1. A flavone-based inhibitor of in vitro human rhinovirus replication induces resistant mutations in capsid protein VP4. Céline Lacroixa, Hari Babu Bollikollac, David Francoa, Vasu Babu Alac, Surender Singh Jadavb, Barij Nayan Sinhab, Venkatesan Jayaprakashb, Johan Neytsa and Pieter Leyssena aLaboratory of Virology and Experimental Chemotherapy, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium bDepartment of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi , India cDepartment of Chemistry, Acharya Nagarjuna University, Nagarjunanagar, Guntur , India
Broad-spectrum anti-rhinovirus activity of A0019
Figure 3. A0019 (■) does not protect HRV14IC wild-type (●) from heat-inactivation in a thermostability assay. This in contrast to pleconaril (▲), a capsid binder that stabilize the capsid upon binding. Compound and virus were incubated for 15’ at 37°C, 2’ at temperatures ranging from 37-57°C, followed by a rapid cool down to 4°C. Infectious viral loads were subsequently quantified by end-point titration (HeLa cells, 35°C, 3 days). Data in duplicate from three independent assays (mean ± stdev).
A flavone skeleton-based compound, A0019 (Figure 1), inhibits the replication of both human rhinovirus group A and B serotypes (Table 1). No activity against enteroviruses A, B or C was observed (Table 2). For reference, activity of capsid binder pleconaril is shown.
Table 1. Antiviral activity against HRV-A and HRV-B
EC50 (µM) ± Med abs dev
 A0019
 pleconaril
HRV A2 ND
0.2 ± 0.1*
HRV A9
10.4 ± 1.0 *
HRV A15
7.6 ± 0.9*
0.6 ± 0.5*
HRV A16
0.2 ± 0.03*
HRV A29
2.9 ± 0.9
0.1 ± 0.07*
HRV A41
5.1 ± 1.1*
0.7 ± 0.2*
HRV A59
9.6 ± 2.0
0.5 ± 0.3*
HRV A63
4.9 ± 3.3
0.1 ± 0.08*
HRV A85
1.7 ± 0.8*
0.1 ± 0.1*
HRV A89
>56
0.7 ± 0.3*
HRV B14
4 ± 1.4*
0.3 ± 0.2*
HRV B42
1.1 ± 0.09*
>26
HRV B70
2.1 ± 0.6*
4 ± 3.6
HRV B72
1.0 ± 0.1*
1.1 ± 0.7*
HRV B86
0.8 ± 0.4*
1.6 ± 1.4*
A0019-resistant HRV14 variants acquired mutations in capsid protein VP4
Two drug-resistant virus variants, obtained independently by clonal selection, proved >4-fold less sensitive to the compound than the parent viruses. Both virus variants were shown to
Table 4. A0019-resistant variants acquired mutations in VP4.
Figure 1. Chemical structure of A0019
HRV14 genotype
HRV14 wild-type / 
A0019-resistant
HRV14
variant 1
VP4_T19S
variant 2
VP4_T19S_3C_L123F
Table 2. A0019 is not active against a selection of EV-A, EV-B and EV-C prototype-strains.
EC50 (µM) ± Med abs dev
 A0019
 pleconaril
EV71 (EV-A)
>56
>131
CVB4 (EV-B)
>21
0.3 ± 0.2
PV-1 (EV-C)
>262
have acquired a mutation at position 19 of the internal capsid protein VP4 (Table 4). The T19S mutation was reverse-engineered into an infectious clone of HRV14 (HRV14IC). In a CPE reduction assay, the mutant proved 3- fold less susceptible to the antiviral effect of A0019 (Table 5). Residue VP4_T19 was found to be conserved in the HRV-B stains included in the screening panel, but not in the HRV-A strains, and in other enteroviruses (Figure 4). The naturally resistant HRV-A serotypes included in the screen are carrying a serine at position 19 of VP4.
Table 5. The mutation VP4_T19S resulted in low-level resistance to A0019.
HRV14IC
A0019
pleconaril
EC50 (µM) RR
wild-type
1.2 ± 0.06 /
0.08 ± 0.007
VP4_T19S
3.2 ± 0.7 3
0.17 ±.0.05 2
Activity was determined in a CPE-reduction assay with MTS read-out. EC50 = median 50% effective concentration ± Med abs dev from dose response curves set up in at least four experiments of which at least two independent, ND = EC50 not reached. * = 100% inhibition of virus-induced cytopathic effect is observed in the tested concentration range.
Activity was determined in a CPE-reduction assay with MTS read-out. EC50 = median 50% effective concentration ± Med abs dev. Data in duplicate from three independent assays. RR = relative resistance (EC50 of resistant strain / EC50 of wild-type).
A0019 interferes with an early stage event in the viral life cycle
Figure 4. The residue T19 in VP4 is conserved in all HRV-B strains that were included in the broad-spectrum screen.
Figure 2. A0019 inhibits viral replication during the early steps of the virus life cycle, as does the early-stage inhibitor pleconaril. A. Viral HRV14 intracellular RNA increases from baseline input (0h) till 8h post-infection. This time point was therefore selected for quantification of the inhibitory effect on viral replication during the time-of-drug-addition study. B. Compounds were added prior, at the time of, or after viral infection at indicated time points and viral RNA was quantified 8h pi. Viral RNA load was expressed as equivalents of 50% tissue culture infective dose/mL (log (TCID50/mL) eq) (mean ± stdev from three independent assays).
Future experiments
Currenlty we are:
selecting virus variants with increased resistance to the compound,
(ii) initiating studies employing density centrifugation to explore whether A0019 alters (the kinetics of) rhinovirus uncoating, and
(iii) studying the effect of the compound on capsid ‘breathing’ by means of protein mass spectrometry mapping.
Mode-of-action of A0019 differs from that of capsid binder pleconaril
Conclusion
Activity against pleconaril-resistant HRV14 variants compared to HRV14 wild-type was not altered for A0019 (Table 3) and the compound does not, like pleconaril, prevent heat-inactivation of HRV14 (Figure 3). These two observations suggests that the mode-of-action of A0019 substantially differs from that of capsid binder pleconaril, which stabilizes the capsid structure upon binding.
We identified a broad-spectrum rhinovirus inhibitor that interferes with an early-stage event during the viral replication cycle. However, its mode-of-action differs from that of the capsid binder pleconaril. Low-level A0019 HRV14 variants acquired mutations in the small, internal capsid protein VP4. High level A0019-resistant variants, together with uncoating studies, will provide more information about the mechanism-of-action of this compound and its interaction with VP4.
Table 3. Pleconaril-resistant HRV14 variants remained equally sensitive to A0019.
HRV14 genotype
A0019
pleconaril
EC50 (µM) RR
HRV14 wild-type / 
3.1 ± 0.2 /
0.3 ± 0.08
pleconaril-resistant
HRV14
variant 1
VP1_L106W_A150T_VP2_N20T
3.3 ± 0.2 1
>26*
>95
variant 2
VP1_N105S_A150V_P154S
3.1 ± 0.08
Activity was determined in a CPE-reduction assay with MTS read-out. EC50 = median 50% effective concentration ± Med abs dev. Data in duplicate from two independent assays. RR = relative resistance (EC50 of resistant strain / EC50 of wild-type). *p<0.0001
KU Leuven Rega Institute