Zika virus NS2B-NS3 protease

In this work, researchers elucidate new structure-activity relationships of benzo[d]thiazole-based allosteric NS2B/NS3 inhibitors. They developed a new series of Y-shaped inhibitors, which, with its larger hydrophobic contact surface, should bind to previously unaddressed regions of the allosteric NS2B/NS3 binding pocket. By scaffold-hopping, researchers varied the benzo[d]thiazole core and identified benzofuran as a new lead scaffold shifting the selectivity of initially ZIKV-targeting inhibitors to higher activities towards the DENV protease. In addition, researchers were able to increase the ligand efficiency from 0.27 to 0.41 by subsequent inhibitor truncation and identified N-(5,6-dihydroxybenzo[d]thiazol-2-yl)-4-iodobenzamide as a novel sub-micromolar NS2B/NS3 inhibitor. Utilizing cell-based assays, researchers could prove the antiviral activity in cellulo. Overall, researchers report new series of sub-micromolar allosteric DENV and ZIKV inhibitors with good efficacy profile in terms of cytotoxicity and protease inhibition selectivity. [1]

Fluorometric assays [1]
The determination of the inhibitory activity of the compounds against the proteases was performed with an assay based on the fluorogenic substrates or FRET-based substrates. The inhibitors and the substrate were prepared as stock solutions in DMSO. The fluorescence was measured in white flat-bottom 96-well microtiter plates using a Tecan Infinite F2000 PRO plate reader. Measurements were performed in at least three independent experiments. In each well a total volume of 200 µL was used, consisting of 180 µL buffer, 5 µL enzyme solution, 10 µL inhibitor in DMSO or pure DMSO as control, and 5 µL solution of the corresponding substrate. Initial screenings were performed at inhibitor concentrations of 20 µM. IC50 values were determined with dilution series between 0.01 µM and 100 µM. The fluorescence was measured every 30 s for 10 min at 25 °C with the corresponding excitation and emission wavelengths. IC50 values were calculated with GraFit by fitting the remaining enzymatic activity to the four-parameter IC50 equation, with Y [ΔF/min] as the substrate hydrolysis rate, Ymax as the maximum value of the dose–response curve, measured at inhibitor concentrations of [I] = 0 µM, Ymin as the minimum value, obtained at high inhibitor concentrations, and s as the Hill coefficient.

Replication analysis and luciferase assays [1]
Quantification of luciferase activity was used to determine ZIKV and DENV2 RNA replication as described previously.68, 69 In brief, single-cell suspensions of Huh7 cells were prepared by trypsinization and washed once with phosphate-buffered saline. Cells were resuspended at a concentration of 1 × 10~7 cells per mL in Cytomix containing 2 mM ATP and 5 mM glutathione. 10 μg of in vitro transcribed RNA was mixed with 400 μL of the cell suspension (4x10~6 cells) and transfected by electroporation using a Gene Pulser system in a cuvette with a gap width of 0.4 cm at 975 μF and 270 V.82 Cells were resuspended in 15 mL culture medium. Cells were seeded in duplicate wells in a 12-well plate: 500 µL for the time points 4 h and 24 h and 1 mL for the time points 48 h, 72 h, and 96 h. 4 h after electroporation, cells were treated with 10 µM compound or an equivalent volume of DMSO, as vehicle control, in culture medium supplemented with 15 mM HEPES. Compound- or DMSO-containing culture medium was replenished after 48 h. Cells were lysed at 4, 24, 48, 72, and 96 h after electroporation by addition of 250 µL luciferase lysis buffer (0.1% (v/v) TritonX-100, 25 mM glycylglycine, 15 mM MgSO4, 15 mM K3PO4 pH 7.8, 4 mM EGTA, 10% (v/v) glycerol, and 1 mM DTT). For detection of Renilla luciferase activity, 100 μL lysate was mixed with 200 µL luciferase assay buffer (25 mM glycylglycine, 15 mM MgSO4, 15 mM K3PO4 pH 7.8, and 4 mM EGTA) supplemented with 14 or 28 nM coelanterazine (P.J.K). For detection of firefly luciferase activity, 100 μL lysate was mixed with 350 μL luciferase assay buffer freshly supplemented with 1 mM DTT and 2 mM ATP, and d-luciferin substrate (200 µM d-Luciferin, P.J.K.) in 25 mM glycylglycine.

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Cell viability assay [1]
To determine the impact of compound treatment on cell viability, Huh7 cells were seeded at a density of 4x10~3 cells per well in white-walled 96-well plates and one day after seeding cells were treated with 1.25, 2.5, 5, 10, 20, and 40 µM compound or equivalent volumes of DMSO for 96 h. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay following manufacturer instructions. Cell viability was determined by measurement with a plate luminometer and values normalized to untreated cells.

[1]. SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B/NS3 proteases. Bioorg Med Chem. 2021 Oct 1;47:116392.

In recent years, dengue virus (DENV) and Zika virus (ZIKV) – both mosquito-borne viruses of the Flaviviridae family – have become transcontinental health problems as climate change and globalization have led to the spread of their vectors from tropical to temperate regions. DENV and ZIKV are both positive-sense single-stranded RNA viruses whose genomes consist of three structural proteins (capsid protein, membrane precursor protein, and envelope protein) and seven non-structural proteins (NS proteins), all of which are initially expressed as a single precursor polyprotein. During viral maturation, the processing of the polyprotein is accomplished by the host protease and the viral NS2B/NS3 protease complex. Studies have shown that inhibitors of the NS2B/NS3 protease complex are effective antiviral drugs and can reduce viral pathogenicity. [1]

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This study optimized allosteric inhibitors of DENV2 and ZIKV NS2B/NS3 proteases and discovered novel lead compounds with good inhibitory properties. Different series of inhibitors were synthesized by exchanging parts of lead compounds 1a and 1b, and their inhibitory effects on dengue virus type 2 (DENV2) and Zika virus (ZIKV) NS2B/NS3 proteases were investigated. Replacing the amino acid linker with (R/S)-alanine (8a, 8b), isoleucine (8c), tert-leucine (8d), phenylalanine (8e), tyrosine (12a), and aspartic acid (12b) did not significantly improve the inhibitory effect. However, compounds 8a–f and 12a inhibited the Zika virus protease in the low micromolar concentration range (IC50 = 2.13–6.48 µM). Among the newly designed Y-type inhibitor series (Table 2), the 2,2-diphenylacetic acid derivative 23b is the most promising compound, with an IC50 value for Zika virus (ZIKV) below the micromolar level (0.95 µM). The IC50 values of Y-type compounds 23b and 20b, as well as AA-type compounds 8c-f with hydrophobic side chains, are all in the low micromolar range, indicating that using Y-type inhibitors to simultaneously target two binding subsites is a promising strategy that could help further improve inhibitory efficacy. By truncating the inhibitor backbone (Table 3), we achieved, for the first time, low micromolar inhibition of dengue virus type 2 (DENV2) protease while improving ligand potency, providing a new starting point for further DENV2 drug development. The iodine-substituted inhibitor 25b is the most promising in this series, with an IC50 of 4.38 µM for DENV2 and a submicromolar IC50 (0.67 µM) for ZIKV. By replacing the benzo[d]thiazole core heterocycle, we demonstrated that other heteroaromatic ring systems besides benzo[d]thiazole can also serve as backbones. In this structure-activity relationship series, the benzofuran derivative 34e exhibited the best inhibitory activity (IC50(DENV2) = 0.69 µM, IC50(ZIKV) = 1.04 µM). Compared with benzo[d]thiazole compounds, 34e showed inhibitory activity against the NS2B/NS3 proteases of ZIKV and DENV2 on the same order of magnitude. Screening of the selected compounds against various serine and cysteine proteases demonstrated excellent off-target selectivity for our inhibitors. We employed a cell-based antiviral assay to investigate the potential of the most active inhibitors and their corresponding methoxy prodrugs to interfere with dengue virus type 2 (DENV2) and Zika virus (ZIKV) replication. Compounds 22b, 23b, and 25b significantly inhibited DENV2 replication, highlighting their antiviral potential; while compounds 25b and 34e reduced ZIKV replication. In conclusion, we identified two promising compounds that could serve as a suitable starting point for future drug development. First, N-(5,6-dihydroxybenzo[d]thiazolyl)-4-iodobenzamide (25b) and second, 5,6-dihydroxy-N-phenylbenzofuran-2-carboxamide (34e) contain only 20 and 21 heavy atoms, respectively, and have ligand efficiencies higher than 0.4, making them a good starting point for further development of NS2B/NS3 inhibitors. [1]

C17H13N3O2S

323.37

323.072

1021221-94-2

45283583

White to off-white solid powder

4.3

1

5

2

23

463

0

O1C2=CC=CC=C2C=C1C(NC1=NC2=NC(C)=CC(C)=C2S1)=O

MIATZSYTBCVGSR-UHFFFAOYSA-N

InChI=1S/C17H13N3O2S/c1-9-7-10(2)18-15-14(9)23-17(19-15)20-16(21)13-8-11-5-3-4-6-12(11)22-13/h3-8H,1-2H3,(H,18,19,20,21)

N-(5,7-dimethyl-[1,3]thiazolo[4,5-b]pyridin-2-yl)-1-benzofuran-2-carboxamide

NS2B/NS3-IN-8; 1021221-94-2; N-(5,7-dimethylthiazolo[4,5-b]pyridin-2-yl)benzofuran-2-carboxamide; N-(5,7-dimethyl-[1,3]thiazolo[4,5-b]pyridin-2-yl)-1-benzofuran-2-carboxamide; N-{5,7-dimethyl-[1,3]thiazolo[4,5-b]pyridin-2-yl}-1-benzofuran-2-carboxamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)