Discovery of 2-phenoxyacetamides as inhibitors of the Wnt-depalmitoleating enzyme NOTUM from an X-ray fragment screen

Abstract

NOTUM is a carboxylesterase that has been shown to act by mediating the <i>O</i>-depalmitoleoylation of Wnt proteins resulting in suppression of Wnt signaling. Here, we describe the development of NOTUM inhibitors that restore Wnt signaling for use in <i>in vitro</i> disease models where NOTUM over activity is an underlying cause. A crystallographic fragment screen with NOTUM identified 2-phenoxyacetamide <b>3</b> as binding in the palmitoleate pocket with modest inhibition activity (IC₅₀ 33 μM). Optimization of hit <b>3</b> by SAR studies guided by SBDD identified indazole <b>38</b> (IC₅₀ 0.032 μM) and isoquinoline <b>45</b> (IC₅₀ 0.085 μM) as potent inhibitors of NOTUM. The binding of <b>45</b> to NOTUM was rationalized through an X-ray co-crystal structure determination which showed a flipped binding orientation compared to <b>3</b>. However, it was not possible to combine NOTUM inhibition activity with metabolic stability as the majority of the compounds tested were rapidly metabolized in an NADPH-independent manner.