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ChEMBL-NTD Compound Activity Search

Deposited Set 1: GSK TCAMS Dataset

Assay Min Value Adjust Range Max Value
TCMDC: % inhibition of P. falciparum strain 3D7 (at 2uM)
TCMDC: % inhibition of P. falciparum Dd2 (at 2uM)
TCMDC: pXC50 determination of P. falciparum 3D7 growth
TCMDC: % inhibition of human HepG2 cell line (at 10uM)
TCMDC: Inhibition Frequency Index (IFI)

Predicted Target:  


Available Commercially


Name: % inhibition of P. falciparum 3D7
Type: Cellular assay
Description: This assay uses levels of P. falciparum lactate dehydrogenase as surrogate of parasite growth. Inhibition of 3D7 growth by compounds has been determined in this assay.

Readout Definitions: PCT_INHIB_3D7
Unit: percent
Description: Percent inhibition of P. falciparum 3D7 growth

Conditions

Preparation of assay plates
Assay plates (384 well plates) are prepared by stamping 0.05 uL of compound from master plates at 1mM in each well. Final volume assay is 25 uL and final compound concentration is 2 uM. In the 6th column 0.05 uL of DMSO are dispensed (positive control). In the 18th column 0.05 uL of a mix of a 50 uM chloroquine, 50 uM artemisinin stock solution are dispensed (negative controls).

Preparation of parasite inoculum.
P. falciparum 3D7 strain is cultivated using standard procedures as described in Trager et al. (1976). Science 193:673-675. An inoculum of parasitized red blood cells (pRBC) with a parasitaemia of 0.25% and 2% haematocrit in RPMI-1640, 5% albumax, 2% D-sucrose, 0.3% glutamine and 150 uM hypoxanthine is prepared and used for the assay.

Whole cell assay
25 ul of parasite inoculum is dispensed in assay plates with pre-dispensed compounds using a multidrop combi dispenser. Plates are shaken for 10 sec. to ensure mixing of the compound with the parasites. Plates are incubated at 37ºC for 72h in an atmosphere of 5% CO2, 5% O2, 95% N2.

Evaluation of LDH activity as surrogate of parasite growth
After 72 hours of incubation, plates are frozen at -70ºC overnight. Next morning, plates are thawed at room temperature for at least 4 hours. To evaluate LDH activity, 70 uL of reaction mix solution (143 mM Sodium L-Lactate - 143 uM APAD, 178.75 uM NBT, 286 ug/ml Diaphorase (2.83 U/ml), 0.7% Tween 20, 100 mM Tris-HCl pH 8.0) freshly made is dispensed using a multidrop. Plates are shaken to ensure mixing and absorbance at 650nm is monitorized after 10 minutes of incubation at room temperature. Data are normalized to percentage of growth inhibition using positive, negative controls and next equation: % inhibition growth = 100- (Abs. in well-Abs. neg. control/Abs. pos. control- Abs. neg. control)

Name: % inhibition of P. falciparum Dd2
Type: Cellular assay
Description: This assay uses levels of P. falciparum lactate dehydrogenase as surrogate of parasite growth. Inhibition of Dd2 growth by compounds has been determined in this assay.

Readout Definitions: PCT_INHIB_Dd2
Unit: percent
Description: Percent inhibition of P. falciparum Dd2 growth

Conditions

Preparation of assay plates
Assay plates (384 well plates) are prepared by stamping 0.05 uL of compound from master plates at 1mM in each well. Final volume assay is 25 uL and final compound concentrations is 2 uM. In the 6th column 0.05 uL of DMSO are dispensed (positive controls). In the 18th column 0.05 uL of a mix of a 50 uM chloroquine, 50 uM artemisinin stock solution are dispensed (negative controls).

Preparation of parasite inoculum
P. falciparum Dd2 strain is cultivated using standard procedures as described in Trager et al. (1976). Science 193:673-675. An inoculum of parasitized red blood cells (pRBC) with a parasitaemia of 0.25% and 2% haematocrit in RPMI-1640, 5% albumax, 2% D-sucrose, 0.3% glutamine and 150 uM hypoxanthine is prepared and used for the assay.

Whole cell assay
25 ul of parasite inoculum is dispensed in assay plates with pre-dispensed compounds using a multidrop combi dispenser. Plates are shaken for 10 sec. to ensure mixing of the compound with the parasites. Plates are incubated at 37ºC for 72h in an atmosphere of 5% CO2, 5% O2, 95% N2.

Evaluation of LDH activity as surrogate of parasite growth
After 72 hours of incubation, plates are frozen at -70ºC overnight. Next morning, plates are thawed at room temperature for at least 4 hours. To evaluate LDH activity, 70 uL of reaction mix solution (143 mM Sodium L-Lactate - 143 uM APAD, 178.75 uM NBT, 286 ug/ml Diaphorase (2.83 U/ml), 0.7% Tween 20, 100 mM Tris-HCl pH 8.0) freshly made is dispensed using a multidrop. Plates are shaken to ensure mixing and absorbance at 650nm is monitorized after 10 minutes of incubation at room temperature. Data are normalized to percentage of growth inhibition using positive, negative controls and next equation: % inhibition growth = 100- (Abs. in well-Abs. neg. control/Abs. pos. control- Abs. neg. control)

Name: % inhibition of P. falciparum LDH Reporter Assay
Type: Biochemical assay
Description: Inhibition of 3D7 lactate dehydrogenase (LDH) enzymatic activity by compounds has been determined in this assay. LDH activity has been evaluated in crude parasite extracts.

Readout Definitions: PCT_INHIB_LDH
Unit: percent
Description: Percent inhibition of P. falciparum lactate dehydrogenase activity

Conditions

Preparation of assay plates
Assay plates (384 well plates) are prepared by stamping 0.05 uL of compound from master plates at 1mM in each well. Final volume assay is 25 uL and final compound concentrations is 2 uM. In the 6th column 0.05 uL of DMSO are dispensed (positive controls). In the 18th column 0.005 mL of oxamic acid 500mM is dispensed (negative controls).

Preparation of parasite inoculum
P. falciparum 3D7 strain is cultivated using standard procedures as described in Trager et al. (1976). Science 193:673-675. An culture of parasitized red blood cells (pRBC) with a parasitaemia of 0.25% and 2% haematocrit in RPMI-1640, 5% albumax, 2% D-sucrose, 0.3% glutamine and 150 uM hypoxanthine is growth at 37ºC for 72h in an atmosphere of 5% CO2, 5% O2, 95% N2. Final culture is frozen at -70ºC overnight. Next morning culture is thawed at room temperature for at least 4 hours and is used as inoculum (crude extract) for this assay.

Evaluation of LDH activity
25 ul of parasite inoculum is dispensed in assay plates with pre-dispensed compounds using a multidrop combi dispenser. Plates are shaken for 10 sec. to ensure mixing of the compound. Immediately 70 uL of reaction mix solution (143 mM Sodium L-Lactate - 143 uM APAD, 178.75 uM NBT, 286 ug/ml Diaphorase (2.83 U/ml), 0.7% Tween 20, 100 mM Tris-HCl pH 8.0) freshly made is dispensed using a multidrop. Plates are shaken to ensure mixing and absorbance at 650nm is monitorized after 10 minutes of incubation at room temperature. Data are normalized to percentage of activity inhibition using positive, negative controls and next equation: % inhibition = 100- (Abs. in well-Abs. neg. control/Abs. pos. control- Abs. neg. control)

Name: XC50 for P. falciparum 3D7
Type: Cellular assay
Description: This assay uses levels of P. falciparum lactate dehydrogenase as surrogate of parasite growth. An estimation of potencies of compounds for inhibition of 3D7 growth (XC50) has been determined in this assay.

Readout Definitions: XC50_3D7
Unit: uM
Description: XC50 determination of P. falciparum 3D7 growth

NOTE: XC50 values have been converted to pXC50, where pXC50 = -log10 XC50. Example where XC50 = 1uM, then pXC50 = 6

Conditions

Preparation of the compounds to be tested. Preparation of assay plates
Compounds are prepared from stock solutions at 1mM in DMSO. Master plates of serial dilutions (5-fold dilution, 5 different concentrations) are prepared using an inter-plate approach. Assay plates (384 well plates) are prepared by stamping 0.05 uL of compound from master plates in each well. Final volume assay is 25 uL and final compound concentrations are 2 uM, 0.4 uM, 0.08 uM, 0.016 uM and 0.0032 uM. In the 6th column 0.05 uL of DMSO are dispensed (positive controls). In the 18th column 0.05 uL of a mix of a 50 uM chloroquine, 50 uM artemisinin stock solution are dispensed (negative controls).

Preparation of parasite inoculum
P. falciparum 3D7 strain is cultivated using standard procedures as described in Trager et al. (1976). Science 193:673-675. An inoculum of parasitized red blood cells (pRBC) with a parasitaemia of 0.25% and 2% haematocrit in RPMI-1640, 5% albumax, 2% D-sucrose, 0.3% glutamine and 150 uM hypoxanthine is prepared and used for the assay.

Whole cell assay
25 ul of parasite inoculum is dispensed in assay plates with pre-dispensed compounds using a multidrop combi dispenser. Plates are shaken for 10 sec. to ensure mixing of the compound with the parasites. Plates are incubated at 37ºC for 72h in an atmosphere of 5% CO2, 5% O2, 95% N2.

Evaluation of LDH activity as surrogate of parasite growth
After 72 hours of incubation, plates are frozen at -70ºC overnight. Next morning, plates are thawed at room temperature for at least 4 hours. To evaluate LDH activity, 70 uL of reaction mix solution (143 mM Sodium L-Lactate - 143 uM APAD, 178.75 uM NBT, 286 ug/ml Diaphorase (2.83 U/ml), 0.7% Tween 20, 100 mM Tris-HCl pH 8.0) freshly made is dispensed using a multidrop. Plates are shaken to ensure mixing and absorbance at 650nm is monitorized after 10 minutes of incubation at room temperature. Data are normalized to percentage of growth inhibition using positive, negative controls and next equation % inhibition = 100- (Abs. in well-Abs. neg. control/Abs. pos. control- Abs. neg. control). XC50 is calculated as compound concentration giving 50% inhibition of positive control growth using excel XC50 data analysis worksheet.

Name: % inhibition of HepG2 cell line
Type: Cellular assay
Description: This assay uses intracellular ATP levels as surrogate of cell viability

Readout Definitions: PCT_INHIB_HepG2
Unit: percent
Description: Percent inhibition of Homo sapiens HepG2 growth

Conditions

Preparation of assay plates
Assay plates (384 well plates) are prepared by stamping 0.25 uL of compound from master plates at 1mM in each well. Final volume assay is 25 uL and final compound concentrations is 10 uM. In the 6th column 0.25 uL of DMSO are dispensed (positive controls). In the 18th column 0.25 uL of 10mM digitonin are dispensed (negative controls).

Preparation of cells and cytotox assay
Actively growing HepG2 cells are removed from a T-175 TC flask using Cell Dispersion Medium (5ml), and made as disperse as possible by repeated pipetting. Cell suspension is added to 500 ml (Eagles MEM / 10 % FBS / 1% NEAA / 1% Penicillin + Streptomycin) and 25 ul dispensed into wells of compound containing TC treated, Greiner black 384-well clear bottomed plates using a Multidrop. Seeding density should be checked to ensure that new monolayers are not more than ~ 50% confluent at the time of seeding (typically 3000 cells per well), before completing preparation of plates. Cells are left at 37C, 5% CO2 in a humidified incubator in the presence of compound for 24-48 hours.

Evaluation of ATP levels as surrogate of cell growth
Promega CellTiter Glo Luminescent Cell viability Assay Kit is used. To assay ATP levels CellTiter Glo Buffer is thawed and equilibrated to room temperature prior to use (this can be done up to 48hrs prior to use); CellTiter Glo Substrate and the compound plates are also equilibrated to room temperature before use (it typically takes around 60 minutes and 30 minutes respectively). When ready, the contents of the CellTiter Glo Buffer is added to the amber bottle containing the Substrate, and the Reagent is mixed by inverting the bottle 3-5 times. Using the MultiDrop, 25ul of the CellTiter Glo Reagent is added to each well of the compound plates, and the contents are mixed for 2 minutes on a plate orbital shaker to induce cell lysis. The plates are left for 10 minutes at room temperature to stabilise signal before the luminescence is read. Data are normalized to percentage of growth inhibition using positive, negative controls and next equation % inhibition = 100- (Lum. in well-Lum. neg. control/Lum. pos. control- Lum. neg. control).

Name: Inhibition Frequency Index (IFI)
Unit: percent
Definition: Inhibition Frequency Index (IFI) = B/A, where

B = Number of non-kinase HTS assays (median result per assay and compound) where a compound showed > 50 % inhibition

A = Number of non-kinase HTS assays (median result per assay and compound) where a compound was tested for inhibition

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