Biological activity of parathyroid hormone antagonists substituted at position 13

Peptides,Vol. 12, pp. 57-62. ©PergamonPress plc, 1991. Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Biological Activity of Parathyroid Hormone Antagonists Substituted at Position 13 M I C H A E L C H O R E V , * E L I A H U R O U B I N I , R O B E R T A L. M c K E E , S U S A N W . G I B B O N S , J A N E E. R E A G A N , M A R K E, G O L D M A N , M I C H A E L P. C A U L F I E L D A N D M I C H A E L R O S E N B L A T ' I "l

*The Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem 91120, Israel Merck Sharp and Dohme Research Laboratories, West Point, PA 19486 Received 6 A u g u s t 1990

CHOREV, M., E. ROUBINI, R. L. McKEE, S. W. GIBBONS, J. E. REAGAN, M. E. GOLDMAN, M. P. CAULFIELD AND M. ROSENBLATT. Biological activity of parathyroid hormone antagonists substituted at position 13. PEPTIDES 12(1) 57-62, 1991.--Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [NleS 'i S ,Tyr34 ]bPTH(7-34)NH 2. Acylation of the t-amino group in lysine13 by a hydrophobic moiety is well tolerated in terms ofbioactivity: the analog [Nle8 ' lS ,D-Trp12 ,Lys 13 (~3-phenylpropanoyl),Tyra4]bPTH(7-34)NH2is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal a-amino group yielded the analog desarnino[Nles'ls,D-Trp12,Lyst3(~3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2,an antagonist of high potency in vitro (Kb=4 and 9 nM, Ki=73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems. Parathyroid hormone antagonists Receptor binding Adenylate cyclase Human bone derived (B10) cells

Solid-phase peptide synthesis

PARATHYROID hormone (PTH) is a linear peptide, 84 amino acids in length. In normal physiology, PTH acts on two major target tissues, bone and kidney, to prevent hypocalcemia and to maintain blood calcium levels within a narrow physiological range. This is achieved through stimulation of bone resorption and renal calcium reabsorption, as well as through actions on the metabolism of the hormone vitamin D via activation of the 25(OH)-vitamin D 3 1-a-hydroxylase. PTH antagonists have the potential to be therapeutically useful in clinical disorders of excess PTH secretion. Such agents also have potential utility for treatment of certain cases of humoral hypercalcemia of malignancy in which high levels of a PTH-related protein (PTHrP), which possesses limited structural homology to PTH, are present in circulation as a result of secretion by tumors. While full PTH-like agonist activity resides in the N-terminal portion of PTH (1-34 sequence), truncation of 6 amino acid residues from the N-terminus generates a PTH antagonist (25) which can inhibit PTH-stimulated adenylate cyclase activity and bind to PTH receptors in vitro (11). At least one such antagonist has been demonstrated to be effective in vivo (14). In structure-activity studies directed at designing more potent antagonists, we have identified structurally "tolerant" sites within the 7-34 sequence which can be substituted by a variety of amino acids without altering bioactivity (7). Previously, the glycine at position 12 had been proposed to be part of a B-turn. However,

Bovine renal cortical membranes

we found that position 12 accommodates a wide range of modifications, provided that the substitution does not disrupt an a-helix, such as occurs with N-substituted amino acids. Our working hypothesis is that substitution of "tolerant" sites by hydrophobic amino acid residues will provide auxiliary hydrophobic interactions with the receptor which increase binding affinity without conferring agonist-like properties. Along these lines our earlier investigations found that substitution of Gly 12 by D-Trp, as in [NleS'lS,D-Trp12,Tyr34]bPTH(734)NH/, resulted in a 10-fold increase in antagonist potency (12). Following a similar rationale, D-Trp substitution at position 18 was also found to be well tolerated and in combination, substitution of both position 12 and 18 by D-Trp resulted in a highly potent antagonist [Nle8 ,D-Trp 12 ' 18 ,Tyr 3 4 ]bPTH(7-34)NH2 (8). To further test our hypothesis and to examine the region 1218 in greater detail, we undertook hydrophobic modification of lysine 13. We also combined this modification with elimination of the N-terminal a-amino group, an alteration which may afford resistance to amino-terminal-oriented proteolysis in certain biological systems. METHOD

Materials p-Methyl benzhydrylamine resin HCI 0 % cross-linked, 0.57

1Requests for reprints should be addressed to Dr. Michael Rosenblatt.

57

58

CHOREV ET AL

N -Boc-[side-chainprotected Nle18,Tyr34] bPTH(14-34)-pMBHA-Q l

TFA DCC, N -Boc-Lys(£-Fmoc) OH(I:2) 20% piperidine

N -Boc-Lys-[side-chainprotected Nle~8,Tyr34] bPTH (14-34)-pMBHA-Q

/

\

DCC, O-CH2-CH2CO2H(2:1) /

kk~(CH3)2 CH-CHO (excess, NaCNBH3) N Boc [Lys13(E-N,N-diisobutyl), Nlela,Tyr34) bPTH(13-34)-pMBHA-Q

N Boc [Lys13(£,-3-phenylpropanoyl),Nlela, Tyr34] bPTH (13-34)-pMBHA-Q FIG. 1. Flow chart for the incorporation of N~-Boc-Lys(e-Fmoc)OHonto the side-chain protected peptide-resin and the subsequent acylation or alkylation of the e-amino group of Lys~3.

mM nitrogen/g 100-200 mesh) (pMHBA-R) was obtained from United States Biochem. Inc. (Cleveland, OH). The analogs [Nlea'lS,Tyr34]bPTH(1-34)NH2 and [NleS'lS,Tyr34]bFrH(7-34) NH 2 and N-Boc protected amino acids N-Boc-L-Asp(OcHex)OH, N-Boc-N'~-Bom-L-His-OH, N-Boc-L-Nle-OH, N-Boc-N inFor-D-Trp-OH and N-Boc-L-Lys(e-Fmoc)-OH were obtained from Bachem Inc. (Torrance, CA). The remaining protected amino acid derivatives, N,N'-dicyclohexyl carbodiimide (DCC), 1-hydroxybenzotfiazole, diisopropylethylamine, trifluoroacetic acid, N,Ndimethylformamide and dichloromethane were purchased from Applied Biosystems Inc. (Foster City, CA). Hydrogen fluoride was purchased from Matheson (Secaucus, NJ). p-Thiocresol, pcresol, isobutyraldehyde, isovaleric acid, 3-phenylpropionic acid, NaCNBH 3 and methyl sulfide were obtained from Aldrich Chemical Co. (Milwaukee, WI). Bovine kidneys were the gift of Baum's Meat Packing Inc. (Hatfield, PA). Bovine serum albumin, Tris-HC1, phosphocreatine, creatine phosphokinase, GTP, isobutylmethylxanthine and magnesium-ATP were obtained from Sigma (St. Louis, MO). Modified Ham's F-12 medium and calcium/magnesium-free Hank's balanced salt solution were obtained from Mediatech (Herndon, VA). Fetal bovine serum was purchased from Hazelton (Lenexa, KS). Plastic cultureware was purchased from Costar (Cambridge, MA). [3H]Adenine was purchased from Amersham Corp. (Arlington Heights, IL).

Peptide Synthesis, Purification and Analytical Procedures [Nle8'18,D_Trp12,Lys13(e.phenylpropanoyl),Tyr34]bPTH(7_34) NH 2, analog 1, its desamino counterpart analog 3, desamino[Nle8"~8 D-Trpl 2,Lys 13(e-3-phenylpropanoyl),Tyr34]bFrH(8-34)NH2, analog 4, and [Nle8 •1 8 ,D-Trp 12 ,Lys 13 (e-N,N-diisobutyl),Tyr34 ]bPTH (7-34)NH2, analog 2 were synthesized by a modification of the solid-phase peptide synthesis methodology (18) on an Applied Biosystems 430A automated peptide synthesizer using version 1.2 of the software. Incorporation of the Not-Boc-Lys(e-Fmoc)-OH in position 13 and the subsequent modification of the t-amino group were performed as follows and as outlined in Fig. 1.

Preparation of the Boc-Lys(e-Fmoc)-[side-chain protected NlelS, Tyr34]bPTH(14-34)pMBHA-R. Coupling of Boc-Lys(~Fmoc)-OH (0.94 g, 2.0 mmol) to free amino terminus of [sidechain protected Nle ~8,Tyr34]bPTH(14-34)pMBHA-R (0.25 mmol) was carried out in the standard manner (1 mmol of preformed symmetrical anhydride). The recoupling of Boc-Lys(e-Fmoc)-OH was performed in the presence of 5% diisopropylethylamine (DIPEA) in dimethylformamide (DMF) followed with consecutive washes: CH2C12 (1 × 1 min) and DMF (1 × 1 min). Removal of e-Fmoc protecting group. The protected resinbound peptide was treated with 20% piperidine in DMF (1 × 1 min followed by 1 x 20 min). The resin was consecutively washed with methanol (1 x 1 min), CH2C12 (4 × 1 min) and DMF (2 × 1 min) (10).

Acylation of t-amino in Lys 13 with 3-phenylpropionic anhydride. Two consecutive couplings (2 mmol each) of preformed 3-phenylpropionic anhydride (prepared by DCC treatment) to the free t-amino protected resin-bound peptide were carried out, followed by serial washing: CH2C1z (1 x 1 min), DMF (1 × 1 min), MeOH (1 × 1 min), CHzC12 (1 × 1 min) and MeOH (1 × 1 rain). The ninhydrin test (15) was used to indicate completion of the reaction. The resin-bound peptide was then further washed: CH2C12 (4 × 1 rain) and DMF (2 × 1 rain).

Reductive alkylation of the t-amino group in Lys 13 with isobutyraldehyde. The free t-amino protected resin-bound peptide, [Nle~8,Tyr34]bPTH(13-34), was treated with isobutyraldehyde (0.072 ml, 1.25 mmol) in 1% acetic acid/DMF (10 ml) followed by staged addition of NaCNBH 3 (79 mg, 1.25 mmol) over 40 min. After 1 h, the reaction mixture was filtered and the procedure repeated with a 20-fold excess of reagent which was allowed to react overnight. The resin-bound peptide was then washed: CH2C12 (3x 1 min), MeOH (1 x 1 min) [negative ninhydrin test (15)], CH2C12 (4× 1 min), ethanol (2× 1 min), and DMF (2 × 1 min). The final coupling step in the synthesis of the a-desamino analogs 3 and 4 was carried out with 2 equivalents of preformed symmetrical anhydride of isovaleric acid. The details of the purification and characterization were reported previously (4).

PARATHYROID HORMONE ANTAGONISTS

59

TABLE 1 AMINO ACID ANALYSES OF PEPTIDE ANTAGONISTS, ANALOGS 1-,4

Analog 1

Observed Expected* Observed Expected* Observed Expected* Observed Expected*

2 3 4

Asx

Ser

Glx

Val

Leu

Nle

Tyr

Phe

His

Lys

Arg

2.97 3 3.11 3 2.95 3 3.09 3

2.04 2 2.20 2 1.98 2 2.01 2

3.13 3 3.24 3 3.08 3 3.19 3

1.86 2 1.97 2 1.92 2 1.93 2

4.14 4 4.09 4 4.15 4 4.19 4

2.00 2 2.07 2 2.01 2 1.02 1

1.00 1 1.01 1 0.98 1 0.99 1

1.06 1 0.95 1 -----

2.96 3 3.00 3 2.98 3 3.02 3

2.91 3 2.03t 3 3.00 3 3.06 3

1.92 2 2.34 2 1.91 2 1.98 2

*The analyses represent an average of two analyses performed on hydrolysates of two samples. Trp residues were destroyed in the acidic hydrolysis. t¢-Amino-N,N-diisobutyrl-Lys ~3 does not yield lysine upon hydrolysis.

Receptor Binding and Adenylate Cyclase Assays The procedures used for testing inhibition of PTH-stimulated adenylate cyclase and PTH receptor binding in bovine renal cortical membranes (BRCMs) were described previously (11). Human osteosarcoma B10 cells (23) were cultured in RPMI 1640 medium supplemented with 0.1 mg/ml kanamycin and 10% FBS and subcultured every 7 days. In preparation for assay, cells were passaged into 24-well plates. Confluent cultures were assayed 1-3 days after a change in medium. Cyclic AMP levels, after exposure to peptides, in the cellbased assay were measured using the procedure of Rodan et al. (22). Briefly, cells were prelabeled with [3H]adenine for 2 h, washed twice with calcium/magnesium-free Hank's buffer and incubated with modified Ham's F-12 medium supplemented with 2% FBS and 1 mM IBMX for 10 rain at 37°C. Reactions were stopped by the addition of 0.1 ml of 1.2 M TCA and the [3H]cAMP was isolated by Dowex/alumina chromatography according to Solomon et al. (29).

Data Analysis Binding constants (Kb) and inhibitory constants for PTH-stimulated adenylated cyclase (Ki) were calculated according to Cheng and Prusoff (6).

in this work is summarized in Tables 1 and 2. The purity of peptide analogs as determined by RP-HPLC is greater than 99.5%. Amino acid analyses (Table 1), and determinations of molecular weight by fast atom bombardment-mass spectrometry (FAB-MS) (Table 2) are in agreement with the anticipated values. Table 3 summarizes the biological activities of the Lys 13 substituted bPTH analogs and the corresponding nonmodified parent compound, [NleS'lS,D-Trp12,Tyr34]bPTH(7-34)NH2, in BRCMs and B10 cells in inhibiting PTH binding and antagonizing PTHstimulated adenylate cyclase activation. Analog 2, which was modified at Lys ~3 by ~-N,N-diisobutyl (Fig. 2), was 2- to 3-fold less potent than the parent peptide in BRCMs and 6- to 10-fold less potent than the parent peptide in B10 cells. Modifications of the lysine ~3 by 3-phenylpropanoyl (Fig. 2) resulted in a peptide, analog 1, with increased potency, relative to the parent peptide, of 2- to 4-fold in BRCMs and equivalent potency in B10 cells. However, removal of the a-amino group from analog 1, to yield analog 3, resulted in decreased potency, relative to analog 1, but with similar potency to that seen with the parent peptide in BRCMs. In B10 cells, analog 3 was equipotent to the parent peptide in antagonizing PTH binding. Surprisingly, removal of phenylalanine7 together with the a-amino group resulted in an analog, analog 4, which had a similar potency profile to analog 1, i.e., 2- to 4-fold more potent than the parent peptide in BRCMs and equipotent to the parent peptide in B10 cells.

RESULTS

The physiochemical characterization of the analogs described TABLE 2 MOLECULAR WEIGHTS OF PTH ANTAGONISTS, ANALOGS 1--4, DETERMINED BY FAST ATOM BOMBARDMENT-MASS SPECTROMETRY (FAB-MS) AND RP-HPLC CHARACTERISTICS

Emperical Molecular Weight Analog 1 2 3 4

Formula

Ct78H257N4sO41 CI77H265N4aO4o El7sH265N47041 CI69H.~TN4604o

RP-I-IPLC*

Calculated

Observed

k'

%Bto-%Bt3o

3721 3701 3707 3559

3720 3702 3708 3558

14.0 11.0 14.2 13.1

20--40 20-80 25--40 20--40

*The peptides were analyzed on a Vydac Protein C18 column (15 x 0.42 cm) using a linear gradient system of the following composition: A, 0.1% TFA in acetonitdle:H20 (1:19); B, 0.1% TFA in aeetonitrile. Flow rate was 1.5 ml/min and the column effluent was monitored at 214 rim.

DISCUSSION

The analogs synthesized and evaluated in this investigation were generated by selectively modifying only one of three potentially reactive amino side-chain functional groups present in the full peptide structure. Selective side-chain modification of the protected resin-bound peptide was achieved by employing an orthogonal protection strategy. Introduction of the Fmoc protecting group on the t-amino of Lys ~3 together with the Boc on the oramino and Born, cHex, 2-C1-Z, Tos, For, Bzl, and 4-Br-Z on the other side-chains (listed in the Method section) enabled selective removal of Fmoc with retention of all the others (see Fig. 1). The only free amino function was then either acylated by 3-phenylproionic acid to yield analogs 1, 3, and 4, or was exhaustively reductively alkylated with isobutyraldehyde to yield analog 2. A similar approach was used to modify the t-amino group of DLys 6 in luteinizing hormone-releasing antagonists by alkylating with various aldehydes or ketones (13). Reductive alkylation has also been used in solid-phase peptide synthesis to obtain the CH2NH peptide bond surrogate (26) or an N-alkylated peptide bond (24).

60

CHOREV ET AL

TABLE 3 BINDING AFFINITIES AND INHIBITORY POTENCIES OF ANTAGONISTS IN RENAL- AND BONE-BASED ASSAYS

Renal Bovine Cortical Membranes

Analogs [X,NIe s'ls,D-Trp12, Tyr34]bPTH(7-34)NHz X=[None] 1. [Lys13(~-R)] 2. [Lys~3(~-R')] 3. desamino[Lys~3(~-R)] 4. desamino[desPheT,Lys13(¢-R)]'~

Binding* Kb (nM)

Cyclaset Ki (nM)

15 4 40 13 4

125 60 230 138 73

__. 5 __. 1 _+ 4 _+ 2 _+ 1

+ 10 + 10 _+ 20 _+ 27 _+ 22

Bone Human Osteosarcoma (B10) Cells Bindings Kb (nM) 5.3 --- 0.6 6.5 - 0.7 25.0 + 4.0 N.D. 8.0 ___ 1.8

Cyclase§ Ki (nM) 2.0 7.0 20.0 5.3 3.5

- 0.3 _+ 0.2 _ 2.0 __. 0.9 _+ 0.4

R = 3-phenylpropanoyl. R' = N,N-diisobutyl. Values are the mean --- SEM from at least three separate experiments. *Inhibiting binding of 25 pM [NleS'la,(mono-12~I)Tyr34]bPTH(l-34)NH2. ?Antagonizing 3 nM [NleS'la,Tyr34]bgrH(1-34)NH2. :[:Inhibiting binding of 95 pM [NleS'lS,(mono-125I)Tyr34]bPTH(1-34)NH2. §Antagonizing 0.25 nM of [NleS'~8,Tyr34]bFrH(1-34)NH2 . ¶desamino[NleS'la,D-Trp12,Lys13(¢-phenylpropanoyl),Tyr34]bPTH(8-34)NH2 . N.D. = not determined.

The stimulus for synthesizing the analogs used in this study came from models of the thermodynamics of receptor-ligand interactions, which suggest that the difference in the force driving binding of agonists versus antagonists to the receptor is mostly an increase in enthalpy for the former versus an increase in entropy for the latter. This increase in enthalpy comes from conformational changes in the receptor, induced by the agonist, leading to receptor signal transduction. Therefore, the hydrophobic interactions of antagonists with receptors are considered to be the predominant contributors to receptor affinity (2). By example, many antagonists of peptide hormone/neurotransmitter systems contain several substitutions of aromatic and highly hydrophobic amino acids (21). Once a lead structure (which has the property of binding to receptors without producing biological activity) is defined, a systematic effort can be undertaken toward the design of improved antagonists using hydrophobic substitutions. For F r H , the localization of the activation domain to a sequence proximal to the amino terminus (20,25) provided the framework for studies such as those described in this paper. In order to select sites for hydrophobic substitution, we first identified positions in the sequence that tolerate an extensive range of substitution without substantial change in bioactivity. We previ-

O

A

O

B

FIG. 2. Structure of the two lysine modified residues; (A) Lys(e-3-phenylpropanoyl); (B) Lys(~-N,N-diisobutyl).

ously demonstrated that positions 12 and 18 are such "tolerant" sites (7,8). Substitution for Gly 12 in [NleS'18,Tyr34]bgrH(7 34)NH 2 with hydrophobic amino acids, such as D-Trp, Da-naphthylalanine, or D-13-naphthylalanine, results in 7- to 10fold increases in binding affinity and 5- to 10-fold increases in antagonist potency in BRCMs (7). Substitution at position 18 by L-Trp yields approximately a 4-fold increase in potency. Furthermore, cooperativity was found between positions 12 and 18: the analog [Nle 8 ,D-Trp 12 •18 ,Tyr 34]bPTH(7-34)NH 2 had about 40fold higher affinity and about 50-fold higher antagonist potency than the parent peptide [Nle8'18,Tyr34]bPTH(7-34)NH2 in BRCMs (8). Other studies with PTH agonist analogs, modified to serve as photoaffinity ligands for labeling of PTH receptors, suggested that Lys 13 may also tolerate hydrophobic substitution. Biotinylation of the e-amino functions of lysine side-chains in [NleS'lS,Tyr34] bPTH(1-34)NH 2 gave mixtures of biotinylated derivatives (3,19). It was suggested that analogs biotinylated at one of the three lysines (positions 13, 26 or 27) maintain the biological potency. Similarly, photoreactive analogs of g r H were prepared (9, 27, 28). The most potent derivative, [Nle8'lS,Lys13(N-e-4-azido 2-nitrophenyl),Tyr34]bPTH(1-34)NH2, was more than twice as potent as the unmodified analog in canine renal cortical membranes (9). In order to study the effects of hydrophobic substitution for lysine at position 13, we selected [NleS'18,D-Trp12,Tyr 34] bPTH(7-34)NH 2 as the parent antagonist. Both acylation and exhaustive reductive alkylation were used to modify the e-amino group in Lys 13. The neutral 3-phenylpropanoyl moiety (analog 1) was better tolerated than alkylation by isobutyraldehyde (analog 2). Analog 1 was 4- to 10-fold more potent than analog 2 in antagonizing PTH-stimulated adenylate cyclase activity and inhibiting PTH binding, respectively. A similar trend was observed in the human bone cell-based assay. Finally, we attempted to stabilize the antagonist against potential amino-terminal-directed proteolytic degradation by eliminating the amino-terminal amino group, a modification which simultaneously enhances hydrophobicity. Recently, we have shown that both desamino[Tyr34]bPTH(7 34)NH 2 and [N-MeMetS,Tyr34]bPTH(7-34)NH2 are slightly more

PARATHYROID HORMONE ANTAGONISTS

61

potent than unmodified [Tyr34]bPTH(7-34)NH2 (11). However, evaluation of the desamino analog, analog 3, and its N-terminal truncated version, desamino[NleS'lS,D-Trp12,Lysla(~-3-phenyl propanoyl),Tyr34]bPTH(8-34)NH2 (analog 4) yielded unexpected results. As seen in Table 3, changes at the N-terminus had different effects on potency, depending on the extent of truncation. Analog 3, missing only the a-amino function, was, unexpectedly, somewhat less potent than analog 4, in which Phe 7 was deleted. This could be the consequence of either steric or conformational effects attributed to the multiplicity of specific modifications at the amino terminus in the desamino[NleS'lS,D-Trp12,Lys13(e 3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2, analog 3. Further truncation of PTH beyond position 6 has been reported previously to result in a slight decrease in antagonist activity compared to the corresponding 7-34 sequences. [Nle~S,Tyr34]

bPTH(10-34)NH 2 had an IC5o of 7 × 10 - 6 M and K b = 2 . 7 × 10-6 M inhibiting bPTH(1-84)-stimulated adenylate cyclase (17) and 125I-[NleS'1S,Tyr34]bPTH(1-34)NH2 binding (20), respectively. [Tyr34]hPTH(8-34)NH2 and [Tyra4]hPTH(9-34)NH2 were only slightly less potent than the corresponding 7-34 sequence (16). A more substantial decrease in antagonist activity is observed for [Tyr34]hPTH(14-34)NH2 (Kb= 260 and 910 nM and Ki=840 and 6050 nM for the 7-34 and 14-34 sequences in bovine renal cortical membrane, respectively) (5). Similarly, [Tyra'*]bPTH(14-34)NH2 competes with I25I-[NleS'lS,Tyr34] bPTH(1-34)NH 2 for binding sites on ROS 17/2.8 cells with a K b = 50 nM. Therefore, it is possible that introducing hydrophobic moieties into "tolerant" positions within PTH will generate potent, shorter antagonists, such as analog 4 described in this study.

REFERENCES 1. Abramson, S. N.; Molinoff, P. B. In vitro interactions of agonists and antagonists with 13-adrenergic receptors. Biochem. Pharmacol. 33:869-875; 1984. 2. Abu-Samara, A.-B.; Uneno, S.; Jueppner, H.; Kentmann, H.; Potts, J. T., Jr.; Segre, G. V.; Nussbaum, S. R. Non-homologous sequences of parathyroid hormone and parathyroid hormone-related peptide bind to a common receptor on ROS 17/2.8 cells. Endocrinology 125: 2215-2217; 1989. 3. Abu-Samra, A.-B.; Freeman, M.; Juppner, H.; Uneno, S.; Segre, G. V. Characterization of fully active biotinylated parathyroid hormone analogs. J. Biol. Chem. 265:58-62; 1990. 4. Caporale, L.; Nutt, R. F.; Levy, J. J.; Smith, J.; Afison, B.; Bennett, C.; Albers-Schonberg, G.; Pitzenberger, S.; Rosenhlatt, M.; Hirschmann, R. Characterization of synthetic parathyroid hormone analogues and of synthetic byproducts. J. Org. Chem. 54:343-346; 1989. 5. Caulfield, M. P.; McKee, R. L.; Goldman, M. E.; Duong, L. T.; Fisher, J. E.; Gay, C. T.; DeHaven, P. A.; Levy, J. J.; Roubini, E.; Nutt, R. F.; Chorev, M.; Rosenblatt, M. The bovine renal parathyroid hormone (PTH) receptor has equal affinity to two different amino acid sequences: The receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. Endocrinology 127: 83-87; 1990. 6. Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibiton constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I5o) of an enzymatic reaction. Biochem. Pharmacol. 22:3099-3108; 1973. 7. Chorev, M.; Goldman, M. E.; McKee, R. L.; Roubini, E.; Levy, J. J.; Gay, C. T.; Reagan, J. E.; Fisher, J. E.; Caporale, L. H.; Golub, E. E.; Caulfield, M. P.; Nntt, R. F.; Rosenblatt, M. Modification of position 12 in parathyroid hormone and parathyroid hormone-related protein: Toward the design of highly potent hormone antagonists. Biochemistry 29:1580-1586; 1990. 8. Chorev, M.; Roubini, E.; Goldman, M. E.; McKee, R. L.; Gibbons, S. W.; Reagan, J. E.; Caulfield, M. P.; Rosenhlatt, M. Effects of hydrophobic substitutions at position 18 on the potency of parathyroid hormone antagonists. Int. J. Pept. Protein Res.; in press. 9. Coltrera, M. D.; Potts, J. T., Jr.; Rosenblatt, M. Identification of a renal receptor for parathyroid hormone by photoaffinity radiolabeling using a synthetic analogue. J. Biol. Chem. 256:10555-10559; 1981. 10. Felix, A. M.; Wang, C.-T.; Heimer, E. P.; Fournier, A. Application of BOP reagent in solid-phase synthesis. U. Solid phase side-chain to side-chain cyclization using BOP reagent. Int. J. Pept. Protein Res. 31:231-238; 1988. 11. Goldman, M. E.; Chorev, M.; Reagan, J. E.; Nutt, R. F.; Levy, J. J.; Rosenblatt, M. Evaluation of novel parathyroid hormone analogs using a bovine renal membrane receptor binding assay. Endocrinology 123:1468-1475; 1988. 12. Goldman, M. E.; McKee, R. L.; Caulfield, M. P.; Reagan, J. E.; Levy, J. J.; Gay, C. T.; DeHaven, P. A.; Rosenblatt, M.; Chorev, M. A new highly potent parathyroid hormone antagonist: [D-

Trp12,Tyr34]bPTH(7-34)NH2. Endocrinology 123:2597-2599; 1988. 13. Hocart, S. J.; Nekola, M. V.; Coy, D. H. Effect of reductive alkylation of D-lysine in position 6 on the histamine-releasing activity of luteinizing hormone-releasing hormone antagonists. J. Med. Chem. 30:739-743; 1987. 14. Horiuchi, N.; Holick, M. F.; Potts, J. T., Jr.; Rosenhlatt, M. A parathyroid hormone inhibitor in vivo: Design and biological evaluation of a hormone analog. Science 220:1053-1055; 1983. 15. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34:595-598; 1970. 16. Kubota, M.; Ng, K. W.; Murase, J.; Nods, T.; Moseley, J. M.; Martin, T. J. Efficacy and specificity of human parathyroid hormone analogues as antagonists in intact clonal osteogenic sarcoma cells. J. Endocrinol. 108:261-265; 1986. 17. Mahaffey, J. E.; Rosenblatt, M.; Shepard, G. L.; Potts, J. T., Jr. Parathyroid hormone inhibitors, determination of minimal sequence requirements. J. Biol. Chem. 254:6496-6498; 1979. 18. Merrifield, R. B. Solid phase synthesis. Adv. Enzymol. 32:221-296; 1969. 19. Newman, W.; Beall, L. D.; Levine, M. A.; Cone, J. L.; Randhawa, Z. I.; Bertolini, D. R. Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. J. Biol. Chem. 264:1635916366; 1989. 20. Nussbaum, S. R.; Rosenblatt, M.; Potts, J. T., Jr. Parathyroid hormone-renal receptor interactions: Demonstration of two receptorbinding domains. J. Biol. Chem. 255:10183-10187; 1980. 21. Regoli, D. Peptide antagonists. Trends Pharm. Sci. 7:481-484; 1985. 22. Rodan, S. B.; Insogna, K. L.; Vignery, A. M.-C.; Stewart, A. F.; Broadus, A. E.; D'Souza, S. M.; Betolini, D. R.; Mundy, G. R.; Redan, G. A. Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoblastic cells. J. Clin. Invest. 72:1511-1515; 1983. 23. Rodan, S. B.; Wesolowski, G.; lanacone, J.; Thiede, M. A.; Rodan, G. A. Production of parathyroid hormone-like peptide in human osteosarcoma cell line: Stimulation by phorbol esters and epidermal growth factor. J. Endocrinol. 122:219-227; 1989. 24. Ron, D.; Laufer, R.; Frey, J.; Selinger, Z.; Chorev, M. Synthesis and biological activity of agonists for the neuronal tachykinin receptor in the guinea pig ileum. In: Henry, J. L.; Coutre, R.; Cuello, A. C.; Pelletier, G.; Quirion, R.; Regoli, D., eds. Substance P and neurokinins. New York: Springer-Verlag; 1987:144-145. 25. Rosenhlatt, M.; Segre, G. V.; Tyler, G. A.; Shepard, G. L.; Nussbaum, S. R.; Pntts, J. T., Jr. Identification of a receptor-binding region in parathyroid hormone. Endocrinology 107:545-550; 1980. 26. Sasaki, Y.; Coy, D. H. Solid phase synthesis of peptides containing the CH2NH peptide bond isostere. Peptides 8:119-121; 1987. 27. Shigeno, C.; Hiraki, Y.; Westerberg, D. P.; Potts, J. T., Jr.; Segre, G. V. Photoaffinity labeling of parathyroid hormone receptors in clonal rat osteosarcoma cells. J. Biol. Chem. 263:3864-3871; 1988. 28. Shigeno, C.; Hiraki, Y.; Keutmann, H. T.; Stem, A. M.; Potts, J.

62

CHOREV ET AL

T., Jr.; Segre, G. V. Preparation of a photoreactive analog of parathyroid hormone [Nlea,Lys(N-e-azido-2-nitrophenyl)13,Nle~S,Tyr34] bovine parathyroid hormone (I-34)NH 2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors. Anal.

Biochem. 179:268-273; 1989. 29. Solomon, Y.; Londos, C,; Rodbell, M. A highly selective adenylatc cyclase assay. Anal. Biochem. 58:541-548; 1974.