External ID: Sarm1 TIR NADase inhibitors

Protocol: Sarm1 "Sam-Tir" (Sarm1 without N-terminus, including residues 410-721 of human Sarm1 protein) was expressed in NRK1-HEK/293T cells by transient transfection: 15 million cells were transfected with 15microg plasmid DNA (Kow et al., 2018. Current Biology, 28:421), supplementing with 1mM nicotinamide riboside (NR). After 48 hours, cells were harvested and lysed. Reaction mixtures were prepared by mixing SARM1 SAM-TIR cell lysate (0.13 microg total protein), compound stock (5 microM final concentration, NCI diversity IV), and PBS (pH 7.4) to a final volume of 12 microl. NAD (5 microM final concentration) was then added for a final reaction volume of 20 microl. The reaction was incubated at 37 degrees C for 60 min, and then stopped by addition of 180 microl of 0.55 M perchloric acid (HClO4) and placed on ice. After 10 min on ice, the reaction plates were centrifuged for 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). The supernatant (120 microl) was transferred to a new plate and 10 microl of 3M K2CO3 was added to neutralize the solution. Precipitated salts were removed by centrifugation 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). The supernatant was transferred and analyzed by HPLC (Shimadzu Nexera X2) to quantify the amounts of NAD and the enzymatically cleaved product ADPR in each sample. From these values, the NAD/ADPR ratio was calculated for each compound as a measure of NAD cleavage activity (Note: there is a residual but detectable ADPR in control samples). This ratio was compared to the ratio generated in the absence of compound inhibitors. A reduction of NADase activity (NAD/ADPR ratio > 4) was used to identify compounds that inhibited the SAM-TIR catalyzed NAD cleavage.

Comment: The ratio of NAD/ADPR at time 0 is 15-16. The compounds are listed with their NSC numbers.

External ID: CHEMBL993817

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J. Nat. Prod.
Year: 2003
Volume: 66
Issue: 11
First Page: 1486
Last Page: 1489
DOI: 10.1021/np030107a

Target ChEMBL ID: CHEMBL398
ChEMBL Target Name: KB
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL993816

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J. Nat. Prod.
Year: 2003
Volume: 66
Issue: 11
First Page: 1486
Last Page: 1489
DOI: 10.1021/np030107a

Target ChEMBL ID: CHEMBL364
ChEMBL Target Name: Plasmodium falciparum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL993818

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 2003
Volume: 66
Issue: 11
First Page: 1486
Last Page: 1489
DOI: 10.1021/np030107a