An analogue of the antibiotic teicoplanin prevents flavivirus entry in vitro.

There is an urgent need for potent inhibitors of dengue virus (DENV) replication for the treatment and/or prophylaxis of infections with this virus. We here report on an aglycon analogue of the antibiotic teicoplanin (code name LCTA-949) that inhibits DENV-induced cytopathic effect (CPE) in a dose-dependent manner. Virus infection was completely inhibited at concentrations that had no adverse effect on the host cells. These findings were corroborated by quantification of viral RNA levels in culture supernatant. Antiviral activity was also observed against other flaviviruses such as the yellow fever virus and the tick-borne encephalitis virus (TBEV). In particular, potent antiviral activity was observed against TBEV. Time-of-drug-addition experiments indicated that LCTA-949 inhibits an early stage in the DENV replication cycle; however, a virucidal effect was excluded. This observation was corroborated by the fact that LCTA-949 lacks activity on DENV subgenomic replicon (that does not encode structural proteins) replication. Using a microsopy-based binding and fusion assay employing DiD-labeled viruses, it was shown that LCTA-949 targets the early stage (binding/entry) of the infection. Moreover, LCTA-949 efficiently inhibits infectivity of DENV particles pre-opsonized with antibodies, thus potentially also inhibiting antibody-dependent enhancement (ADE). In conclusion, LCTA-949 exerts in vitro activity against several flaviviruses and does so (as shown for DENV) by interfering with an early step in the viral replication cycle.Fil: De Burghgraeve, Tine. Katholikie Universiteit Leuven; BélgicaFil: Kaptein, Suzanne J. F.. Katholikie Universiteit Leuven; BélgicaFil: Ayala Nunez, Nilda V.. University of Groningen; Países BajosFil: Mondotte, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pastorino, Boris. Université de la Méditerranée; FranciaFil: Printsevskaya, Svetlana S.. Russian Academy of Medical Sciences; RusiaFil: de Lamballerie, Xavier. Université de la Méditerranée; FranciaFil: Jacobs, Michael. Royal Free & University College Medical School; Reino UnidoFil: Preobrazhenskaya, Maria. Russian Academy of Medical Sciences; RusiaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Smit, Jolanda M.. University of Groningen; Países BajosFil: Neyts, Johan. Katholikie Universiteit Leuven; Bélgic

Dose-dependent inhibition of flavivirus replication by LCTA-949 and ribavirin.

<p>Vero-B cell cultures infected with DENV-2 (panels A and B) or YFV-17D (panels C and D) were treated with different concentrations of LCTA-949 (panels A and C) or ribavirin (panels B and D). Viral RNA levels were quantified on day 4 p.i. by means of RNA RT-qPCR (bars). Mock-infected cells were treated with the same dilution series of LCTA-949 or ribavirin. Cell viability was determined by the MTS/PMS method (lines). Data represent mean values ± standard deviations (SD) for three independent experiments.</p

Dose-dependent inhibition of virus-induced CPE formation by LCTA-949 and effect of LCTA-949 on flavivirus protein expression.

<p>A: Vero-B cell cultures infected with DENV-2 were treated with different concentrations of LCTA-949. CPE formation was monitored at day 8 p.i. B: Vero-B cell cultures (panels A and D) were treated with 12.5 µM LCTA-949 (panels C and F) and infected with DENV-2 (panels B and C) or YFV-17D (panels E and F). DENV-2 E protein and YFV-17D NS1 protein expression was visualized on day 3 p.i.</p

<i>In vitro</i> antiviral effect of LCTA-949 against selected flaviviruses.

*<p>Data are mean values ± standard deviations (SD) for three independent experiments.</p><p>EC₅₀: 50% effective concentration, CC₅₀: 50% cytostatic concentration.</p

LCTA-949 prevents antibody-dependent enhancement of DENV infection.

<p>P388D1 cells were infected with DENV pre-opsonized with anti-E mAb DV2-104 at an MOI of 10 in presence of increasing concentrations of LCTA-949. Anti-E DV2-104 was used at a concentration of 400 ng/ml. The percentage of inhibition of infection after LCTA-949 treatment was calculated with respect to the positive control of untreated opsonized virus. Data are expressed as the means ± SD from two separate experiments, each of which was carried out in duplicate.</p

Time-of-drug-addition assay.

<p>LCTA-949 [10 µM] was added to DEN reporter virus (A, circles) or YFV-17D-infected cultures (A, squares) at 2-h intervals starting 2 hours before infection. The luciferase activity (DEN reporter virus) or the intracellular viral RNA content (YFV-17D) was measured at 24 hours p.i. Values were standardized to untreated virus controls. The data represent averages ± SD for 3 independent experiments. B: Similar conditions for YFV as in A, but time-intervals are now 20 min, starting 60 min before infection. The data represent mean values ± SD for 2 independent experiments. C: Effect of LCTA-949 on protein expression at 2-h intervals starting 2 hours before infection. DENV-2 E protein expression was visualized on day 3 p.i.</p