1. The ester-containing prodrug NT-0796 enhances delivery of the NLRP3 inflammasome inhibitor NDT-19795 to monocytic cells expressing carboxylesterase-1.
- Author
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Doedens JR, Diamond C, Harrison D, Bock MG, Clarke N, Watt AP, and Gabel CA
- Subjects
- Humans, Esters chemistry, THP-1 Cells, Prodrugs pharmacology, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, NLR Family, Pyrin Domain-Containing 3 Protein antagonists & inhibitors, Monocytes metabolism, Monocytes drug effects, Inflammasomes metabolism, Carboxylesterase metabolism, Carboxylesterase antagonists & inhibitors, Carboxylic Ester Hydrolases metabolism, Carboxylic Ester Hydrolases antagonists & inhibitors
- Abstract
NT-0796 is an ester prodrug which is metabolized by carboxylesterase-1 (CES1) to yield the carboxylic acid NDT-19795, an inhibitor of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome. When applied to human monocytes/macrophages which express CES1, however, NT-0796 is much more potent at inhibiting NLRP3 inflammasome activation than is NDT-19795. Comparison of the binding of NDT-19795 and NT-0796 in a cell-based NLRP3 target engagement assay confirms that NDT-19795 is the active species. Moreover, microsomes expressing CES1 efficiently convert NT-0796 to NDT-19795, confirming CES1-dependent activation. To understand the basis for the enhanced potency of the ester prodrug species in human monocytes, we analyzed the accumulation and de-esterification of NT-0796 in cultured cells. Our studies reveal NT-0796 rapidly accumulates in cells, achieving estimated cellular concentrations above those applied to the medium, with concomitant metabolism to NDT-19795 in CES1-expressing cells. Using cells lacking CES1 or a poorly hydrolysable NT-0796 analog demonstrated that de-esterification is not required for NT-0796 to achieve high cellular levels. As a result of a dynamic equilibrium whereby NDT-19795 formed intracellularly is subsequently released to the medium, concentrations of NT-0796 sufficient to inhibit NLRP3 can be completely metabolized to NDT-19795 resulting in a transient pharmacodynamic response. In contrast, when NDT-19795 is applied directly to cells, observed cell-associated levels are below those present in the medium and remain stable over time. Dynamics observed within the context of a closed tissue culture system highlight the utility of NT-0796 as a vehicle for delivering the NDT-19795 acid payload to CES1 expressing cells., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [JRD, CD, NL, DH, MGB, NC, APW and CAG are current or former employees of NodThera. This work was funded entirely by Nodthera]., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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