapeptide, nous avons modifié sa structure en synthétisant des dendrimères multi-branchés AaTs-1-2B, AaTs-1-4B et AaTs-1-8B. Tous les dendrimères synthétisés ont montré une meilleure activité, qui augmente avec le nombre des branches, sur la prolifération des cellules U87. AaTs-1-4B et AaTs-1-8B ont aussi montré un effet inhibiteur sur la migration des cellules U87. L’étude du mécanisme d’action de AaTs-1-8B a montré qu’il agit en diminuant le taux calcique intracellulaire et en augmentant la phosphorylation des protéines ERK1/2 et AKT et l’expression de P53.Glioblastoma multiforme (GBM) remains the most aggressive brain cancers. Despite the progress made in recent decades in understanding the pathogenesis of GBM, the available therapies are not effective against the malignancy of the disease, hence the need to find new and more effective treatments. In this context, the screening of the Tunisian scorpion venom Androctonus australis hector has allowed the identification of two small molecules with anticancer activities on U87 glioblastoma cells. The first native molecule, named H8, acts on the migration of these cells. Structural characterization of this peptide yielded 5 possible sequences that we synthesized and tested for their effects on the same cell line. One of the five peptides synthesized showed activity close to the native peptide but still less effective. Thus we concluded that none of the molecules synthesized corresponds to H8. The second molecule, called AaTs-1, is a tetrapeptide that has an antiproliferative activity on U87 cells with an IC50 of 0.5 mM. In order to increase the efficiency of this tetrapeptide we modified its structure by synthesizing multi-branched dendrimers AaTs-1-2B, AaTs-1-4B and AaTs-1-8B. All synthesized dendrimers have shown better activity, which increases with the number of branches, on the proliferation of U87 cells. Interestingly, the dendrimers AaTs-1-4B and AaTs-1-8B also showed an inhibitory effect on the migration of the U87 cells. The study of the mechanism of action of AaTs-1-8B showed that it acts by decreasing intracellular calcium levels and increasing phosphorylation of ERK1/2 and AKT proteins and P53 protein expression

Expression of the First Recombinant Anti-Tumoral Snake Venom Kunitz-Type Serine Protease Inhibitor

International audiencePIVL is a Kunitz-type serine protease inhibitor that was previously characterized from Tunisian snake venom, Macrovipera lebetina transmediterranea. It reduced glioblastoma cells’ development and significantly blocked angiogenesis in in-vitro and ex-vivo models. PIVL exerted these effects by interfering with αvβ3 integrin. In order to produce a biological active recombinant, the cDNA cloning and expression of PIVL was performed in Escherichia coli (BL21)-DE3 cells using pET-22b (+) vector. The recombinant PIVL protein (rPIVL) was purified by nickel affinity chromatography and has recognized monoclonal anti-His antibody. Functionally, rPIVL exhibited potent anti-tumor cell effects as well as anti-angiogenesis properties. Interestingly, we found that both native PIVL (nPIVL) and rPIVL modulated PI3/AKT and MAPK signaling pathways. In all, our results showed that we have successfully expressed the first active anti-oncogenic snake venom Kunitz-type protease inhibitor that can be a potential therapeutic drug against glioblastoma, in its native or recombinant form

The Synthesis of 2′-Hydroxychalcones under Ball Mill Conditions and Their Biological Activities

International audienceChalcones are polyphenols that belong to the flavonoids family, known for their broad pharmacological properties. They have thus attracted the attention of chemists for their obtention and potential activities. In our study, a library of compounds from 2′-hydroxychalcone’s family was first synthesized. A one-step mechanochemical synthesis via Claisen–Schmidt condensation reaction under ball mill conditions was studied, first in a model reaction between a 5′-fluoro-2′-hydroxyacetophenone and 3,4-dimethoxybenzaldehyde. The reaction was optimized in terms of catalysts, ratio of reagents, reaction time, and influence of additives. Among all assays, we retained the best one, which gave the highest yield of 96% when operating in the presence of 1 + 1 eq. of substituted benzaldehyde and 2 eq. of KOH under two grinding cycles of 30 min. Thus, this protocol was adopted for the synthesis of the selected library of 2′-hydroxychalcones derivatives. The biological activities of 17 compounds were then assessed against Plasmodium falciparum, Leishmania donovani parasite development, as well as IGR-39 melanoma cell lines by inhibiting their viability and proliferation. Compounds 6 and 11 are the most potent against L. donovani, exhibiting IC50 values of 2.33 µM and 2.82 µM, respectively, better than the reference drug Miltefosine (3.66 µM). Compound 15 presented the most interesting antimalarial activity against the 3D7 strain, with IC50 = 3.21 µM. Finally, chalcone 12 gave the best result against IGR-39 melanoma cell lines, with an IC50 value of 12 µM better than the reference drug Dacarbazine (IC50 = 25 µM)

Synthesis and characterization of Ni (II), Cu (II), Fe (II) and Fe 3 O 4 nanoparticle complexes with tetraaza macrocyclic Schiff base ligand for antimicrobial activity and cytotoxic activity against cancer and normal cells

International audienceThis work describes a simple synthesis of complexes of the type [M(C32H28N4)Cl2], where M = Ni (II), Cu (II) and Fe (II) and a novel complex of magnetite nanoparticle (Fe3O4NP) inside (INS) tetraaza macrocyclic Schiff base ligand (C32H28N4): [Fe3O4NP‐INS‐(C32H28N4)], which was prepared by using a novel co‐precipitation method of coordinated ferric ion (Fe3+) in the complex [Fe(C32H28N4)Cl2] under mild conditions. The synthesized compounds were characterized and compared with a various physic‐chemical techniques like: Fourier transform infrared (FT‐IR), ultraviolet–visible spectroscopic techniques (UV–Vis), 1‐dimensional (1D) 1H‐NMR, 13C‐NMR spectroscopic techniques, mass spectra, Powder X‐ray diffraction (PXRD), Vibrating sample magnetometer (VSM), Scanning electron microscopy (SEM), elemental analysis and molar conductance measurements. Furthermore, the highest saturation magnetization was 26.56 emu.g−1 obtained from [Fe3O4NP‐INS‐(C32H28N4)] (diameter of Fe3O4NPs~20.87 nm) that prove easy separation by an external magnetic field. In vitro screening of all the compounds against different species of bacteria and fungi shows that [Fe3O4NP‐INS‐(C32H28N4)] is effective against the tested strains as compared to the tetraaza macrocyclic ligand and selected complexes. The cytotoxic activity of the all compounds was also examined in 3 human tumor cell lines as U87, MDA‐MB‐231 and LS‐174. The complex [Fe3O4NP‐INS‐(C32H28N4)] shows moderate and strong cytotoxic activity against brain cancer, colon cancer and breast cancer (U87, MDA‐MB‐231 and LS‐174 respectively), without showing cytotoxicity towards peripheral blood mononucleocyte (PBMC) cells

Helix aspersa maxima mucus exhibits antimelanogenic and antitumoral effects against melanoma cells

International audienceSnail secretion is currently revolutionizing the world of cosmetics and human skin care. The efficacy of snail secretion in wounds healing has been proven both in vitro and by clinical studies. However, the potential anti-tumor effect of snail secretion was poorly investigated. In this report, our in vitro study showed that Helix aspersa maxima species snail slime (SS) could not only treat melanogenesis but also endowed with anti-tumoral activity against human melanoma cells. Indeed, SS reduced melanin content and tyrosinase activity on B16F10 cells with IC50 values of 288 μg/mL and 286 μg/mL, respectively, without altering cell viability. This effect was also observed, at a lesser extent, on human melanoma IGR-39 and SK-MEL-28 cell lines. On another hand, SS specifically inhibited the viability of IGR-39 and SK-MEL-28 cells associated to an apoptotic effect highlighted by PARP cleavage. It is worth to note that SS did not affect the viability of B16F10 cells and non tumorigenic HaCaT cells. Interestingly, this extract was found to inhibit migration and invasion of both human melanoma cells through reducing the expression of Matrix metalloproteinase MMP2. Snail slime also exerted a high inhibitory effect on IGR-39 cell adhesion through blocking the function of α2β1 (45%), αvβ3 (38%) integrins and by reducing the expression levels of αv and β1 integrins. The presented results shed light on the potential anti-melanoma effect of SS and support its use against skin diseases

Functional role of Kv1.1 and Kv1.3 channels in the neoplastic progression steps of three cancer cell lines, elucidated by scorpion peptides

International audienceVoltage-gated potassium (Kv) channels are known to play a pivotal role in the progression of various cancer types and considered as new targets for designing anti-cancer therapy. However, the fact that many Kv channels are expressed in different cell lines makes it difficult to ascribe a functional role for a given Kv channel on a specific aspect of the tumorogenesis. In this work, we showed that although both Kv1.1 and Kv1.3 channels are expressed in U87 (glioblastoma), MDA-MB-231 (breast cancer) and LS174 (colon adenocarcinoma) cells, these respond differently to KAaH1 or KAaH2, two homologous Kv1 blockers from scorpion venom. KAaH1 is active on Kv1.1 and Kv1.3 and was found to inhibit migration and adhesion of U87 cells whereas KAaH2 which is slightly active only on Kv1.1 channel, inhibits their proliferation via the EGFR signaling pathway. The correlation between the electro-physiological activity of the scorpion peptides and their anti-migratory effects suggests the involvement of the Kv1.1 and Kv1.3 channels in the mobility of the three cancer cell lines. Our results showed that besides they can elucidate the implication of Kv1.1 and Kv1.3 channels in molecular mechanisms of neoplastic progression, KAaH1 and KAaH2 may be used as therapeutic tools against glioblastoma

Strengthening Anti-Glioblastoma Effect by Multi-Branched Dendrimers Design of a Scorpion Venom Tetrapeptide

International audienceGlioblastoma is the most aggressive and invasive form of central nervous system tumors due to the complexity of the intracellular mechanisms and molecular alterations involved in its progression. Unfortunately, current therapies are unable to stop its neoplastic development. In this context, we previously identified and characterized AaTs-1, a tetrapeptide (IWKS) from Androctonus autralis scorpion venom, which displayed an anti-proliferative effect against U87 cells with an IC50 value of 0.57 mM. This peptide affects the MAPK pathway, enhancing the expression of p53 and altering the cytosolic calcium concentration balance, likely via FPRL-1 receptor modulation. In this work, we designed and synthesized new dendrimers multi-branched molecules based on the sequence of AaTs-1 and showed that the di-branched (AaTs-1-2B), tetra-branched (AaTs-1-4B) and octo-branched (AaTs-1-8B) dendrimers displayed 10- to 25-fold higher effects on the proliferation of U87 cells than AaTs-1. We also found that the effects of the newly designed molecules are mediated by the enhancement of the ERK1/2 and AKT phosphorylated forms and by the increase in p53 expression. Unlike AaTs-1, AaTs-1-8B and especially AaTs-1-4B affected the migration of the U87 cells. Thus, the multi-branched peptide synthesis strategy allowed us to make molecules more active than the linear peptide against the proliferation of U87 glioblastoma cells

AaTs-1: A Tetrapeptide from Androctonus australis Scorpion Venom, Inhibiting U87 Glioblastoma Cells Proliferation by p53 and FPRL-1 Up-Regulations

International audienceGlioblastoma is an aggressive cancer, against which medical professionals are still quite helpless, due to its resistance to current treatments. Scorpion toxins have been proposed as a promising alternative for the development of effective targeted glioblastoma therapy and diagnostic. However, the exploitation of the long peptides could present disadvantages. In this work, we identified and synthetized AaTs-1, the first tetrapeptide from Androctonus australis scorpion venom (Aa), which exhibited an antiproliferative effect specifically against human glioblastoma cells. Both the native and synthetic AaTs-1 were endowed with the same inhibiting effect on the proliferation of U87 cells with an IC50 of 0.56 mM. Interestingly, AaTs-1 was about two times more active than the anti-glioblastoma conventional chemotherapeutic drug, temozolomide (TMZ), and enhanced its efficacy on U87 cells. AaTs-1 showed a significant similarity with the synthetic peptide WKYMVm, an agonist of a G-coupled formyl-peptide receptor, FPRL-1, known to be involved in the proliferation of glioma cells. Interestingly, the tetrapeptide triggered the dephosphorylation of ERK, p38, and JNK kinases. It also enhanced the expression of p53 and FPRL-1, likely leading to the inhibition of the store operated calcium entry. Overall, our work uncovered AaTs-1 as a first natural potential FPRL-1 antagonist, which could be proposed as a promising target to develop new generation of innovative molecules used alone or in combination with TMZ to improve glioblastoma treatment response. Its chemical synthesis in non-limiting quantity represents a valuable advantage to design and develop low-cost active analogues to treat glioblastoma cancer