Etude de l'activité antivirale d'extraits d'Euphorbia de Corse : recherche de nouveaux diterpènes d'intérêt biologique

摘要

Chikungunya fever is caused by an arthropod-borne virus that is associated with massive epidemics and severe morbidity (virus-induced arthralgia, fever, myalgia and rashes). Worldwide expansion of the mosquito vectors, such as Aedes albopictus ("Tiger moquito) is responsible for the spread of Chikungunya virus (CHIKV) throughout the world. A. albopictus has spread throughout Mediterranean areas, which could lead to epidemics outbreaks. Currently, no antiviral drugs or vaccines are available for the treatment or prevention of CHIKV infection. Since ten years, however, recent results showed that diterpene esters from Trignostemon (Euphorbiaceae) possess inhibiting activity of CHIKV replication.With the objective to discover new compounds with antiviral activities, 45 extracts from various plant parts of 11 Euphorbiaceae species native to Corsica were evaluated for selective inhibition of CHIKV replication. In collaboration with Dr. Leyssen (KU Leuven, Belgium), several extracts made from 10 Euphorbia species exhibited significant and selective anti-CHIKV activity in a virus-cell-based assay. The antiviral activities of 29 commercially available phorboïds were studied. Some phorboïds were potent inhibitors of CHIKV and human immunodeficiency virus (VIH) replication. Results allowed drawing new structure-activity relationships, which supported the hypothesis that PKC may be an important target in CHIKV replication. In order to confirm or infirm the presence of phorboïds with anti-CHIKV activity in Euphorbia extracts, a liquid chromatography (LC) coupled to linear ion trap mass spectrometry (MSn) method was developed using standard compounds. Application of this methodology indicated that none anti-CHIKV phorboïds was present in Euphorbia extracts. A second LC-MSn procedure was developed to profile untargeted phorboïdes. Results suggested that numerous other diterpene esters were present in the Euphorbia extracts. The species Euphorbia amygdaloides ssp. semiperfoliata was selected to perform a bioassay-guided purification procedure, which led to the isolation and identification of 14 jatrophane esters, including eight new components. Among them, antiviral evaluation indicated that one jatrophane ester was possessing anti-CHIKV and anti-HIV activities. Furthermore, the structure of an atypical jatrophane ester derivative, jatrohemiketal, was determined unambiguously through an original strategy combining NMR spectroscopy and molecular modeling. Finally, an original tandem mass spectrometry (MS/MS)-targeted supercritical fluid chromatography (SFC) method was developed and used to study bioactive fractions of E. amygdaloïdes ssp. semiperfoliata. The MS/MS data were analyzed by molecular networking. Thanks to this approach, four 4-deoxyphorbol esters and two new jatrophane esters were targeted, isolated and identified. Collaboration with the group of Pr. Alcami (CNM, Espagne) was started to explore the anti-HIV properties of the isolated diterpene esters. Thus, one compound derived from 4-deoxyphorbol esters proved to possess a potent inhibiting activity of HIV-1 replication (IC50 = 8 nM, and selectivity index > 6250). The investigation of the mechanism of this component indicated that it acted like prostratin, but with antiviral effect more than 28-fold. Furthermore, the evaluation of the anti-CHIKV activity indicated that another 4-deoxyphorbol derivative was one of the strongest inhibitor of CHIKV replication isolated up to date (EC50 = 0.34 ± 0.12 µM and selectivity index > 638).