Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

Figures & data

Table 1 Protein sequences used in the homology modeling of the NS5 RdRp for all four dengue virus serotypes

Serotype	UniProt – RdRp	Structures of NS5 RdRp at Protein Data Bank	NS5 RdRp homology model	PDB template for homology modeling
DENV-1	POLG_DEN 1B P27909|2733-3392	None	POLG_DEN1B-1.pdb	4C11
			POLG_DEN1B-2.pdb	3VWS
			POLG_DEN1B-3.pdb	4C11
	POLG_DEN1W P17763|2733-3392	None	POLG_DEN1W-1.pdb	4C11
			POLG_DEN1W-2.pdb	3VWS
			POLG_DEN1W-3.pdb	4C11
DENV-2	POLG_DEN2J P07564|2732-3391	None	POLG_DEN2J-1.pdb	4C11
			POLG_DEN2J-2.pdb	4C11
			POLG_DEN2J-3.pdb	3VWS
	POLG_DEN2N P14340|2732-3391	None	POLG_DEN2N-1.pdb	4C11
			POLG_DEN2N-2.pdb	4C11
			POLG_DEN2N-3.pdb	3VWS
	POLG_DEN2P P12823|2729-3388	None	POLG_DEN2P-1.pdb	4C11
			POLG_DEN2P-2.pdb	3VWS
			POLG_DEN2P-3.pdb	4C11
	POLG_DEN26 P29990|2732-3391	None	POLG_DEN26-1.pdb	4C11
			POLG_DEN26-2.pdb	4C11
			POLG_DEN26-3.pdb	3VWS
	POLG_DEN27 P29991|2732-3391	None	POLG_DEN27-1.pdb	4C11
			POLG_DEN27-2.pdb	4C11
			POLG_DEN27-3.pdb	3VWS
	POLG_DEN28 P14337|2732-3391	None	POLG_DEN28-1.pdb	4C11
			POLG_DEN28-2.pdb	4C11
			POLG_DEN28-3.pdb	3VWS
DENV-3	POLG_DEN3I Q5UB51|2730-3390	4V0Q, 4V0R, 5DTO	POLG_DEN3-1.pdb	4V0Q
			POLG_DEN3-2.pdb	4V0R
			POLG_DEN3-3.pdb	5DTO
	Q6DLV0_9FLAV Q6DLV0|2730-3390	2J7U, 2J7W, 3VWS, 4C11, 4HHJ	Q6DLV0_9FLAV-1.pdb	5DTO
			Q6DLV0_9FLAV-2.pdb	4V0R
			Q6DLV0_9FLAV-3.pdb	4V0R
DENV-4	POLG_DEN4P Q58HT7|2728-3387	None	POLG_DEN4D-1.pdb	4C11
			POLG_DEN4D-2.pdb	4V0R
			POLG_DEN4D-3.pdb	4V0Q
	POLG_DEN4D P09866|2728-3387	None	POLG_DEN4P-1.pdb	4C11
			POLG_DEN4P-2.pdb	4V0R
			POLG_DEN4P-3.pdb	4V0Q
	POLG_DEN4T Q2YHF0|2728-3387	None	POLG_DEN4T-1.pdb	5DTO
			POLG_DEN4T-2.pdb	4V0R
			POLG_DEN4T-3.pdb	4C11

Notes: UniProt code includes information of the number of aminoacids for the polymerase into the full-length polyprotein. Models are available at http://docking.umh.es/

Abbreviations: NS5, nonstructural protein 5; RdRp, RNA-dependent RNA polymerase; PDB, Protein Data Bank.

Figure 1 Multiple sequence alignment of NS5 RNA-dependent RNA polymerases of the different dengue virus serotypes (above) and the corresponding percent identitity matrix (below).

Notes: Pink boxes indicate the aminoacid position of M343, T413, and R737 for DENV-3 considering the full length of NS5 (MTase N-terminal + RdRp C-terminal) or the equivalent position for the other serotypes. Yellow boxes indicate missing residues in the crystallographic data. Orange boxes indicate the GDD motif. For each sequence, the UniProt code for the full polyprotein of the virus | SEROTYPE is indicated.

Abbreviation: NS5, nonstructural protein 5.

Table 2 Molecular docking analysis for NITD-related and potential inhibitor compounds (candidate molecules in this study) at the binding site of NS5 RdRp located in the RNA template tunnel

Ligand name	ΔG (kcal/mol) mean ± SD	Ligand name	ΔG (kcal/mol) mean ± SD	Ligand name	ΔG (kcal/mol) mean ± SD	Ligand name	ΔG (kcal/mol) mean ± SD
NS5 RdRp of DENV-1		NS5 RdRp of DENV-2
NITD-1	−8.6±0.5	NITD-1	−8.5±0.6	11999940	−11.2±0.9	11612450	−11.0±0.7
NITD-2	−9.6±0.5	NITD-2	−9.9±0.8	71815510	−11.2±0.6	22867347	−11.0±1.0
NITD-29	−10.1±0.8	NITD-29	−10.8±1.2	53019114	−11.2±1.3	53834657	−11.0±0.7
91166410	−11.5±1.2	53019095	−11.8±1.2	68563659	−11.2±0.9	86608192	−11.0±1.1
88881717	−11.5±1.1	53019000	−11.7±1.6	90644640	−11.2±0.8	58550574	−11.0±0.6
53019095	−11.4±1.0	58847065	−11.6±0.6	74937125	−11.2±1.0	87254969	−11.0±1.0
58509828	−11.4±1.3	58847008	−11.6±0.6	5330687	−11.2±1.0	91982228	−11.0±0.8
86592137	−11.4±0.7	59558635	−11.5±0.6	53019157	−11.1±1.0	58117326	−11.0±1.0
87254969	−11.4±0.7	53019136	−11.5±1.7	53019118	−11.1±1.1	86592137	−11.0±1.0
58847008	−11.4±0.6	91166410	−11.5±1.3	57483735	−11.1±0.9	46228828	−11.0±0.8
24320265	−11.4±0.8	53019046	−11.5±1.2	42623761	−11.1±1.1	57590479	−11.0±0.8
5330687	−11.3±1.0	58509828	−11.5±1.3	58847188	−11.1±0.6	24818217	−11.0±1.1
71815510	−11.3±0.6	57883147	−11.4±0.8	11612953	−11.1±1.0	58716236	−11.0±0.9
53019000	−11.3±1.0	88881717	−11.4±1.2	59120485	−11.1±0.9	53019023	−11.0±1.4
58847188	−11.3±0.3	76724063	−11.4±0.8	71815570	−11.1±0.7	99173129	−11.0±1.0
58847065	−11.3±0.7	4982894	−11.4±1.1	60118013	−11.1±0.9	16749692	−11.0±0.6
46228732	−11.3±0.9	90644642	−11.4±1.0	46228732	−11.1±0.9	53019026	−11.0±1.5
4982894	−11.2±1.1	75237511	−11.3±1.1	90644637	−11.1±1.0	10163912	−11.0±0.9
58550574	−11.2±0.7	60138427	−11.3±0.9	53098093	−11.1±0.9	53019135	−11.0±1.6
71815377	−11.2±0.5	21114517	−11.3±0.9	99437562	−11.1±1.2	11503697	−11.0±1.1
16749628	−11.2±0.5	60118830	−11.3±0.9	68490845	−11.1±0.8	58117469	−11.0±0.9
53019136	−11.2±0.9	53019143	−11.3±1.3	17240799	−11.1±0.9	67017052	−11.0±1.1
66966190	−11.1±0.3	11591299	−11.3±1.0	10116388	−11.1±0.9	83275648	−11.0±1.3
53019046	−11.1±0.9	46926790	−11.2±0.8	60402568	−11.1±1.0	21598341	−11.0±1.0
66661763	−11.1±0.6	67023200	−11.2±0.8	59508095	−11.1±1.2	44419986	−11.0±0.9
57955372	−11.1±0.3	44605230	−11.2±1.4	51038723	−11.1±1.1	58518208	−11.0±0.4
16749692	−11.1±0.6	60250081	−11.2±1.1	69707773	−11.0±0.9	11547552	−11.0±0.8
71815441	−11.1±0.5	59558530	−11.2±0.6	57955382	−11.0±0.6	59558378	−11.0±0.9
58044822	−11.1±0.8	66662216	−11.2±0.7	22556722	−11.0±1.0	59508029	−11.0±1.1
21598341	−11.0±1.2	24672975	−11.2±0.6	22587546	−11.0±0.9	53019073	−11.0±1.1
22556747	−11.0±1.0	59069323	−11.2±1.2	58044822	−11.0±0.6	53310399	−11.0±1.0
42598694	−11.0±0.5	9804722	−11.2±1.2	10239163	−11.0±0.7	86608190	−11.0±1.0
71815570	−11.0±0.6	5330694	−11.2±1.1	56085441	−11.0±1.4	59113200	−11.0±1.1
11591299	−11.0±0.7	66661763	−11.2±0.7	44068150	−11.0±1.2	2307085	−11.0±0.9
90644637	−11.0±0.9	88881725	−11.2±0.2	2408779	−11.0±0.8	58847051	−11.0±0.6
53239274	−11.0±1.0	71815377	−11.2±0.4	60117771	−11.0±1.0
90644642	−11.0±1.0	88884325	−11.2±0.4	58094574	−11.0±0.9
NS5 RdRp of DENV-4
NITD-1	−8.9±0.3	22556722	−11.3±1.3	5330659	−11.1±1.1	56244330	−11.0±1.0
NITD-2	−10.2±0.5	58187960	−11.3±0.7	53041346	−11.1±1.0	4498761	−11.0±0.9
NITD-29	−10.8±0.7	71016043	−11.3±0.6	58117268	−11.1±0.6	6129755	−11.0±0.8
4982894	−12.2±1.4	58117308	−11.3±0.5	66605204	−11.1±0.7	10410064	−11.0±0.7
2408779	−12.0±1.1	59121600	−11.3±0.8	25065563	−11.1±0.9	23627602	−11.0±0.8
59558635	−11.9±1.0	6524590	−11.2±1.1	53884638	−11.1±1.3	26283419	−11.0±0.9
44605230	−11.9±0.5	55797299	−11.2±0.8	57483798	−11.1±0.8	45167069	−11.0±1.2
71815510	−11.8±0.8	11525541	−11.2±1.2	9930411	−11.1±1.3	51925674	−11.0±0.6
59558530	−11.8±0.6	76810150	−11.2±1.8	25138236	−11.1±0.7	55790271	−11.0±1.0
88881717	−11.8±1.3	56085441	−11.2±0.9	25154812	−11.1±0.9	57467031	−11.0±1.0
91166410	−11.8±1.3	68698259	−11.2±1.0	58117332	−11.1±0.4	59558574	−11.0±0.8
58509828	−11.7±1.2	99173125	−11.2±0.7	59962443	−11.1±0.6	60118484	−11.0±0.4
16749692	−11.6±1.1	11999813	−11.2±0.9	77152204	−11.1±0.7	66662021	−11.0±1.1
46228732	−11.6±1.0	25138230	−11.2±0.8	99501210	−11.1±1.3	69707178	−11.0±0.7
68701918	−11.6±1.2	53098094	−11.2±0.3	16749694	−11.1±1.0	22063077	−11.0±0.6
60402568	−11.6±0.9	59875861	−11.2±1.1	19288807	−11.1±1.0	56022032	−11.0±0.4
25065772	−11.6±1.0	68496294	−11.2±1.3	46926790	−11.1±1.2	59558479	−11.0±0.9
68702576	−11.6±1.1	60205770	−11.2±0.9	58787557	−11.1±1.3	66662543	−11.0±0.6
66661763	−11.6±1.0	21114517	−11.2±0.6	76541785	−11.1±0.7	4498763	−11.0±0.8
58094574	−11.6±0.9	25065362	−11.2±1.0	20765416	−11.1±1.0	10392307	−11.0±0.9
76724063	−11.6±1.4	54127944	−11.2±0.8	25066204	−11.1±1.1	46377852	−11.0±0.7
66662686	−11.6±0.7	44303726	−11.2±1.0	27563621	−11.1±1.1	50848671	−11.0±0.9
57955382	−11.5±1.3	53098093	−11.2±0.4	46602120	−11.1±1.1	59212388	−11.0±0.9
88884325	−11.5±1.8	56925033	−11.2±1.9	53098102	−11.1±0.5	66604901	−11.0±0.8
57883147	−11.5±1.4	66606315	−11.2±1.0	16431302	−11.1±1.0	68894072	−11.0±1.1
60250081	−11.5±0.8	86608190	−11.2±1.2	3554749	−11.1±0.8	89886799	−11.0±0.9
11591299	−11.5±1.1	89361165	−11.2±0.6	25174057	−11.1±1.0	44605022	−11.0±0.7
17240799	−11.5±0.6	89361072	−11.2±0.6	58509782	−11.1±1.3	60402552	−11.0±0.9
25065775	−11.5±1.0	20765428	−11.2±1.0	69144161	−11.1±0.9	76808085	−11.0±1.2
44176508	−11.5±1.2	58044822	−11.2±0.8	23627601	−11.1±1.1	17359718	−11.0±0.6
46885243	−11.5±0.9	59239811	−11.2±1.0	69579276	−11.1±0.9	35767226	−11.0±1.1
15605185	−11.5±0.6	146429	−11.2±1.3	10116388	−11.0±0.9	58768495	−11.0±0.9
5330687	−11.5±1.3	11612450	−11.2±1.0	25065360	−11.0±1.0	4710567	−11.0±0.7
25065774	−11.5±1.0	44068150	−11.2±0.9	76310566	−11.0±0.8	9799922	−11.0±1.2
66662216	−11.5±1.1	53098204	−11.2±0.4	46833812	−11.0±1.8	11756868	−11.0±1.0
60118013	−11.5±1.1	25065776	−11.2±0.9	10206429	−11.0±0.8	16589824	−11.0±1.0
88881725	−11.5±1.1	53310399	−11.2±0.8	30870527	−11.0±0.8	44155098	−11.0±0.7
53019000	−11.4±1.0	18710168	−11.2±0.7	44580357	−11.0±1.1	44159652	−11.0±0.7
60118830	−11.4±0.5	50848824	−11.2±1.0	59304633	−11.0±0.5	57483735	−11.0±0.7
11612953	−11.4±0.9	98939207	−11.2±0.6	66694881	−11.0±0.9	59121601	−11.0±1.0
86592137	−11.4±0.4	59375041	−11.2±1.8	9537955	−11.0±1.6	58117321	−11.0±0.4
87254969	−11.4±0.4	60494790	−11.2±0.6	10069263	−11.0±0.8	9886055	−11.0±1.2
60117771	−11.4±0.4	5330699	−11.2±1.4	41273686	−11.0±1.1	52886452	−11.0±0.5
25138229	−11.4±0.8	25065773	−11.2±0.8	46228828	−11.0±0.9	66604312	−11.0±0.9
46228865	−11.4±0.7	56057109	−11.2±0.6	57483762	−11.0±0.4	68714559	−11.0±1.1
10342132	−11.4±1.0	58550574	−11.2±0.9	68701232	−11.0±1.0	6129075	−11.0±0.6
67133415	−11.4±1.4	10228015	−11.2±0.9	53018993	−11.0±0.5	11752081	−11.0±0.6
71815570	−11.4±0.6	39906246	−11.2±0.5	58069426	−11.0±1.0	16065158	−11.0±1.2
16675341	−11.4±1.0	58064300	−11.2±1.2	11760627	−11.0±1.1	18710165	−11.0±0.7
24320265	−11.4±1.0	60402580	−11.2±1.1	16583630	−11.0±0.6	19665288	−11.0±1.0
68695702	−11.4±1.2	68698167	−11.2±0.9	20637893	−11.0±1.0	22556747	−11.0±1.3
50848923	−11.4±0.9	20765438	−11.2±1.1	22909360	−11.0±0.9	24512675	−11.0±0.8
87254968	−11.4±1.3	51038723	−11.2±0.5	57483761	−11.0±0.4	25138237	−11.0±0.9
16749628	−11.4±1.1	60481456	−11.2±0.8	58607039	−11.0±1.1	57483734	−11.0±0.5
25066203	−11.4±0.9	56085277	−11.2±1.2	59615666	−11.0±0.9	57878492	−11.0±1.0
86608192	−11.4±1.3	56025347	−11.1±1.3	59986516	−11.0±0.9	88042824	−11.0±0.8
362451	−11.3±1.3	11999940	−11.1±0.7	60481455	−11.0±0.8	90299314	−11.0±0.9
10477276	−11.3±0.9	53019143	−11.1±0.9	66599533	−11.0±0.6	5330694	−11.0±1.4
71815377	−11.3±0.5	58117326	−11.1±0.2	69138857	−11.0±0.4	45856807	−11.0±0.6
59375107	−11.3±1.9	67023384	−11.1±0.7	76541767	−11.0±0.9	2647295	−11.0±0.8
67023200	−11.3±0.7	44251670	−11.1±1.0	89856954	−11.0±1.4	6197109	−11.0±0.7
68490845	−11.3±0.9	44178168	−11.1±1.0	10002281	−11.0±0.8	9804722	−11.0±1.3
44419986	−11.3±0.8	53019026	−11.1±1.1	42623761	−11.0±0.9	20637894	−11.0±0.7
5280143	−11.3±0.8	58064137	−11.1±1.1	51035546	−11.0±0.6	21378092	−11.0±0.6
57878498	−11.3±0.8	16327670	−11.1±0.8	51035547	−11.0±0.4	25070620	−11.0±1.5
18710166	−11.3±1.2	24548686	−11.1±0.9	56214202	−11.0±1.1	31895387	−11.0±0.8
59880286	−11.3±1.2	86608206	−11.1±1.3	59558378	−11.0±1.0	54347717	−11.0±1.3
59144489	−11.3±1.2	68700123	−11.1±1.2	71149319	−11.0±0.6	55760732	−11.0±1.0
18710167	−11.3±1.5	46885245	−11.1±1.0	25065560	−11.0±1.1	57955372	−11.0±1.0
60402567	−11.3±0.9	53019136	−11.1±0.9	58117279	−11.0±0.6	58975131	−11.0±0.5
71815441	−11.3±0.6	60621794	−11.1±0.8	90731410	−11.0±1.0	59121588	−11.0±1.1
25065987	−11.3±1.2	68559862	−11.1±1.0	102119533	−11.0±1.1	59558337	−11.0±0.8
11317629	−11.3±1.0	85874649	−11.1±1.0	44605023	−11.0±0.7	59880283	−11.0±1.2
44419982	−11.3±0.9	5330697	−11.1±1.4	46231533	−11.0±0.6	66605319	−11.0±0.7
47037799	−11.3±0.7	16376567	−11.1±0.8	54087042	−11.0±0.5	66661799	−11.0±0.9
50848762	−11.3±0.8	53019095	−11.1±0.9	57391501	−11.0±0.5	74937104	−11.0±0.5
53019046	−11.3±0.9	20765417	−11.1±1.2	59973115	−11.0±1.4
58518208	−11.3±1.8	22556882	−11.1±1.2	69707773	−11.0±0.9
89856935	−11.3±1.2	99455350	−11.1±0.9	59212517	−11.0±0.6
10239163	−11.3±1.1	2947808	−11.1±0.8	41927843	−11.0±0.7

Notes: The name of each ligand was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).²² The name of the compounds NITD-1, -2, and -29 was taken from Niyomrattanakit et al.²¹ The table shows the estimated binding free energy variation³¹ (mean ± SD). The maps of the interacting residues of the protein with the best ligands and ΔG values among the candidate compounds are available at http://docking.umh.es/

Abbreviations: NS5, nonstructural protein 5; RdRp, RNA-dependent RNA polymerase; SD, standard deviation.

Table 3 Molecular docking analysis for natural compounds

RdRp of DENV-1		RdRp of DENV-2		RdRp of DENV-4
Ligand name	ΔG (kcal/mol) mean ± SD	Ligand name	ΔG (kcal/mol) mean ± SD	Ligand name	ΔG (kcal/mol) mean ± SD
SN00091933	−10.8±0.7	SN00151425	−11.1±0.8	SN00016053	−11.2±1.0
SN00074091	−10.7±0.7	SN00010280	−10.9±0.8	SN00057073	−11.1±1.1
SN00091667	−10.7±0.8	SN00023794	−10.9±0.7	SN00063622	−11.1±1.2
SN00306679	−10.6±1.0	SN00372243	−10.9±0.9	SN00058424	−11.1±1.1
SN00151425	−10.6±0.9	SN00115885	−10.8±1.1	SN00058828	−11.1±1.1
SN00366028	−10.5±1.0	SN00018927	−10.8±0.5	SN00057669	−11.1±1.1
SN00372243	−10.5±1.0	SN00016053	−10.8±0.5	SN00230471	−11.1±1.3
SN00074088	−10.5±0.9	SN00057073	−10.7±1.1	SN00057220	−11.0±1.2
SN00010280	−10.5±0.6	SN00081933	−10.6±1.1	SN00057061	−11.0±1.2
SN00127203	−10.5±1.1	SN00127203	−10.6±1.3	SN00303561	−11.0±1.2
SN00016053	−10.5±0.6	SN00058424	−10.6±1.1	SN00058587	−10.9±1.1
SN00230471	−10.5±0.9	SN00063622	−10.6±1.0	SN00366028	−10.9±0.8
SN00245001	−10.5±0.7	SN00379696	−10.6±0.6	SN00127203	−10.8±0.8
		SN00396968	−10.6±0.8	SN00303525	−10.8±1.0
		SN00014343	−10.6±0.9	SN00016042	−10.7±1.4
		SN00057669	−10.5±1.0	SN00010280	−10.7±0.6
		SN00282274	−10.5±0.9	SN00111882	−10.6±1.0
		SN00381633	−10.5±1.2	SN00282274	−10.6±1.0
		SN00004095	−10.5±0.6	SN00317979	−10.6±1.0
		SN00245001	−10.5±0.4	SN00018896	−10.6±0.9
		SN00058828	−10.5±1.0	SN00026414	−10.6±1.2
		SN00397486	−10.5±0.9	SN00151956	−10.6±0.6
		SN00282224	−10.5±0.9	SN00282224	−10.6±1.2
		SN00009726	−10.5±0.9	SN00004146	−10.6±0.9
				SN00151425	−10.6±1.1
				SN00379696	−10.6±1.0
				SN00012056	−10.5±0.7
				SN00381633	−10.5±0.7
				SN00115885	−10.5±0.8
				SN00148919	−10.5±0.6
				SN00276030	−10.5±0.8
				SN00249137	−10.5±0.7
				SN00091933	−10.5±0.9

Notes: The maps of the interacting residues of the protein with the best ligands and ΔG values among the candidate compounds are available at http://docking.umh.es/. The name of each ligand was obtained from the SuperNatural database (http://bioinf-applied.charite.de/supernatural_new/index.php?site=home). The table shows estimated binding free energy variation³¹ (mean ± SD) of potential inhibitors at the binding site of NS5 RdRp located in the RNA template tunnel.

Abbreviations: NS5, nonstructural protein 5; RdRp, RNA-dependent RNA polymerase; SD, standard deviation.

Table 4 Physicochemical parameters for selected compounds based on molecular docking analysis

Compound	% ABS	TPSA (Å^2)	MW	clogS	clogP	HBA	HBD	Ro5 violations	Drug score	Drug likeness
NITD-1	66.05	124.5	373.1	−3.39	0.544	8	2	0	0.21153	−0.2761
NITD-2	71.91	107.5	433.1	−4.61	2.41	7	2	0	0.608586	1.9599
NITD-29	56.01	153.6	656.2	−8.22	2.602	10	3	1	0.11867	−3.1308
SN00115885	70.36	112	478.5	−2.872	4.763	8	2	0	0.571559	3.9196
71815570	70.67	111.1	478.2	−4.08	3.935	8	3	0	0.613687	2.1441
89856954	70.67	111.1	478.2	−4.08	4.146	8	3	0	0.613687	2.1441
SN00018896	71.60	108.4	487.6	−3.608	−0.06	8	3	0	0.682805	3.8613
SN00303525	71.60	108.4	482.5	−3.791	0.732	8	1	0	0.542329	0.3641
SN00379696	72.57	105.6	443.5	−0.855	2.249	9	2	0	0.800132	7.1765
SN00282274	72.78	105	418.4	−1.829	1.623	9	1	0	0.725687	1.0679
SN00372243	72.78	105	432.4	−1.938	1.921	9	1	0	0.707052	1.0873
SN00396968	72.78	105	418.4	−1.829	1.623	9	1	0	0.725687	1.0679
SN00057061	73.19	103.8	463.5	−3.33	−0.003	7	0	0	0.736422	5.5881
SN00057220	73.19	103.8	493.9	−3.876	0.942	7	0	0	0.660014	6.9371
SN00057669	73.19	103.8	477.5	−3.454	0.437	7	0	0	0.709263	5.2853
SN00058587	73.19	103.8	479.9	−3.752	0.502	7	0	0	0.688934	7.2363
SN00081933	73.19	103.8	473.5	−3.689	1.987	7	0	0	0.689051	12.562
SN00276030	73.36	103.3	453.5	−3.312	2.68	6	4	0	0.642133	1.2805
SN00151425	73.50	102.9	483.5	−2.059	2.768	8	2	0	0.579584	0.4603
SN00004146	74.91	98.8	476.9	−3.894	1.441	8	2	0	0.660046	2.9203
SN00009726	75.47	97.2	465.5	−2.537	4.503	8	1	0	0.587977	2.0354
SN00306679	75.78	96.3	413.4	−1.491	1.835	8	2	0	0.838983	7.1078
50848824	76.57	94	489.2	−3.75	0.864	8	0	0	0.638051	8.2672
55760732	76.57	94	475.2	−2.95	0.661	8	0	0	0.710746	9.1186
SN00282224	76.74	93.5	402.4	−2.051	2.283	8	1	0	0.815395	3.1154
SN00397486	76.74	93.5	402.4	−2.051	2.283	8	1	0	0.815395	3.1154
50848671	77.64	90.9	494.2	−3.25	1.125	8	0	0	0.533224	4.7277
57590479	77.95	90	486.2	−3.49	2.117	8	1	0	0.675188	3.0453
SN00018927	79.26	86.2	496.6	−3.01	−2.63	9	4	0	0.708905	6.3156
SN00023794	79.26	86.2	494.6	−3.28	−0.002	8	3	0	0.697577	6.3156
SN00111882	79.43	85.7	458.5	−3.072	4.922	8	1	0	0.569557	3.6342
39906246	80.81	81.7	464.1	−3.46	1.655	7	0	0	0.693327	5.794
SN00012056	81.37	80.1	477.6	−2.58	4.976	7	1	0	0.563559	4.8245
SN00317979	81.47	79.8	491.5	−2.988	3.963	8	0	0	0.605967	2.8022
SN00016042	81.57	79.5	473.5	−2.96	4.68	7	2	0	0.569749	2.744
SN00074088	81.64	79.3	418.4	−3.072	3.241	7	1	0	0.69883	1.8624
SN00074091	81.64	79.3	418.4	−3.072	3.241	7	1	0	0.69883	1.8624
SN00016053	82.81	75.9	485.6	−3.12	3.398	7	2	0	0.629475	2.2823
SN00014343	85.57	67.9	488.6	−3.285	0.458	8	2	0	0.697442	3.8483
SN00026414	85.71	67.5	454.5	−3.046	4.801	6	1	0	0.589066	4.2039
SN00366028	87.09	63.5	364.4	−3.959	3.49	5	1	0	0.621615	0.9079
SN00010280	92.85	46.8	442.6	−3.694	3.91	5	1	0	0.586606	1.4089

Note: The NITD-1, -2, and -29 compounds, tested experimentally,²¹ are included for comparison.

Abbreviations: % ABS, percentage of absorption; clogS, the estimated logarithm (base 10) of the solubility measured in mol/L; clogP, calculated logarithm of partition coefficient between n-octanol and water; HBA, number of hydrogen bond acceptors; HBD, number of hydrogen bond donors; MW, molecular weight; Ro5 violations, violation of Lipinski’s rules; TPSA, topological polar surface area.

Figure 2 Comparison of the free energy variation (ΔG) for selected compounds based on molecular docking analysis against NS5 RdRps of all four serotypes of dengue virus (A). ΔG for NITD compounds is also included. (B) The molecular structures of the compounds represented in (A); the PubChem ID or the SuperNatural 2 ID is close to the structure of each compound.

Abbreviation: NS5, nonstructural protein 5.

Figure 3 3D structure of the proteins NS5 RNA-dependent RNA polymerases of DENV-4 (A and B) and DENV-2 (C and D) showing the binding sites (left), the binding cavity (middle), and the main residues involved in the ligand–protein interaction of compound 50848824 (A), 71815570 (B), SN00151425 (C), and SN00010280 (D). Structure visualization was by PyMol 1.8.2.0.

Abbreviations: 3D, three dimensional; NS5, nonstructural protein 5.

Table 5 Predicted molecular pharmacokinetic properties of selected compounds against the NS5 RdRp

Compound	ADME
	BBB	HIA	Caco-2 permeability	Caco-2 permeability (logPapp, cm/s)	P-gp substrate	P-gp inhibitor I	P-gp inhibitor II	CYP450 2C9 substrate	CYP450 2D6 substrate	CYP450 3A4 substrate	CYP450 1A2 inhibitor	CYP450 2C9 inhibitor	CYP450 2D6 inhibitor	CYP450 2C19 inhibitor	CYP450 3A4 inhibitor	CYP IP	ROCT
NITD-1	BBB−	HIA+	Caco-2−	0.1784	−	−	−	+	−	−	−	−	−	−	−	Low	−
NITD-2	BBB+	HIA+	Caco-2−	−0.0001	−	−	+	−	−	−	−	+	−	−	−	Low	−
NITD-29	BBB+	HIA+	Caco-2−	0.2513	−	−	+	+	−	−	−	+	−	−	+	High	−
SN00115885	BBB+	HIA+	Caco-2−	0.0438	+	+	−	−	−	+	−	−	−	−	+	High	+
71815570	BBB−	HIA+	Caco-2−	0.2006	+	−	−	−	−	+	−	+	−	−	+	High	−
89856954	BBB−	HIA+	Caco-2−	0.246	+	−	−	−	−	+	−	+	−	−	+	High	−
SN00018896	BBB+	HIA−	Caco-2−	0.0127	+	+	−	−	−	+	−	−	−	−	−	High	−
SN00303525	BBB+	HIA+	Caco-2−	1.0792	+	+	−	−	−	+	−	−	−	−	−	Low	−
SN00379696	BBB−	HIA+	Caco-2−	0.8915	+	+	−	−	−	+	−	−	−	−	−	Low	−
SN00282274	BBB−	HIA+	Caco-2−	0.711	+	−	−	−	−	+	−	+	−	−	+	Low	−
SN00372243	BBB−	HIA+	Caco-2−	0.7707	+	+	−	−	−	+	−	+	−	−	−	Low	−
SN00396968	BBB−	HIA+	Caco-2−	0.711	+	−	−	−	−	+	−	+	−	−	+	Low	−
SN00057061	BBB+	HIA+	Caco-2+	0.83	+	−	−	−	−	+	−	−	−	−	−	Low	−
SN00057220	BBB+	HIA+	Caco-2+	0.9218	+	−	−	−	−	+	−	−	−	−	−	Low	−
SN00057669	BBB+	HIA+	Caco-2+	0.8885	+	−	−	−	−	+	−	−	−	−	−	Low	−
SN00058587	BBB+	HIA+	Caco-2+	0.8622	+	−	−	−	−	+	+	−	−	−	−	Low	+
SN00081933	BBB+	HIA+	Caco-2+	1.1446	+	−	+	−	−	+	−	−	−	−	+	Low	−
SN00276030	BBB+	HIA+	Caco-2−	0.8668	−	−	−	−	−	−	+	+	−	+	+	High	−
SN00151425	BBB−	HIA+	Caco-2−	0.1968	+	−	−	−	−	−	−	−	−	−	−	Low	−
SN00004146	BBB+	HIA+	Caco-2−	0.3751	+	−	−	−	−	+	−	−	−	+	+	High	−
SN00009726	BBB+	HIA+	Caco-2−	0.7022	+	+	−	−	−	+	−	−	−	−	+	High	−
SN00306679	BBB+	HIA+	Caco-2−	0.7746	+	+	−	−	−	+	+	−	−	+	−	High	−
50848824	BBB+	HIA+	Caco-2−	0.7676	−	−	+	−	−	−	−	+	−	+	+	High	+
55760732	BBB+	HIA+	Caco-2−	0.9103	−	+	+	−	−	+	−	+	−	+	+	High	−
SN00282224	BBB+	HIA+	Caco-2−	0.7294	+	+	−	−	−	+	−	+	−	+	−	High	−
SN00397486	BBB+	HIA+	Caco-2−	0.7294	+	+	−	−	−	+	−	+	−	+	−	High	−
50848671	BBB+	HIA+	Caco-2−	0.753	−	+	−	−	−	+	−	+	−	−	+	High	−
57590479	BBB+	HIA+	Caco-2−	0.5167	+	+	+	−	−	+	−	−	−	−	−	High	−
SN00018927	BBB+	HIA+	Caco-2−	−0.2237	+	+	−	−	−	+	−	−	−	−	−	Low	−
SN00023794	BBB+	HIA+	Caco-2−	0.0423	+	+	−	−	−	+	−	−	−	−	−	Low	−
SN00111882	BBB+	HIA+	Caco-2−	0.7627	+	+	−	−	−	+	−	−	−	−	+	Low	−
39906246	BBB+	HIA+	Caco-2−	0.7821	−	+	+	−	−	+	−	+	−	+	+	High	−
SN00012056	BBB+	HIA+	Caco-2−	0.6186	+	+	+	−	−	+	−	−	−	−	+	High	−
SN00317979	BBB+	HIA+	Caco-2−	0.823	+	+	+	−	−	+	−	+	−	+	+	High	−
SN00016042	BBB+	HIA+	Caco-2−	0.9035	+	+	−	−	−	+	−	−	−	+	+	High	−
SN00074088	BBB−	HIA+	Caco-2+	1.175	+	−	−	−	−	+	−	−	−	−	−	Low	−
SN00074091	BBB−	HIA+	Caco-2+	1.175	+	−	−	−	−	+	−	−	−	−	−	Low	−
SN00016053	BBB+	HIA+	Caco-2−	0.4032	+	+	−	−	−	+	−	−	−	−	−	Low	−
SN00014343	BBB+	HIA+	Caco-2−	0.6715	+	+	+	−	−	+	−	−	−	−	−	High	−
SN00026414	BBB+	HIA+	Caco-2+	0.9295	+	+	+	−	−	+	−	+	−	+	+	High	−
SN00366028	BBB+	HIA+	Caco-2+	1.5359	−	+	+	−	−	+	+	+	−	+	−	High	−
SN00010280	BBB+	HIA+	Caco-2−	0.7316	+	+	−	−	−	+	−	−	−	−	−	Low	+

Notes: Compound names were obtained from PubChem²² and SuperNatural II²³ databases, respectively. ADME profiles of all compounds tested in this study are available at http://docking.umh.es/. All parameters have been calculated using the http://lmmd.ecust.edu.cn:8000/predict/site.³⁴

Abbreviations: ADME, absorption, distribution, metabolism, and elimination; BBB, blood–brain barrier; CYP450, cytochrome P450; CYP IP, cytochrome P450 inhibitory promiscuity; HIA, human intestinal absorption; NS5, nonstructural protein 5; P-gp, P-glycoprotein; RdRp, RNA-dependent RNA polymerase; ROCT, renal organic cation transporter.

Table 6 Predicted toxicity assessment of selected compounds against the NS5 RdRp

Toxicity profile	NITD-1	NITD-2	NITD-29	SN00115885	71815570	89856954	SN00018896	SN00303525	SN00379696	SN00282274	SN00372243
Mutagenica	None	None	None	None	None	None	None	None	None	None	None
Tumorigenica	High	None	None	None	None	None	None	None	None	None	None
Rea	None	None	None	None	None	None	None	None	None	None	None
Irritanta	High	None	None	None	None	None	None	None	None	None	None
HEaggRGI Ib	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak
HEaggRGI IIb	−	−	−	−	+	+	+	−	−	−	−
AMES toxicityb	−	−	−	−	+	+	−	−	−	−	−
Carcinogensb	+	−	−	−	−	−	−	−	−	−	−
FT (pLC50, mg/L)b	High, 1.6887	High, 1.5982	High, 1.6493	Low, 1.3346	High, 0.9852	High, 0.9753	High, 1.5741	High, 0.8064	High, 1.0384	High, 1.0351	High, 1.0048
TPT (pIGC50, µg/L)b	High, 0.3722	Low, 0.3255	Low, 0.3255	High, 0.4624	High, 0.573	High, 0.5913	High, 0.5162	High, 0.5102	High, 0.545	High, 0.564	High, 0.5765
Honey bee toxicityb	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	High HBT	Low HBT	Low HBT	Low HBT
Biodegradationb	−	−	−	−	−	−	−	−	−	−	−
Acute oral toxicityb	III	III	III	III	III	III	III	III	III	III	III
Carcinogenicity (three class)b	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required
RAT (LD50, mol/kg)b	2.2495	2.1577	2.2287	2.5523	2.4901	2.4789	2.4839	2.8557	2.7548	2.631	2.6183
Toxicity profile	SN00396968	SN00057061	SN00057220	SN00057669	SN00058587	SN00081933	SN00276030	SN00151425	SN00004146	SN00009726	SN00306679
Mutagenica	None	None	None	None	None	None	None	None	None	None	None
Tumorigenica	None	None	None	None	None	None	None	None	None	None	None
Rea	None	None	None	None	None	None	None	None	None	None	None
Irritanta	None	None	None	None	None	None	None	None	None	None	None
HEaggRGI Ib	Weak	Weak	Weak	Weak	Strong	Weak	Weak	Weak	Weak	Weak	Weak
HEaggRGI IIb	−	−	−	−	−	−	−	−	+	−	−
AMES toxicityb	−	−	−	−	−	−	+	−	−	−	+
Carcinogensb	−	−	−	−	−	−	−	−	−	−	−
FT (pLC50, mg/L)b	High, 1.0351	High, 0.5057	High, 0.4518	High, 0.4706	High, 0.4841	High, 0.4376	High, −0.5511	High, 1.0836	High, 1.4651	High, 1.4614	Low, 1.3386
TPT (pIGC50, µg/L)b	High, 0.564	High, 0.5344	High, 0.6176	High, 0.5524	High, 0.6036	High, 0.6474	High, 1.1988	High, 0.4314	High, 0.5149	High, 0.4394	High, 0.5624
Honey bee toxicityb	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	High HBT	Low HBT	Low HBT	Low HBT	Low HBT
Biodegradationb	−	−	−	−	−	−	−	−	−	−	−
Acute oral toxicityb	III	III	III	III	III	III	III	III	III	III	III
Carcinogenicity (three class)b	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required
RAT (LD50, mol/kg)b	2.631	2.6238	2.6179	2.622	2.6159	3.0664	2.886	2.6718	2.2887	2.5842	2.7611
Toxicity profile	50848824	55760732	SN00282224	SN00397486	50848671	57590479	SN00018927	SN00023794	SN00111882	39906246	SN00012056
Mutagenica	None	None	None	None	None	None	None	None	None	None	None
Tumorigenica	None	None	None	None	None	None	None	None	None	None	None
Rea	None	None	None	None	Low	None	None	None	None	None	None
Irritanta	None	None	None	None	None	None	None	None	None	None	None
HEaggRGI Ib	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak	Weak
HEaggRGI IIb	−	+	−	−	−	+	+	+	−	+	−
AMES toxicityb	−	−	−	−	−	−	−	−	−	−	−
Carcinogensb	−	−	−	−	−	−	−	−	−	−	−
FT (pLC50, mg/L)b	High, 1.7586	High, 1.614	High, 0.929	High, 0.929	High, 1.6041	High, 0.9916	High, 1.5079	High, 1.4629	High, 1.0684	High, 1.5133	High, 1.3947
TPT (pIGC50, µg/L)b	High, 0.4819	High, 0.4803	High, 0.511	High, 0.511	High, 0.4817	High, 0.6474	High, 0.4237	High, 0.403	High, 0.5731	High, 0.5618	High, 0.4454
Honey bee toxicityb	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT
Biodegradationb	−	−	−	−	−	−	−	−	−	−	−
Acute oral toxicityb	III	III	III	III	III	III	III	III	III	III	III
Carcinogenicity (three class)b	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required
RAT (LD50, mol/kg)b	2.4555	2.4339	2.6944	2.6944	2.4526	2.4894	2.5869	2.6161	2.5259	2.4869	2.6562
Toxicity profile	SN00317979	SN00016042	SN00074088	SN00074091	SN00016053	SN00014343	SN00026414	SN00366028	SN00010280
Mutagenica	None	None	None	None	None	None	None	None	None
Tumorigenica	None	None	None	None	None	None	None	None	None
Rea	None	None	None	None	None	None	None	None	None
Irritanta	None	None	None	None	None	None	None	None	None
HEaggRGI Ib	Weak	Weak	Weak	Weak	Weak	Strong	Weak	Weak	Weak
HEaggRGI IIb	−	+	−	−	+	+	−	−	+
AMES toxicityb	+	−	−	−	−	+	−	+	−
Carcinogensb	−	−	−	−	−	−	−	−	−
FT (pLC50, mg/L)b	High, 1.0835	High, 1.2478	High, 0.6637	High, 0.6637	High, 1.5817	High, 1.4206	High, 0.88	High, 0.8777	High, 1.3743
TPT (pIGC50, µg/L)b	High, 0.4373	High, 0.5876	High, 0.5422	High, 0.5422	High, 0.4925	High, 0.3772	High, 0.575	High, 0.5326	High, 0.4375
Honey bee toxicityb	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT	Low HBT
Biodegradationb	−	−	−	−	−	−	−	−	−
Acute oral toxicityb	III	III	III	III	III	III	III	II	III
Carcinogenicity (three class)b	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required	Non-required
RAT (LD50, mol/kg)b	2.4919	2.5861	2.6706	2.6706	2.5654	2.6011	2.6371	2.9304	2.6376

Notes: LD50 is the amount of a compound, given all at once, which causes the death of 50% (one-half) of a group of test rats. Toxicity profile of all compounds tested in this study is available http://docking.umh.es/.

a These parameters were calculated using the DataWarrior v4.2.2 software (http://www.openmolecules.org/datawarrior/download.html).³³

b These parameters were calculated using the http://lmmd.ecust.edu.cn:8000/predict/site.³⁴ Compound names are from PubChem²² and SuperNatural II²³ databases, respectively.

Abbreviations: FT, fish toxicity; HEaggRGI I, human ether-a-go-go-related gene inhibition I; HEaggRGI II, human ether-a-go-go-related gene inhibition II; NS5, nonstructural protein 5; RAT, rat acute toxicity; RdRp, RNA-dependent RNA polymerase; Re, reproductive effectiveness; TPT, Tetrahymena pyriformis toxicity; HBT, Honey Bee Toxicity.

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