Elsevier

Bone

Volume 44, Issue 6, June 2009, Pages 1063-1068
Bone

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation

https://doi.org/10.1016/j.bone.2009.02.013Get rights and content

Abstract

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a KD of 0.35 μM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC50 of 3.9 μM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a KD of 0.08 μM and inhibited it with an EC50 of 0.65 μM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 μM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.

Introduction

Genetic studies in humans and mice have established the importance of canonical Wnt signaling in osteoblast physiology and bone remodeling [1], [2], [3], [4], [5], [6], [7]. For example, loss-of-function mutations of secreted Wnt antagonists like dickkopf (Dkk)1 and SOST/sclerostin result in increased bone formation due to changes in a variety of osteoblast parameters like proliferation, differentiation, recruitment/longevity and function [8], [9], while deletion of the β-catenin-activated transcription factor T-cell factor (TCF)-1 causes osteopenia that arises from a reduction in osteoprotegrin expression by the osteoblast [10]. These observations have focused attention on this pathway as an important one for the development of new therapies for metabolic bone diseases including osteoporosis [11], [12], [13], [14].

We uncovered the Wnt antagonist secreted frizzled-related protein (sFRP)-1 during an investigation to identify genes whose expression changed as a function of human osteoblast differentiation and in response to treatment with bone forming agents like prostaglandin E2 (PGE2), transforming growth factor (TGF)-β1 and parathyroid hormone (PTH) 1–34 [15]. Levels of sFRP-1 mRNA increased with advancing human osteoblast development in vitro, and this change correlated with elevated apoptosis. These in vitro observations were confirmed in vivo using a knockout mouse model. Deletion of sFRP-1 in mice led to increased trabecular bone volume in aged animals that resulted from enhanced osteoblast proliferation, differentiation and function, but suppressed programmed cell death [16]. At the molecular level, ablation of murine sFRP-1 elevated canonical Wnt pathway activity in bone and enhanced Runx2 expression [17]. These data demonstrate that sFRP-1 suppresses canonical signaling in osteoblasts, presumably by binding and sequestering Wnts, which leads to reduced bone accrual through a combination of decreased cellular proliferation, differentiation and activity, as well as increased apoptosis. Loss of this Wnt antagonist in mice also leads to accelerated chondrocyte differentiation and endochondral ossification with few extra-skeletal effects [18], [19]. Thus, sFRP-1 is a potential drug target for skeletal diseases like osteoporosis and osteoarthritis, since inhibitors of this protein could activate the canonical Wnt pathway and elevate bone and cartilage formation.

In this report, we describe the discovery of a class of small molecule inhibitors of sFRP-1. These compounds preferentially bind to sFRP-1 and suppress its ability to antagonize canonical Wnt signaling and increase osteoblast apoptosis. In addition, treatment of murine calvarial bone with optimized molecules in an organ culture assay stimulates bone formation. This work establishes the importance of sFRP-1 as a negative regulator of bone accrual and its potential as a drug target for anabolic therapies.

Section snippets

Compounds

sFRP-1 inhibitors were synthesized as previously described [20], [21]. The glycogen synthase kinase (GSK)-3 inhibitor was obtained from Wyeth Research. The caspase inhibitor I was purchased from Calbiochem (San Diego, CA, USA).

Transient transfection and luciferase reporter assays

U2-OS human osteosarcoma cell-based transient transfection and luciferase reporter assays were performed as previously described [22], [23].

Fluorescence binding assay

Compound binding to purified sFRP was determined by spectroscopy methods. Tryptophan residues in proteins emit fluorescence at a

Discovery and characterization of diphenylsulfone sulfonamide sFRP-1 inhibitors

In order to identify small molecule sFRP-1 inhibitors, we used a cell-based reporter gene assay to screen a chemical library of over 440,000 drug-like compounds. This assay utilized U2-OS human osteosarcoma cells transfected with Wnt-3, human sFRP-1 and an optimized TCF-luciferase reporter plasmid that measures activation of the canonical Wnt/β-catenin pathway. From this high throughput screen (HTS), we found 685 confirmed hits. These confirmed hits were reduced to 65 sFRP-1 inhibitors using a

Discussion

Wnt proteins play vital roles in many fundamental biological processes [33], [34]. Due to the importance of aberrant canonical Wnt/β-catenin signaling in oncogenesis, most drug discovery research on this pathway has focused towards the development of anti-cancer agents, which have ranged from small molecule inhibitors to antisense oligonucleotides, RNA interference and gene therapy [34], [35], [36], [37]. GSK-3β inhibitors have been in development for a variety of indications including type 2

Acknowledgments

This manuscript is dedicated to the memory of Dr. Ronald L. Magolda, whose passion and dedication to science and drug discovery will be greatly missed by all who knew him.

References (47)

  • J.M. Chong et al.

    Disulfide bond assignments of secreted Frizzled-related protein-1 provide insights about Frizzled homology and netrin modules

    J. Biol. Chem.

    (2002)
  • R.S. Jope

    Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes

    Trends Pharmacol. Sci.

    (2003)
  • I. Wilting et al.

    Lithium use and risk of fractures

    Bone

    (2007)
  • M.L. Johnson et al.

    LRP5 and Wnt signaling: a union made for bone

    J. Bone Miner. Res.

    (2004)
  • P.V.N. Bodine et al.

    Wnt signaling and osteoblastogenesis

    Rev. Endocr. Metab. Disord.

    (2006)
  • V. Krishnan et al.

    Regulation of bone mass by Wnt signaling

    J. Clin. Invest.

    (2006)
  • F. Morvan et al.

    Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass

    J. Bone Miner. Res.

    (2006)
  • X. Li et al.

    Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength

    J. Bone Miner. Res.

    (2008)
  • M. Grisanti et al.

    Dkk-1 inhibition increases bone mineral density in rodents

    J.Bone Miner. Res.

    (2006)
  • N.H. Kulkarni et al.

    Orally bioavailable GSK-3a/b dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo

    J. Bone Miner. Res.

    (2006)
  • P. Liu et al.

    Enhancement of bone formation by small molecule compounds that disrupt Dkk-LRP5/6 interaction

    J. Bone Miner. Res.

    (2005)
  • D. Padhi et al.

    Anti-sclerostin antibody increases markers of bone formation in healthy postmenopausal women

    J. Bone Miner. Res.

    (2007)
  • P.V.N. Bodine et al.

    The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis

    J. Cell. Biochem.

    (2005)
  • Cited by (0)

    View full text