Immunosuppressive activity in the series of cycloamanide peptides from mushrooms

Immunosuppressive activity in the series of cycloamanide peptides from mushrooms

Peptides.Vol. 14, pp. 1-5, 1993 0196-9781/93$6.00 + .00 Copyright© 1993PergamonPressLtd. Printed in the USA. Immunosuppressive Activity in the Seri...

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Peptides.Vol. 14, pp. 1-5, 1993

0196-9781/93$6.00 + .00 Copyright© 1993PergamonPressLtd.

Printed in the USA.

Immunosuppressive Activity in the Series of Cycloamanide Peptides From Mushrooms Z B I G N I E W W I E C Z O R E K , * I G N A C Y Z. S I E M I O N , t ~ M I C H A L Z I M E C K I , * E L E O N O R A B O L E W S K A - P E D Y C Z A K t A N D T H E O D O R WIELANDJ~

*Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroc~aw, Poland, -#Institute of Chemistry, Wrocbaw University, 14 F. Joliot-Curie str., 50-383 Wrockaw, Poland, and ¢Max-Planck Institut j~r Medizinische Forschung, 6900-Heidelberg, Germany Received 22 J u n e 1992 WIECZOREK, Z., I. Z. SIEMION, M. ZIMECKI, E. BOLEWSKA-PEDYCZAK AND T. WIELAND. Immunosuppressive activity in the series of cycloamanidepeptidesfrom mushrooms. PEPTIDES 14(1) 1-5, 1993.--The immunosuppressor activity of the cycloamanides A, B, C, and D, and two of their D-amino acid residue-containing analogues, was examined using PFC (plaque forming cell) and DTH (delayed type hypersensitivity)tests. It was found that cycloamanide A (CyA A, II) [c-(Phe-PheAla-Gly-Pro-Val-)]and its D-Phe-containinganalogueIII [c-(Phe-D-Phe-Ala-Gly-Pro-Val-)]are the most potent immunosuppressors of the whole series. The retroanalogue of III [C-(D-Phe-Phe-Val-Pro-Gly-Ala-)]was found to be less active than III. The immunosuppressor activity of O-carboxymethyl-Tyrr-antamanide(I) was also tested. It was found that the substitution of one of the Phe residues of ANT by O-carboxymethyl-Tyrdoes not substantially affect the immunosuppressor activity. Cycloamanides

O-Carboxymethyl-Tyr6-antamanide Peptideimmunosuppressors

RECENTLY we found that both cyclolinopeptide A (CLA) (6,9) and antamanide (ANT) (6,8) possess quite strong immunosuppressive activities comparable, at low doses, with that of cyclosporin A (CSA). The activity of these peptides was measured by using different models such as: plaque forming cell (PFC) test, performed in vitro as well as in vivo; graft-versus-host (GvH) reaction; delayed type hypersensitivity (DTH) test; and autologous rosette forming cell (ARFC) test. It can be seen from the formulas shown below that CLA and ANT demonstrate a definite similarity in their structures. Both compounds share similar sequences: -Val-Pro-Pro-Phe-Phe- in CLA, and -ValPro-Pro-Ala-Phe-Phe- in ANT. In both peptides the amide bonds, located between two Pro residues, are of the cis configuration:

length was obtained (1). These peptides, for which the name cycloamanides was proposed, show definite sequential similarities to CLA and/or ANT. Therefore, it seemed interesting to us to examine these peptides with respect to their potential immunosuppressor properties. The application of CLA and ANT for immunologic experiments is restricted by a low solubility of both peptides in water. Therefore, we decided to include into the investigated series Ocarboxymethyl-Tyr6-antamanide(I), in the hope that the O-carboxymethylation of phenolic hydroxyl group of Tyr would enhance the solubility of the compound. In addition to this peptide, the following cycloamanides and their structural analogues were investigated: Pro-Val-Phe 2

I 8

9

1

2

/ Ile-Leu-Val-Pro 7 lie,, I cis \ Leu-Phe-Phe-Pro 6 5 4 3 CLA

8

9

10

1

2

Pro-Phe-Phe-Val-Pro

cis I

[cis

Pro-Phe-Phe-Ala-Pro 7 6 5 4 3

Cycloamanide A (CyA A) (II)

Gly-Ala-Phe3 Pro-Val-Phe

I

[D-Phe3]CyA A (III)

Gly-Ala-D-Phe Pro-Ser-Phe-Phe

ANT

Cycloamanide B (CyA B) (IV) L Ile_Pro_P~he

Antamanide was isolated in 1968 from the mushroom

Pro-D-Ser-Phe-Phe

Amanita phalloides (11). From the same material the series of cyclic peptides with reduced, as compared to ANT, peptide chain

Requests for reprints should be addressed to Dr. Ignacy Z. Siemion.

[D-Ser]CyA B (V) Ile-Pro-Phe

2

WIECZOREK ET AL.

Leu-Val-Leu-Pro [ ~ Phe-Gly-Leu-Met-O (R, S) Leu-Pro-Leu-Pro [ ~

Cycloamanide C (R and S sulfoxide) (VI) Cycloamanide D (R and S sulfoxide) (VII)

Phe-Gly-Leu-Met-O (R, S) P~o-Val-Phe--] ~1 Gly-Ala-D-Phe

Retro-[D-Phe3]CyA A (VIII)

D-Phe-Phe-Val-Pro-Gly-Ala

linear precursor of VIII (IX).

The sequence -Ala-Gly-Pro-Val-Phe-Phe-ofII closely resembles the retrosequence of the fragment -Phe-Phe-Val-Pro-ProAla- of ANT. The fragment -Gly-Pro-Val-Phe-, forming one side of the ring of II, is almost the same as that present in the linear peptide Tyr-Gly-Pro-Leu-Phe-Pro investigated by us recently. We have found (7) that the immunostimulatory peptide TyrVal-Pro-Leu-Phe-Pro can be transformed into an immunosuppressive one by simple substitution of Val or/and Leu residue by Gly. The sequence -Ile-Pro-Ser-Phe-Phe- of IV is very similar to the sequence -Val-Pro-Pro-Phe-Phe- present in CLA; the sequence -Ser-Phe-Phe-Phe-Pro- of this peptide is similar to the sequence -Ala-Phe-Phe-Pro-Pro- of ANT. The same is true for VI and VII: the fragment -Phe-Leu-Ile-Ile- of CLA corresponds to the fragment -Phe-Leu-Vai-Leu- in VI, and also to the fragment -Phe-Leu-Pro-Leu- in VII. There is also definite similarity in the sequence of-Phe-Leu-Pro-Leu-Pro- of VII and the sequence -Phe-Val-Pro-Pro- of ANT. From the structural analogues ofcycloamanides studied, the cyclic peptide VIII seemed to be a very interesting one, because the retro-arrangement (in respect to II) of amino acid residues makes it more similar to ANT. The substances examined were tested for their effects on the primary humoral immune response to sheep red blood cells (SRBC) in vitro, as well as in vivo (PFC test), and on the cellular immune response to SRBC (DTH, i.e., foot pad test). METHOD

Peptides The peptides II, IV, VI, and VII were isolated from the biological material (1). The synthesis of I was described in (12). Compound III (m.p. 165-170°C) was obtained from II (m.p. 150-155°C) by mild alkaline treatment (0.1 N ethanol-NaOH, 50°C, 1 h; A. Gauhe, unpublished) or by total solid phase synthesis (10). Partial epimerization was likewise the method for preparing V from IV. Cyclolinopeptide A was of synthetic origin (9). Cyclosporin A (Sandimmun, Sandoz, Basel, Switzerland), in ampules, was used as the solution in the Cremophor/ethanol mixture.

Syntheses of Peptides VIII and IX The linear peptide D-Phe-Phe-Val-Pro-Gly-Ala (IX) was synthesized on solid support by using a Milligen/Biosearch 9500 automatic peptide synthesizer (Merrifield resin, t-Boc protection, coupling with DCCI). The peptide was cleaved off the resin by 10% sulfuric acid in trifluoroacetic acid (0°C, 2 h). The crude product was purified by preparative HPLC (RP-CI8, Ultrasphere, Beckman). Amino acid analysis (calc./found) Phe 2/2.15, Val 1/1.03, Pro 1/0.95, Gly 1/0.98, Ala 1/1.06; FAB-MS 637.5 (calc. 636.74).

Cyclo(D-Phe-Phe-Val-Pro-Gly-Ala-) (VIII) Two hundred mg (0.31 mmol) of linear peptide was dissolved in 5 ml of dimethylformamide (DMF) and diluted with 1500 ml of dichloromethane (DCM). To the resulting solution 525.4 mg (1.24 mmol) of benzotriazolyl-N-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) and 148 mg ( 1.24 mmol) ofdimethylaminopyridine were added and the mixture was stirred at room temperature for 5 days. Then the solvents were removed in vacuo, and the remaining oil was precipitated with water and filtered off. The precipitate was dissolved in 1 N HCI, and left for crystallization overnight in the refrigerator. The crystals were filtered off, and washed with water and acetone. The crude product was purified by preparative HPLC (RP-CI8, Ultrasphere, Beckman); yield was 45 mg

(24.3%). Amino acid analysis (calc./found) Phe 2/2.04, Val 1/1.05, Pro 1/0.96, Gly 1/0.97, Ala 1/1.03; FAB-MS 619.2 (calc. 618.8).

Biological Tests The immunosuppressive activities of peptides I-IX were tested in the humoral immune response to SRBC, performed in vitro as well as in vivo on mice [PFC test (4)], and in the cellular response to SRBC in mice [DTH, i.e., foot pad test (3)].

Animals" The PFC test was performed with CBA/Iiw mice 8-10 weeks old. Mice of the same strain were also used in the DTH experiments.

Solvents and Culture Medium A mixture of Cremophor-El (Sigma) and 96% ethanol (6.5: 3.5), and 0.9% saline solution were used as solvents. RPMI (Rockwell Park Memorial Institute) medium supplemented with 10% of FCS (fetal calf serum) was used for PFC in vitro experiments. The antigen was sheep red blood cells (SRBC).

Preparation of the Probes The peptides were dissolved, sometimes by weak heating, in 0.2 ml of 96% ethanol, then the same volume of Cremophor/ ethanol mixture was added and the solution was diluted to the desired concentration in 0.9% saline solution (for the in vivo experiments), or in culture medium (for the experiments in vitro).

PFC In Vivo Test Treatment of mice with reagents. Peptides were dissolved in solvents indicated in the text, and diluted to the required concentration; 0.2 ml of each solution were administered twice into the animal. The first dose was applied 3 h before the antigen administration, and the second dose 24 h later. Immunization of mice. Mice were treated IP with 0.2 ml of 10% SRBC in PBS (phosphate buffer solution) 3 h after introduction of the first dose of reagent. After 4 days, the number of PFC producing IgM antibodies against SRBC in the spleen was determined by the Jerne test, using the technique presented in (4). The magnitude of the humoral response was expressed as PFC number per l0 6 splenocytes. PFC In Vitro Test Mice were primed IV with 0.2 ml of 1% suspension of SRBC in 0.9% NaCI. Four days later, the animals were killed; their spleens were minced and then pressed through a plastic screen

CYCLOAMANIDE IMMUNOSUPPRESSORS

into 0.83% NH4C! and buffered with 0.017 M Tris buffer to remove erythrocytes. Then the cells were washed three times with PBS and, finally, resuspended in RPMI medium supplemented with 10% of FCS. To 1 ml of spleen cell suspension (5 X 106 cells/ml), 0.1 ml of the reagent was added, followed by the addition of 0.1 ml of 0.005% suspension of SRBC in the culture. This was incubated for 4 days at 37°C. The PFC number was estimated by the above-mentioned procedure. The experiments were performed using Falcon B-D Labware 24-well tissue culture plates. The viability of the splenocytes in the cultures was estimated by using the MTT (Thiazolyl blue) colorimetric assay for cell growth and kill (5) modified by (2). The cultures were assayed after their 4-day incubation; for the optical density measuring a DYNATECH 5000 recorder was used.

DTH Test Mice were treated IP with two doses of the reagents. A first dose was applied 3 h before sensitization with SRBC, the second dose 24 h later. Mice were sensitized IV with 105 SRBC in 0.1 ml of PBS. After 4 days, the effector reaction was elicited by an intradermal introduction of 108 SRBC into the left hind foot pad. The magnitude of the reaction was measured as the increase of the sensitizing dose of the antigen. The results are expressed in units (1 unit = 10-3 cm of the increase of foot pad thickness).

Statistical Evaluation of the Results The results were evaluated by using analysis of variance (ANOVA) and the Scheffe procedure. In the case of the results presented in Table 1, the correspondent calculations were performed (because of computational reasons) for peptides I-IV and peptides V-VII separately. In both these cases, however, the same controls and references (CLA and CSA) were used. RESULTS

The results of the determination of immunosuppressor activities of peptides I-IX are summarized in Tables 1-4. The effects exerted by these peptides were compared with those produced by cyclosporin A (CSA) or/and cyclolinopeptide A (CLA). The data given in Tables 1-4 show that the substitution of one Phe residue ofantamanide (ANT) by O-carboxymethyl-tyrosine does not substantially affect the immunosuppressor properties of the peptide. The analogue I demonstrates a low activity in the humoral immune response in vitro (Table 1). However, when tested in vivo, it was equally as active as CLA, but weaker than CLA in the cellular response test (Table 3). All cycloamanides, and also their structural analogues investigated, showed in the humoral immune response in vitro (Table 1) a definite immunosuppressor activity. The amplitudes of the effects produced were comparable at the dose of 5 #g with that of CLA, excluding the peptides II and VII, which were distinctly less active. The inversion of the configuration of one Phe residue in peptide II, however, leads to the compound (peptide III) with the highest activity (at the dose of 5 ~tg) in the whole cycloamanide series. The results obtained were statistically significant: for the peptides I-IV, F(14, 75) = 55.00, p = 0.0001; for the peptides V-VII, F(12, 65) = 29.79, p = 0.0001. In PFC in vitro experiments, no toxicity effects were observed at the dose of 1 #g. At the dose of 5/zg, peptides III, V, and VI produced relatively low toxicity effects in the cultures of mouse spleen cells (5 to 10% of the cells died during the experiments), whereas peptide IV was found to be more toxic: at the dose of 5 #g 30-40% of the cells died. No toxic effects were observed for peptides I and II.

3 TABLE 1 THE NUMBER OF PFC IN THE MOUSE SPLEEN CELL CULTURES, TREATED WITH CLA, CSA, AND THE PEPTIDES I-VII, DISSOLVED IN C R E M O P H O R / E T H A N O L MIXTURE AND DILUTED AT THE DESIRED CONCENTRATION IN THE CULTURE MEDIUM

Group

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Substance

Control NaCI Control Cremophort CLA CSA 1 I1 II1 IV V VI VII

Dose (#g/well)

PFC/10 6 *

1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5

2782 3298 3065 883 132 323 26 2416 786 2472 1388 1758 90 1863 129 1642 209 2325 227 1794 953

SD

712 481 371 673 76 166 10 373 361 427 454 395 27 305 29 339 154 661 81 1220 554

* Mean of the values obtained from 6 wells. t In the controls the amounts of Cremophor/ethanol mixture required for the preparation of the solutions containing 1 ttg and 5 /ag of the peptides examined, respectively,were used.

In the experiments performed in vivo [Table 2 (top), F(8, 45) = 44.43, p = 0.0001; Table 2 (bottom), F(10, 44) = 20.62, p = 0.0001] the peptides IV, VI, and VII evoked a decrease of PFC number comparable in its range with that found for CLA. Peptide V, however, was found to be distinctly less active than its counterpart IV and CLA at the dose of 1 ~tg. The inversion of the L-configuration of the Set residue in IV to D-Set (V) does not lead to an enhancement of the immunosuppressive activity as in peptide II, where the change of the configuration of the LPhe 3 residue results in a stronger immunosuppressively active compound (III). It should also be noted that both these peptides, II and III, are even more potent than CLA in their ability to reduce the PFC number in the spleen cells of mice. In the DTH test, the equal doses of 10 ~zg were used in all experiments performed. Table 3 shows that the peptides IV to VII reduce the DTH response to the level similar to that observed after cyclosporin A (CSA) injection. Cyclolinopeptide A is more active than CSA in this test: the largest reduction of DTH response was, however, observed for peptides II and III [in this series of experiments, F(9, 57) = 6.32, p = 0.0001 ]. Peptide VIII and its linear precursor IX both show immunosuppressive effects in the humoral immune response in vivo [see Table 4, F(10, 44) = 16.16, p = 0.0001]. At the dose of l0 ~zg, peptides VIII and IX influence the immune system to the same extent as CSA, and both are even more active than CSA at the low dose of l ~tg. The effects produced by the cyclic peptide VIII are, however, distinctly stronger than those exerted by its linear precursor IX. In the case of VIII, practically no concentration dependence on the effects is visible: PFC numbers are almost the same for the dose of 1 ~tg, l0/~g, and 100 tzg. In the case of IX, the PFC

4

W I E C Z O R E K ET AL.

TABLE 2 PFC NUMBER IN THE SPLEEN OF MICE TREATED WITH TWO DOSES OF CLA AND PEPTIDES l-VII* Group

Substance

1 2 3 4 5 6 7 8 9

Control NaCI

1 2 3 4 5 6 7 8 9

Control NaCI

CLA ! II III

PFC/106

SD

1 10 1 10 1 5 1 5

1875 639 348 714 382 291 251 137 86

449 139 142 317 73 102 54 41 46

1602 731 386 770 312 1337 549 558 501 914 443

303 114 54 165 176 308 74 69 109 361 167

VI

1 10 1 10 1 10 1 10

VII

10

CLA IV V

10

1t

Dose (tag/well)

1

* IP administration; n = 6.

n u m b e r decreases slightly with the initial doses (1 #g to 10/zg) b u t then increases for the dose of 100 ug. DISCUSSION The data presented show that cycloamanides, natural cyclic peptides a c c o m p a n y i n g a n t a m a n i d e in the tissues o f Amanita phalloides, possess i m m u n o s u p p r e s s o r activity c o m p a r a b l e to that of CLA a n d ANT. However, there appeared to be no clear correlation between the results obtained for these peptides during the in vivo a n d in vitro experiments. For example, peptide II, which was equally potent in vivo at the same m o l a r dose as CLA, was markedly less active t h a n CLA in the e x p e r i m e n t in vitro. There was also n o proportionality between the doses o f peptides a n d the amplitudes o f effects. For example, in the investigated range of doses ( 1 - 1 0 0 ~g per animal), no dose-response d e p e n d e n c e was observed in the case of peptides VIII a n d IX (see Table 4). T h e a m p l i t u d e o f effects hardly changed over the whole range o f doses. W e have n o explanation for this p h e n o m enon. However, similar p h e n o m e n a were also observed by us in the case of CLA: the i m m u n o s u p p r e s s o r activity of this peptide (measured in vivo) d i m i n i s h e d at very high doses. The molecular m e c h a n i s m of action o f cycloamanides was not investigated. However, close structural similarity of this group of peptides with CLA a n d A N T suggests t h a t they should also be similar in the m e c h a n i s m o f action. T h e m e c h a n i s m o f action o f CLA consists in the inhibition o f the action o f interleukin-1 a n d interleukin-2 (9). T h e inhibitory activity of ANT, with respect to i n t e r l e u k i n - l , is lower t h a n that o f CLA, b u t remains sufficient (8). There appeared, however, to be n o direct correspondence between the activity of cycloamanides a n d the degree o f their structural similarity with CLA a n d ANT. Thus, when peptide III, the most active from the whole series, was t r a n s f o r m e d into the retro-analogue VIII (which is, from the

TABLE 3 THE INFLUENCE OF CLA AND PEPTIDES I-VII ON THE INDUCTIVE PHASE OF THE DTH TEST IN MICE* Group

Peptide

DTH Unitst

SD

l 2 3 4 5 6 7 8 9 10

Control NaCI CLA CSA I II III IV V VI VII

16.84 8.30 10.74 10.50 7.82 7.70 10.33 10.74 11.13 10.58

4.22 2.41 2.48 1.26 1.84 2.24 2.05 3.69 2.87 2.61

* Substances administered IP in two doses 10 ;zg each; n-7. t 1 unit 10-3 cm of the increase of foot pad thickness. =

structural point of view, m u c h more similar to A N T t h a n the p a r e n t c o m p o u n d ) , a substance with d i m i n i s h i n g i m m u n o s u p pressor activity was obtained. T h e doses of peptides used in PFC in vivo, as well as in the D T H tests, were of the same range as those used previously in the investigation of CLA (9) a n d A N T (8). It enables a direct c o m p a r i s o n o f the data reported here with those previously shown. To have some perspective a b o u t the influence of cycloamanides on the viability of splenocytes, the P F C in vitro experiments were performed with comparatively high doses ( 15 #g per well) of peptides. We f o u n d that at a dose of 1 ug, n o n e o f the peptides investigated exerted toxic effects. This result, together with the results o b t a i n e d in the in vivo experiments, where no toxicity was observed, d e m o n s t r a t e s clearly that the i m m u n o s u p p r e s s i v e effects o f the substances are connected to their own i m m u n o m o d u l a t i n g potencies a n d not to their possible cytotoxicity. At the dose equal to 5 #g, no toxicity effect was observed for the peptide II only. In the case o f peptides III, V, a n d VI, very low toxicity effects, comprising 5-10% of the cells, were observed at this dose. It suggests that at the dose o f 5 #g

TABLE 4 PFC NUMBER IN THE SPLEEN OF MICE TREATED WITH TWO DOSES OF CSA AND PEPTIDES VIII AND IX* Group

Substance

1 2 3 4 5 6 7 8 9 10 11

Control NaCI Control Cremophor t CSA

VIII

IX

Dose (tag/well)

PFC/106

SD

1.0 10.0 100.0 1.0 10.0 100.0 1.0 10.0 100.0

1094 1212 860 511 94 321 334 283 434 318 385

352 267 309 101 56 119 31 87 293 128 128

* IP administration; n = 5. t In the control the amount of Cremophor/ethanol mixture required for the preparing of the solutions containing 10 #g of the peptides was used.

CYCLOAMANIDE I M M U N O S U P P R E S S O R S

5

the cytotoxicity does not participate to a sufficient degree in the immunosuppression. However, the peptide IV (CyA B) at this dose killed 30 to 40% of the cells. Thus, in the last case, the suppression of the i m m u n e response may be distinctly influenced by the toxicity of the compound. In this connection, the toxic effects also appeared in the case of linear precursors o f a n t a m a n i d e and its symmetrical analogue (8). The cyclic peptides obtained from these precursors were not toxic at the comparable doses. A separate problem in this paper concerns peptide I, i.e., the antamanide analogue containing O-carboxymethyl-tyrosine residue instead of the one of two phenylalanine residues. We

included this c o m p o u n d in the investigated series in the hope that the enhancement of hydrophilicity of the peptide evoked by a such substitution would enhance its i m m u n o m o d u l a t o r y potency. Our investigation showed, however, that this does not take place. In the search for c o m p o u n d s interesting from a practical point of view, at least two factors are of importance: the high i m m u n o m o d u l a t i n g potency a n d the low cytotoxicity of the substance. The evaluation of the presented results from this point of view suggests that cycloamanide A (CyA A, II) m a y be of interest as a start in the search for new peptide i m m u noeffectors.

REFERENCES 1. Gauhe, A.; Wieland, T. Components of the green deathcap toadstool, Amanita phalloides. LI. The cycloamanides, monocyclic peptides: Isolation and elucidation of a cyclic heptapeptide (CyA B) and of two cyclic octapeptides (CyA C and CyA D). Liebigs Ann. 1977: 859-868; 1977. 2. Hansen, M. B.; Nielsen, S. E.; Berg, K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119:203-210; 1989. 3. Lagrange, P. H.; Macksaness, G. B.; Miller, T. E.; Pardon, P. Influence of dose and route of antigen injection on the immunological induction ofT cells. J. Exp. Med. 139:528-542; 1974. 4. Mishell, R. 1.; Dutton, R. W. Immunization of dissociated spleen cell cultures from normal mice. J. Exp. Med. 126:423-428; 1967. 5. Mosman, T. Rapid colorimetric assay for cellular growth and cytotoxicity assays. J. Immunol. Methods 65:55-63; 1983. 6. Siemion, I. Z.; Bengtsson, B.; Trojnar, J.; Wieczorek, Z. New peptide immunosuppressors. In: Girault, E.; Andreu, D., eds. Peptides 1990. Leiden: Escom Science Publ.; 1991:882-884. 7. Siemion, I. Z.; Kubik, A.; Lisowski, M.; Szewczuk, Z.; Zimecki, M.; Wieczorek, Z. Immunosuppressive analogues of hexapeptide

8. 9. 10. 11.

12.

TyrValProLeuPhePro an immune system stimulant. Int. J. Pept. Protein Res. 38:54-61; 1991. Siemion, I. Z.; Pedyczak, A.; Trojnar, J.; Zimecki, M.; Wieczorek, Z. Immunosuppressive activity of antamanide and some of its analogues. Peptides (in press). Wieczorek, Z.; Bengtsson, B.; Trojnar, J.; Siemion, I. Z. The immunosuppressive activity of cyclolinopeptide A. Peptide Res. 4:275283; 1991. Wieland, T.; Birr, C.; Flor, F. Peptide syntheses. XLI. Synthesis of antamanide with the Merrifield technique. Liebigs Ann. 727:130137; 1969. Wieland, T.; Luben, G.; Ottenheym, H.; Faesel, J.; De Vries, J. X.; Prox, A.; Schmid, J. Chemical composition ofAmanita phalIoides (XXXVI). Discovery, isolation, elucidation of structure, and synthesis of antamanide. Angew. Chem. (Int.) 7:204-208; 1968. Wieland, T.; Rietzel, C.; Seelinger A. Antamanide X V. Derivatives at the phenolic side chain of tyrosine6-antamanide. Liebigs Ann. 759:52-70; 1972.