Discovery of a small molecule antagonist of theparathyroid hormone receptor by using an N-terminalparathyroid hormone peptide probePercy H. Carter*, Rui-Qin Liu, William R. Foster, Joseph A. Tamasi, Andrew J. Tebben, Margaret Favata, Ada Staal,Mary Ellen Cvijic, Michele H. French, Vanessa Dell, Donald Apanovitch, Ming Lei, Qihong Zhao, Mark Cunningham,Carl P. Decicco, James M. Trzaskos, and Jean H. M. Feyen

Pharmaceutical Research Institute, Bristol–Myers Squibb Company, Princeton, NJ 08543-4000

Edited by John T. Potts, Massachusetts General Hospital, Charlestown, MA, and approved February 9, 2007 (received for review July 18, 2006)

Once-daily s.c. administration of either human parathyroid hor-mone (PTH)-(1–84) or recombinant human PTH-(1–34) provides fordramatic increases in bone mass in women with postmenopausalosteoporosis. We initiated a program to discover orally bioavail-able small molecule equivalents of these peptides. A traditionalhigh-throughput screening approach using cAMP activation of thePTH/PTH-related peptide receptor (PPR) as a readout failed toprovide any lead compounds. Accordingly, we designed a newscreen for this receptor that used a modified N-terminal fragmentof PTH as a probe for small molecule binding to the transmembraneregion of the PPR, driven by the assumption that the pharmaco-logical properties (agonist/antagonist) of compounds that boundto this putative signaling domain of the PPR could be altered bychemical modification. We developed DPC-AJ1951, a 14 amino acidpeptide that acts as a potent agonist of the PPR, and characterizedits activity in ex vivo and in vivo assays of bone resorption. Inaddition, we studied its ability to initiate gene transcription byusing microarray technology. Together, these experiments indi-cated that the highly modified 14 amino acid peptide inducesqualitatively similar biological responses to those produced byPTH-(1–34), albeit with lower potency relative to the parent pep-tide. Encouraged by these data, we performed a screen of a smallcompound collection by using DPC-AJ1951 as the ligand. Thesestudies led to the identification of the benzoxazepinone SW106, apreviously unrecognized small molecule antagonist for the PPR.The binding of SW106 to the PPR was rationalized by using ahomology receptor model.

gene microarray � homology model � osteoporosis � bone

Parathyroid hormone (PTH), a principal regulator of boneremodeling and calcium ion homeostasis, exerts its effects by

binding and activating the PTH/PTH-related peptide receptor(PPR), a family B G protein-coupled receptor (GPCR). Notably,once-daily s.c. injection of either recombinant human PTH-(1–34) or PTH-(1–84) provides for increases in bone mineraldensity and decreased fracture risk in postmenopausal women.This observation has prompted broad interest in identifyingorally bioavailable equivalents of PTH (reviewed in ref. 1).Herein, we disclose our initial efforts to find a small moleculeligand of the PPR.

We began our efforts by screening for the ability to activatecAMP production in UMR106 cells, but this approach did notyield any tractable lead structures. Although this initial screenindicated that our compound collection did not contain anysmall molecule PPR agonists, it did not reveal any informationabout antagonist compounds. Extensive work with the family AGPCR small molecules, which typically bind to the juxtamem-brane region of their receptors, has shown that trivial chemicalmodifications of antagonists can convert them into agonists (2).On the basis of this literature precedent, we assumed that a smallmolecule antagonist of the PPR could be a viable lead structure

for the discovery of a small molecule agonist, provided that theantagonist bound to the juxtamembrane domain of the PPR.

PTH-(1–34) is thought to bind by using a two-step sequence(1) in which the carboxyl-terminal domain of the peptide firstbinds to the extracellular domain of the PPR (Fig. 1, step A toB) and then the peptide N-terminal domain activates the recep-tor through interaction with its juxtamembrane domain (Fig. 1,step B to C). Thus, antagonists of a radiolabeled PTH-(1–34)might bind either to the N-terminal domain (and thereby blockPTH binding) or to the juxtamembrane domain (and therebyblock PTH function and perhaps allosterically modulate PTHbinding), making PTH an unsuitable probe molecule for ourpurposes. We thus turned to examine the modified PTH-(1–14)derivatives first disclosed by Gardella and colleagues (leadingrefs. 3 and 4), which interact primarily with the juxtamembraneregion of the PPR (Fig. 1, step A to D); we assumed thatcompetitive antagonists of such peptides would themselves bindto the juxtamembrane region of the PPR. We set three short-term objectives for the research program: (i) synthesize atruncated peptide that could serve as a viable tracer ligand in abinding assay, (ii) document that the peptide was biologicallyequivalent to PTH-(1–34) in vitro and in vivo, and (iii) validatethat screening with this peptide could provide PPR antagonists.The successful results of our attempt to meet these threeobjectives are described in the sections that follow.

Results and DiscussionDevelopment of the Probe Peptide. Modification of the knownPTH-(1–14) derivative 1 (3) such that it was suitable for use ina fluorescent spin polarization assay or standard radioactivebinding assay provided peptides with nearly equivalent agonistpotency (2–4, Table 1). However, our attempts to use 2–4 in theappropriately formatted binding assays were unsuccessful. Werationalized that this failure could be attributed to the subop-timal potency of the peptides, and so we examined the effects ofsubstitution of aminoisobutryic acid (Aib) for Ala at positions 1and 3, which has been reported by Gardella and colleagues (4)as potency-enhancing. As shown in the comparison of 4 and 5,

Author contributions: P.H.C., R.-Q.L., W.R.F., A.S., M.E.C., D.A., C.P.D., J.M.T., and J.H.M.F.designed research; P.H.C., R.-Q.L., W.R.F., J.A.T., A.J.T., M.F., M.E.C., M.H.F., V.D., M.L., andQ.Z. performed research; P.H.C., A.J.T., and M.C. contributed new reagents/analytic tools;P.H.C., R.-Q.L., W.R.F., J.A.T., A.J.T., A.S., M.E.C., M.H.F., V.D., D.A., M.L., Q.Z., J.M.T., andJ.H.M.F. analyzed data; and P.H.C., R.-Q.L., W.R.F., and J.H.M.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: Aib, aminoisobutryic acid; GLP-1, glucagon-like peptide 1; GPCR, G protein-coupled receptor; PPR, PTH-related peptide receptor; PTH, parathyroid hormone.

*To whom correspondence should be addressed. E-mail: percy.carter@bms.com.

This article contains supporting information online at www.pnas.org/cgi/content/full/0605125104/DC1.

© 2007 by The National Academy of Sciences of the USA

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this simple change, consisting of the addition of 28 Da toa 1,600-Da peptide, afforded a dramatic increase in agonistpotency.

Encouraged by these results, we synthesized the three [Aib1,3,Arg11]-PTH derivatives 6–8 (Table 1), all of which were found tobe potent agonists. Although we could not format a fluorescentspin polarization-based assay with 7, 8, or a more elaboratePTH-(1–21) analog (data not shown), we were able to use 125I-6in a standard radioactive binding assay in B28 cells [supportinginformation (SI) Fig. 6], in which PTH-(1–34) and cold 6afforded apparent IC50 values of 0.69 � 0.15 nM and 6.87 � 0.90nM, respectively. In saturation binding experiments, peptide 6exhibited a Kd of 21.6 � 5.2 nM and a Bmax of 603,000 receptorsper cell. Importantly, 125I-6 afforded the expected potencieswhen studied with our other peptide analogs in intact B28 cells(Table 1), suggesting that the potency for cAMP formation of

these peptides was likely related to their affinity for the peptide6 binding site. The overlap of this binding site with the classicalPTH binding site was shown both through displacement of 125I-6by PTH-(1–34) (SI Fig. 6) and through the competitive antag-onism of 6 by PTH-(3–34) in the cAMP assay (SI Fig. 7A). Sincethe completion of our work, detailed pharmacological studiesfrom other laboratories (5, 6) have shown that 14-mer and15-mer peptides related to DPC-AJ1951 interact predominantlywith the juxtamembrane and not the N-terminal domain of thePPR. These studies further strengthen the hypothesis that DPC-AJ1951 can serve as a probe of the juxtamembrane domain.

Biological Characterization of the Probe Peptide. Because peptide 6(hereafter referred to as DPC-AJ1951) met our basic criteria asa probe of the juxtamembrane domain of the PPR, we moved tostudy it further. As shown above (Table 1), DPC-AJ1951 was afull agonist of cAMP production in cell lines containing eitherhuman or rat PPR. In addition, DPC-AJ1951 induced intracel-lular Ca2� mobilization in HEK 293 cells transfected with thehuman PPR (EC50 26 � 14 nM) (see SI Fig. 7B). As expected,DPC-AJ1951 did not elicit responses from cells that did notcontain the PPR. Likewise, DPC-AJ1951 did not show anysignificant binding to the following selection of family A and BGPCRs when studied at concentrations up to 10 �M: calcitoninreceptor, glucagon-like peptide 1 (GLP-1) receptor, adrenergic�1A, adrenergic �1B, histamine H1, dopamine D2, and 5HT2A.

To broaden the comparison of signaling induced by DPC-AJ1951 and PTH-(1–34), we turned to microarray analysis.When 100 nM PTH-(1–34) was administered to UMR106 cellsunder low serum conditions, gene expression changes weretime-dependent, with global expression changes increasing overtime with 279, 416, and 514 reporters of 8,799 seen changing atP � 0.01 at 2, 8, and 24 h, respectively; likewise, 100 nMDPC-AJ1951 induced time-dependent changes, with 264, 472,and 479 reporters of 8,799 changing at P � 0.01 at 2, 8, and 24 h,respectively [see SI Table 3 for a listing of the significanttranscriptional changes elicited by PTH-(1–34) and DPC-AJ1951]. The microarray results with DPC-AJ1951 were con-firmed with real-time PCR for a selection of seven knownmarkers of PTH action by using RNA from an experimentperformed subsequent to that analyzed with microarrays, withUMR106 cells grown freshly from frozen stock (Table 2).Notably, the transcriptional activity of DPC-AJ1951 was mod-ulated by the PDE4b inhibitor rolipram (SI Fig. 8), consistentwith the inhibition of a negative feedback pathway.

The patterns of transcriptional activity elicited by probe

Extra

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Fig. 1. The pharmacological basis of our approach. PTH-(1–34) binds in atwo-step mechanism (A to B to C), and PTH-(1–14) derivatives bind primarily tothe juxtamembrane domain (step A to D). Thus, PTH-(1–14) derivatives shouldbe superior probe reagents for the discovery of juxtamembrane-specific smallmolecule ligands.

Table 1. Structure–activity relationships of peptides used in this study

Compoundno. Peptide sequence (N to C)

CRE-luc*EC50, nM

SaOS-2†

EC50, nMUMR-106‡

EC50, nMB28 Bnd§

IC50, nM

1 Ala-Val-Ala-Glu-lle-Gln-Leu-Met-His-Gln-Har-Ala-Lys-Trp 32 800 � 120 155 � 23 10,484 � 5,1022 Ala-Val-Ala-Glu-lle-Gln-Leu-Met-His-Gln-Har-Ala-Lys-Trp-Fly 53 1,000 � 265 251 � 42 10,1303 Ala-Val-Ala-Glu-lle-Gln-Leu-Nle-His-Gln-Har-Ala-Lys-Trp-Tyr 26 1,460 � 321 129 � 15 2,187 � 2094 Ala-Val-Ala-Glu-lle-Gln-Leu-Nle-His-Gln-Har-Ala-Lys-Tyr 13 1,430 � 157 344 � 43 10,560 � 4535 Aib-Val-Aib-Glu-lle-Gln-Leu-Nle-His-Gln-Har-Ala-Lys-Tyr 0.06 N.T. N.T. 7.2 � 0.46 Aib-Val-Aib-Glu-lle-Gln-Leu-Nle-His-Gln-Arg-Ala-Lys-Tyr 0.15 � 0.08 2.2 � 0.38 1.1 � 0.44 6.87 � 0.97 Aib-Val-Aib-Glu-lle-Gln-Leu-Nle-His-Gln-Arg-Ala-Lys-Tyr-Fly 1.1 20 � 4.2 N.T. 98.2 � 14.68 Aib-Val-Aib-Glu-lle-Gln-Leu-Nle-His-Gln-Arg-Ala-Fly-Tyr 0.92 7.2 � 1.2 N.T. 13.5 � 1.8

Peptide sequences are shown with the standard three-letter code, with the following codes used for unnatural amino acids: Har, homoarginine; Fly,�-fluorescein lysine; Nle, norleucine; Aib, aminoisobutyric acid. Residues that differ from the native PTI-(1–14) sequence are shown in bold. Those that areunderlined differ from both the native sequence and the control peptide 1. All peptides are C-terminally amidated and have a free amine at the N terminus.The assays are described in detail in Materials and Methods. Relative to PTH-(1–34), peptides 1–8 all induced a full response. N.T., not tested.*EC50 for induction of luciferase activity in HEK 293 cells expressing both human PPR and a CRE-luc reporter.†EC50 for cAMP formation in SAOS-2 cells, which endogenously express human PPR.‡EC50 for cAMP formation in UMR106 cells, which endogenously express rat PPR.§IC50 for inhibition of binding of 125I–6 to B28 cells.

Carter et al. PNAS � April 17, 2007 � vol. 104 � no. 16 � 6847

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peptide DPC-AJ1951 and PTH-(1–34) after 2 h were highlysimilar (Fig. 2): The responses showed a 98% correlation fortranscripts having a P value of �0.01 in both treatments. Theexcellent correlation was maintained after 8 h of treatment, butthe difference in potency between the peptides became evidentby 24 h (SI Fig. 9). Significantly, the correlation between theresponses to 14-mer DPC-AJ1951 and PTH-(1–34) was higherthan between the responses to partial agonist PTH-(3–34) andPTH-(1–34) (SI Fig. 10 and Table 4). The transcriptionalregulation induced by both DPC-AJ1951 and PTH-(1–34) wasconsistent with that reported for PTH-(1–34) after our work wascompleted (7) (SI Table 4). The microarray data speak both tothe activity and the selectivity of the 14-mer peptide: Not only

does DPC-AJ1951 induce almost all of the changes induced byPTH-(1–34), it does not induce any substantial changes notinduced by PTH-(1–34) (see SI Fig. 11 and Table 3), consistentwith our initial in vitro GPCR counterscreening. Thus, eventhough the sequence of DPC-AJ1951, (Aib1,3, Nle8, Gln10, Arg11,Ala12, Tyr14)-PTH-(1–14)NH2, is only 50% identical to that ofnative PTH-(1–14)NH2, it is �100,000-fold more potent thanPTH-(1–14) in stimulating cAMP formation in SaOS-2 cells andappears to have equivalent selectivity to the parent peptide,PTH-(1–34).

To extend the in vitro findings described above, we studied14-mer peptide DPC-AJ1951 in classical ex vivo models of PTHaction. DPC-AJ1951 stimulated osteoclast-mediated bone re-sorption, as measured by the release of 45Ca from fetal ratlong-bone explant cultures prelabeled with 45Ca in utero (Fig.3A). Likewise, in neonatal mouse parietal bone explants, DPC-AJ1951 caused a concentration-dependent decrease in collagensynthesis (incorporation of [3H]proline, Fig. 3B) and stimulationof cell proliferation (increase in [3H]thymidine, Fig. 3C). Thesedata substantially extend the earlier report by Shimizu et al. (4)that a different Aib1,3-PTH-(1–14) fragment can inhibit bonemineralization in embryonic mouse metatarsals in vitro.

Although DPC-AJ1951 was perfectly stable in buffer or salinesolution, it exhibited a relatively short half-life in rat plasma (�20min) or rat whole blood (�15 min) when incubated at 37°C.Accordingly, we focused our attention on an acute in vivo modelof PTH action in the rat. The peptide DPC-AJ1951 normalizedserum calcium levels in a dose- and time-dependent fashionwhen administered to thyroidparathyroidectomized rats by con-tinuous s.c. infusion (Fig. 3D). The 14-mer DPC-AJ1951 was�40-fold less potent (molar basis) than PTH-(1–34) in this invivo model of bone resorption (cf. Fig. 3D and SI Fig. 12), whichis consistent with its �10-fold weaker potency in the aforemen-tioned in vitro and ex vivo assays of PTH action (Table 1 and Fig.3 A–C). This result demonstrates in vivo activity for peptide inthe PTH-(1–14) class.

Taken together, the results from our biological characteriza-tion of DPC-AJ1951 suggest that the extensive structural mod-ifications embodied in DPC-AJ1951 enable this shortened pep-tide to bind and fully activate the PPR in a selective manner.Thus, although the overall potency and bioavailability of 14-merDPC-AJ1951 are not equivalent to those of PTH-(1–34),† itappears to exhibit the same complement of pharmacologicalactivity (Figs. 2 and 3). Accordingly, we consider DPC-AJ1951to be a validated probe of the agonist-binding site on the PPR.

Use of the Probe Peptide in Small Molecule Screening. Havingconfirmed through several different mechanisms that DPC-AJ1951 induced qualitatively similar biological responses to theclinically efficacious PTH-(1–34), we used it to screen a com-pound collection consisting of structurally diverse chemotypesknown to inhibit multiple protein target classes (GPCRs, pro-teases, kinases, etc). Herein we describe the initial character-ization of the most potent compound identified in this initialscreen, r-5-(2-E-cyclopropylvinyl)-t-3-ethyl-6,7-dif luoro-5-(trif luoromethyl)benzo[e][1,4]-oxazepin-2(1H,3H,5H)-one

†For example, whereas a daily s.c. 0.2 mg/kg dose of DPC-AJ1951 induced a statisticallysignificant increase in trabecular bone mineral density in the ovariectomized rat model(�21%, AJ1951 � 392.1 � 10.7 mg/cm3, vehicle � 323.3 � 12.9 mg/cm3), a daily s.c. 0.02mg/kg dose of PTH-(1–34) induced a more profound response (��100%, �640 mg/cm3).Likewise, single s.c. administration of DPC-AJ1951 produced an unexpectedly smallerincrease in plasma cAMP relative to PTH-(1–34): 1.0 mg/kg s.c. AJ1951 �150% at 30 min;0.1 mg/kg s.c. PTH-(1–34) �400% at 30 min. Together with the plasma stability data citedin the text, these preliminary data (J.H.M.F. and J.A.T., unpublished data) suggest that,although DPC-AJ1951 is indeed capable of inducing an anabolic response, the plasmastability and/or pharmacokinetics of DPC-AJ1951 need to be optimized to improve its invivo activity by s.c. administration. This finding can be contrasted with the ability ofDPC-AJ1951 to provide the expected result when administered by infusion (Fig. 3D).

Table 2. Confirmation of microarray results with real-time PCR

Real-timePCR Microarray analysis

AJ19561 DPC-AJ1951 PTH-(1–34)

Marker 2 h 2 h 8 h 2 h 8 h

c-fos 66 48 11 40 14Ptpn16 15 9 6 9 7IL-18 6 18 15Crem 5 13 14Pde4b 4 11 9 13 8Pthr1 �2 �2 �3 �2 �2Bmp3 �14 �4 �3

Real-time PCR was performed on chosen markers with RNA from separatetreatment of UMR106 cells with DPC-AJ1951. The table shows the real-time PCRresults to match the microarray findings, but with real-time PCR fold changestypically larger than those seen from the microarrays (real-time PCR is moresensitive and has a greater linear range than the Affymetrix microarrays). Blanksindicate that the microarray P value was �0.01. Where the array had multipleprobe sets to the same gene values, one of the probe sets was used in the table.Specifically, values from the following probe sets are reported in the table: c-fos,X06769cds�at;Ptpn16,S81478�s�at; IL-18,AJ222813�s�at;Crem,U04835�at;Pde4b,M25350�s�at; Pthr1, AB012944cds�s�at; Bmp3, D63860�s�at.

DPC-AJ1951 (100 nM) 2 hours

Unchanged (0/8,376)

Query Signature (179/264)

Target Signature (179/279)

Common Signature (179/179)

Anticorrelated (2/2)

Selection (30/125)

LegendP<=0.01

Non-Weighted Weighted0.99

0.9970.99

0.9540.788

0.9750.9950.9750.8470.791

VisibleSelected

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Fig. 2. Comparison of gene up-regulation by PTH-(1–34) and 14-mer DPC-AJ1951. UMR106 cells were treated with either PTH-(1–34) or DPC-AJ1951, andchanges in gene expression were measured at 2 h. Shown is the log offold-change relative to control of PTH-(1–34) vs. DPC-AJ1951. Each point is atranscript on the RGU34A microarray that changed at P � 0.01 with bothPTH-(1–34) and DPC-AJ1951. Transcripts shown in red are 30 transcripts in thelist of 125 published by Qin et al. (7). At t � 2 h, all but 2 of 179 commontranscriptional changes show as correlated between PTH-(1–34) and DPC-AJ1951, with overall correlation being 98%.

6848 � www.pnas.org�cgi�doi�10.1073�pnas.0605125104 Carter et al.

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(hereafter SW106). Compound SW106 displaced 125I-DPC-AJ1951 with an IC50 of 0.99 �M in B28 cells (Fig. 4A). Whenapplied to B28 cells or SaOS-2 cells alone, SW106 did not inducea cAMP response, suggesting that it was an antagonist. Consis-tent with this assumption, SW106 antagonized the cAMP re-sponse induced by DPC-AJ1951 in several cell lines, includingthose containing human and rodent variants of the PPR (see Fig.4B for B28 cells; see SI Fig. 13 for others). SW106 was also foundto be an antagonist of PTH-(1–34), but it exhibited �10-foldweaker potency against the longer peptide (Fig. 4B). WhereasSW106 is not as potent as DPC-AJ1951 in the binding assay, itis more potent than several of the peptides listed in Table 1,despite its smaller size (molecular mass of 343 Da for SW106 vs.�1,600 Da for a typical PTH 14-mer), and is only �10-foldweaker than the most optimized PTH-(1–14) antagonist re-ported in the literature (8).

We also studied the mode of antagonism by SW106. In aSchild-type analysis, SW106 weakened the apparent cAMP EC50of DPC-AJ1951 without changing the cAMP Emax (Fig. 4C),suggesting that it was a competitive antagonist. Consistent withthis proposed competitive mechanism, the addition of 5 �MSW106 weakened the apparent affinity of DPC-AJ1951 without

altering the total bound: the IC50 values of DPC-AJ1951 fordisplacing 125I-DPC-AJ1951 were 6.9 � 0.9 nM and 55.4 � 9.2nM in the presence of 0 �M and 5 �M of SW106, respectively(SI Fig. 14).

Given the promiscuity of many small molecule GPCR antag-onists, we examined the selectivity profile of SW106. Whenstudied at concentrations of 10 �M and above, we found thatSW106 neither activated the human GLP-1 receptor nor blockedthe actions of GLP-1 or nonnative peptide ligands (includingtruncated variants). Given that the GLP-1 receptor is a family BGPCR that couples through G�S, these data suggest that theability of SW106 to block PTH-initiated cAMP accumulation isspecific to its actions on the PPR and not related to its ability tointerfere with other signaling mechanisms. We also studied thebinding of SW106 to a panel of family A GPCRs. When testedat concentrations of 20 �M, the compound did not exhibit anyactivity against a number of these receptors (CC chemokinereceptors 1, 2, 3, 4, and 5; CXC chemokine receptors 2 and 3;adrenergic �1B and �2A; dopamine D2; histamine H1; musca-rinic M5; and serotonin 5HT2C), but did exhibit weak binding toserotonin 5HT2A (IC50 � 15 �M), adrenergic �1D (IC50 � 13.8�M), neurokinin-2 (IC50 � 28 �M), and adrenergic �2C

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Fig. 3. Comparison of the ex vivo and in vivo actions of 14-mer DPC-AJ1951 and PTH-(1–34). (A) 45Ca-labeled fetal rat radii/ulnae were dissected and cultured.Radioactive calcium release from the bone explants into the culture medium was measured on day 5 and is expressed as the percentage of total 45Ca (n � 3, eachexperiment with six explants). One representative experiment is shown (mean � SEM). Apparent EC50 values for PTH-(1–34) and 14-mer DPC-AJ1951 were 0.7and 30 nM, respectively. *, P � 0.05. (B and C) Calvaria from 2- to 4-day-old mice were dissected and treated with PTH peptides for 2 consecutive days as indicated.After 2 days of treatment, the calvaria were pulsed with either [3H]proline (B) or [3H]thymidine (C). Shown are the incorporation of [3H]proline into theacid-insoluble bone protein pool and the incorporation of [3H]thymidine into the acid-insoluble bone DNA fraction (n � 3, each experiment with six explants).One representative experiment is shown (disintegration per micrograms of dry bone weight � SEM). Apparent EC50 values for PTH-(1–34) and 14-mer DPC-AJ1951were 0.02 and 0.2 nM in the proline incorporation assay and 0.5 and 6.0 nM in the thymidine incorporation assay, respectively. *, P � 0.05. (D) The ability ofDPC-AJ1951 to normalize serum calcium in thyroidparathyroidectomized rats was studied (n � 2, five rats per experimental group). Serum calcium levels (meanvalue � SEM) are expressed as percent change over timepoint 0 min. Baseline reading of plasma calcium is 7.76 � 0.03 mg/dl and is set to 100%. *, P � 0.05.

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(IC50 � 10.4 �M). Thus, SW106 displays higher affinity for thePPR relative to the other GPCRs studied.

The lead compound SW106 embodies an unusual 4,1-benzoxazepinone ring structure as a constrained dipeptide motifand has been described as a member of an orally bioavailableseries of HIV reverse transcriptase inhibitors (9). After identi-fying it as a PPR antagonist, we screened �60 additionalrepresentatives of this specific compound class to establish theinitial structure-activity relationships in this series (data notshown). These data confirmed that multiple members of thestructural class had activity against the PPR and that thechemotype was relatively tolerant to certain changes (e.g.,halogenation pattern on aromatic ring, terminal appendage onolefin, alkyne exchange for olefin). Moreover, this screen iden-tified the ethyl substituent as a key determinant of PPR binding:Although it could be altered to other small alkyl groups (methyl,n-propyl, isopropyl, cyclopropyl), alteration to phenyl (large,aromatic) or hydrogen (small) reduced activity dramatically. Inaddition, the relative stereochemical projection of the ethylrelative to trif luoromethyl was important, in that the cis-relationship allowed for maintenance of PPR activity, whereasthe trans-relationship ablated that activity.

To understand better the actions of SW106 at the PPR, weconstructed a new homology model of the PPR by using the x-raystructure of bovine rhodopsin [Protein Databank (PDB) ID code1F88] as a template (SI Fig. 15). The PPR antagonist SW106 wasmanually docked into a hydrophobic/aromatic pocket betweentransmembrane domains 3, 4, 5, and 6 of the modeled PPR (Fig.5; see also SI Fig. 16). The illustrated binding pose is consistentwith the structure-activity relationships of SW106 (vide supra); itis also consistent with the aforementioned selectivity of SW106for the PPR over the GLP-1 receptor, as five of eight key contactresidues vary in the illustrated pocket between the two receptors(see SI Fig. 17 for the sequence alignment).

The hypothesis that SW106 might bind to residues on TM6(specifically Met-425 and Tyr-429) allows us to rationalize otherexperimental observations. First, because Met-425 has beenidentified in photochemical cross-linking and site-directed mu-tagenesis experiments as a contact site for the first two N-terminal residues of PTH and/or PTH-related protein (10–12),it suggests that SW106 blocks the binding of the N terminus ofPTH; this is consistent with the ability of SW106 to antagonizethe function of the C-terminally truncated series PTH-(1–34),PTH-(1–14), and PTH-(1–11) (data with the 11-mer not shown).

Second, it is in accord with the ability of SW106 to modifysignaling by the PPR, as TM6 has been shown to be a keymediator of PPR activation (13, 14).

The hypothesis that SW106 binds to a site contacted by Val-2of PTH, the modification of which is known to enable switchingbetween agonism and inverse agonism/antagonism (8, 10, 12),suggests that chemical modification of SW106 may enable thisstructure to activate the PPR rather than blocking its activation.Indeed, several independent lines of research with family AGPCRs have documented that this antagonist/agonist ‘‘pharma-cological switching’’ can be achieved through sometimes sur-prisingly minor chemical modifications (2). Further explorationof the structure-activity relationships and pharmacological be-

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Fig. 4. Characterization of small molecule PPR antagonist SW106. (A) The chemical structure of SW106, which was tested as a racemate in this study,superimposed on its binding isotherm (B28 cells, 125I-DPC-AJ1951 tracer). An average IC50 of 0.99 � 0.16 �M (n � 3) was obtained for SW106. Shown is onerepresentative experiment performed in triplicate. (B) Illustrated is the ability of increasing concentrations of SW106 to block the activity of either 5.0 nMDPC-AJ1951 (squares, IC50 � 5.78 � 1.35 �M) or 0.5 nM PTH-(1–34) (diamonds, IC50 � 68 � 11 �M). Shown is one representative experiment of three experiments,each performed in triplicate. The antagonism of DPC-AJ1951 by SW106 in UMR106, CL0153, and SaOS-2 cells is illustrated in SI Fig. 13. (C) Illustrated is the abilityof increasing concentrations of SW106 to antagonize competitively the cAMP production induced by 14-mer DPC-AJ1951. The EC50 values for DPC-AJ1951 were0.17 � 0.02 nM, 1.97 � 0.45 nM, 21.2 � 4.75 nM, and 750 � 200 nM in the presence of 0, 10, 50, and 100 �M SW106, respectively. Shown is one representativeexperiment of two experiments, each performed in duplicate.

Fig. 5. Predicted binding of SW106 to PPR. This figure highlights the receptorresidues predicted to be in contact with SW106 from a homology model of thehuman PPR (constructed by using rhodopsin as a template; see SI Materials andMethods). The seven transmembrane (TM) helices are colored as follows: TM1,blue; TM2, cyan; TM3, green; TM4, yellow–green; TM5, yellow; TM6, orange;and TM7, red.

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havior of the SW106 chemical class is warranted: Given that thePPR plays a key role in a number of human disease states,optimization of SW106 to a potent, orally bioavailable agonist,antagonist, or inverse agonist could provide a compound ofpotential clinical utility.

Materials and MethodsPeptide Synthesis. The reference peptide 1 and previously unrec-ognized analogs 2–8 were synthesized on an automated peptidesynthesizer, globally deprotected, purified by reverse-phaseHPLC, and characterized by electrospray MS and 1H-NMR.DPC-AJ1951 was iodinated to a specific activity of 2,000Ci/mmol (1 Ci � 37 GBq) by using the lactoperoxidase method(Amersham Biosciences, Piscataway, NJ).

Cell Culture. Cells were cultured at 37°C in T-75 flasks in DMEMsupplemented with 10% FBS. Cells were treated with freshmedia for 12–24 h before assay.

cAMP Stimulation. Culture f lasks containing nonconf luentSaOS-2 (or UMR106, B28, CLO153, or COS-7 with transientlytransfected PPR) were rinsed with two or three washes of warm(37°C) PBS. Prewarmed EDTA (10–15 ml, 37°C) was added, andthe flask was incubated (37°C, 5–10 min). The cells were countedwhile being centrifuged at 300 � g for 5 min and then resus-pended in stimulation buffer (Hanks’ balanced salt solution/5mM Hepes Cellgro/0.1% BSA/0.5 mM 3-isobutyl-1-methylxan-thine, pH 7.4). The AlphaScreen used to detect cAMP wasperformed as described by the manufacturer (Packard, Gro-ningen, The Netherlands).

Competition Binding. HKRK-B28 cells were harvested at 80–95%confluency by trypsinization and then plated at 30,000 cells perwell in 96-well plates in 100 �l of media (high-glucose DMEMand 10% FBS supplemented with Hepes and Pen/Strep). Plateswere incubated overnight at 37°C and 5% CO2. Compound (3 �lof DMSO solution, 50X) was added to 110 �l of binding buffer(50 mM Tris/100 mM NaCl/2 mM CaCl2/5 mM KCl/5.5% FBS,pH 7.7) and mixed. Media was discarded, and 75 �l of compoundin binding buffer was added per well. 125I-6 was prepared inbinding buffer so that �100,000 cpm (100 pM) was added perwell in 25 �l. The plates were incubated for 3 h at roomtemperature before being harvested with three washes of 100-�lbinding buffer. Fifty microliters of Microscint20 (Packard) wasadded per well, and the plates were covered with TopSeal(Packard). The plates were counted after �1 h on a TopCount(Packard) instrument.

Microarray Cell Culture and Treatment. UMR106 cells were grownin DMEM and 10% FBS to confluency and switched to mediumwith 0.1% FBS 24 h before treatment. Solutions of compound,PTH-(1–34), and peptide were added to the media to achieve theconcentrations indicated in the results.

Expression Profiling. Total RNA isolations were performed byusing the RNeasy (Qiagen, Valencia, CA) purification systemaccording to the manufacturer’s instructions. Expression profil-ing of RNA samples was performed by using the Affymetrix(Santa Clara, CA) U34A array following standard protocols.Image acquisition used GeneChip 5.0 (Affymetrix). ResultingCEL-file outputs were imported into Resolver 4.0 (RosettaBiosoftware, Seattle, WA) for analysis.

Real-Time PCR. Fluorescence-based real-time PCR was per-formed with primers and probes manufactured by BiosearchTechnologies (Novato, CA) and targeting the following Gen-Bank accessions and sequence ranges: NM�053769, 1685–1766;NM�013086, 158–227; NM�017031, 2871–2948; NM�019165,178–252; NM�017105, 1806–1872; NM�022197, 1226–1293;NM�020073, 1504–1575; and M11188, 463–649.

Template cDNA was generated by using the AdvantageRT-PCR kit according to the manufacturer’s (Clontech, Moun-tain View, CA) instructions by using random hexamers and 1 �gof DNaseI-treated total RNA. Taqman-based RT-PCR wasperformed with a 7900HT according to the manufacturer’s(Applied Biosystems) instructions with relative expression levelsdetermined by serial dilution.

In Vivo and ex Vivo Assays. Ex vivo bone resorption and formationassays were performed as described by us in ref. 15. Thyropara-thyroidectomy surgery was performed on male Sprague–Dawleyrats (200 g). Animals were supplemented with thyroxin (4 �g perrat, s.c.) three times a week. Animals with serum calcium levelsbetween 5 and 8 mg/dl were used. One week after surgery, Alzet(Palo Alto, CA) minipumps were implanted s.c. for delivery ofpeptides at the concentrations indicated (see Fig. 3D legend).Serum samples were analyzed for total calcium by using theCalcium System Pack kit (Roche Diagnostics, Indianapolis, IN).

More detailed procedures are provided in SI Materials andMethods.

We thank Prof. Thomas Gardella (Harvard University, Boston, MA) forproviding the B28 cells; Dr. Nilsa Graciani for the resynthesis ofDPC-AJ1951; Dr. Dean Wacker for the resynthesis of SW106; Dr.Pamela A. Benfield and D. Ellis for the cell culture used to obtain RNA;J. Bunville for design of primers; and Jing Chen, Melissa Yarde, CelesteTawmley, and Ding Ren Shen for GPCR selectivity screening.

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Carter et al. PNAS � April 17, 2007 � vol. 104 � no. 16 � 6851

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