Supplementary data

Figure S1. In vitro inhibition of flavivirus helicases activity by an increasing amount of ivermectin
measured using a FRET-based helicase assay. (a) Percentage of YFV helicase activity (in red) at
different concentrations of the inhibitor (0, 1, 40, 80, 160, 320 and 640 nM); the YFV-double mutant
(Thr413Ile and Asp414Glu, in blue) does not show any activity inhibition at all the concentrations of
Ivermectin tested (up to 5 M, not shown). (b) Percentage of DENV helicase activity (in red) at
different concentrations of the inhibitor (0, 1, 30, 200, 400, 800 and 1000 nM); the DENV-double
mutant (Thr408Ile and Asp409Glu, in blue) does not show any activity inhibition at all the
concentrations tested (up to 5 M, not shown). (c) Percentage of WNV helicase activity (in red) at
different concentrations of the inhibitor (0, 1, 30, 200, 400, 800 and 1000 nM); the WNV-double
mutant (Thr409Ile and Asp410Glu, in blue) does not show any activity inhibition at all the
concentrations tested (up to 5 M, not shown). Hel, helicase.

(a)
YFV Hel
120

% Residual Activity

100

80

60

40

20

IC50=12210 (nM)

0
1

25

125

625

[Ivermectin](nM)

(b)
DENV Hel
120

% Residual Activity

100
80
60
40

20

IC50=50070 (nM)

0
1

25

125

[Ivermectin](nM)

625

(c)
WNV Hel
120

% Residual Activity

100

80

60

40

20

IC 50=35040 (nM)

0
1

25

125

[Ivermectin](nM)

625

Figure S2. ATPase activity of the DENV helicase domain. The ATPase activity of the helicase domain
was assessed using an ATP bioluminescent assay. Decrease in the luminescence signal, caused by the
consumption of ATP by the DENV helicase in the absence (blue) or presence (red) of 1 M ivermectin
(I). HEL, helicase.

Figure S3. Polymerase activity of the DENV RdRp domain. The polymerase activity of DENV is not
affected by the presence of ivermectin (I). Increase in fluorescence due to in vitro polyC-G dsRNA
synthesis in the absence (blue) or presence (red) of 1 M ivermectin.