NOTUM inhibition increases endocortical bone formation and bone strength

Brommage, Robert; Liu, Jeff; Vogel, Peter; Mseeh, Faika; Thompson, Andrea Y.; Potter, David; Shadoan, Melanie K.; Hansen, Gwenn M.; Jeter-Jones, Sabrina; Cui, Jie; Bright, Dawn; Bardenhagen, Jennifer; Doree, Deon; Movérare‐Skrtic, Sofia; Nilsson, Karin; Henning, Petra; Lerner, Ulf H.; Ohlsson, Claes; Sands, Arthur; Tarver, James E.; Powell, David R.; Zambrowicz, Brian; Liu, Qingyun

doi:10.1038/s41413-018-0038-3

Cited by 62 publications

(65 citation statements)

References 55 publications

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“…4a). They include the Wnt-regulator notum (Notum), which regulates bone formation [46][47][48] and a distintegrin and metalloproteinase like member (Adamtsl2), which is implicated in geleophysic dysplasia 49 . The majority of osteocyte transcriptome signature genes (78%, n=968) had not previously been shown to have a role in the skeleton ('unannotated' in Fig.…”

Section: A Unique Transcriptome Signature Defines the Osteocytementioning

confidence: 99%

Osteocyte Transcriptome Mapping Identifies a Molecular Landscape Controlling Skeletal Homeostasis and Susceptibility to Skeletal Disease

Youlten

Kemp

Logan

et al. 2020

Preprint

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Osteocytes are master regulators of the skeleton. We map the transcriptome of osteocytes at different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cell-network. We define an osteocyte transcriptome signature, 1239 genes that distinguishes osteocytes from other cells. 77% have no known role in the skeleton.We show they are enriched for genes controlling neuronal network formation, suggesting this program is important in the osteocyte network. We evaluated 19 skeletal parameters in 733 mouse lines with functional-gene-deletions and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human homologues that cause monogenic skeletal dysplasias (P=6x10 -17 ), and associated with polygenic diseases, osteoporosis (P=1.8×10 -13 ), and osteoarthritis (P=2.6×10 -6 ). This reveals the molecular landscape that regulates osteocyte network formation and function, and establishes the importance of osteocytes in human skeletal disease. IntroductionThe skeleton is a highly dynamic structure that changes in shape and composition throughout life. Osteocytes are the most abundant cell type in bone and have emerged as master regulators of the skeleton. These enigmatic cells are connected via ramifying dendritic processes that form a complex multicellular network distributed throughout mineralized bone 1,2 .The scale and complexity of the osteocyte network is comparable to neurons in the brain, with 42 billion osteocytes present in the human skeleton forming 23 trillion connections 2,3 . This network enables osteocytes to detect and respond to mechanical strain, hormones and local growth factors and cytokines 1 . The network responds by regulating the formation and activity of osteoclasts and osteoblasts, instructing these cells to repair damaged bone, controlling bone mass and composition, and ensuring the optimal distribution of bone tissue in response to mechanical stress. Osteocytes also remove and replace bone surrounding the osteocyte network by the process of perilacunar remodeling, liberating calcium and phosphate in response to endocrine demands 4 . These features allow the osteocyte network to maintain both the structural integrity of the skeleton and mineral homeostasis. Osteocytes also have regulatory functions beyond the skeleton, including in skeletal muscle, adipose tissue, the central nervous system and in the control of phosphate homeostasis and energy expenditure, indicating the network acts as an important and endocrine organ 5-8 .Although osteocytes are pivotal in controlling the skeleton, the molecular programs that regulate their formation and function are poorly defined. Osteocytes are entombed in bone making them challenging to isolate and study. As a result osteocytes have been omitted from large-scale efforts to map tissue-specific transcriptomes 9-12 and studies of their transcriptome are limited [13][14][15][16] . Consequently, the influenc...

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Section: A Unique Transcriptome Signature Defines the Osteocytementioning

confidence: 99%

Osteocyte Transcriptome Mapping Identifies a Molecular Landscape Controlling Skeletal Homeostasis and Susceptibility to Skeletal Disease

Youlten

Kemp

Logan

et al. 2020

Preprint

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“…Their research with 1 has shown that Notum is a potential drug target for stimulating bone formation and treating osteoporosis [7]. However, although 1 demonstrates low plasma clearance, the structure contains an essential carboxylic acid and acids tend to have low passive brain penetration [8][9][10][11][12].…”

Section: Lp-922056 (1)mentioning

confidence: 99%

“…Pharmacokinetic (PK) data for 1 was generated in vivo in mouse to evaluate brain penetration (Table 1; Figure 1) (see Supporting Information Tables S1-S3 (File 1)). The route of administration and dose were selected to most closely match relevant published mouse disease model studies [5,7]. Following single oral dose (p.o.)…”

Section: Improved Synthesis Ofmentioning

confidence: 99%

An improved, scalable synthesis of Notum inhibitor LP-922056 using 1-chloro-1,2-benziodoxol-3-one as a superior electrophilic chlorinating agent

Willis¹,

Bayle²,

Papageorgiou³

et al. 2019

Preprint

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Background: The carboxylesterase Notum has been shown to act as a key negative regulator of the Wnt signalling pathway by mediating the depalmitoleoylation of Wnt proteins. LP-922056 (1) is an orally active inhibitor of Notum. We are investigating the role of Notum in modulating Wnt signalling in the central nervous system and wished to establish if 1 would serve as a peripherally restricted control. An accessible and improved synthetic route would allow 1 to become more readily available as a chemical tool to explore the fundamental biology of Notum and build target validation to underpin new drug discovery programs. Results: An improved, scalable synthesis of 1 is reported. Key modifications include: (1) the introduction of the C7-cyclopropyl group was most effectively achieved with a Suzuki–Miyaura cross-coupling reaction with MIDA-boronate 11 (5 → 6); and (2) C6 chlorination was performed with 1-chloro-1,2-benziodoxol-3-one (12) (6 → 7) as a mild selective electrophilic chlorination agent. This 7-step route has been reliably performed on large scale to produce multigram quantities of 1 in good efficiency and high purity. Pharmacokinetic studies in mouse showed CNS penetration of 1 is very low with brain:plasma concentration ratio of just 0.01. A small library of amides 17 were prepared from acid 1 to explore if 1 could be modified to deliver a CNS penetrant tool by capping off the acid as an amide. Although significant Notum inhibition activity could be achieved, none of these amides demonstrated the required combination of metabolic stability along with cell permeability without evidence of P-gp mediated efflux. Conclusion: Mouse pharmacokinetic studies demonstrate that 1 is unsuitable for use in models of disease where brain penetration is an essential requirement of the compound but would be an ideal peripherally restricted control. These data will contribute to the understanding of drug levels of 1 to overlay with appropriate in vivo efficacy endpoints, i.e. the PK-PD relationship. The identification of a suitable analogue of 1 (or 17) which combines Notum inhibition with CNS penetration would be a valuable chemical probe for investigating the role of Notum in disease models.

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“…Wnt ligands are post‐translationally modified by a palmitoleate moiety attached to a serine, while the recently discovered Wnt deacylase Notum can remove this moiety and acts as a secreted feedback antagonist . Notum belongs to the α/β‐hydrolase superfamily of enzymes, and its structure reveals a large hydrophobic pocket at the active site, suggesting its enzyme activity can be blocked by small molecule inhibitors, and inhibition of Notum can increase bone density and may have the potential to rejuvenate stem cells …”

Section: Introductionmentioning

confidence: 99%

“…We recently reported the development of 2‐phenoxyacetamides as Notum inhibitors . One of the biggest limitations of these inhibitors is their inability or limited ability to cross the blood‐brain barrier, even though they are found to be effective in vitro . In order to identify new chemically divergent small molecules and generate structural information for rational design of new inhibitors, we used a crystallography‐based fragment screening approach .…”

Section: Introductionmentioning

confidence: 99%

Structural characterization of melatonin as an inhibitor of the Wnt deacylase Notum

Zhao

Ren

Hillier

et al. 2020

Journal of Pineal Research

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The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β‐catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N‐[2‐(5‐fluoro‐1H‐indol‐3‐yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N‐acetylserotonin into Notum crystals and obtained high‐resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial.

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NOTUM inhibition increases endocortical bone formation and bone strength

Cited by 62 publications

References 55 publications

Osteocyte Transcriptome Mapping Identifies a Molecular Landscape Controlling Skeletal Homeostasis and Susceptibility to Skeletal Disease

Osteocyte Transcriptome Mapping Identifies a Molecular Landscape Controlling Skeletal Homeostasis and Susceptibility to Skeletal Disease

An improved, scalable synthesis of Notum inhibitor LP-922056 using 1-chloro-1,2-benziodoxol-3-one as a superior electrophilic chlorinating agent

Structural characterization of melatonin as an inhibitor of the Wnt deacylase Notum

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