WAY-316606 – Usp25 28 Inhibitoraz1

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation
Peter V.N. Bodine a,⁎, Barbara Stauffer a, Helga Ponce-de-Leon a, Ramesh A. Bhat a, Annamarie Mangine a, Laura M. Seestaller-Wehr a, Robert A. Moran a, Julia Billiard a, Shoichi Fukayama b, Barry S. Komm a, Keith Pitts c, Girija Krishnamurthy c, Ariamala Gopalsamy c, Mengxiao Shi c, Jeffrey C. Kern d,
Thomas J. Commons d, Richard P. Woodworth d, Matthew A. Wilson d, Gregory S. Welmaker d,
Eugene J. Trybulski d, William J. Moore d
a Women’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
b Women’s Health and Musculoskeletal Biology, Wyeth Research, Cambridge, MA 02140, USA
c Chemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965, USA
d Chemical and Screening Sciences, Wyeth Research, Collegeville, PA 19426, USA

a r t i c l e i n f o

Article history:
Received 15 September 2008
Revised 13 December 2008
Accepted 11 February 2009
Available online 27 February 2009 Edited by: R. Baron
Keywords:
sFRP-1
Wnt Bone Inhibitors
Diarylsulfone sulfonamide

a b s t r a c t

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 [1–7]. 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

⁎ Corresponding author. Fax: +1 484 865 9395.
E-mail address: [email protected] (P.V.N. Bodine).

important one for the development of new therapies for metabolic bone diseases including osteoporosis [11–14].
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 hor- mone (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.

Materials and methods

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 maximum wavelength of 335 nm due to excitation at 295 nm. Changes in the quantum yield and emission maximum of tryptophan residues are good probes for determining compound binding affinity and to detect conformational changes that can occur in the presence of inhibitors.
Purified human sFRP-1 was either purchased from R&D Systems (Minneapolis, MN, USA) or prepared as previously described [24]. Purified murine sFRP-2 was purchased from R&D Systems. The sFRP-1 or -2 stock solutions were diluted to 1 μM in a buffered solution and the initial ﬂuorescence was measured. Increasing concentrations of inhibitor (0 to 50 μM) were added to the protein in the cuvette and incubated for 5 min prior to assessing ﬂuorescence intensity using a Fluoromax-2 ﬂuorometer (Jobin-Yvon Horiba Inc, Edison, NJ, USA). In control experiments, the dimethyl sulfoxide (DMSO, vehicle control)- matched buffer solution was used. Fluorescence spectra were scanned in the ratio mode (S/R, signal/reference) to compensate for variations in lamp output as a function of wavelength. Fluorescence changes were fitted to a quadratic equation to obtain apparent dissociation constants as previously described [25].

Apoptosis assay

The human HOB-01-09 sFRP-1 apoptosis assay was performed as previously described [15].

Ex vivo bone formation assay

Neonatal mouse calvaria were prepared from 4-day-old pups as described previously [26,27]. Brieﬂy, calvaria were excised and cut in

half along the sagittal suture. Calvaria were incubated overnight in serum-free BGJ medium containing 0.1% (w/v) bovine serum albumin (BSA). Each half calvaria was placed with the concave surface downward on a stainless steel grid (Small Parts Inc, Miami, FL, USA) in a 12-well tissue culture dish (Becton Dickinson, Oxnard, CA, USA). Each well contained 1.0 mL of BGJ medium with 1.0% (v/v) fetal bovine serum (FBS). Calvaria were incubated for 7 days with 0.1% (v/ v) DMSO (vehicle control) or with compounds in a humidified atmosphere of 95% air and 5% CO2, and the medium was changed on day 4 with fresh DMSO or compounds added.
After organ culture, calvaria were fixed in 10% neutral phosphate- buffered formaldehyde at room temperature for at least 72 h, then decalcified for 6 h in 10% EDTA in phosphate-buffered saline (PBS). Calvaria were embedded in parallel in the same paraffin block, and 4 mm sections were stained with hematoxylin–eosin. Consistent bone areas (200 mm away from the frontal sutures) were selected for histomorphometric analysis. A 200 μm square grid was placed on each calvarium, and total bone area, as well as the number of osteoblasts within the grid, was determined with the Osteomeasure System (Osteometrics Inc, Atlanta, GA, USA). All cells on the bone surface were counted as osteoblasts.

Statistical analyses

Data were analyzed for statistical significance by one-way ANOVA using the Dunnett’s test with JMP software (SAS Institute, Cary, NC, USA).

Results

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 reporter gene control assay, which eliminated compounds that activated canonical signaling in the absence of sFRP-1 and Wnt-3. One of these inhibitors was an sFRP-1 selective diphenylsulfone sulfonamide. This compound was optimized for in vitro sFRP-1 potency, ex vivo bone forming efficacy, chemical novelty and pharmaceutical properties to produce WAY-316606, a lead triﬂuoromethyl analog.
As shown in Fig. 1A, the diphenylsulfone sulfonamide HTS hit
inhibited human sFRP-1 activity in the U2-OS cell-based transient transfection assay and increased TCF-dependent luciferase expression 2–3-fold at a concentration of 15.0 μM. However, the compound had no effect on luciferase expression in the absence of sFRP-1 and Wnt-3. In contrast, a GSK-3β inhibitor increased TCF-dependent luciferase expression 3–9-fold in the presence or absence of sFRP-1 and Wnt-3, since it acts downstream of these pathway components [28]. The HTS hit had a reasonable potency against sFRP-1 and increased luciferase expression in a dose-dependent manner with an EC50 of about 3.9 μM (Fig. 1B). On the other hand, WAY-316606 has improved potency and efficacy against sFRP-1 and increased TCF-dependent luciferase expres- sion in a dose-dependent manner up to 9-fold with and EC50 of about
0.65 μM, while retaining its selectivity for sFRP-1 (Figs. 1C and D).
As shown in Fig. 2, both the HTS hit and WAY-316606 bound saturably and stoichiometrically to purified human sFRP-1 protein. Using a tryptophan ﬂuorescence quenching binding assay that measures direct binding of compounds to proteins, the KD for the HTS hit was about 0.35 μM (Fig. 2A), while WAY-316606 bound

Fig. 1. Diphenylsulfone sulfonamides inhibit sFRP-1 function in a cell-based transient transfection reporter gene assay. U2-OS cells were transiently transfected with Wnt-3, human sFRP-1 and an optimized TCF-luciferase reporter plasmid as previously described [21,22]. The cells were treated overnight with vehicle (1.0%, v/v, DMSO), HTS hit or WAY- 316606, or a glycogen synthase kinase-3 inhibitor (GSKi) [15]. The results are presented as the mean ±SEM, n =3–12 (A and C) or mean ±SD, n =4–7 (B and D); ⁎P b 0.05 versus the vehicle treated cells.

approximately 5 times tighter with a KD of 0.08 μM (Fig. 2B). WAY- 316606 also bound to sFRP-2, albeit over 10 times weaker with a KD of
1.0 μM. Using a ﬂuorescence polarization binding assay that employed a ﬂuorescent probe compound and purified human sFRP-1 protein in a competitive-binding format, the IC50 for WAY-316606 was 0.5 μM [20].

Diphenylsulfone sulfonamides selectively inhibit sFRP-1 via the netrin domain

sFRP-1 contains 2 domains and shares sequence identity with sFRP-2 to -5 [29–32]. sFRP-1, -2 and -5 form a sub-domain within this

family and are more distantly related to sFRP-3 and -4 [29–32]. The Wnt-binding cysteine-rich domain (CRD) is located in the amino- terminus and shares 56–74% identity with sFRP-2 and -5, respectively. The netrin domain is found in the carboxy-terminus and has 52–62% identity with sFRP-2 and -5. The function of this domain is unknown. As shown in Fig. 3, both the HTS hit and WAY-316606 are specific inhibitors of sFRP-1 versus other sFRP family members. In the U2-OS cell-based transient transfection assay, the HTS hit inhibited human, mouse and rat sFRP-1 by about 30% when tested at 15.0 μM (Fig. 3A). The compound also antagonized human sFRP-1 function with either Wnt-3 or Wnt-1 indicating that it is not Wnt selective. However, this

Fig. 2. Diphenylsulfone sulfonamides preferentially bind to sFRP-1 as measured by tryptophan ﬂuorescence quenching. The results are presented as means of duplicates.

Fig. 3. Diphenylsulfone sulfonamides preferentially inhibit sFRP-1 function and appear to act via the netrin domain. (A) U2-OS cells were transiently transfected with combinations of Wnt-3 or Wnt-1, human (h), mouse (m) or rat (r) sFRP-1, a CRD construct of hsFRP-1, hsFRP-2, hsFRP-5 and an optimized TCF-luciferase reporter plasmid as previously described [21,22]. The cells were treated overnight with vehicle (1.0%, v/v, DMSO) or the HTS hit. (B) U2-OS cells were transiently transfected with Wnt-3 and an optimized TCF-luciferase reporter plasmid and incubated with conditioned medium containing either hsFRP-1, -2 or -5 as previously described [21]. The cells were treated overnight with vehicle (1.0%, v/v, DMSO) or WAY-316606. The results are presented as the mean ±SEM, n =3–9; ⁎P b 0.05 versus the vehicle treated cells.

compound did not inhibit sFRP-2 or -5, which completely blocked Wnt signaling in the presence of the inhibitor. Moreover, the compound did not blunt the activity of the sFRP-1 CRD, suggesting that it acts outside of the CRD and perhaps via the netrin domain. Using a similar assay that utilized human sFRP-1, -2 and -5 conditioned media, WAY- 316606 also specifically inhibited sFRP-1 by about 40% when tested at
2.0 μM (Fig. 3B).

Diphenylsulfone sulfonamide sFRP-1 inhibitors suppress apoptosis

As we reported previously, deletion of sFRP-1 in mice results in about a 50% decrease in calvarial osteoblast and osteocyte apoptosis when compared to wild-type controls [16]. As shown in Fig. 4, the diphenylsulfone sulfonamide sFRP-1 inhibitors also suppressed osteocyte apoptosis. Using a DNA fragmentation assay and a human pre-osteocytic cell line that over-expressed sFRP-1, the HTS hit reduced apoptosis by about 70% when tested at 10.0 μM. The GSK- 3β inhibitor also suppressed apoptosis, indicating that this process involves the canonical Wnt pathway.

Fig. 4. The diphenylsulfone sulfonamide HTS hit suppresses osteocytic apoptosis induced by sFRP-1. The HOB-01-09 sFRP-1 apoptosis assay was performed as previously described [15]. This human pre-osteocytic cell line stably over-expresses human sFRP-1. The cells were treated for 3 days with vehicle (0.1%, v/v, DMSO), caspase inhibitor I (CASPi), GSKi or the HTS hit. The results are presented as the mean±SEM, n =3;
⁎P b 0.05 versus the vehicle treated cells.

Diphenylsulfone sulfonamide sFRP-1 inhibitors stimulate bone formation

As we reported previously, deletion of sFRP-1 in mice increases trabecular bone mineral apposition rate by about 30% when compared to wild-type controls [16]. This result indicates that osteoblast activity is enhanced upon loss of sFRP-1. Likewise, WAY-316606 increased bone formation when tested in a neonatal murine ex vivo calvarial assay. As shown in Fig. 5, WAY-316606 increased total bone area up to 60% in a dose-dependent manner with an EC50 of about 0.001 μM. In contrast, the HTS hit did not increase bone formation when tested at a concentration of 5.0 μM in this assay.
WAY-316606 had good aqueous solubility, moderate to low inhibition of cytochrome p450 isozymes (3A4, 2D6, 2C9) and good stability in rat and human liver microsomes (t1/2 N 60 min in each species) [20]. However, this favorable in vitro profile did not translate into good in vivo pharmacokinetics. In female Sprague- Dawley rats, WAY-316606 exhibited high plasma clearance (77 mL/ min/kg, greater than hepatic blood ﬂow) following a single intravenous bolus dose (2 mg/kg), which resulted in a rapid decline of drug exposure in the plasma despite the route of administration. This pharmacokinetic profile prohibited sustained exposure and limited the compound’s advancement as an orally administered anabolic bone agent.

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 [34–37]. GSK-3β inhibitors have been in development for a variety of indications including type 2 diabetes and Alzheimer’s disease [28]; while LiCl, which inhibits this enzyme, has been used to treat bipolar disorder for decades [38]. GSK-3β inhibitors may also have utility in embryonic stem cell self-renewal and in regenerative medicine [39].
In addition to cancer, diabetes and Alzheimer’s disease, research over the past 7 years has established a key role for canonical Wnt signaling in bone metabolism [1–7]. These investigations have identified and validated several potential drug targets for the

Fig. 5. WAY-316606 stimulates bone formation in an organ culture assay. (A) Quantification of a dose–response experiment with WAY-316606. (B) Photomicrograph of a hematoxylin–eosin stained vehicle-treated (0.1%, v/v, DMSO) calvarium. (C) Photomicrograph of a hematoxylin–eosin stained WAY-316606-treated (100 nM) calvarium. The results are presented as the mean ±SEM, n =5; ⁎P b 0.01 versus the vehicle treated cells.

treatment of osteoporosis and other skeletal pathologies [40,41], and some of these therapies are beginning to enter clinical trials. These targets include extracellular modulators of Wnt signaling such as Dkk1, SOST/sclerostin and sFRP-1 as well as intracellular regulators like GSK-3β.
Since sclerostin and Dkk1 are secreted proteins, they are good targets for neutralizing antibodies, which are currently in clinical development for treatment of osteoporosis as well as the skeletal complications of multiple myeloma. A monoclonal antibody that inhibits sclerostin has recently been shown to increase serum markers of bone formation in healthy postmenopausal women [14], while a neutralizing antibody to Dkk1 was reported to increase bone formation and bone mineral density in intact rats and mice [11]. Dkk1 antibodies have also exhibited efficacy in mouse models of rheumatoid arthritis and multiple myeloma [42,43].
In addition to biotherapeutic approaches, some Wnt pathway components are being targeted for orally active, small molecular weight compounds. The GSK-3β inhibitor LiCl has been shown to elevate bone formation in mice [44], and there is some clinical data to suggest that its use may reduce fracture risk [45], although this is controversial [46]. In addition, a potent and orally active small organic compound that inhibits GSK-3β has been reported to reverse the effects of ovariectomy-induced bone loss in rats [12]. Moreover, small molecular weight inhibitors of Dkk1 have also been described [13].
Even though sFRP-1 is a secreted protein that would not typically be considered as a drug target for orally active compounds [47], we chose to develop small molecular weight inhibitors of this Wnt pathway antagonist, as these kinds of drugs are the most cost effective therapies for diseases like osteoporosis. We discovered diphenylsulfone sulfonamide sFRP-1 inhibitors using a cell-based HTS of over 440,000 compounds. Although sFRPs are not known to interact with small molecule ligands, these inhibitors bound saturably and stoichiometrically to human sFRP-1 with KDs below 500 nM. These molecules also selectively reversed the inhibition of canonical Wnt signaling by sFRP-1 and appeared to act via the netrin domain of the receptor. While the function of the netrin domain is unknown, we have previously reported that it plays an important role in Wnt antagonism [23]. Moreover, it shares sequence and disulfide bonding pattern similarity with the axon guidance proteins netrins as well as type I procollagen C-proteinase enhancer (PCOLCE) proteins and tissue inhibitors of metalloproteinases (TIMPs) [29]. Finally, these compounds suppressed the ability of sFRP-1 to induce human bone cell apoptosis in vitro and stimulated ex vivo murine bone formation in an organ culture assay. Further optimization of the

diphenylsulfone sulfonamide scaffold has resulted in small mole- cules with improved in vitro and pharmaceutical profiles and will be reported in future publications.

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.

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