human Notum Pectinacetylesterase (EC50 = 21 nM); mouse Notum Pectinacetylesterase (EC50 = 55 nM)

LP-922056 and LP-935001 emerged as three advanced leads for the program and were used to demonstrate in rodent pharmacology studies, along with complementary approaches, that inhibition of Notum activity is a potential novel anabolic therapy for strengthening cortical bone and preventing nonvertebral fractures.
Regulation of the Wnt signaling pathway is critically important for a number of cellular processes in both development and adult mammalian biology. This Perspective will provide a summary of current and emerging therapeutic opportunities in modulating Wnt signaling, especially through inhibition of Notum carboxylesterase activity. Notum was recently shown to act as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group. Inhibition of Notum activity may represent a new approach to treat disease where aberrant Notum activity has been identified as the underlying cause. Reliable screening technologies are available to identify inhibitors of Notum, and structural studies are accelerating the discovery of new inhibitors. A selection of these hits have been optimized to give fit-for-purpose small molecule inhibitors of Notum. Three noteworthy examples are LP-922056 (26), ABC99 (27), and ARUK3001185 (28), which are complementary chemical tools for exploring the role of Notum in Wnt signaling. [3]

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By acting as a WNT lipase, NOTUM can render WNTs inactive [2].

LP-922056 (Compound 44; 3-30 mg/kg; interfacial gavage; daily; for 25 days) resulted in increased local bone thickness at all doses [1]. LP-922056 (10 mg/kg; interface) LP-922056 (10 mg/kg; daily diet; for 4 weeks) improves local bone thickness of the femur and achieves high Cmax (129 μM) and AUC ( 1533 μM?h)[1].

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The in vivo effect of LP-922056/2-((6-chloro-7-cyclopropylthieno[3,2-d]pyrimidin-4-yl)thio)acetic acid 44 was determined by treating F1 male hybrid (129xC57) mice for 25 days with the compound, starting at 8.7 weeks of age. The compound was administered by daily oral gavage (vehicle = 0.1% Tween 80 in water). Four groups of mice (N = 13) were used: control, 3 mg/kg compound, 10 mg/kg compound, and 30 mg/kg compound. Midshaft femur cortical thickness was measured by microCT (Scanco μCT40). As shown in Figure 2, an increase in cortical bone thickness was observed at all doses compared to control: 7% (p = 0.003) at 3 mg/kg; 10% (p <0.001) at 10 mg/kg; and 13% (p <0.001) at 30 mg/kg. [1]

NOTUM activity [2]
Enzymatic and cell-based assays of NOTUM activity were employed to screen both a chemical library and antibodies generated against mouse NOTUM. The enzymatic assay was employed for HTS, with the cell-based assay providing confirmation.

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Enzymatic assay [2]
NOTUM activity was first quantified in an enzymatic assay employing 5 mmol·L−1 OPTS (8-octanoyloxypyrene-1,3,6-trisulfonic acid) as the substrate and monitoring the increase in fluorescence (485 nm excitation and 520 nm emission) upon cleavage of the octanoyl group.

Cell-based assay [2]
NOTUM inhibition of WNT activity was quantified in a cell-based TCF/LEF CellSensor® assay having a β-lactamase reporter gene under control of the β-catenin/LEF/TCF response element. β-lactamase activity was measured using a FRET-based substrate technology. LEF/TCF-bla FreeStyle 293F cells were incubated in medium overnight with WNT3A-conditioned medium, NOTUM conditioned medium and either small molecule NOTUM inhibitors or NOTUM neutralizing antibodies.

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Effects of NOTUM and NOTUM inhibition on mineralization of MC3T3-E1 cells [2]
MC3T3-E1 cells were seeded at 40 000 cells/16 mm wells in growth medium (αMEM with 10% FBS) for 3 days to reach confluency. Differentiation and mineralization were induced by adding ascorbic acid (50 μg·mL−1), β-glycerophosphate (10 mmol·L−1) and dexamethasone (100 nmol·L−1). Conditioned media from parental HEK-293F cells, a mouse Notum HEK-293F line or a human NOTUM HEK-293F line were added to the cell medium to examine the effects of NOTUM on differentiation and mineralization. NOTUM inhibition was evaluated by addition of LP-914822 or LP-922056. Cultures were fed every 3 to 4 days with complete change of medium.

Animal/Disease Models: 8.7weeks old F1 male hybrid (129xC57) mouse[1]
Doses: 3, 10, 30 mg/kg
Route of Administration: po (oral gavage); strength, femoral neck and vertebral body control the bone mass of the shell[2] . Daily; continued for 25 days
Experimental Results: All doses resulted in increased cortical bone thickness.

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Animal/Disease Models: Mouse[1]
Doses: 10 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: Oral
Experimental Results: Achieved high Cmax (129 μM) and AUC (1533 μM·h) with low clearance ( 0.49 mL/min·kg) and volume of distribution (0.13 L/kg).

To assess the brain permeability of LP-922056, we obtained additional mouse pharmacokinetic data. Plasma parameters (Cmax, AUC, and t1/2) in these experiments were consistent with published data after a single oral administration (10 mg/kg, po). The brain permeability of LP-922056 was very low, with brain/plasma concentration ratios of approximately 0.01 at all measurement time points over 24 hours, and the ratio based on AUC(0→inf) was also 0.01. Therefore, LP-922056 is not suitable for disease models where brain permeability is a necessary condition. [3]

[1]. Stimulation of cortical bone formation with thienopyrimidine based inhibitors of Notum Pectinacetylesterase. Bioorg Med Chem Lett. 2016 Mar 15;26(6):1525-1528.

[2]. NOTUM inhibition increases endocortical bone formation and bone strength. Bone Res. 2019 Jan 8;7:2.

[3]. Carboxylesterase Notum Is a Druggable Target to Modulate Wnt Signaling. J Med Chem . 2021 Apr 22;64(8):4289-4311.

This article describes a class of small molecule thienopyrimidine notum pectin acetylesterase inhibitors. We investigated 2-((5,6-thieno[2,3-d]pyrimidin-4-yl)thio)acetic acid and 2-((6,7-thieno[3,2-d]pyrimidin-4-yl)thio)acetic acid. In both series, highly potent and orally effective notum pectin acetylesterase inhibitors were found. In summary, researchers have developed novel notum pectin acetylesterase inhibitors that have shown significant efficacy in increasing mid-fracture cortical bone thickness in mice and rats in in vivo experiments. [1] Non-vertebral osteoporotic fractures result in significant disability, death, and economic losses. Existing osteoporosis therapies are very effective in reducing vertebral fractures but less effective in reducing non-vertebral fractures. Cortical bone is the main factor determining the strength of non-vertebral bone. To find new targets for osteoporosis drugs, we performed cortical bone phenotypic analysis on 3366 surviving mouse strains with systemic druggable gene knockout. Notum-/- mice showed a significant increase in cortical bone thickness. NOTUM is a secreted WNT lipase, and we observed high expression of NOTUM in cortical bone and osteoblasts, but no expression in osteoclasts. We developed three orally active small molecule drugs and a neutralizing antibody that inhibits NOTUM lipase activity. These drugs increased cortical bone thickness and strength in multiple skeletal sites in rodents with intact gonads and ovariectomy by stimulating endosteal bone formation. Therefore, inhibiting NOTUM activity may be a potential novel anabolic therapy for enhancing cortical bone strength and preventing non-vertebral fractures. [2] Currently available anabolic therapies for osteoporosis are daily subcutaneous parathyroid hormone analogs (teriparatide and abalopate). Monthly subcutaneous injections of the sclerosing agent antibody romosomab can reduce fractures, but its regulatory approval has been delayed due to ongoing evaluation of cardiovascular safety concerns. Based on our experience, teriparatide and NOTUM inhibitors (Notum−/− mice, orally active small molecule LP-922056 and neutralizing antibody 2.78.33) have similar efficacy in increasing cortical bone thickness, while other anabolic therapies also increase cancellous bone mass. The advantage of NOTUM inhibitors over clinically used parathyroid hormone analogs is that they can be taken orally and the frequency of administration may be less than once daily. [2] In summary, by performing cortical bone thickness phenotypic analysis on 3366 mouse strains with knocked-out druggable genes, we identified a promising new drug target for cortical bone osteoporosis—NOTUM. To validate the concept of the drug target, we characterized three orally active small molecules and a neutralizing antibody that inhibits NOTUM lipase activity. They increased cortical bone thickness and strength in multiple skeletal sites in ovariectomized rodents by stimulating intracortical bone formation. Therefore, inhibition of NOTUM activity is a potential new anabolic therapy for strengthening cortical bone and may help prevent non-vertebral osteoporotic fractures. [2]

C11H9CLN2O2S2

300.784358739853

299.979

C, 43.93; H, 3.02; Cl, 11.79; N, 9.31; O, 10.64; S, 21.32

1365060-22-5

56932967

White to light yellow solid powder

3.398

1

6

4

18

343

0

OC(CSC1=C2SC(Cl)=C(C2=NC=N1)C3CC3)=O

LJYRIWUQISYYHA-UHFFFAOYSA-N

InChI=1S/C11H9ClN2O2S2/c12-10-7(5-1-2-5)8-9(18-10)11(14-4-13-8)17-3-6(15)16/h4-5H,1-3H2,(H,15,16)

2-(6-chloro-7-cyclopropylthieno[3,2-d]pyrimidin-4-yl)sulfanylacetic acid

1365060-22-5; LP-922056; LX-5061; CHEMBL3774760; 2-((6-Chloro-7-cyclopropylthieno[3,2-d]pyrimidin-4-yl)thio)acetic acid; 2-[(6-Chloro-7-cyclopropylthieno[3,2-d]pyrimidin-4-yl)thio]acetic acid; 2-(6-chloranyl-7-cyclopropyl-thieno[3,2-d]pyrimidin-4-yl)sulfanylethanoic acid; LX5061; LP 922056;

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 : ~125 mg/mL (~415.59 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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