Table 5.
Summary of NS2B/NS3 protease inhibitors recent development applying in vitro and in silico method.
| Compound | In vitro method | In silico method | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Method | Cell type | EC50 (µM) | CC50 (µM) | IC50 (µM) | Method | Docking energy (kcal/mol) |
Residues interacting with Ligand | Ref | ||
| 1 | MB21 | i. Protease inhibition assays ii. Cell-based DENV inhibition assay iii. MTT assay iv. Molecular docking |
Vero | ND | ND | 5.95 μ | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compound from ‘In-house’ library iii. Molecular docking program: Glide v5.7 |
ND | Ile123, Val147, Tro83, Leu85, Ile165, Leu76, Met46, Ala164, Val154, Val155 | (Raut et al., 2015a) |
| 2 | T5341917 (Compound 14) |
i) Molecular docking ii) Protease inhibition assay iii) Cell-based flavivirus immune detection iv) Cell viability assay |
Huh-7 and BHK21 | 5.0 ± 0.2 (HuH7), 5.0 ± 1.1 (BHK21) |
>300 (HuH7), 55.0 (BHK21) |
85% mean inhibition | i. Protein PDB ID: 3U1I ii. Ligands: Synthesized compounds from ChemBridge library iii. Molecular docking program: MOE, AutoDock |
-10.65 | Pro132, Val155, Tyr161, Met84, Gly153, Ile86, Val165 | (Li et al., 2015) |
| 3 | C35H27NO9
(CID 54681617) |
i) Molecular docking ii) Fluorimetric enzyme activity assay iii) MTT assay iv) Virus yield reduction assay |
HepG-2 | ND | 58.6 ± 3.0 | 14.9 ± 2.9 | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compounds from PubChem iii. Molecular docking program: AutoDock Vina |
-11.6 | Ile65, Trp69, Lys74, Leu76, Thr120, Ile123, Val154, Ala164, Ile165, and Ala166 | (Cabarcas-Montalvo et al., 2016) |
| 4 | C30H25NO5
(CID 54692801) |
i) Molecular docking ii) Fluorimetric enzyme activity assay iii) MTT assay iv) Virus yield reduction assay |
ND | 42.1 ± 1.6 | 11.8 ± 0.2 | -13.5 | Ile65, Trp69, Lys74, Leu76, Thr120, Ile123, Val154, Ala164, Ile165, and Ala166 | |||
| 5 | C34H23NO7S2
(CID 54715399) |
i) Molecular docking ii) Fluorimetric enzyme activity assay iii) MTT assay iv) Virus yield reduction assay |
ND | 162.4 ± 0.9 | 61.5 ± 4.6 | -11.4 | Ile65, Trp69, Lys74, Leu76, Thr120, Ile123, Val154, Ala164, Ile165, and Ala166 | |||
| 6 | Nitro derivatives of 3,5-bis(arylidene)-4-piperidones (Compound 4e) |
i) Compound synthesis ii) Molecular docking iii) Protease assay |
ND | ND | ND | 15.22 | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compounds iii. Molecular docking program: AutoDock |
11.36 | His51, Pro132, Ser135, Gly153 and Arg54 | (Osman Idris et al., 2017) |
| 7 | Nitro derivatives of 3,5-bis(arylidene)-4-piperidones (Compound 4j) |
ND | ND | ND | 16.23 | 11.09 | His51, Pro132, Ser135, Gly153, Arg54, Trp50 | |||
| 8 | NSC135618 | I) Protease inhibition assay ii) Cytotoxicity assay iii) Viral titer reduction assay iv)Immunofluorescence assay v) qRT-PCR vi)Protein thermal shift assay vii) Western blot viii)Mass spectrometry |
A549 | 0.81 | 48.8 | 1.8 | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compounds from Diversity Set II library from the National Cancer Institute Developmental Therapeutics Program (NCI DTP) iii. Molecular docking program: AutoDock Vina |
ND | Lys74, Asn152, Trp89, V147, Ala164, Val154, Ile123, Asn167, Trp89, Ile165, Ile147, Trp83, Leu149 and Leu76 | (Brecher et al., 2017) |
| 9 | Calmodulin antagonist: N-(6-aminohexyl) - 5- chloro-1-naphthalene-sulfonamide hydrochloride (W-7) |
i) Cell-based assay ii)Western blot iii) Confocal microscopy and flow cytometry (FACS) assays iv)qRT-PCR v)Molecular docking |
Huh-7 | ND | (W7 is not inducing apoptosis in Huh-7 cells) | (64% secretion reduction of NS3) | i. Protein PDB ID: ND ii. Ligands: Synthesized compound iii. Molecular docking program: Molegro Virtual Docker |
92.502 | His51, Asp75, and Ser135 | (Bautista-Carbajal et al., 2017) |
| 10 | 6-fluoro-4-(2-((5-nitrobenzo[d]thiazol-2-yl) amino)-2-oxoethoxy) quinoline-2- carboxylic acid (BT24) |
i) Protease inhibition assay ii) Cell-based DENV inhibition assay iii) RT-PCR iii) plaque assay iv) MTT assay v) Molecular docking |
Vero | ND | 75.00 | 0.50 | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compound from ‘in-house’ library, iii. Molecular docking program: Glide v5.7 |
ND | Trp83, Thr120 and Asn152 | (Beesetti et al., 2018) |
| 11 | Diasarone-I | i. Virus-induced cytopathic effect and measurement of viral infection ii.Plaque assay iii. Time of drug addition assay iv. NS2B/NS3 enzyme inhibition assay v. Reactive oxygen species assay vi. Western blotting vii. Immunofluorescence assay viii. Quantitative real-time PCR (qRT-PCR) ix. Molecular docking |
C6/36 | 4.5 | >80 | ND | i. Protein PDB ID: ND ii. Ligands: Natural product compounds iii. Molecular docking program: AutoDock Vina |
-7.200 | Lys105, Thr104, Gly83, Cys82, Gly81, Val132, Phe133, Ile141 | (Yao et al., 2018) |
| 12 | N-(adamantan-1-yl)-4-[(adamantan-1-yl) sulfamoyl] benzamide) (Compound 3) |
i. Compound synthesis ii. Cell-Based Flavivirus Immunodetection (CFI) Assay iii. Cytotoxicity Assay iv. Molecular docking |
A549 | ND | <100 | 22.4 ± 7.7 | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compounds iii. Molecular docking program: MOE |
-7.413 | His51, Gly153 | (Joubert Foxen and Malan, 2018) |
| 13 | N-(adamantan-1-yl)-4-sulfamoyl benzamide (Compound 7) |
A549 | ND | <100 | 42.8 ± 8.6 | -7.123 | Val72, Asp75, Gly153 | |||
| 14 | Erythrosin B | i. Protease inhibition assay ii. MTT assay iii. Viral reduction assay iv. IF assay v. qRT-PCR vi. Western blot vii. Molecular docking viii. Protein thermal shift assay (PTSA) |
A549 | 1.2 ± 0.2 | > 150 | 15 | i. Protein PDB ID: 3U1I ii. Ligands: Synthesized compound iii. Molecular docking program: Schrodinger |
ND | ND | (Li et al., 2018) |
| 15 | Thiosemicarbazones derived phenyl-acetyl ketones (DB-TYR-TSC) |
i. Cytotoxicity assay ii. Indirect immunofluorescence assay iii. In silico method iv. Plaque formation unit reduction assay v. Molecular docking |
Vero | ND | 350 | 50 | i. Protein PDB ID: 3U1I ii. Ligands: Synthesized compound iii. Molecular docking program: AutoDock 4.2.6 and Rasmol |
-6.36 | Ser135, Gly151, Pro132, Asp 75 | (Padmapriya et al., 2018) |
| 16 | Thioguanine derivatives (Compound 18) | 1. Compound synthesis 2. Molecular docking 3. Protease Inhibition assay Molecular dynamic simulation |
ND | ND | ND | 0.38 | i. Protein PDB ID: 2FOM ii. Ligands: Synthesized compound from National Cancer Institute database, Hyperchem 8.0 iii. Molecular docking program: AutoDock4.2 |
-16.10± 2.70 | Gly175, Asn174, Tyr183, Asp97, Tyr183, Ser157, Gly35, Ser36, His73, Asp34, Met37, Arg76 | (Hariono et al., 2019) |
| 17 | Thioguanine derivatives (Compound 21) |
ND | ND | ND | 16 | -18.24 ± 4.66 | His73, Ser157, Asp97, Gly175, Asn174 | |||
| 18 | 4-hydroxy-6-(9,13,17-trimethyldodeca- 8,12,16-trienyl)2(3H)-benzofuranone (Compound 1) (Isolated from Endiandra kingiana) |
i) Protease activity assay ii) Molecular docking |
ND | ND | ND | 403.14 ± 33.03 | i. Protein PDB ID: 2FOM ii. Ligands: Natural product compounds iii. Molecular docking program: AutoDock |
ND | Asp129 and Ser135 | (Sulaiman et al., 2019) |
| 19 | (−)-Epicatechin (Compound 2) (Isolated from Endiandra kingiana) |
ND | ND | ND | 170.10 ± 5.94 | ND | Asp129, Ser135, Tyr161 and Asn152 | |||
| 20 | (+)-Catechin (Compound 3) (Isolated from Endiandra kingiana) |
ND | ND | ND | 184.13 ± 2.11 | ND | Asp129, Tyr161 and Asn 152 | |||
| 21 | Hesperetin (From Ganoderma lucidum var. antler) |
i) Protease activity assay ii) Cytotoxicity test iii) Molecular docking |
WRL-68 | 326. | ND | ND | i. Protein PDB ID: 2FOM ii. Ligands: Natural product compounds from numerous molecular databases (ZINC, PubChem etc), GaussView 5.0 iii. Molecular docking program: HADDOCK2 |
- 7.2 | His107, Val128, Pro188, Ser191, Trp106, Gly207, Asn208, Gly209, Tyr217, His107, Val128, Asp131, Leu184, Pro18 8, Gly207, Gly209, Tyr217 and Asp131 | (Lim et al., 2020) |
| 22 | Isobiflorin (Compound 1) (From S. aromaticum) (cloves extract) |
i) Protease activity assay ii)Protease inhibition assay iii) Molecular docking |
ND | ND | ND | 58.9 ± 1.3 | i. Protein PDB ID: 3U1I ii. Ligands: Natural product compounds iii. Molecular docking program: AutoDock |
−6.8 | Trp-50, Arg-54, Asp-75, His-51, Val-72, Asp-81, and Asn-152 | (Saleem et al., 2019) |
| 23 | Biflorin (Compound 2) (From S. aromaticum) (cloves extract) |
ND | ND | ND | 89.6 ± 4.4 μM | −7.2 | Met-84, Ile-86, Asn-152, Gly-153, Tyr-161, Thr-83, Arg-85, Val-154, and Val-155 | |||
| 24 | Eugeniin (Compound 3) (From S. aromaticum) (cloves extract) |
ND | ND | ND | 94.7 ± 2.5 μM | −10.2 | Asp-75, Asp-81, Met-84, Asp-129, Phe-130, Gly-133, Ser-135, His-51, Arg-54, Pro-132, Tyr-150, Val-154, Val-155, and Tyr-161 | |||
| 25 | Kaempferol-3-O-rutinoside (bioflavonoids from Azadirachta indica) | i) Molecular docking ii) Cytotoxicity test iii)Protease inhibition assay iv) IF assay |
BHK-21 | ND | No significant cyto-toxicity till 100 μM concentra-tion | 55.6% in DENV-2 infectivity at lower concentra-tions of 1 and 10μM; Maximum inhibition of 77.7% at 10 and 100 μM concentra-tion |
i. Protein PDB ID: 2FOM ii. Ligands: Natural product compounds iii. Molecular docking program: GLIDE5.8 |
–9.555 | Asp75, Phe130, Gly151, Asn152, Gln153, Trp50, His51, Val72, Lys73, Leu128, Ser131, Pro132, Ser135, Tyr150, Val154, and Try161 | (Dhar Dwivedi et al., 2020) |
| 26 | Epicatechin (Bioflavonoids from Azadirachta indica) |
BHK-21 | ND | 20% cyto-toxicity on the BHK-21 cells at 1 mM (1000 μM) concentra-tion | 47.1% reduction in the DENV-2 infectivity at 0.1 mM (100 μM); Maximum of 66.2% inhibition of DENV-2 infectivity at 1 mM (1000 μM) concentra-tion |
-7.622 | His51, Pro132, Gly151, Phe130, Leu128, Ser131, Gly133, Ser135, Try150, Asn152, Gly123, His51, Ser131, Ser135, Asn152, Gly133, Gly151, and Gly 153 | |||
| 27 | C26H19F3N4O5S2
Compound 8g |
i) Compound synthesis ii) Protease activity assay iii) Protease inhibition assay iv) Molecular dockin |
ND | ND | ND | 13.9 ± 1.4 | i. Protein PDB ID: 3U1I ii. Ligands: Synthesized compounds iii. Molecular docking program: AutoDock Vina |
−8.8 | Thr118, leu85, Trp83, Asn167 | [106] |
| 28 | C27H21F3N4O5S2
Compound 8h |
ND | ND | ND | 15.1 ± 1.3 | −8.8 | Trp83, Asn167 | |||
| 29 | Compound 1 | i. Molecular docking ii. Protease inhibition assay iii. Cell viability assay iv. Western blot, RT-PCR v. IF microscopy |
Huh-7 | ND | 35.4mM | 7.1mM | i. Protein PDB ID: 5YW1 ii. Ligands: Maestro v.11.5 iii. Molecular docking program: Schrodinger Suite v.2018 |
ND | Pro132, Tyr150, Tyr161, Asp129, Asp75. | (Shin et al., 2021) |
ND, Not defined.