Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit

Abstract

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer’s disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

Structural Analysis and Development of Notum Fragment Screening Hits

“…Structurally, the 3-Cl gained hydrophobic interaction with P287 and strengthened the interaction with F268 (Figure A). Triazole 7 has properties consistent with lead-like chemical space and scored highly when assessed by design metrics (LE 0.59; LLE 4.1), including a favorable prediction of brain penetration. − Triazole 7 was further assessed in standard in vitro assays to determine its ADME properties and showed good aqueous solubility (100 mg/mL), moderate stability in liver microsomes (MLM, Cl i 88 mL/min/mg protein; HLM, Cl i 12 mL/min/mg protein), and excellent cell permeability (MDCK-MDR1, AB/BA P app 57/59 × 10 –6 cm/s, ER 1.0) . The development of 26 by modifying the heterocyclic head group, along with the exploration of SAR of the phenyl ring, identified two complementary lead series: 1,3,4-oxadiazol-2­(3 H )-ones ( 7a ) and 1,2,3-triazoles ( 7b ).…”

“…Full details of these SAR studies, along with the profile of 7d, have been presented in a recent publication. 38 Structurally, all members of the 1,2,3-triazole series (7, 7b− d) were crystallographically resolved at high resolution with good data collection and refinement statistics, and their omit maps are shown in Figure 7A-D. Oxadiazole 7a has been published through optimization of 26.…”

“…Triazole 7d was designed to improve pharmacokinetic properties in vivo by reducing clearance through phase 2 metabolism of the primary hydroxyl group, but it also became the most potent inhibitor from this series despite removal of the −CH 2 OH group. 38 From the structure point of view, despite the loss of the hydrogen bond of the −CH 2 OH group compared to 7c, the catalytic residue S232 presents an alternative rotamer conformation and forms a new hydrogen bond with -1N as well as interaction with -2N of the triazole head (Figure 7G). These additional interactions contribute to further increasing potency, and 7d proved to be a potent inhibitor of Notum activity inhibition in both biochemical (OPTS) and cell-based reporter (TCF/LEF) assays (IC 50 0.0067 ± 0.0016 μM and EC 50 0.110 ± 0.100 μM, respectively).…”

“…Thus, hits from this cluster represent clear candidates for optimization. Some hits (21,28,38,56) show atoms that extend toward the entrance of the pocket (where the PAMserine linkage is observed, position b, Figure 1B, D), but retain several interactions with the central pocket residues. Among these, 28 shows a modest IC 50 value and was chosen for optimization.…”

“…40−42 Triazole 7 was further assessed in standard in vitro assays to determine its ADME properties and showed good aqueous solubility (100 mg/mL), moderate stability in liver microsomes (MLM, Cl i 88 mL/min/mg protein; HLM, Cl i 12 mL/min/mg protein), and excellent cell permeability (MDCK-MDR1, AB/BA P app 57/59 × 10 −6 cm/s, ER 1.0). 38 The development of 26 by modifying the heterocyclic head group, along with the exploration of SAR of the phenyl ring, identified two complementary lead series: 1,3,4-oxadiazol-2(3H)-ones (7a) 39 and 1,2,3-triazoles (7b).…”