This summary describes the project approaches used to discover small molecules inhibiting West Nile virus replication in a cell culture system with high specificity. West Nile virus (WNV), a member of flaviviruses, is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Since the first case of WNV was detected in New York City in 1999 the virus spread rapidly west resulting in the largest epidemics of neuorinvasive WNV disease ever reported in the US.

Three lines of experiments were provided to get possible scaffolds for the SAR; 1) to screen a 300K of compounds library of MLSMR, 2) to screen cytotoxic compounds out and select compounds specific for the virus in a dose response study and 3) to confirm the activities in infectious virus reduction assays.
For the SAR, synthesized/analog compounds were built and collected based on the scaffolds found through the processes described above. A dose response study (same format as AID 1650) was used for the assessment of the antiviral and cytotoxic activities of the compounds. Another series of SAR compounds were tested for anti-WNV activity in a dose response format with higher concentration range. A cytotoxicity test was done in a dose response format with a higher range of concentrations for the compounds which did not show definite results in the previous cytotoxicity study. Two scaffolds which met probe criteria were nominated as probes through this study.

Specificity of the nominated probes was tested by assessing antiviral activity for other viruses; Respiratory syncitial virus (AID 2405) and Venezuelan equine encephalitis virus. Any of proposed probe candidates did not show significant antiviral activities for either virus. A cytotoxicity study for Vero 76 cells was performed as a counter screening for anti-VEEV assay.

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Summary of the Primary Screen for antiviral activity - AID 1621
Method: A cell based assay with CPE by an infectious virus
Test concentration: 10 microM
Activity criterion: >3.47% inhibition
Number compounds evaluated: 288,428
Number of active compounds: 3617

The protection of cytopathic effect from WNV infection in Vero E6 cells was measured as an antiviral effect of compounds screened. An end-point assay using the luminescence measuring ATP amount in a well was employed to measure the antiviral effect. A total of 288,428 compounds have been screened and Z values of the screen ranged between 0.62 and 0.91 with the median of 0.83 Three thousand six hundred seventeen compounds were evaluated as active with criteria of the average of negative control + 3 times of SD (3.47%) of whole screening. Six hundred one compounds were subjected to the dose response study to verify their activity and cytotoxicity.
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Confirmatory Screen for antiviral activity - AID 1621
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.039 microM to 20 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=30%
Number of compounds evaluated: 601
Number of active compounds: 62

Confirmatory Screen for antiviral activity in synthesized/analog compounds - AID 2238
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.156 microM to 80 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=30%
Number of compounds evaluated: 65
Number of active compounds: 22

Confirmatory Screen for antiviral activity in synthesized/analog compounds - AID 2419
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.2 microM to 100 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=30%
Number of compounds evaluated: 27
Number of active compounds: 7

Confirmatory Screen for antiviral activity in synthesized/analog compounds - AID 588371
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.2 microM to 100 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=30%
Number of compounds evaluated: 64
Number of active compounds: 5

Confirmatory Screen for antiviral activity in synthesized/analog compounds - AID 588372
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.391 microM to 200 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=30%
Number of compounds evaluated: 57
Number of active compounds: 8

Confirmatory Screen for antiviral activity in synthesized/analog compounds - AID 623934
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.78 microM to 100 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=30%
Number of compounds evaluated: 42
Number of active compounds: 13


The same measurement as the primary screening was employed but in the dose response format with a lowered virus infection amount, 0.05 MOI. IC50 value was calculated with data in duplicate (triplicate where applicable).
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Secondary screen for cytotoxicity - AID 1650
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.039 microM to 20 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 601
Number active: 183

Secondary screen of synthesized/analog compounds for cytotoxicity - AID 2244
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.078 microM to 80 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 65
Number active: 45

Secondary screen of synthesized/analog compounds for cytotoxicity - AID 2402
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.195 microM to 200 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 68
Number active: 53

Secondary screen of synthesized/analog compounds for cytotoxicity - AID 2402
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.2 microM to 100 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 115
Number active: 59

Secondary screen of synthesized/analog compounds for cytotoxicity - AID 623930
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.78 microM to 100 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 42
Number active: 34

The cytotoxicity of the compounds was evaluated to rule out cytotoxic compounds which might be inappropriate for antiviral probes. The host cells, Vero E6, about 50% confluent were incubated for three days in a culture media containing different concentration of compounds. The cell viability was measured with an end-point assay using the luminescence measuring ATP amount in a well. Compounds causing cell viability to decrease to less than 70% viability at any concentration were deemed Active.

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Secondary Titer Reduction by TCID50 - AID 2024
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 2.5 microM to 20 microM
Activity criterion: 10 fold reduction in the progeny titer (<-1 log reduction)
Number compounds evaluated: 55
Number active: 12

The inhibitory activities of selected confirmed actives from the confirmatory and cytotoxicity assays were verified in a titer reduction assay format which measures the difference in viral titer between non-treated and treated cells. A compound which inhibits viral replication during the replication process results in less progeny virus compared to control group. The titer reduction assay involves challenging 70~80% confluent Vero cells infected with WNV at a multiplicity of infection (MOI) of 0.01 in the presence of the compounds for 48 hours.
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Secondary Titer Reduction by TCID50 - AID 2065
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 2.5 microM to 20 microM
Activity criterion: 10 fold reduction in the progeny titer (<-1 log reduction)
Number compounds evaluated: 10
Number active: 8

The inhibitory activities of synthesized analogs were verified in a titer reduction assay format which measures the difference in viral titer between non-treated and treated cells. A compound which inhibits viral replication during the replication process results in less progeny virus compared to control group. The titer reduction assay involves challenging 70~80% confluent Vero cells infected with WNV at a multiplicity of infection (MOI) of 0.01 in the presence of the compounds for 48 hours.

Confirmatory Screen for antiviral activity for Respiratory Syncitial virus in probe candidate compounds AID 2405
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.195 microM to 100 microM
Activity criterion: Inhibition >= 50%
Number of compounds evaluated: 83
Number of active compounds: 0

Specificity of the nominated probes was tested by assessing antiviral activity for other viruses; Respiratory syncitial virus. IC50 value was calculated with data in quadriplicate. None of compounds were classified as active.

Confirmatory Screen for antiviral activity for Venezuelan Equine Encephalitis virus in probe candidate compounds - AID 2409
Method: A cell-based assay with lucifererase expression by an infectious virus in dose response format.
Test concentration: 0.13 microM to 133 microM
Activity criterion: Inhibition >= 30%
Number of compounds evaluated: 83
Number of active compounds: 30

Specificity of the nominated probes was tested by assessing antiviral activity for other viruses; Venezuelan Equine Encephalitis virus. IC50 value was calculated with data in quadruplicate. The assay measures the level of a luciferase expressed by V3526-luc in Vero 76 cells.

Secondary screen of Probe candidate compounds for cytotoxicity for Vero 76- AID 2414
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.13 microM to 133 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 68
Number active: 34

A series of probe candidates was subjected to anti-VEEV test for specificity. Some of the compounds showed marginal activity so a further study on the cytotoxicity was performed to rule out cytotoxic compounds which might be inappropriate for antiviral probes. The host cells for the VEEV, Vero 76, about 50% confluent were incubated for three days in a culture media containing different concentration of compounds. The cell viability was measured with an end-point assay using the luminescence measuring ATP amount in a well. Compounds causing cell viability to decrease to less than 70% viability at any concentration were deemed Active.

Grant Number: 1R03 MH084847-01