Structural Analysis and Development of Notum Fragment Screening Hits

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development

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

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

C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggest that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells

Use of antibody–hapten complexes attached to optical sensor surfaces as a substrate for proteases: real-time biosensing of protease activity

Fluorescent antibody protein (IgG) was attached to the surface of an integrated optical glass waveguide chip via specific binding to a covalently attached hapten and used as a substrate for the measurement of protease activities. Exposure of the optical chip to proteases resulted in digestion of the bound fluorescent antibody molecules and proportional decrease in the detectable fluorescence resulting from loss of fluorescence from the evanescent field. The bound fluorescent antibody protein was used as a unique universal protease substrate in which the combined biological activity and fluorescence signal were the basis of measurement. The action of proteases was monitored in real-time mode where the gradual decrease in evanescent fluorescence was recorded. The chip was regenerated by complete digestion of the antibody substrate by excess pepsin and recharged by incubation with a fresh sample of the labelled antibody. The biosensor was used to detect activity of several proteases including a bacterial protease preparation, Pronase E. The linear range of measurable Pronase E activity was from 0.03 to 2 units/mL. A measurement cycle took 40 min for samples with high protease concentration (≥0.5 units/mL), when the concentration of the protease was less measurement times up to 100 min were required. The method demonstrates the principle of a new mode of real-time biosensing of proteases. The modular integrated optical glass waveguide biosensor system used in this study is compact and controlled by a laptop computer and could easily be miniaturised and utilized as a true probe device for detecting proteases with potential applications in a wide range of areas including research, clinical diagnostics, biotechnology processing and food and detergent manufacturing industries

Design of a potent, selective and brain penetrant inhibitor of Wnt-deactivating enzyme Notum by optimization of a crystallographic fragment hit

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 disease such as colorectal cancer and Alzheimer’s disease supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we describe the discovery and profile of 8l (ARUK3001185) as a potent, selective and brain pentrant 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, identifed 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

Detection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate.

A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatin–hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein–hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin–cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody–HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose–response curves for enzyme activities were measured within ranges of 0–550 µunits mL−1 for chymotrypsin, 0–12 µunits mL−1 for type IX, 0–35 µunits mL−1 for type XIV and 0–100 µunits mL−1 for type XXIV. The detection limits of the proteases studied were 89 µunits mL−1 for chymotrypsin, 0.26 µunits mL−1 for type IX, 5.8 µunits mL−1 for type XIV and 6.5 µunits mL−1 for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format