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).

Additional mouse PK data for LP-922056 was generated to evaluate brain penetration.116 Following a single oral dose (10 mg/kg, po), the plasma parameters from these experiments (Cmax, AUC, and t1/2) were consistent with published data.95 Brain penetration of LP-922056 is very low with brain/plasma concentration ratios ∼0.01 at all time points measured up to 24 h and also 0.01 based on AUC(0→inf). Hence, LP-922056 is unsuitable for use in models of disease where brain penetration is an essential requirement. [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.

A group of small molecule thienopyrimidine inhibitors of Notum Pectinacetylesterase are described. We explored both 2-((5,6-thieno[2,3-d]pyrimidin-4-yl)thio)acetic acids and 2-((6,7-thieno[3,2-d]pyrimidin-4-yl)thio)acetic acids. In both series, highly potent, orally active Notum Pectinacetylesterase inhibitors were identified. In summary, researchers have developed novel inhibitors of Notum Pectinacetylesterase showing in vivo efficacy in significantly increasing midshaft femur cortical bone thickness in mice and rats.[1]

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The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum -/- mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures.[2]
Analogs of parathyroid hormone (teriparatide and abaloparatide), given by daily subcutaneous injections, are the currently available anabolic therapies for osteoporosis. The sclerostin antibody romosozumab, given by monthly subcutaneous injections, reduces fractures but regulatory approval has been delayed while cardiovascular safety concerns are evaluated. In our experience treatments with teriparatide and NOTUM inhibitors (Notum−/− mice, the orally active small molecule LP-922056 and the neutralizing antibody 2.78.33) are similarly effective in increasing cortical bone thickness, whereas the other anabolic therapies also increase trabecular bone mass. Compared to the clinically used analogs of parathyroid hormone, NOTUM inhibitors have the advantage that they can be given orally and possibly less frequently than daily.[2]
In summary, a promising novel cortical bone osteoporosis drug target, NOTUM, was identified by phenotyping cortical bone thickness of 3 366 mouse strains with knockout of druggable genes. For drug target proof-of-principle, three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were characterized. They increased cortical bone thickness and strength at multiple skeletal sites in ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and might be useful for preventing 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.)