a new class of potent bradykinin antagonist dimers

ELSEVIER Immunopharmacology 33 (1996) 178 - 182

Immunopharmacology

A new class of potent bradykinin antagonist dimers

Lajos Gera a, John M. Stewart a,*, Eric Whalley b, Michael Burkard b,

John S. Zuzack b a Department of Biochemistry, University of Colorado School of Medicine, Denver, CO 80262, USA

b Cortech, Inc., Denver, CO 80221, USA

Keywords: Bradykinin antagonist; Antagonist dimer; Receptor, B1; Receptor, B 2

1. Introduction

It is a well-established principle in chemistry and pharmacology that bivalent ligands tend to bind much more tightly to their targets than do univalent ligands. Therefore dimeric ligands tend to possess greater potency and duration of action compared to the corresponding monomers. In the bradykinin (BK) field, the Cortech homo- and hetero-dimers are good examples (Cheronis et al., 1992; Cheronis et al., 1994). Cortech dimers are principally Cys-containing BK antagonists linked by bis-maleimidoalkanes. In 1980 Stewart and co-workers (unpublished) made a series of succinyl-bis-dimers of bradykinin and ex- tended bradykinins which showed good agonist activity in all assays and were totally resistant to enzymatic degradation in the rat pulmonary circula-

tion. We report here dimers of our potent new BK antagonists (such as B9430) that contain a-(2-indanyl)glycine and have both B 1 and B 2 receptor antagonist activity (Stewart et al., 1996). In these new dimers, the cross-linkers are generally at the N-terminus of the peptide chain. We have synthesized dimers having succinyl-, suberyl-, suberimidyl- and bis-succinimidohexane linkers. Many of these dimers show high affinities for human and guinea pig B j and B 2 receptors. In addition, certain of these dimers are selectively cytotoxic to small cell lung carcinoma (Chan et al., 1996).

2. Materials and methods

2.1. Synthesis

Abbreviations: BSH, bissuccinimidohexane; Cpg, eL-cyclopentylglycine; Dhq, 2-dehydroquinuclidine-3-carboxyl; Eac, e- aminocaproic acid; Flu, fluorescein thiourea; Gun, guanidyl: -C(=NH)-NH2; Hyp, trans-4-Hydroxy-L-proline; Igl, a-(2-indanyl)glycine; Mosi, methoxy-suberimido: -C(=NH)-(CH2) 6- C(=NH)-OCH3; Nig, N-(2-indanyl)glycine; Oic, octahydroin- dole-2-carboxylic acid; Sub, suberyl: -CO-(CH2)6-CO-; Suc, succinyl: -CO-(CH2)2-CO-; Suim, subefimidyl: -C(=NH) - (CHz)6-C(=NH)-; Tbi, [3-2-thienylalanine; Tic, 1,2,3,4-tetrahy- droisoquinoline-3 -carboxylic acid

* Corresponding author.

Unusual amino acids: a-(2-indanyl)glycine (D-Igl, L-Igl), a-cyclopentylglycine (D-Cpg, L-Cpg) and N- (2-indanyl)glycine (Nig) were prepared in our labo- ratory, and converted to the N-Boc derivatives (Stewart et al., 1996; Gera and Stewart, 1996). Cross-linkers BMH, DMS and DSS were obtained from Pierce Chemical Co.

Peptides were synthesized by standard solid phase methods (Stewart and Young, 1984), purified by countercurrent distribution and/or HPLC and char-

0162-3109/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved Pll S0 162-3 109(96)00099-9

L. Gera et al. / hnmunopharmacology 33 (19961 178 182 179

acterized by HPLC, TLC, mass spectroscopy and amino acid analysis. BOP or BOP-HOBt was used for coupling of amino acids.

2.2. Synthesis of" dimers

Method A: (succinyl dimer synthesis on resin) (B9132 and B9572). A 10-fold excess of succinic anhydride and triethylamine was allowed to react with the peptide-resin having a free amino group to give the succinyl peptide (mono-adduct) on the resin. After washing, the second pure, neutralized peptide monomer was allowed to react in DMF with the BOP-, TBTU- or HATU-activated succinyl-peptide on the resin to give the succinyl-bis peptide dimer still attached to the resin. The finished peptide dimers were cleaved from the resin by HF (Stewart and Young, 1984). The free peptides were extracted with acetic acid, lyophilized, purified and characterized as above.

Method B: (suberyl-dimer synthesis on resin) (B9860-3, B9860-4 and B9876). A 5-fold excess of disuccinimidyl suberate (DSS) and a 2-fold excess of N,N-diisopropyl-ethylamine (DIEA) in DMF were allowed to react with the peptide-resin having a free e-Lys group (B9798) overnight to give the succi- nimidyl-suberyl peptide (mono adduct) on the resin. Then additional pure, neutralized e-Lys-peptide monomer (B9810) was allowed to react with the suberyl-peptide on the resin to give the Sub-bis- peptide dimer still attached to the resin. The finished peptide was cleaved from the resin with HF. Free peptides were extracted and purified as above. HPLC separation gave hetero- (B9860-3) and homo-dimer (B9860-4). The B9876 peptide dimer was made simi- larly, but from the free c~-amino-Lys(e-Fmoc) monomer peptide.

Method C: (suberyl-bis-peptide dimers in solution) (B9832). 1 Equivalent of neutralized peptide, 10 equiv. DIEA and 0.75 equiv, disuccinimidyl suberate (DSS) were allowed to react overnight in DMF at room temperature, and the resulting dimer was purified by preparative reversed-phase HPLC.

Method D: Bis-succinimidohexane peptide dimers in solution) (B9834 and B9836). One equiv, of Cys- containing peptide monomer salt, 10 equiv. DIEA and 0.75 equiv, bismaleimidohexane (BMH) linker were allowed to react overnight in DMF. The result-

ing bis-succinimidoalkane peptide dimers were then purified by preparative reversed-phase HPLC.

Method E: (suberimidyl-bis-peptide dimers in solution) (B9830, B9870, B9872, B9878) One equiv. of peptide monomer salt, 15 equiv. DIEA and 1 equiv, dimethyl suberimidate.2HC1 (DMS) were stirred overnight in DMF at room temperature. The DMF was removed in vacuo and the residue was purified by reversed-phase HPLC. The HPLC separation gave the Mosi-monomers and the Suim-bis-dimers generally in 1:2 ratio.

2.3. Binding to human B 1 and B 2 receptors

Human lung fibroblast IMR-90 cells expressing B~ receptors were obtained from ATCC and propagated in DMEM medium in 85 mm roller bottles until confluent. Three hours prior to harvesting, the cells were treated with interleukin-l[3 (200 pg/ml). Chinese hamster ovary (CHO) cells expressing cloned human B 2 receptors were propagated in F12 medium until confluent. Preparation of membranes for binding assays was carried out by scraping cells from roller bottles in ice-cold phosphate-buffered saline (PBS) and centrifuging at 1000 × g, at 4°C for 15 min. The supernatant was discarded and the pellet was resuspended in Buffer A (25 mM TES, pH 6.8, with 2 p~M 1,10-phenanthroline) and cells were rup- tured with 10 strokes of a Dounce homogenizer. The homogenate was centrifuged at 27 000 X g for 15 min; the centrifugation was repeated. The final pellet was resuspended in Buffer B (Buffer A with 2 txM captopril, 140 Ixg/ml bacitracin, 0.1% bovine serum albumin, BSA), stored in 1 ml aliquots, and frozen at -20°C until needed.

Binding assays were performed by incubating CHO human clone membranes with 0.3 nM [3H]bradykinin or IMR-90 membranes with 0.5 nM [3H][des-Argl°]-kallidin in the presence of the peptides in assay buffer (Buffer B with 1 mM dithiothreitol), at room temperature, for 45 min. All compounds were assayed in triplicate. Membranes were harvested by quick filtration in a Tomtec Harvester 96, using ice-cold wash buffer (10 mM Tris-HC1, pH 7.5, 100 mM NaCI, 0.02% BSA), onto Wallac printed glass fiber Filtermat B, which had been pre-soaked with 0.1% PEI and air-dried. Filtermats were counted

180 L. Gera et al. / lmmunopharmacology 33 (1996) 178-182

Table 1 Structures and activities of bradykinin dimers and their precursors

Number Structure

0 1 2 3 4 5 6 7 8 9

Binding

Bl a B2 b B2 c SM d

BK BK (1-8) Lys-BK (1-8) CP0298 B9132

B9572

B9830

B9832

B9834

B9836

B9860-3

Arg Pro Pro Gly Phe Ser Pro Phe Arg - 9.0 9.2 Arg Pro Pro Gly Phe Ser Pro Phe 5.6 i.a. i.a.

Lys Arg Pro Pro Gly Phe Ser Pro Phe 8.9 4.7 4.2 Lys Arg Pro Pro Gly Phe Ser Pro Leu 8.7 4.1 i.a.

Dhq DArg Arg Pro Hyp Gly Lys Ser DCpg Cpg Arg - - A I Suc I

Dhq DArg Arg Pro Hyp Gly Lys Ser DCpg Cpg Arg Eac DArg Arg Pro Hyp Gly Thi Ser Dlgl Oic Arg - 8.7 8.5 A I Suc DArg Arg Pro Hyp Gly Thi Ser Dlgl Oic Arg

DArg Arg Pro Hyp Gly Thi Ser DTic Nig Arg 6.0 8.3 8.6 E I Suim

DArg Arg Pro Hyp Gly Thi Ser DTic Nig Arg DArg Arg Pro Hyp Gly Thi Ser DTic Nig Arg 6.0 8.4 7.9 C I Sub I IgArg Arg Pro Hyp Gly Thi Ser DTic Nig Arg DArg Arg Pro Hyp Gly Igl Cys Dlgl Oic Arg 7.2 8.4 7.9 D

BSH I

DArg Arg Pro Hyp Gly lgl Cys Dlgl Oic Arg Cys DArg Arg Pro Hyp Gly lgl Ser Dlgl Oic Arg 7.9 9.1 8.8 D

BSH I Cys DArg Arg Pro Hyp Gly Igl Ser olgl Oic Arg (~)-Lys DArg Arg Pro Hyp Gly lgl Ser Dlgl Oic Arg 8.5 9.3 9.0 B I

B9860-4

B9870-1 B9870-2

S u b ' - -

I Gun-Gly Lys Arg Pro Pro Gly Phe Ser Pro Leu (~)-Lys IgArg Arg Pro Hyp Gly Igl Ser Dlgl Oic Arg 8.0 8.8 8.5 B

I Sub I (e)-Lys DArg Arg Pro Hyp Gly Igl Ser Dlgl Oic Arg

Mosi DArg Arg Pro Hyp Gly Igl Ser DIgl Oic Arg 7.8 9.1 9.1 E DArg Arg Pro Hyp Gly Igl Ser DIgl Oic Arg 7.9 8.4 8.3 E I Suim I DArg Arg Pro Hyp Gly Igl Ser DIgl Oic Arg

L. Gera et al. / lmm un opharmacology 33 (1996) 178- 182 181

Table 1 (continued)

Number Structure Binding

1 2 3 4 5 6 7 8 9 BI a B2 b B2 c SM d

B9872-2 Mosi oArg B9872-3 DArg

[ Suim I DArg

B9876 Lys r)Arg I Sub DArg

B9878-2 Mosi DArg B9878-3 DArg

Suim I DArg

B9882 Lys(Flu) DArg I Sub DArg

CP-0364 DArg

Arg Pro Hyp Gly Thi Ser DTic Oic Arg 9.5 9.7 E Arg Pro Hyp Gly Thi Ser DTic Oic Arg - 8.8 9.0 E

Arg Pro Hyp Gly Thi Ser oTic Oic Arg Arg Pro Hyp Gly lgl Set Dlgl Oic Arg

Arg Pro Hyp Gly Igl Ser DIgl Oic Arg Arg Pro Hyp Gly Cpg Ser DCpg Cpg 8.5 6.4 6.1 E Arg Pro Hyp Gly Cpg Ser DCpg Cpg 8.9 6.5 6.5 E

Arg Pro Hyp Gly Cpg Ser DCpg Cpg Arg Pro Hyp Gly Igl Ser DIgl Oic Arg

Arg Pro Hyp Gly lgl Ser Dlgl Oic Arg Arg Pro Hyp Gly Phe Cys Phe Leu Arg

L BSH I

I DArg Cys Pro Hyp Gly Phe Ser Pro Leu

9.1 7,2 7.1 D

a IMR 90, pICs0; b Human clone, pICs0; " GP ileum, pICs0; d Synthesis methods. Mosi-peptides are monomer + linker.

in 9.5 ml Wallac Beta-Plate Scint, in a Wallac 1450 MicroBeta Counter.

2.4. Binding to guinea pig ileum B 2 receptors

Guinea pig ileum membranes were prepared by Analytical Biological Services, inc. Briefly, ilea were finely chopped and combined with TES buffer (25 raM, pH 6.8) containing 1 mM 1,10-phenanthroline, 5 p~g/ml soybean trypsin inhibitor, 100 p.g/ml bacitracin, 1 mM benzamidine, and 100 o~M phenyl- methylsulfonyl fluoride. This mixture was homoge- nized in a Brinkman PT-20 Polytron (setting 7, 4 × 20 s intervals) and centrifuged (1000 × g, 4°C, 10 rain). The pellet was discarded and the supernatant centrifuged for 15 rain at 43000 × g at 4°C. The pellet was washed twice by resuspending in the same buffer and recentrifuging. The pellet was resuspended and stored at -70°C until use.

Guinea pig ileum membrane suspensions were incubated with [3H]bradykinin (final concentration

0.3 riM) with or without test compounds in assay buffer (25 mM TES, pH 6.8 containing 1 mM 1,10- phenanthroline, 1 mM dithiothreitol, 2 txM captopril, 140 Ixg/ml bacitracin, and 0.1% BSA) at room temperature for 45 rain in a final volume of 315 txl. All test compounds were assayed in triplicate. Mem- branes were harvested and counted as for human receptors.

3. R e s u l t s a n d d i s c u s s i o n

Structures and binding data are given in Table 1. We have described here the synthesis and receptor binding activity of a class of BK antagonist dimers based on the highly potent combined B~-B z antagonist, B9430. Cross-linking of the [Cys 6 ]-derivative of B9430 with bismaleimidohexane yielded a dimer which retained high B 1 and B 2 receptor binding. Most other dimers reported here were cross-linked at the N-terminus. Acylation of B9430 with a neutral

182 L. Gera et a l . / Immunopharmacology 33 (1996) 178-182

hydrophobic acyl group reduces significantly the B 1 binding; a similar effect was seen with succinyl-bis- B9430 and suberyl-bis-B9430. Addit ion of another basic amino acid residue and then cross-linking with acylating agents that retain the basic character of the N-terminus of the dimer, such as dimethyl suberimidate, gives dimers that retain high potency at both B l and B 2 receptors. Peptides containing one (B9860-3; B9878-2) or two (B9878-3) chains lacking the C- terminal arginine showed, not surprisingly, high potency at B 1 receptors. Of these, B9878-2 and B9878-3

showed surprisingly high potency at B 2 receptors, considering that they lack the C-terminal arginine,

long considered essential for B 2 binding. B9860-3, which has one B 1 and one B 2 antagonist chain, shows the highest overall potency. B9882 is useful for fluorescent labeling of BK receptors in tissues.

Some of these dimers have been found to be selectively cytotoxic in vitro to human small cell lung carcinoma (Chan et al., 1996).

Acknowledgements

This work was aided by grant HL-26284 from the U.S. NIH. We thank Paul A. Bury for assistance

with chemistry, and G.R. Matseuda for a gift of N,N'-bis-Cbz-guanylpyrazole.

References

Chan D, Gera L, Helfrich B, Stewart JM, Whalley ET, Bunn P. Novel bradykinin antagonist dimers for treatment of human lung cancers. Immunopharmacology 1996; 33: 201-204.

Cheronis JC, Whalley ET, Nguyen KT, Eubanks SR, Allen LG, Duggan MJ, Loy SD, Bonham KA, Blodgett JK. A new class of bradykinin antagonists: synthesis and in vitro activity of bissuccinimidoalkane peptide dimers. J Med Chem 1992; 35: 1563-1572.

Cheronis JC, Whalley ET, Allen LG, Loy SD, Elder MW, Duggan MJ, Gross KL, Blodgett JK. Design, Synthesis, and in vivo activity of bis(succinimido)hexane peptide heterodimers with combined B1 and B2 antagonist activity. J Med Chem 1994; 37: 348-355.

Gera L, Stewart JM. A new class of bradykinin antagonists containing indanylglycine. Immunopharmacology 1996.

Stewart JM, Young JD. Solid Phase Peptide Synthesis, 2nd Edn. Rockford, IL: Pierce Chemical Company, 1984.

Stewart JM, Gera L, Zuzack JS, Burkard M, Hanson W, McCul- lough R, Whalley ET. A new generation of bradykinin antagonists. Immunopharmacology 1996; 33: 51-60.

a new class of potent bradykinin antagonist dimers

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