Journal of Protein Chemistry, Vol. 4, No. 6, 1985

Design of Analogues of Parathyroid Hormone:

A Conformational Approach

S. R. Nussbaum, 1 N. V. Beaudette, 2 G. D. Fasman, 2'4 J. T. Ports, Jr., 1 and M. Rosenblatt 1'3

Received February 10, 1986

An approach to the design of peptide-hormone analogues in which amino acid substitutions are based on predicted effects on secondary structure was investigated. The structural require- ments for parathyroid-hormone (PTH) action are distinct from the determinants necessary for receptor binding alone without subsequent activation of adenylate cyclase. Two analogues of PTH containing substitutions in the principal binding domain of PTH, the region 25-34, were synthesized by the solid-phase method and evaluated for bioactivity. The sequence 25-34 was predicted to have nearly equal conformational potential for both a-helix and fl-sheet using Chou and Fasman parameters. A previously studied analogue, [Tyr34]bPTH(1-34) amide, containing substitutions in this region, was more active than was bPTH-(1-34). The substitution of tyrosine for phenylalanine at position 34 in this analogue is predicted to promote fl-sheet conformation. The analogues [Ile 2s, Tyr 3°, Tyr34]bPTH-(1-34) amide and [Arg 32, Tyr34]bPTH-(1-34) amide each contain substitutions predicted to further enhance or stabilize ~-sheet formation. The solution conformation of these analogues, determined by circular dichroism studies in an aqueous buffer and an organic solvent, indicated promotion of fl-sheet secondary structural content in both analogues in a hydrophobic environment chosen to simulate that of the interaction of the peptide and the membrane receptor. In contrast, the native sequence lacks ~-structure. Biological activity of these analogues in the rat renal adenylate cyclase assay in vitro and binding affinity in a radioreceptor assay were threefold those of unsubstituted PTH-(1-34). Peptide analogue design based on conformational pre- diction, rather than substitution of primary structure alone, offers an attractive alternative approach to the development of hormone analogues and antagonists.

KEY WORDS: parathyroid hormone; analogues; prediction of secondary structure; solid- phase peptide synthesis; circular dichroism; bioactivity.

1. I N T R O D U C T I O N

Trad i t iona l ly , pep t i de ana logue des ign has involved single amino ac id subs t i tu t ions based on p r i m a r y s t ructure to a l ter the charge, size, or shape o f an amino ac id side chain. A l though gu ide l ines for select ing amino ac id subst i tu t ions have been formu- la ted (Rud inger , 1971), h o r m o n e ana logues o f enhanced b io log ica l act ivi ty have

1 Department of Medicine, Harvard Medical School, and the Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts.

2 Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts. 3 Present address: Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania. 4 To whom correspondence should be addressed.

391 0277-8033/85/1200-0391504.50/0 O 1985 Plenum Publishing Corporation

392 Nussbaum et al.

largely been developed through a trial-and-error approach of synthesis followed by bioassay. This approach has been most rewarding for small peptides such as vasopressin and gonadotropin-releasing hormone because of the ability to generate more rapidly short amino acid sequences for biological evaluation (Rivier et aL, 1983). For larger peptide hormones, such as parathyroid hormone (PTH), use of this single-amino acid and sometimes random substitutive approach becomes less attractive because the technical effort, expense, and time required for the synthesis and purification of an analogue severely limit the total number of analogues that can be made and evaluated.

Studies of PTH, an 84-amino acid polypeptide, in vitro and in vivo demonstrated that the amino-terminal 34 amino acids contain all the structural determinants necessary for full biologic activity in multiple bioassay systems (Potts et aI., 1971; Tregear et al., 1973; Parsons et aL, 1975). Receptor-binding (positions 3-34) and adenylate cyclase-activation (positions 1 and 2) domains are separable (Goltzman et al., 1975; Mahaffey et al., 1979; Rosenblatt et aL, 1977; Segre et aL, 1979a, b).

Further structure-activity studies of PTH demonstrated that there are at least two binding domains within the PTH molecule; the regions 10-27 and 25-34. Analysis of binding affinities for synthetic fragments representing these regions has demonstrated that the 25-34 binding region is the principal binding domain (Rosen- blatt et al., 1980a, b; Nussbaum et al., 1980). A further indication of the importance of this region in expression of PTH bioactivity is indicated by the complete conserva- tion of sequence of the 27-34 region across the four species for which the native sequence of PTH has been elucidated (Heinrich et aL, 1984).

A recent approach to peptide analogue design focuses on the larger issues of peptide conformation and topography (Chou and Fasman, 1975; Hruby, 1981; Sternberg and Thornton, 1978). Single amino acid substitutions may have effects broader than those limited to one position in a peptide chain, inasmuch as they now place significant conformational constraints on the hormone molecule or change the environment of conformationally neighboring amino acids. Using a predictive model of secondary structure of PTH in conjunction with our previous structure- activity studies of the biologically active region of PTH, we designed and synthesized two analogues of PTH containing substitutions predicted to enhance and stabilize /?-sheet secondary structure in the region of principal importance for receptor binding: [Arg 32, Tyr34]bPTH-(1-34) amide and [Ile ~8, Tyr 3°, Tyr34]bPYH-(1-34) amide (Chou and Fasrnan, 1974a, b, 1977, 1978a, 1979). /?-Sheet-enhancing modifications were selected because previous PTH analogues in which tyrosine was substituted for phenylalanine at position 34 (a modification that is predicted to promote/3-sheet formation) enhanced biological activity in vitro (Parsons et al., 1975; Rosenblatt et al., 1976). Theoretically,/?-sheet secondary structure might facilitate receptor interaction, inasmuch as hydrogen bonding can occur between hormone and receptor when antiparallel peptide chains in/?-sheet conformation are aligned. In contrast, the a-helix conformation utilizes all hydrogen bonding internally to stabilize the helix, and intermolecular hydrogen bonding is minimal or absent.

The biological activity and circular dichroism (CD) analyses of two PTH analogues, [Arg32,Tyr34]bPTH-(1-34) amide and [Ile28,Tyr3°,Tyr34]bPTH-(1-34) amide, are reported here. The secondary structures of these two peptides were

PTH Conformal Analogues 393

obtained by computer best-fit analysis of CD spectra (Greenfield and Fasman, 1969) obtained in aqueous buffer at physiological pH and in an organic solvent chosen to simulate the hydrophobic lipid environment of the membrane receptor to assess the conformational effects of our modifications.

2. EXPERIMENTAL P R O C E D U R E S

2.1. Synthesis and Purification

Two analogues of bovine parathyroid hormone, [Ile28,Tyr3°,Tyr34]bPTH-(1-34) amide and [Arg32,Tyr34]bPTH-(1-34) amide, were synthesized by a modification of the Merrifield solid-phase technique (Merrifield, 1962, 1963, 1969; Erickson and Merrifield, 1976). The primary amino acid structure of the analogues is depicted in Fig. 1. Syntheses were performed manually with benzhydrylamine resin (polystyrene cross-linked with 1% divinylbenzene) serving as an insoluble support that generated a carboxyamide carboxy-terminal modification in the final product. The t-butyloxy- carbonyl group (Boc) was used for a-amino group protection during coupling for each amino acid, except arginine, which was protected by the amyloxycarbonyl group. Amino acid side-chain protecting groups were identical with those used previously (Nussbaum et aL, 1980; Rosenblatt et al., 1977, 1980a, b). Amino acids were incorporated by using dicyclohexyl carbodiimide as the coupling agent, except for glutamine and asparagine, which were coupled as "active" p-nitrophenyt esters. All amino acid-coupling reactions were monitored by the fluorescamine test (Felix and Jimenez, 1973). Removal of the peptide from the resin and simultaneous deprotection of side-chain functions were achieved by treatment with anhydrous hydrogen fluoride (0°C, 60min). Purification was by gel filtration, followed by ion-exchange chromatography in 8 M urea (Rosenblatt et al., 1977). The purified peptides conformed to theoretically expected amino acid composition and appeared homogeneous by thin-layer and high-pressure liquid chromatography and Edman sequence analysis.

2.2. Secondary Structural Analyses

The secondary structure of PTH was predicted from the amino acid sequence by the protein conformational predictive method of Chou and Fasman (1974a, b, 1977, 1979). Amino acid modifications were based on these secondary-structure parameters for the likelihood of promoting/3-sheet conformation.

CD measurements were made with a Cary 60 recording spectropolarimeter equipped with model 6001 CD accessory, as described by Adler et aL (1971), with the original photomultiplier tube replaced with an end-on Hamamatsu tube, # R375. All measurements were made at 23°C in a 0.2- to 0.5-mm (path length) cell (Optical Cell, Woodbine, MD) at a full-scale sensitivity of 0.04 deg. Peptide concentration was 0.441-1.66 mg/ml. Secondary structure (% a-helix,/3-sheet, and random-coil conformation) was derived from the CD spectra by the method of Greenfield and Fasman (1969) by using a- and /3-spectra of poly-L-lysine and the random-coil spectrum of the histones (Fulmer, 1979) in an iterative computer program to select

394 Nussbaum et ai.

a best-fit curve. Ellipticity, [0], is expressed in deg cm2/dmole of amino acid residue by using a mean residue molecular weight of 113.

The peptide was dissolved in either 2.5 mM sodium phosphate buffer (pH 7.4) or 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) (Eastman). Peptide concentration was determined on duplicate samples of the peptide-containing solutions by amino acid analysis. After lyophilization, aliquots were acid hydrolyzed (5.7 M HC1, 100°C, 24 h) in the presenceof 1 : 2000 mercaptoethanol in an evacuated dessicator. Amino acid analyses were performed using a Beckman model 121 MB analyzer. Amino acid content was determined by comparison of sample composition performed in duplicate with mixtures of independently quantified standards. Mean peptide con- tent was used to calculate peptide concentration in the samples used for CD analysis.

2.3. Bioassay

Assessment of biological activity in vi tro was performed by a ~nodification of the rat renal cortical adenylate cyclase assay (Krishna et al., 1968; Marcus and Aurbach, 1969, 1971). [32p]ATP and [3H]cAMP were obtained from New England Nuclear (Boston, MA). The bPTH standard used in the assay was Medical Research Council Standard (Great Britain), lot no. MRC 72/286. Each preparation was assayed at least three times at multiple concentrations. The separate potency esti- mates were combined to yield the mean potency of each analogue.

2.4. Radioligand-Binding Assay

An assay for the binding of PTH to the membrane receptor, based on canine renal cortical membranes and the sulfur-free, 125I-labeled hormone analogue [NleS,Nle18,Tyr34]bPTH-(1-34) amide was employed (Segre et al., 1979a, b; Nussbaum et al., 1980; Rosenblatt et al., 1980a, b). The assay is specific and saturable with regard to PTH binding. Binding affinity in this system correlates closely with biologic activity for multiple PTH fragments, analogues, and antagonists (Segre et al., 1979a, b; Nussbaum et al., 1980; Rosenblatt et al., 1980a, b). Radioactive specific activity of the 125I-labelled ligand was 500+75mCi/mg. Canine renal cortical membranes were purified by ultracentrifugation by use of discontinuous gradients of sucrose in a manner identical with that used in previously conducted studies of inhibitory potency (Mahaffey et al., 1979). Inhibition of specific binding of radio- ligand was assessed for each peptide over several log orders of magnitude. The concentration range employed was 1.0 × 10 -1° to 1.0 X 10 -14 M. Inhibition of radio- ligand binding at each dose of peptide was determined in triplicate. Each peptide was assayed at least three times in this system.

3. RESULTS

Chou and Fasman parameters used to predict secondary structures are presented in Table I. The secondary structure of PTH based on this predictive index is represented in Fig. 2. For the biologically active 1-34 region of PTH, it is predicted that there are three conformationally discrete regions. The a-helix conformation is

PTH Conformal Analogues 395

A 5 /0

N H 2 - ~

/5

25 2 0

3oGs L ¢ S ST, T,ON

B

5 /0

N H 2 - ~ / 5

25 2 0

~ ) ~ O NATURAL SEQUENCE (~) SUBSTITUTION

Fig. 1. Amino acid sequence of analogues of parathyroid hormone fragment 1-34: (A) [mrg 32, Tyr34]bPTH-(1-34) amide and (B) [Ile aS, Tyr s°, Tyr34]bPTH - (1-34) amide. The naturally occurring bovine sequence is indicated by open circles. Shaded residues represent amino acid substitutions in these analogues.

favored for positions 1-9 and 18-29 (with a tribasic Arg-Lys-Lys sequence at positions 25-27). The sequence 12-15 has a high probability of forming a fl-turn. The sequence 30-34 has nearly equivalent potentials for a-helix or /3-sheet, although/~-sheet formation is slightly favored.

396 Nussbaum et al.

< 0

..=

<

e ~

[-

v

v

v

~ u z n l - ~

+ ÷

PTH Conformal Analogues 397

~-~ot = <~>-~

I ~ o~~ = ( ~ )

I + I + + + I

° ~

~.~ ~ + ~ ~ ' ~

.~ .~ ~ ~, c ~ . ~

~ . ~ . ~

r~

398 Nussbaum et al.

9 [

~ N H 2

15 18 30 g4

Fig. 2. Predicted secondary structure of parathyroid hor- mone. Conformation for fl-sheet is predicted between residues 28-34, a region important for receptor binding. Curled lines, c~-helix; zigzag lines, /3-sheet.

The carboxymethyl-cellulose (CMC) chromatographic purification profile of [Ile 28, Tyr 3°, Tyr34]bPTH-(1-34) amide, following Biogel P-6 gel filtration, strongly suggests that peptide aggregation occurs that might be promoted by the presence of a/3-sheet secondary structure. This elution profile was altered when CMC column chromatography was performed under denaturing conditions of 8 M urea (Fig. 3).

These two conformational analogues of bPTH-(1-34) were assayed in vitro and in vivo. The potency of [Ile28,Tyr3°,Tyr34]bPTH-(1-34) amide in the in vitro renal adenylate cyclase assay using native bPTH-(1-84) (MRC # 74/286) (potency 3000, 100% activity) as the assay standard was 13,000 MRC units/mg (280%). The [Arg32,Tyr34]bPTH-(1-34) amide, with a potency of 15,000, also was more active than the MRC standard (Fig. 4, Table II). Both of these analogues had renal adenylate cyclase activity comparable with [Tyr34]bPTH-(1-34) amide. The substitu- tions predicted to enhance /3-sheet formation led to activity greater than that of unsubstituted bPTH-(1-34). However, neither analogue possessed greater potency than [Tyr34]bPTH-(1-34) amide.

Each analogue caused complete inhibition of radioligand-specific binding in a dose-dependent manner that paralleled the inhibition observed for the assay stan- dard, the peptide [Nle 8, Nle is, Tyr34]bPTH-(1-34) amide. The concentration of each peptide that inhibited radioligand-specific binding to a 50% maximal level was taken as the apparent binding constant KB. The KB values of [Nle 8, Nle 18, Tyr34]bPTH-(1-34) amide and the analogues [Ile 28, Tyr 3°, Tyr34]bPTH-(1-34) amide, and [Arg 32,Tyr34]bPTH-(1-34) amide, as demonstrated in Fig. 5, are virtually identical and equal to 1.2 x 10 -8 M, indicating equal affinities for the renal-receptor binding site.

Representative CD spectra of bPTH-(1-34) amide and the two conformational analogues in phosphate buffer and HFP are shown in Fig. 6. In HFP, chosen to simulate the hydrophobic lipid environment of the plasma membrane, the three polypeptides showed a higher helical content than in phosphate buffer. The bPTH- (1-34) amide has ellipticity extrema of [0]207 = -28,000 deg cm2/dmole and [0]220 = -22,400 deg cm2/dmole in HFP. The CD values for [Arg 32, Tyr34]bPTH-(1-34) amide in HFP were found to be [ 0]zo7 = -18,000 deg cm2/dmole and [01221 = -14,500 deg cm2/dmole. The analogue [Ile 28, Tyr 3°, Tyr34]bPTH-(1-34) amide yielded ellip-" ticity values in HFP of [0]207 =-17,500 deg cm2/dmole and [0]220 = -14,400 deg cm2/dmole. The derived secondary structure based on computer best-fit programs

P T H Conformal Analogues 399

A

06!

I (35

O44

o os4

a 02 o

0L

70 8'o gb Ioo lio kio 130 140 150 160 TUBE NUMBER

. q-

O

~6 E

>

• 4 2 Z O O

I E C

Od K3 O

127 I

,o t 1

os i q

0 61 04

t O I

. ?

/ / / I -~ --:J \ / v \ J2 ~

6'0 7'0 sb 9'0 k6o lio 12o TUBE NUMBER

Fig. 3. Chromatographic profile of [Ile 28, Tyr 3°, Tyra4]bPTH- (1-34) amide on polyacrylamide gel (Bio-Gel P6) in (A) 1.0 M acetic acid and (B) 8 M urea.

(Fulmer, 1979) indicates that /3-sheet structure is present in both conformational analogues in HFP (Table III). In addition, there is enhancement of a-helix structure in the organic solvent. However, in contrast, the derived secondary structure of the unsubstituted hormone fragment bPTH-(1-34) in HFP shows an increase in a-helix structure, a frequently observed effect of organic solvents, but no/3-sheet secondary structure. Furthermore, neither analogue in HFP contains the a-helix content found in native bPTH-(1-34), but both show conformational shifts toward/3-sheet secon- dary structures. Amino acid substitutions, selected by predictive indices to enhance /3-sheet secondary structure, have been demonstrated by CD to increase /3-sheet structure in an organic nonpolar solvent that simulates the membrane-receptor environment (Table III). For bPTH-(1-34) in phosphate buffer at physiological pH, negative troughs were observed at 203 and 222nm, with [0]2o3 being -16,000 deg cm2/dmole and [0]222 =-6600 deg cm2/dmole at a peptide concentration of 0.57 mg/ml. The CD spectra and derived secondary structure ofbPTH-(1-34), [Ile 28, Tyr 3°, Tyr34]bPTH-(1-34) amide, and [Arg 32, Tyr34]bPTH-(1-34) amide are presen- ted in Fig. 6 and Table III. The secondary structures 2.5 mM phosphate buffer,

400 Nussbaum et al.

t2:

rO

o10- x

~s-

° i 0_6

<[ 0 4

cJ 2 if3

0

--BASAL-

PEPTIDE (~g or MRC units)

Fig. 4. Composite of representative rat renal cortical adenylate cyclase assays of (0) [Ile 28, Tyr 3°, Tyr34]bPTH-(1-34) NH2, (A) [Arg 32, Tyr34]bPTH - (1-34) NH2, and (O) native bovine hormone and stan- dard bPTH-(1-84), 2500 MRC units/mg. Each point is the mean of triplicate determinations. Each peptide was quantitated by amino acid analysis.

pH 7.4, for the above polypeptides, as well as [Tyr34]bPTH-(1-34) amide, are also given in Table III. All analogues have a similar percentage of o-helix (12-18%) and negligible amounts of #-structure.

4. D I S C U S S I O N

T h e p r e d i c t i v e a l g o r i t h m o f C h o u a n d F a s m a n (1974a , b; 1979) f o r t h e s e c o n d a r y

s t r u c t u r e o f p o l y p e p t i d e s a n d p r o t e i n s h a s b e e n a p p l i e d to s e l ec t s e v e r a l a n a l o g u e s

o f b P T H - ( 1 - 3 4 ) t h a t h a v e t h e p o t e n t i a l f o r a l t e r e d c o n f o r m a t i o n s . T h e c o n f o r m a t i o n

o f t h e s e a n a l o g u e s w as d e t e r m i n e d b y C D in a q u e o u s a n d n o n p o l a r e n v i r o n m e n t s

a n d t h e i r b i o l o g i c a l ac t iv i ty e v a l u a t e d . T h e r a t i o n a l e f o r a p p l y i n g t h i s p r o c e d u r e

Table II. Biological Activity of PTH Conformational Analogues

PTH analogue

In vitro cyclase Activitya, b

Binding constant MRC units/mg c % Activity Renal membranes

1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4

1 ......................... 34 1 ...................... Wyr 34 1 ...................... Tyr34-amide 1----Ile2S--Tyr 3° ...... Tyr34-amide 1 ............... Arg3Z--Tyr34-amide

3,000 100 1.2 x 10 8 M 5,400 100 1.2 x 10 -s M 7,500 140 1.2 × 10 -s M

16,000 300 1.2 x 10 -8 M 13,000 240 1.2 x 10 -8 M 15,000 280 1.2 x 10 -8 M

a Molar activities are shown as percent of that of bPTH-(1-84). MRC units are shown as the activities in units/rag by comparison with a house standard of bPTH-(1-84).

bSee Table I of Potts et aL (1982) for comparative values. c MRC 72/286.

P T H Conformal Analogues 401

I00 (_9 • z 8o z

o -

½ 40- 0 EL CO 20-

0 I0-'0 10 9 10-8 LO-7 /0-6 I0-5 10-4

PEPTIDE CONCENTRATION [M]

Fig. 5. Plot of competitive inhibition or radioligand binding to PTH-specific canine renal binding sites by the analogues (0 ) [Ile 28, Tyr 3°, Tyr34]bPTH-(1-34) NH 2 and (&) [Arg 32, Tyr34]bPTH-(1-34) NH 2. Also shown are (11) the analogue fragment [Nle 8, Nle TM, Tyr34]bPTH-(1-34) NH2 and (O) the native bovine sequence bPTH-(1-34). (Fq) The peptides hPTH-(44- 68) and hPTH-(53-84) were used as control peptides, which do not bind to the PTH receptor. Concentration of PTH fragments is indicated on the horizontal axis, percent specific binding of radioligand on the vertical axis. Each point represents the mean of triplicate determinations. The figure is a composite of representative assays for each peptide.

was based on previous successful applications of the method. When utilized with a synthetic precursor peptide of PTH, it predicted two possible conformations that correlated well with the conformations found by CD analysis in two different solvent systems (Rosenblatt et al., 1980a, b).

Structure-activity studies of PTH, utilizing two series of analogue fragments, have demonstrated at least two binding domains within the fully biologically active 1-34 region of the hormone molecule. Comparison of affinity constants of the 10-27

Table IIL Secondary Structure Derived from Circular Dichroism in HFP a and Phosphate Buffer pH 7.4 b (23°C)

HFP 2.5 mM PO 4

Peptide a % /3% R a n d o m % a % /3% Random%

bPTH-(1-34) 68 0 lIle 28, Tyr 3°, Tyr34)bPTH-(1-34) amide 29 18 [Arg 32, Tyr34]bPTH-(1-34) amide 33 12 [Tyr34]bPTH-(1-34) amide c __

32 12 0 88 53 18 2 80 55 12 6 82 - - 15 0 85

HFP, hexaflouroisopropanol. b 2.5 mM sodium phosphate buffer, pH 7.4. c Value not measured because of solubility problems.

402 Nussbaum et al.

-6

E ¢.0

(2) 723

'_o x

-0.8

- I . 6 j

-2"4 t

-3.2

A

/

1 /

/ /

/ /

i It i /

'1 / / '1 i I

C

I L

/ /

200 2~0 T 240 ' 2d@ -I 220 240 2&O ' 220 240 nm

Fig. 6. CD spectra: (A) unsubstituted bovine PTH-(1-34); (B) [Ile 28, Tyr 3°, Tyr34]bPTH - (1-34) NH2; (C) [Arg 32, Tyr34]bPTH-(1-34) NH 2 in two solvents; (--) HFP and (--) 2.5 mM phosphate buffer pH 7.40. Concentrations of peptides ranged from 0.2 to 0.6 mg/ml in phosphate buffer and HFP. Ellipticity 0 is calculated per mole of amino acid residue.

and 25-34 binding domains demonstrated that the 25-34 sequence is the principal binding region (Nussbaum et al., 1980). There is, in addition, evolutionary conserva- tion of the 25-34 region in the porcine, bovine, human, and rat sequences for PTH (Niall et al., 1970; Sauer et aI., 1974; Keutmann et al., 1978; Heinrich et al., 1984), indicating the importance of this region. Previous studies showed that amino acid substitution of Tyr for Phe at position 34 enhances biologic activity in the in vitro rat renal adenylate cyclase assay and the in vivo chicken hypercalcemia assay (Parsons et al., 1975; Rosenblatt et al., 1976). This substitution is predicted to enhance/3-sheet conformation.

The predicted secondary structure of PTH-(1-34) (Table I, Fig. 2) showed that the 30-34 region had nearly equal conformational potential for both/3-sheet and

-helix. On the basis of the biological properties of modifications previously evalu- ated (Potts et aL, 1982), it was decided to synthesize two peptides that would conformationally favor/3-sheet formation in this region. [Ile 28, Tyr 3°, Tyr34]bPTH - (1-34) amide includes substitution of Ile (Pt3 = 1.60) for Leu (Pt~ = 1.30) at position 28, Tyr (PC = 1.47) for Asp (P~ = 0.54) at position 30 (Asp is a strong/3 breaker), and Tyr (P~ = 1.47) for Phe (PC = 1.38) at position 34. By substitution of tyrosine for aspartic acid, a change of considerable magnitude (altering charge, shape, and hydrophobicity) in this biologically important region was made. Despite these substitutions, [ Ile 2s, Tyr 3°, Tyr34]bPTH-(1-34) amide bound to purified renal cortical membranes with an affinity equal to that of the unsubstituted 1-34 sequence (Table II). In addition, the analogue had a biological activity comparable with that of the Tyr34-substituted 1-34 sequence, which has enhanced biological acivity.

The second hormone analogue, [Arg 32, Tyr34]bPTH-(1-34) amide, involved substitution of Arg at position 32 for His (Pt~ = 0.93 vs. PC = 0.87) and maintained the Tyr-for-Phe substitution at position 34. This analogue both has enhanced biological activity in vitro in generation of adenylate cyclase when compared with

PTH Conformal Analogues 403

the native bovine PTH-(1-34) fragment, and bound to renal membranes with an affinity comparable with that of PTH-(1-34) (Table II).

An implication that the amino acid substitutions had altered conformation was suggested by the ~ column chromatographic purification of [Ile 2s, Tyr 3°, Tyr34]bPTH- (1-34) amide. The elution pattern (Fig. 3A) displayed widely spread peaks, suggest- ing product heterogeneity, peptide aggregation, or column-support interaction. The latter two phenomena are often associated with/3-sheet formation. Use of 8 M urea in the eluant buffer to disaggregate and denature peptides produces a chromato- graphic profile composed of sharp, well-defined peaks (Fig. 3B), suggesting that product heterogeneity was not responsible for the widely spread peaks of the chromatographic profile. The secondary structure of both [Ile 28, Tyr 3°, Tyr34]bPTH - (1-34) amide and [Arg 32, Tyr34]bPTH-(1-34) amide, as well as [Tyr34]bPTH-(1-34) amide and unsubstituted bPTH-(1-34), were analyzed by CD (Table III). In phos- phate buffer, at physiological pH, used to approximate the extracellular-fluid environment, bPTH-(1-34) was not highly structured, with 88% random structure and only 12% a-helix, which is similar to the conformation of [Tyr34]bPTH amide. These data support the NMR studies of Bundi et al. (1976) and the CD values of Brewer et al. (1975), whose ellipticity values were slightly lower. Zull and Lev (1980), using hydrophobicity profiles and Chou-Fasman conformational predictive indices and integrating knowledge of the biological activity of analogues of parathyroid hormone, suggested a model for PTH containing two hydrophobic domains separ- ated by a linker region. Although no direct measurements of secondary structure were performed, Zull and Lev's conformational model further extended the earlier dark-field electron microscopic observations of Fiskin et al. (1977a, b) that had been used to develop a model of the three-dimensional structure of PTH. In HFP, used to simulate the hydrophobic environment of the membrane (a low dielectric constant) receptor/peptide interface, the unsubstituted peptide became highly ordered with a predominant a-helical structure (Table III). In contrast, both analogues demon- strated significant amounts of/3-sheet structure in HFP, suggesting that the amino acid substitutions promoted/3-sheet in an environment known to promote secondary stracture.

The importance of secondary structure and tertiary structure to peptide and protein function is illustrated in the evolutionary process. Families of homologous proteins, e.g., cytochromes c, have almost identical conformations despite extensive changes in amino acid sequences (Dickerson and Timkovich, 1975). Similarly, examination of natural substitutions in the same protein as it occurs in different species reveals disparate amino acid sequences, but preservation of similar secondary structures, suggesting that protein conformation evolves more slowly and is func- tionally more important than sequence per se (Doolittle, 1979).

A thoroughly studied system is that of signal or leader sequences of proteins, which are amino-terminal extensions of secreted proteins. These amino-terminal extensions, critical for translocation across the rough endoplasmic reticulum, all have a hydrophobic sequence of from 15 to 31 amino acids, and have been predicted to have a high c~-helical probability followed by a C-terminal/3-turn ending at the site of posttranslational cleavage that generates the mature peptide or protein (Garnier et al., 1980). It appears that a common cellular apparatus and signal

404 Nussbaum et al.

peptidase recognizes the secondary structure of these sequences rather than the dissimilar primary amino acid sequence (Austen, 1979; DiMaio and Schiller, 1980; Garnier et al., 1980; Majzoub et al., 1980). The necessity for the a-helical potential of the signal for transport was clearly demonstrated by the work of Gierasch and co-workers (Briggs et aL, 1985; Briggs and Gierasch, 1984).

The idea for producing conformationally altered sequences was suggested in 1975 for glucagon, shortly after the predictive scheme was published (Chou and Fasman, 1975). The concept of conformational alterations to design biologically active peptides has been successfully utilized in other hormonal systems. Conforma- tional requirements for biological activity were defined for enkephalins (DiMaio and Schiller, 1980), somatostatin (Arison et al., 1978; Veber et al., 1978), oxytocin (Hechter et al., 1975; Walter et al., 1971; Walter, 1977), vasopressin (Smith and Walter, 1978), and a-MSH (Sawyer et aL, 1982) by use of semirigid synthetic peptide-hormone analogues. Amino acid substitutions selected for conformational constraint on a segment of RNAse-S caused profound reductions in binding affinity (Dunn and Chaiken, 1975). Recently, the theory that amphiphilic helices are optimally suited for receptor interaction has stimulated considerable efforts in analogue design for several protein and hormonal systems (Kaiser and K6zdy, 1984; Sparrow and Gotto, 1982). The application of this approach to the corticotropin- releasing factor is of interest. For corticotropin-releasing factor (Lau et aL, 1983; Rivier et al., 1983), which is a highly amphiphilic a-helix, substitution of amino acids that promote the a-helical conformation has led to the development of an antagonist analogue.

Altering the conformation of bPTH-(1-34) by synthesis of peptide analogues produced an increase in in vitro biological activity and maintained the affinity for renal membrane receptors. This strongly suggests that the transition toward/3-sheet is well-tolerated and perhaps the favored conformation recognized by the PTH receptor. Thus, the analogues that are predicted to have a higher/3-potential were indeed found to have the higher biological activity, and this/3-potential was shown to favor/3-structure by CD analysis.

It is worth emphasizing that it should not be surmised that a higher biological activity will result from a greater/3-potential. A greater E-potent ial infers a more probable stable/3-structure, but activity and stability of the active conformation are not synonymous, and thus a sequencing that favors /3-sheet need not enhance biological activity. Rather, if bioactivity declined dramatically as a result of such substitutions, then it could be inferred that the change disrupted a conformation in the hormone more favored for receptor interaction.

For PTH, design of analogues that are based on conformation, rather than on single amino acid substitution in the primary sequence, offers an important approach to the generation of hormone analogues that are biologically active, and should facilitate exploration of the nature of hormone-receptor interaction.

ACKNOWLEDGMENTS

This is publication no. 1585 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts. This research was generously suppor-

PTH Conformal Analogues 405

ted in part by grants from the U.S. Public Health (GM 17533), the American Cancer Society (P-577), and the Department of Energy (EP-78-S-02-4962.A000). G.D.F. is Rosenfield Professor of Biochemistry, Brandeis University.

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