Structural permutation of potent cytotoxin, polytheonamide B: Discovery of cytotoxic peptide with altered activity

Article abstract of DOI:10.1021/ml300264c

Polytheonamide B (1) is an ion-channel forming natural peptide with a d,l-alternating 48 amino acid sequence, which is an exceedingly potent cytotoxin. We recently designed and synthesized a simplified dansylated polytheonamide mimic 2, in which six amino

Full text of DOI:10.1021/ml300264c

Letter
pubs.acs.org/acsmedchemlett
Structural Permutation of Potent Cytotoxin, Polytheonamide B:
Discovery of Cytotoxic Peptide with Altered Activity
Hiroaki Itoh and Masayuki Inoue*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
S
* Supporting Information
ABSTRACT: Polytheonamide B (1) is an ion-channel forming natural peptide with a D,L-
alternating 48 amino acid sequence, which is an exceedingly potent cytotoxin. We recently
designed and synthesized a simplified dansylated polytheonamide mimic 2, in which six amino
acid residues were modified from 1, and demonstrated that 2 emulated the functions of 1. Here
we report a comprehensive structure−activity relationship study of substructures of 2. A unified
synthetic strategy was developed for highly automated syntheses of 13 peptide sequences of 27
to 39 amino acid residues, and the artificial 37-mer peptide 6 was discovered to be significantly
more toxic than the other 12 compounds toward P388 mouse leukemia cells (IC50 = 3.7 nM).
Ion exchange activity experiments of 6 using the liposome and P388 cells both demonstrated
that 6 did not possess ion-channel activity, strongly suggesting that 6 exerted its potent cytoxicity
through a distinct mode of action from 1 and 2.
KEYWORDS: Cytotoxic agents, natural products, structure−activity relationships, solid-phase synthesis, synthetic ion channels
evident that 2 can emulate the functions of 1 despite structural
modifications at six amino acid residues of 1.
olytheonamide B (1, Figure 1) is a giant natural peptide
P(MW 5030) and a potent cytotoxic agent (IC
= 0.098
50
nM, mouse leukemia P388 cells).1,2 Linearly aligned residues of
1 with alternating D- and L-chiralities consist of 13 non-
proteinogenic and 6 proteinogenic amino acids, and 5,5-
dimethyl-2-oxohexanoate (Ncap) at the N-terminus. The
secondary structure of 1 is reported to be a 45 Å-long β6.3-
helix,3 which is believed to function as a transmembrane ion
channel. In fact, monomeric 1 was shown to selectively
transport monovalent cations (e.g., H+, Na+, K+) across the
membrane,4,5 while the structurally related gramicidin D (3)6,7
must form a head-to-head dimer to act as a similar channel. The
exceedingly high toxicity of 1 has been attributed to its ability to
efficiently form a highly stable ion channel.
In the total synthesis of the 48-residue sequence 2, assembly
of the D,L-alternating 37 amino acid sequence from residues 12
to 48 was realized through a single automatic solid-phase
synthesis. Synthesis of compound 2 was completed via dansyl
group introduction, one fragment-coupling reaction between
residues 11 and 12 and global deprotection. The optimized
solid-phase synthesis technology permitted us to initiate a
program directed toward efficient automated preparation of
various sequences of 2 in the search for biologically active
compounds with minimum molecular complexity. Herein we
report the chemical synthesis of 13 substructures of
polytheonamide mimic 2, which resulted in the discovery of a
compound of 37 amino acid residues with nanomolar level
toxicity (IC50 = 3.7 nM), along with five submicromolar level
cytotoxins, and distinct activities between natural product 1,
mimic 2, and the 37-mer fragment of 2.
The 13 N-terminal peptides 4−16 (H-[X-48]-OH, X = 10−
22) were targeted for comprehensive SAR studies (Table 1).
These sequences were designed to be elongated only by
automatic solid-phase synthesis, in that the compounds do not
possess sterically demanding β-methyl valines (residues 2, 4, 5,
6, 8, 9), which significantly decrease the condensation yields.
The synthesis of all the compounds 4−16 was started from
H-Thr(t-Bu)-2-chloro trityl resin 1714,15 using Fmoc-based
solid-phase peptide synthesis.16 To maximize the overall yield, a
powerful reagent system HATU/HOAt17 was applied for
condensation of the amino acids, and t-Bu, Tr, and Tmb were
We recently reported the total synthesis of polytheonamide
B (1), which involved 161 total steps from commercially
available materials.8−10 Subsequent biological evaluations of the
substructure11 and N-terminal derivatives of 1 provided
valuable information on the structural elements relating to
the function of 1.12 However, the length of the total synthesis
limited the supply of 1 to perform further systematic structure−
activity relationship (SAR) studies.
To overcome this issue, we designed an artificial ion channel
molecule, designated as dansylated polytheonamide mimic 2.13
The structure of 1 was simplified by removing or changing the
β-substituents of residues 2, 22, 29, 37, and 47, and the
dansylated amino acid was placed at residue 44 as a reporter of
the localization of 2. Compound 2 not only required
significantly fewer synthetic steps in comparison to original 1
(127 steps vs 161 steps) but also successfully exhibited potent
cytotoxicity toward P388 mouse leukemia cells (IC50 = 12 nM)
and displayed H+ and Na+ ion channel activities. Therefore, it is
Received: September 1, 2012
Accepted: November 1, 2012
Published: November 1, 2012
© 2012 American Chemical Society
52
dx.doi.org/10.1021/ml300264c | ACS Med. Chem. Lett. 2013, 4, 52−56

ACS Medicinal Chemistry Letters
Letter
Figure 1. Structures of polytheonamide B, dansylated polytheonamide mimic, and gramicidin D.
the 36 monomers, the peptide was cleaved from the 2-chloro
trityl resin under mild acidic conditions [(CF3)2CHOH/
CH2Cl2 = 1:3] and purified with HPLC, and then the dansyl
moiety 1818 was incorporated into the alkyne group of residue
44 via 1,3-dipolar addition,19−22 giving rise to 19. Finally,
simultaneous removal of the side chain protective groups (the
eight Tmb,23 four Tr, and three t-Bu groups) from 19 in TFA/
i-Pr3SiH24/H2O (95:2.5:2.5) successfully delivered 6 (0.07%
overall yield from 17 after HPLC purification). Synthesis of 6
required 98 total steps, comprising 73 steps of the solid-phase
synthesis and 25 steps of the solution-phase synthesis
(monomer syntheses, dansyl introduction, and global depro-
tection), demonstrating the highly automated nature of the
strategy. Most importantly, the developed procedure was
applicable for efficient preparations of longer 4−5 and shorter
7−16 in a unified fashion (0.1−0.3% overall yields, Table 1).
A collection of the analogues 4−16 allowed us to determine
the cytotoxicities of this series toward P388 mouse leukemia
cells (Table 1).25,26 Six out of 13 compounds showed
detectable toxicities, and submicromolar level activities (IC50
= 81−390 nM) were observed for five of them (7, 8, 10, 12,
and 14). Surprisingly, compound 6 (H-[12−48]-OH) was
found to be at least 20-fold more toxic (IC50 = 3.7 nM) than
the other 12 analogues, and its toxicity was even 3 times higher
than that of the longer polytheonamide mimic 2 (Ncap-[1−
48]-OH; IC50 = 12 nM) and comparable to that of gramicidin
D 3 (IC50 = 4.3 nM). It is worthy to note that a drastic increase
Table 1. Overall Yields and Cytotoxicity Data of the
Synthesized Peptides
a
a
compd
overall yield from 17 (%)
IC50 (nM)
1: polytheonamide B
2: Ncap-[1−48]-OH
3: gramicidin D
0.098
12
4.3
4: H-[10−48]-OH
5: H-[11−48]-OH
6: H-[12−48]-OH
7: H-[13−48]-OH
8: H-[14−48]-OH
9: H-[15−48]-OH
10: H-[16−48]-OH
11: H-[17−48]-OH
12: H-[18−48]-OH
13: H-[19−48]-OH
14: H-[20−48]-OH
15: H-[21−48]-OH
16: H-[22−48]-OH
0.30
0.20
0.07
0.10
0.09
0.17
0.14
0.14
0.14
0.18
0.32
0.28
0.10
>420
>420
3.7
81
100
>420
140
>450
190
>410
390
>250
>420
a
IC50 values were determined from the results of growth inhibition
assays (XTTmethod) performed on P388 mouse leukemia cells.
used as the protective groups for the hydroxy, primary amide,
and methyl amide functionalities of the side chains, respectively.
The synthetic route to 6 (H-[12−48]-OH) is illustrated as a
representative example in Scheme 1. After stepwise assembly of
53
dx.doi.org/10.1021/ml300264c | ACS Med. Chem. Lett. 2013, 4, 52−56

ACS Medicinal Chemistry Letters
Letter
Scheme 1. Representative Synthetic Scheme of the Substructures of 2
in potency was observed when only one amino acid was
detached from 5 (H-[11−48]-OH) or attached to 7 (H-[13−
48]-OH). These results strongly suggested the significance of
the specific structure of 37-mer 6 for the potent cytotoxicity.
A key question was whether this new cytotoxic agent 6
shared the same mechanism of action as the parent natural
product 1, mimic 2 and gramicidin D 3. Since 1−3 are known
to form ion channels in lipid bilayers, the ion transport activity
of 6 was compared to those of 1−3 using liposomes, which are
models of cell membranes (Figure 2). The liposomes were
and 3, which all induced an increase of the fluorescence over
time through the H+/Na+ exchange, addition of peptide 6
resulted in no change. The lack of effect of 6 on the liposome
membranes demonstrated that it had no ion transport activity
or no membrane disruption activity in this assay.29−31
To further investigate that 6 has a different activity from the
ion channel forming natural products 1 and 3, an ion exchange
assay was performed using the P388 cells, toward which 1, 3,
and 6 all exhibited strong toxicities. In this assay, membrane
permeable BCECF-AM 20 (Figure 3) is externally applied to
Figure 3. Structures of BCECF-AM (20) and BCECF (21).
the P388 cells, and esterase-mediated hydrolysis of the ester
moieties of 20 selectively occurs within the cell membranes to
lead to encapsulation of a hydrophilic pH indicator, BCECF
21.32 Next, the resultant cells are suspended in pH 7.8 MOPS
buffer containing 130 mM NaCl to apply a Na+ gradient, and
treated with the peptides (125 nM) to measure the ion
channel-promoted fluorescent change of 21.33 The addition of
the peptide channels 1 and 3 indeed caused influx of Na+
cations from the external buffer and efflux of protons from the
cells, leading to a pH change over time (Figure 4).34 Higher
efficiency of polytheonamide B 1 in the H+/Na+ exchange in
comparison to gramicidin D 3 correlated to more potent
cytotoxicity of 1 (Table 1) as well as higher activity of 1 in the
liposome experiments (Figure 2). In contrast, addition of
strongly cytotoxic 6 did not affect the intracellular pH of the
P388 cells. This result again indicated that the cytotoxicity of 6
was not induced by ion transport activity. The data in Figures 2
and 4 together corroborated that compound 6 exerted its
cytotoxicity through a different mechanism of action from that
Figure 2. Time-course of H+/Na+ exchange across lipid bilayers of pH-
gradient liposomes (EYPC/cholesterol = 2:1) caused by 1, 2, 3, and 6.
The ion transport was evaluated as the pH-dependent fluorescence
from pyranine standardized against the maximum exchange by Triton
X-100. In all the experiments, the peptides were added at 60 s.
prepared in HEPES buffer containing 200 mM NaCl with a pH
gradient, a pH of 6.5 inside the liposome, and a pH of 7.5
outside. A fluorescent pH indicator, pyranine, was incorporated
in the liposomes.27,28 Addition of the peptide channels (125
nM) then permitted exchange between protons and Na+ ions
across the lipid bilayer of the vesicles to preserve the ionic
milieu, leading to a fluorescence increase caused by
deprotonation of pyranine. In contrast to the cases of 1, 2,
54
dx.doi.org/10.1021/ml300264c | ACS Med. Chem. Lett. 2013, 4, 52−56

ACS Medicinal Chemistry Letters
Letter
phate; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
acid; HOAt, 1-hydroxy-7-azabenzotriazole; MOPS, 3-morpho-
linopropanesulfonic acid; pyranine, trisodium 8-hydroxypyrene-
1,3,6-trisulfonate; SAR, structure−activity relationship; t-Bu,
tert-butyl; TFA, trifluoroacetic acid; Tmb, 2,4,6-trimethoxy
benzyl; Tr, triphenylmethyl; XTT, 3′-[1-[(phenylamino)-
carbonyl]-3,4-tetrazolium]bis(4-methoxy-6-nitro)-
benzenesulfonic acid hydrate
REFERENCES
■
(1) Hamada, T.; Matsunaga, S.; Yano, G.; Fusetani, N.
Polytheonamides A and B, Highly Cytotoxic, Linear Polypeptides
with Unprecedented Structural Features, from the Marine Sponge
Theonella swinhoei. J. Am. Chem. Soc. 2005, 127, 110−118.
(2) Hamada, T.; Sugawara, T.; Matsunaga, S.; Fusetani, N.
Polytheonamides, Unprecedented Highly Cytotoxic Polypeptides,
from the Marine Sponge Theonella swinhoei: 1. Isolation and
Component Amino Acids. Tetrahedron Lett. 1994, 35, 719−720.
(3) Hamada, T.; Matsunaga, S.; Fujiwara, M.; Fujita, K.; Hirota, H.;
Figure 4. Time-course of H+/Na+ exchange across cell membranes of
the P388 leukemia cells caused by 1, 3, and 6. Intracellular pH values
were evaluated by the pH-dependent fluorescence of 21. In all the
experiments, peptides were added at 0 s.
Schmucki, R.; Guntert, P.; Fusetani, N. Solution Structure of
̈
Polytheonamide B, a Highly Cytotoxic Nonribosomal Polypeptide
from Marine Sponge. J. Am. Chem. Soc. 2010, 132, 12941−12945.
(4) Iwamoto, M.; Shimizu, H.; Muramatsu, I.; Oiki, S. A Cytotoxic
Peptide from a Marine Sponge Exhibits Ion Channel Activity through
Vectorial-Insertion into the Membrane. FEBS Lett. 2010, 584, 3995−
3999.
(5) Oiki S.; Muramatsu I.; Matsunaga S.; Fusetani N. A Channel-
Forming Peptide Toxin: Polytheonamide from Marine Sponge
(Theonella swinhoei). Folia Pharmacol. Jpn. 1997, 110 (Suppl. 1),
195−198.
(6) Hotchkiss, R. D.; Dubos, R. J. Fractionation of the Bactericidal
Agent from Cultures of a Soil Bacillus. J. Biol. Chem. 1940, 132, 791−
792.
(7) Gramicidin and related ion channel-forming peptides; Chadwick, D.
J., Cardew, G., Eds.; Wiley & Sons: Chichester, 1999.
(8) Inoue, M.; Shinohara, N.; Tanabe, S.; Takahashi, T.; Okura, K.;
Itoh, H.; Mizoguchi, Y.; Iida, M.; Lee, N.; Matsuoka, S. Total Synthesis
of the Large Non-Ribosomal Peptide Polytheonamide B. Nat. Chem.
2010, 2, 280−285.
(9) Inoue, M. Total Synthesis and Functional Analysis of Non-
Ribosomal Peptides. Chem. Rec. 2011, 11, 284−294.
(10) Inoue, M.; Matsuoka, S. Convergent Total Synthesis of the
Complex Non-Ribosomal Peptide Polytheonamide B. Isr. J. Chem.
2011, 51, 346−358.
2
of 1−3, although 6 shares /3 of its sequence with ion-channel
forming 2.35 Future detailed biological studies remain to be
performed to determine this potential new mode of action of 6.
In conclusion, we developed a unified and highly automated
synthetic procedure for construction of the 13 substructures 4−
16 of ion-channel forming dansylated polytheonamide mimic 2,
and we discovered that D,L-alternating 37-mer peptide 6 was
more toxic than 2 and the other 12 substructures toward P388
mouse leukemia cells. Two types of functional assays of 6
revealed that 6 did not possess ion-channel activity, unlike
polytheonamide B (1), the mimic 2, and gramicidin D (3),
suggesting that 6 exhibited its toxicity through a mode of action
distinct from that of 1−3. The discovery of 6 through structural
permutations of polytheonamide B 1 demonstrates the benefits
of total synthesis endeavors on complex molecule construction,
and offers a unique opportunity for further exploration in
chemical biology studies and drug discovery efforts. Detailed
studies to elucidate its mode of action are currently underway
in our laboratory.
ASSOCIATED CONTENT
* Supporting Information
Characterization data of all new compounds, synthetic
procedures, assay data, and spectroscopic data. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
(11) Matsuoka, S.; Shinohara, N.; Takahashi, T.; Iida, M.; Inoue, M.
Functional Analysis of Synthetic Substructures of Polytheonamide B:
A Transmembrane Channel-Forming Peptide. Angew. Chem., Int. Ed.
2011, 50, 4879−4883.
(12) Shinohara, N.; Itoh, H.; Matsuoka, S.; Inoue, M. Selective
Modification of the N-Terminal Structure of Polytheonamide B
Significantly Changes its Cytotoxicity and Activity as an Ion Channel.
ChemMedChem 2012, 7, 1770−1773.
(13) Itoh, H.; Matsuoka, S.; Kreir, M.; Inoue, M. Design, Synthesis
and Functional Analysis of Dansylated Polytheonamide Mimic: An
Artificial Peptide Ion Channel. J. Am. Chem. Soc. 2012, 134, 14011−
14018.
(14) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. A New
Reagent for the Cleavage of Fully Protected Peptides Synthesised on
2-Chlorotrityl Chloride Resin. J. Chem. Soc., Chem. Commun. 1994,
2559−2560.
(15) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. 2-Chlorotrityl
Chloride Resin. Studies on Anchoring of Fmoc-Amino Acids and
Peptide Cleavage. Int. J. Peptide Protein Res. 1991, 37, 513−520.
(16) Fmoc Solid Phase Peptide Synthesis; Chan, W. C., White, P. D.,
Eds.; Oxford University Press: New York, 2000.
AUTHOR INFORMATION
Corresponding Author
*E-mail: inoue@mol.f.u-tokyo.ac.jp.
Funding
This work was supported financially by Funding Program for
Next Generation World-Leading Researchers [Japan Society for
the Promotion of Science (JSPS)] to M.I. The fellowship from
JSPS to H.I. is gratefully acknowledged.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
AM, acetoxymethylester; BCECF, 2′,7′-bis(2-carboxyethyl)-
5(6)-carboxyfluorescein; EYPC, egg yolk phosphatidylcholine;
Fmoc, 9-fluorenylmethoxycarbonyl; HATU, O-(7-azabenzotria-
zole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophos-
(17) Carpino, L. A. 1-Hydroxy-7-azabenzotriazole. An Efficient
Peptide Coupling Additive. J. Am. Chem. Soc. 1993, 115, 4397−4398.
55
dx.doi.org/10.1021/ml300264c | ACS Med. Chem. Lett. 2013, 4, 52−56

ACS Medicinal Chemistry Letters
Letter
(18) Inverarity, I. A.; Hulme, A. N. Marked Small Molecule Libraries:
A Truncated Approach to Molecular Probe Design. Org. Biomol. Chem.
2007, 5, 636−643.
(19) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed
1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. J. Org.
Chem. 2002, 67, 3057−3064.
to the cytotoxicity assay (4 days). See the Supporting Information for
the experimental details.
(35) The CD spectrum of 6 in liposomes was found to be different
from that of mimic 2, suggesting the differences in their three-
dimensional structures (see Figure S6 in the Supporting Information).
(20) Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba,
G.; Genazzani, A. A. Click Chemistry Reactions in Medicinal
Chemistry: Applications of the 1,3-dipolar Cycloaddition Between
Azides and Alkynes. Med. Res. Rev. 2008, 28, 278−308.
(21) Kolb, H. C.; Sharpless, K. B. The Growing Impact of Click
Chemistry on Drug Discovery. Drug Discovery Today 2003, 8, 1128−
1137.
(22) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry:
Diverse Chemical Function from a Few Good Reactions. Angew.
Chem., Int. Ed. 2001, 40, 2004−2021.
(23) Weygand, F.; Steglich, W.; Bjarnason, J. Leicht abspaltbare
Schutzgruppen fur die Saureamidfunktion, 3. Derivate des Asparagins
̈
̈
und Glutamins mit 2.4-dimethoxy-benzyl-und 2.4.6-trimethoxy-benzyl-
geschutzten Amidgruppen. Chem. Ber. 1968, 101, 3642−3648.
̈
(24) Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C. A.
Trialkylsilanes as Scavengers for the Trifluoroacetic Acid Deblocking
of Protecting Groups in Peptide Synthesis. Tetrahedron Lett. 1989, 30,
2739−2742.
(25) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R.
Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth
and Drug Sensitivity in Culture Using Human and Other Tumor Cell
Lines. Cancer Res. 1988, 48, 4827−4833.
(26) Roehm, N. W.; Rodgers, G. H.; Hatfield, S. M.; Glasebrook, A.
L. An Improved Colorimetric Assay for Cell Proliferation and Viability
Utilizing the Tetrazolium Salt XTT. J. Immunol. Methods 1991, 142,
257−265.
(27) Otis, F.; Racine-Berthiaume, C.; Voyer, N. How Far Can a
Sodium Ion Travel within a Lipid Bilayer? J. Am. Chem. Soc. 2011, 133,
6481−6483.
(28) Clement, N. R.; Gould, J. M. Pyranine (8-Hydroxy-1,3,6-
pyrenetrisulfonate) as a Probe of Internal Aqueous Hydrogen Ion
Concentration in Phospholipid Vesicles. Biochemistry 1981, 20, 1534−
1538.
(29) Membrane disruption also causes the fluorescent change in this
liposome assay. A different set of experiments demonstrated that 1, 2,
and 3 caused no membrane disruption, confirming that the fluorescent
changes in Figure 2 were attributed to their potent ion transport
activities. See refs 11 and 13.
(30) The 37 amino acid sequence from residues 12−48 of
polytheonamide B 1 exhibited the potent H+/Na+ ion exchange
activity and the weak cytotoxic activity (IC50 = 860 nM). Thus, the
molecular behavior of 6 was also different from that of the
corresponding substructure of 1. See ref 11.
(31) The Stokes shift values of the dansyl groups of 2 (see ref 13)
and 6 in liposomes were determined to be close to that in methanol
and in buffer, respectively. The data indicated that the microenviron-
ments of their residues were slightly different. See the Supporting
Information for the details.
(32) Rink, T. J.; Tsien, R. Y.; Pozzan, T. Cytoplasmic pH and Free
Mg2+ in Lymphocytes. J. Cell Biol. 1982, 95, 189−196.
(33) The fluorescence intensity was converted to the intracellular pH
values according to the calibration curve obtained by using ion
transporter nigericin. See: Thomas, J. A.; Buchsbaum, R. N.; Zimniak,
A.; Racker, E. Intracellular pH Measurements in Ehrlich Ascites
Tumor Cells Utilizing Spectroscopic Probes Generated in Situ.
Biochemistry 1979, 18, 2210−2218.
(34) Although the concentrations of the peptides (125 nM) were at
least 10 times higher than their IC50 values, the P388 cells were viable
under these experimental conditions. This is because the cells were
exposed to the peptides for a much shorter time (1.5 h) in comparison
56
dx.doi.org/10.1021/ml300264c | ACS Med. Chem. Lett. 2013, 4, 52−56