Target Cell Activation of a Structurally Novel NOD-Like Receptor Pyrin Domain-Containing Protein 3 Inhibitor NT-0796 Enhances PotencyS

The NOD -like receptor pyrin domain -containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1b and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid -containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl -ester (NT0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester -containing pharmacophore being more cell -penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT -0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human -like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte -specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT -0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the coexistence of CES-1 in these same cells affords a unique opportunity to direct ester -containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT -0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally -novel, ester -containing NLRP3 inhibitor NT -0796 are characterized. To study pharmacodynamics of NT -0796 in vivo, a mouse line was engineered possessing more human -like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT -0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.