IC50: 13 nM (Notum)[1]

In a concentration-dependent manner, ABC99 (0.1~10 μM; 2 hours; SW620 cells) suppresses NOTUM[1]. In a concentration-dependent manner, ABC99 maintains Wnt-3A activity when NOTUM is present[1].
ABC99 inhibited NOTUM with an IC50 value of 13 nM (Figure 2B and Table 1) and showed excellent selectivity across the serine hydrolase family, as initially assessed by gel-based ABPP of soluble and membrane proteomes from SW620 cells (Figure 2C,D). These ABPP experiments were performed using both FP-Rh and ABC45 probes and thus confirmed that PPT1 also did not cross-react with ABC99 (Figure 2D). We next evaluated the selectivity of ABC99 (0.5 and 10 μM) by quantitative mass spectrometry (MS)-based ABPP in both the CM and SW620 in situ treated cells. These data confirmed the inhibition of NOTUM (Figure 2E) with virtually no cross-reactivity with the 64 additional serine hydrolases quantified by MS-ABPP (Figure 2E and Supplementary Figure 3 and Supplementary Data Set 1). A partial, concentration-dependent blockade of ABHD6 was observed, but only ∼50% of this enzyme was inhibited at concentrations of ABC99 (0.5 μM) that fully blocked NOTUM. We also noted that ABC99 more completely inhibited secreted (Figure 2E) compared to cellular (Supplementary Figure 3) NOTUM, which could reflect that the latter fraction contains a greater proportion of incompletely processed NOTUM. [1]
Using a luciferase reporter cell line responsive to Wnt activation (HEK293T-STF),25 we found that exposure of cells to a combination of NOTUM-expressing CM (harvested from SW620 cells) preincubated with either ABC99 or the control compound ABC101 and CM from Wnt3A-producing L-cells26 resulted in the concentration-dependent preservation of Wnt signaling in the presence of ABC99, but not ABC101 (Figure 2F). The IC50 value for ABC99-mediated preservation of Wnt3A activity (Figure 2F and Table 2) was moderately higher than the IC50 value for inhibition of NOTUM measured by ABPP (Figure 2B and Table 1), which could indicate that substantially more than 50% of NOTUM needs to be inhibited for protection of Wnt-mediated cell signaling. [1]

Gel-based ABPP [1]
In vitro and in situ competitive gel-based ABPP was performed as described previously. 3, 6 Briefly, after in vitro (30 min, 37 °C) or in situ (2 hours, 37 °C) inhibitor or DMSO treatment, cell lysates were treated with 1 µM FP-Rhodamine (FP-Rh) probe for 30 minutes at room temperature. Samples were then quenched with 4X SDS loading buffer, separated by SDS-PAGE on a 10% acrylamide gel and in-gel fluorescence was imaged using a ChemiDoc MP system.

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PPT1 Activity Profiling [1]
Gel-based determination of PPT1 inhibition was carried out as described previously. 1 Briefly, SW620 cells were grown in 6 cm plates to ~80% confluency, and then treated with inhibitors or DMSO in situ for 2 hours. Cells were then scraped on ice, washed once with cold PBS, snap frozen and stored at -80 °C. Cell pellets were then thawed on ice, re-suspended in cold PBS and lysed with a probe sonicator. The lysates were pre-cleared (1,400g, 1 min) and the supernantant was fractionated by ultracentrifugation (100,000g, 45 min, 4 °C). The soluble fraction was collected, and sample concentrations were adjusted to 2 mg/mL. Samples were then incubated with 1 µM of the PPT1 probe ABC45 for 30 minutes at room temperature, and for an additional 30 minutes at 37 °C following addition of the de-glycosylating enzyme PNGaseF. A rhodamineazide (Rh-N3) fluorophore reporter was conjugated onto the alkyne probe using CuAAC conditions as reported previously.

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ABC99yne Labeling [1]
Proteome derived from either SW620 whole cell lysates (1 mg/mL), or from SW620 conditioned media (0.1 mg/mL) was treated with varying concentrations of ABC99yne for 30 minutes at room temperature, followed by clicking the fluorophore reporter rhodamine azide (Rh-N3) with conditions as described above for PPT1 Activity Profiling.

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Wnt Activity Assay [1]
Wnt activity was assayed using the HEK293-STF cell line as described previously, 12 with some modifications. SW620 cells were seeded at a concentration of 5 × 106 in 10 cm plates. Two days later, media was collected, sterile filtered through a 22 µm syringe filter, and immediately incubated with inhibitors or DMSO for 1 hour at 37 °C, prior to being mixed 1:1 with freshly collected media from L, or L-Wnt3A cells (seeded at 2 × 106 per 10 cm plate, and processed the same way as media from SW620 cells). The mixture was then incubated at 37 °C for two hours, before being added to HEK293-STF (seeded at 3 × 104 per well of a 96-well plate 24 hours earlier).Luminescence was then measured 24 hours later following replacement of media with 80 µL of Bright-Glo reagent. Luminescence measurements for each condition were performed in quadruplicate

Western Blot Analysis[1]
Cell Types: SW620 cells
Tested Concentrations: 0.1~10 μΜ
Incubation Duration: 2 hrs (hours)
Experimental Results: Inhibited NOTUM in a concentration-dependent manner.

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Western Blotting [1]
Following SDS-PAGE, gel samples were transferred onto nitrocellulose (50V, 2 hours), and blots were blocked with 5% milk TBS-T (30 min, RT), then incubated with anti- NOTUM antibody overnight at 4°C. Blots were then washed with TBS-T (3x) and incubated with a secondary antibody (Li-cor IRDye 800CW Donkey anti-rabbit, 1:5000 in TBS-T with 5% milk) for 2 hours. Transfers were then washed again (3x, TBS-T) and imaged on a Li-cor Odyssey.

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Mass-spectrometry ABPP Sample Preparation and Data Analysis [1]
Samples for quantitative mass spectrometric analysis were prepared and analyzed as previously reported with minor modifications specified below. 8 Inhibitor and DMSO treated proteomes from SW620 whole cell lysates (1 mg per condition), or SW620 conditioned media (0.5-0.75 mg per condition) were treated with 4 µM FP-biotin for 1 hour at room temperature. Samples were then precipitated using chloform/methanol, reduced with 10 mM neutral TCEP (30 minutes, 37 °C), alkylated with 40 mM iodoacetamide (30 minutes, room temperature), before being enriched with PBS-washed streptavidin-agarose beads (100 µL slurry) for 1.5 hours at room temperature with end-over-end rotation. Samples were then washed with 0.2% SDS (2 x 10 mL), transferred to Low-bind eppendorf tubes, washed with 3 x 1 mL PBS, and 3 x 1 mL DI H2O, and then digested with sequence-grade trypsin (2 µg) in 2M urea overnight. Samples were then labeled using reductive dimethylation as previously described.

[1]. Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling. ACS Med Chem Lett. 2018;9(6):563-568.

Wnt proteins are a class of secreted morphogens that play a crucial role in embryonic development and adult tissue remodeling. Abnormal Wnt signaling pathways can lead to diseases such as cancer. Wnt proteins require a specific O-fatty acylation modification (O-linked palmitoylation of a conserved serine residue) to bind to the Frizzled receptor. O-palmitoylation of Wnt proteins is catalyzed by porcupine acyltransferase (PORCN) and removed by the serine hydrolase NOTUM. While PORCN inhibitors are in the development stage for cancer therapy, NOTUM inhibitors have the potential to treat degenerative diseases. This article describes the discovery and advancement of a class of highly efficient and selective NOTUM-inhibiting N-hydroxyhydantoin (NHH) carbamates using activity-based proteomics analysis (ABPP). An optimized NHH carbamate inhibitor, ABC99, maintains Wnt-mediated cell signaling in the presence of NOTUM and has been converted into an ABPP probe for visualizing NOTUM in natural biological systems. [1] To our knowledge, researchers have reported for the first time a highly efficient and selective irreversible inhibitor of the Wnt deacylase NOTUM. The state-of-the-art compound ABC99 blocks NOTUM activity with low nanomolar potency (IC50 = 13 nM) and exhibits excellent selectivity, as assessed by a serine hydrolase-guided ABPP probe and a global proteomic reactivity analysis using a clickable probe analog (ABC99yne). Over the past decade, many studies have highlighted the important role of NOTUM as a negative regulator of Wnt signaling in a variety of biological processes, but almost all of these studies have relied on molecular biology (e.g., genetic) approaches to interfere with NOTUM function. We believe that the covalent inhibitors and associated chemical probes (inactive controls and clickable probes) described in this paper will provide valuable complementary tools to the limited number of previously reported non-covalent inhibitors for characterizing the biological effects of pharmacological inactivation of NOTUM in a variety of physiological and disease processes. Future research directions include: using the ABC99yne probe combined with mass spectrometry-based active site proteomics (ABPP) analysis to detect the reactivity of ABC99 across the proteome; directly measuring the modification of Wnt palmitate after biological systems are exposed to ABC99; and assessing the effects of ABC99 on the Wnt signaling pathway in vivo. Regarding the last research objective, we note that NOTUM appears to have tissue distribution limitations in vivo (http://www.humanproteomemap.org/), which may help reveal which specific regions in Wnt biology are most affected by NOTUM-mediated deacylation. [1]

C22H21CLN4O5

456.88

456.12

C, 57.84; H, 4.63; Cl, 7.76; N, 12.26; O, 17.51

2331255-53-7

134817170

White to off-white solid powder

1.5±0.1 g/cm3

565.9±60.0 °C at 760 mmHg

296.1±32.9 °C

0.0±1.5 mmHg at 25°C

1.710

2.24

0

6

4

32

746

0

O=C(N1C2=CC=CC=C2OCC1)ON(C3=O)C(N4C3CN(CC5=CC=C(Cl)C=C5)CC4)=O

OZPZCETZTBBQBO-UHFFFAOYSA-N

InChI=1S/C22H21ClN4O5/c23-16-7-5-15(6-8-16)13-24-9-10-25-18(14-24)20(28)27(21(25)29)32-22(30)26-11-12-31-19-4-2-1-3-17(19)26/h1-8,18H,9-14H2

[7-[(4-chlorophenyl)methyl]-1,3-dioxo-5,6,8,8a-tetrahydroimidazo[1,5-a]pyrazin-2-yl] 2,3-dihydro-1,4-benzoxazine-4-carboxylate

abc99; 2331255-53-7; 7-(4-chlorobenzyl)-1,3-dioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carboxylate; [7-[(4-chlorophenyl)methyl]-1,3-dioxo-5,6,8,8a-tetrahydroimidazo[1,5-a]pyrazin-2-yl] 2,3-dihydro-1,4-benzoxazine-4-carboxylate; CHEMBL4174188; 7-(4-Chlorobenzyl)-1,3-dioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl 2H-benzo[b][1,4]oxazine-4(3H)-carboxylate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 25 mg/mL (54.72 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)