| Assay Method Information | |
| | SAHH-coupled Assay |
| Description: | As the sensitivity of the FAP assay is limited by the binding affinity of the probe for NNMT, the inhibitory concentration was further determined using an orthogonal S-adenosyl-L-homocysteine hydrolase (SAHH)-coupled fluorescence assay through monitoring the production of SAH (Ivamu et al., RSC Med Chem 12: 1254-1261 (2021); (Mondal et al., Angew Chemie Int Ed 58: 12476-12480 (2019); and Iyamu et al., Anal. Biochem. 2020, 604). The bisubstrate inhibitor II399 retained strong inhibition against NNMT with an IC50 of 49±8 nM like LL320, while II542 only exhibited an IC50 of 7.7 μM. The strong inhibition of II399 against NNMT implied that the aforementioned combinatorial changes including acetyl, 4-chloro pyrrolopyrimidine, and cyclopentane successfully secured the interaction of II399 with NNMT. To understand the contribution of the chloro group, the N6 amino analog II642was also synthesized (Scheme 1). Compound II642 displayed an IC50 of 25±7.0 nM in the SAHH-coupled assay. Comparable activities of II399 and II642 supported the feasibility of the chloro substitution for the amino group on the adenosine moiety to maintain the interaction with higher lipophilicity. |
| Affinity data for this assay | |
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