Acyloxymethyl esterification of nodularin-R and microcystin-LA produces inactive protoxins that become reactivated and produce apoptosis inside intact cells

Article abstract of DOI:10.1021/jm900502e

We report the esterification of the carboxyl groups of the cyclic peptide toxins nodularin-R and microcystin-LA to produce stable diacetoxymethyl and dipropionyloxymethyl ester derivatives. The derivatives had no activity but were reactivated upon esteras

Full text of DOI:10.1021/jm900502e

5758 J. Med. Chem. 2009, 52, 5758–5762
DOI: 10.1021/jm900502e
Acyloxymethyl Esterification of Nodularin-R and Microcystin-LA Produces Inactive Protoxins
that Become Reactivated and Produce Apoptosis inside Intact Cells
Lars Herfindal,*,† Franciszek Kasprzykowski,‡ Frank Schwede,§,^ Leszek Lankiewicz,‡ Kari E. Fladmark,†,#
e
Joanna Lukomska,‡ Matti Wahlsten, Kaarina Sivonen, Zbigniew Grzonka,‡ Bernd Jastorff,§ and Stein Ove Doskeland†
e
e
†Translational Signaling Group, Department of Biomedicine, University of Bergen, Jonas Lies Vei 91, N-5009 Bergen, Norway, ‡Faculty of
Chemistry, University of Gdanꢀsk, Sobieskiego 18, 80-952 Gdanꢀsk, Poland, §Centre for Environmental Research and Sustainable Technology,
University of Bremen, Leobenerstrasse, 28359 Bremen, Germany, and Department of Applied Chemistry and Microbiology, P.O. Box 56, Viikki
Biocenter, University of Helsinki, 00014 Helsinki, Finland. ^ Present address: BIOLOG Life Science Institute, Flughafendamm 9a, P.O. Box
107125, D-28071 Bremen, Germany. # Present address: Department of Molecular Biology, Thormohlensgate, N-5008 Bergen, Norway.
e
Received April 21, 2009
We report the esterification of the carboxyl groups of the cyclic peptide toxins nodularin-R and
microcystin-LA to produce stable diacetoxymethyl and dipropionyloxymethyl ester derivatives. The
derivatives had no activity but were reactivated upon esterase treatment. When injected into cells, the
acyloxymethyl moieties were cleaved off and apoptosis induced. Linking the acyloxymethyl-ester
moiety of these potent toxins to carriers destined for endocytosis paves the way for selective apoptosis
induction in target (e.g., cancer) cells.
Introduction
interior of nanoparticles equipped with surface ligands indu-
cing receptor-mediated endocytosis.12 For this, the hydrophi-
lic cyclic peptide toxins must be rendered more lipophilic
without interfering with the biological activity.
Acetoxymethyl (AMa) esterification has been used success-
fully to enhance the cell penetration of polar compounds,
which become trapped intracellularly when the AM group is
removed by ubiquitously expressed nonspecific cellular es-
terases. Well known examples are FURA-AM, used to de-
We reasoned that nodularin and microcystin could be
attached to a carrier via an esterase-sensitive acyloxy-
methyl-ester linkage. We decided, therefore, to modify no-
dularin and microcystin through diacetoxymethyl (AM2) and
dipropyloxymethyl (PM2) esterification. To produce a more
hydrophobic, putatively active toxin, we introduced a hydro-
phobic phenylglyoxal (PGx) substituent onto nodularin. We
achieved the synthesis of reversibly inactivated esters of both
microcystin and nodularin, as well as the synthesis of active
phenylglyoxal-nodularin, all of which were able to induce
apoptotic cell death when injected into intact cells.
termine the intracellular level of free calcium ion,1 the Ca2þ
-
chelator BAPTA-AM,2 AM-esters of cyclic nucleotides, used
to modulate cAMP receptor actions,3,4 and AM esters of
inositol phosphates.5
The cyanobacterial toxins microcystins and nodularin are
cyclic hepta- and penta-peptides (Figure 1), respectively. They
inhibit major protein phosphatases6 and are extremely potent
inducers of cell death.7 When ingested, microcystins and
nodularin are hepatotoxic due to their specific uptake into
parenchymal hepatocytes,8 mediated through the liver-speci-
fic transport proteins OATP1B1 and 1B3.9 When microin-
jected, however, the toxins induce apoptotic cell death within
minutes in all cell types tested, including cancer cells.7 The
toxins are therefore excellent candidates to eradicate un-
wanted cells if they can be targeted to their interior. In fact,
the PP2A-inhibitor fostriecin has entered phase II clinical
studies as an anticancer drug.10 Successful intracellular drug
targeting of leukemia cells has been achieved using the toxin
calicheamine conjugated to anti-CD33 antibody specifically
recognizing acute myelogenic leukemia cells.11 In the case of
microcystin and nodularin, the conjugation should be rever-
sible and allow the intracellular release of active toxin. Ano-
ther avenue is to encapsulate toxins in the hydrophobic
Results and Discussion
Synthesis of Microcystin-LA-AM2, Nodularin-AM2, No-
dularin-PM2, and Phenylglyoxal-Nodularin. The carboxyl
groups of microcystin (MC) and nodularin-R (Nod) are
predicted to be essential for high affinity binding to the toxin
target protein phosphatases 1 (PP1) and 2A (PP2A).13,14
Their reversible modification by an esterase-sensitive substi-
tution would therefore produce a protoxin active only after
exposure to esterases. We found that the conditions used to
synthesize acetoxymethyl esters of cyclic nucleotides3,4
were inefficient for peptide modification and reworked the
conditions based on a number of experiments on the model
peptide Z-Arg-Leu-Val-Gly-OH, which contains the free
carboxylic and guanidine groups found within the nodularin
structure (Figure 1). Successful and rapid (reaction com-
pleted in 1.5-2 min, yield 55-70% already after 30 s of
incubation) esterification of model peptide as well as of MC-
LA and Nod was achieved when the reaction was carried out
in dimethylsulfoxide, and acetoxymethyl bromide was added
*To whom correspondence should be addressed. Phone: þ47 55 58 63
81. Fax: þ47 55 58 63 60. E-mail: Lars.herfindal@biomed.uib.no.
aAbbreviations: AM, acetoxymethyl; PM, propionyloxymethyl;
PGx, phenylglyoxal; PP2A, protein phosphatase 2A; Nod, nodularin-
R; MC, microcystin-LR or microcystin-LA.
r
pubs.acs.org/jmc
Published on Web 08/25/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5759
Figure 2. Nod-AM2 has a low binding affinity to PP2A. Nod-AM2,
Nod, or MC-LR were mixed with radiolabeled MC-YR and
incubated with PP2A. After separation of PP2A-bound from free
MC-YR, the amount of protein-bound radioactivity was measured
and the data plotted as percent displacement of MC-YR. The data
are average and SEM from 3 to 8 independent experiments.
Table 1. In Silico Estimates of Isoelectric Point, Lipophilicity, and
Net Charge Distribution at Physiological Conditions for Microcystin-
LR, -LA, Nodularin-R, and Their Analoguesa
net charge distribution at pH 7.4
LogD logD at
compd
pI at pI pH 7.4
-2
-1
0
1
microcystin-LR 5.7 -7.4 -8.5
microcystin-LA 1.3 0.3 -6.8
0
99
0
92
1
8
0
0
0
0
0
0
MC-LA-AM2
nodularin-R
PGx-Nodb
Nod-AM2
5.3
1.2
1.2
0
100
0
3.2 -3.1 -8.5
4.4 -1.4 -4.8
12.4
12.4
0
99
1
99
0
0
0.4 -2.0
1.6 -0.7
0
0
100
100
Nod-PM2
0
0
0
a MC-LA-AM2: di-acetoxymethyl-microcystin-LA; PGx-Nod: phe-
nylglyoxal-nodularin-R; Nod-AM2: di-acetoxymethyl-nodularin-R;
Nod-PM2: di-propionyloxymethyl-nodularin-R. b Based on structure
IIa of the R2 substituent of Nod in Figure 1.
of only one additional phenyl ring (not shown). For Nod, we
obtained several adducts of phenylglyoxal (R2, IIa-c in
Figure 1), all with adduct stoichiometry of 1:1 (PGx: arginine
guanidyl). LC-MS analysis of the reaction mixture showed
again that the MW of the precursor peptide had increased by
no more than 99 or 117 (data not shown), indicating a 1:1
ratio PGx: arginine guanidyl. For the biological studies, we
used PGx-Nod with the R2 substituent IIa (Figure 1A),
which was separated from the other reaction products by
HPLC and isolated at >95% purity. We conclude that the
arginine residue of Nod could be substituted to produce a
stable, lipophilic analogue.
The estimated isoelectric pH, lipophilicity, and charge
distributions of the original and modified toxins are listed
in Table 1. In agreement with the in silico predictions of
hydrophobicity, all modified toxins were better retained on
C18 columns than the corresponding nonmodified toxin (not
shown).
The AM2 and PM2 Esters of Nod and MC-LA Failed to
Inhibit Protein Phosphatases, But Were Reactivated by Es-
terases, Also in Intact Cells. The displacement of labeled MC-
YR from the major microcystin target PP2A is the most
sensitive assay to detect protein phosphatase inhibitors of the
microcystin, nodularin class.17 We found that Nod-AM2 had
about 4 orders of magnitude lower affinity for PP2A than
the original Nod molecule (Figure 2). This shows that Nod-
AM2 (a) must contain at most 0.01% of free Nod even after
incubation for more than one hour, and (b) had very low affinity
for PP2A. The low affinity is explained by consideration of the
Figure 1. Nonionized structures of nodularin-R (A), microcystin-
LA (B), and various carboxyl (R1) and arginine (R2) substituents
used in the present study. The structures were drawn in Marvin
version 5.0.1 for apple 2008, ChemAxon, www.chemaxon.com.
with diisopropylethylamine. AM2 (R1-II) esters of Nod and
MC-LA and PM2 (R1-III) esters of Nod (I) (Figure 1) were
obtained with purity >95% by RP-HPLC with acetonitrile/
water/TFA as mobile phase. As expected from other AM-
esters,15 the acyloxymethyl-esters of Nod and MC-LA
were stable in physiological buffers. Thus, less than 0.1%
of the nodularin of PM2-Nod was released during more
than 1 h incubation with PP2A (Figure 2; data not shown).
We conclude that the two carboxyl groups common to
MC-LA and Nod can be substituted with acyloxymethyl
to produce stable, potentially esterase cleavable toxin ana-
logues.
The arginine of Nod is not predicted to be essential for
high-affinity binding to PP2A.14 We introduced therefore
the charge neutralizing substituent phenylglyoxal in the
arginine guanidine of Nod to obtain an active compound
with improved hydrophobicity (Figure 1 and Table 1). In
introductory experiments with the model peptide Z-Arg-
Leu-Val-Gly-OH, we obtained consistently an increase of
peptide mass by 99 or 117, corresponding to the incorpora-
tion of one molecule of phenylglyoxal minus one or two
water molecules. This is in contrast to an early study on
peptide arginine modification, which reported a 2:1 ratio,16
and 2D NMR studies were undertaken showing the presence

5760 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Herfindal et al.
(Figure 3A). This fact and its improved lipohilicity relative
to Nod (Table 1) renders it potentially useful for incorpora-
tion into the lipophilic center of nanoparticles destined for
cell internalization.
The Nodularin-R and Microcystin-LA Analogues Had
Decreased, But Not Abolished, Hepatocyte Toxicity. The
intrinsically inactive and stable diacyloxymethyl esters of
Nod and MC-LA could be activated only when exposed to
esterases (Figure 3). Because primary hepatocytes can be
studied at high dilution in synthetic medium with negligible
esterase activity, their eventual death-inducing activity could
be explained only by cellular uptake with subsequent toxin
activation by intracellular esterases. Primary hepatocytes
have efficient transporters, Oatp1b2 in rodents and
OATP1B1/1B3 in humans,9 for cyclic peptides like MC
and Nod. We found that the esterified toxins had at least
1-2 orders of magnitude lower potency than the unmodified
toxins as inducers of apoptotic hepatocyte death. Fifty
percent death (LC50) was obtained with 0.09 μM Nod
(Figure 4A) or 0.27 μM MC-LR (Figure 4B), while Nod-
AM2 and -PM2 had LC50=14 μM, and MC-LA-AM2 had
LC50=8 μM (Figure 4). PGx-Nod was more potent than the
acyloxy-esters, with LC50=1.4 μM (Figure 4). All of the toxin
analogues induced hepatocyte death with similar morphol-
ogy, as did Nod and MC-LR (Figure 4). The morphology is
characteristic with marked polarized cell blebbing (Figure 4;
see also ref 18). We conclude that the esterified toxins were
considerably less well transported into hepatocytes than the
original toxins but otherwise acted as the unmodified toxins.
We considered the possibility that the effects on hepato-
cytes of the AM2 and PM2 esters (Figure 4A,B) could be due
to transporter-independent membrane penetration in view of
the enhanced lipophilicity of the modified toxins (Table 1). If
so, the compounds should induce apoptosis also in fibro-
blasts and other cells lacking hepatocyte-specific transpor-
ters. Fibroblasts were, however, far less sensitive than the
hepatocytes, only modest (less than 20%) apoptosis being
noted after 3 h incubation with up to 120 μM of Nod-AM2,
Nod-PM2, PGx-nodularin (Figure 4C), or MC-LA-AM2
(Figure 4D). In fact, the modified toxins were not more
potent than the unmodified toxins (Figure 4C,D). When
microinjected with 100 μM toxin, an intracellular concentra-
tion of about 2 μM toxin is achieved based on the estimated
50-fold dilution of the injectate.19 This is enough to induce
rapid fibroblasts death (Figure 3). One can therefore assume
that the intracellular concentration in fibroblasts exposed to
120 μM toxin in the medium must be far lower than 2 μM,
and we conclude that the modified toxins had a very poor
ability to enter cells lacking transport channels for cyclic
peptides.
Figure 3. Acyloxymethyl-esters of Nod and MC-LR, and PGx-
Nod induced apoptosis when microinjected. Swiss 3T3 fibroblasts
were injected with 100 μM of Nod, MC-LR, or their analogues in
vehicle with TRITC-dextran, and the percentage of the injected
(fluorescent) cells with apoptotic morphology determined as a
function of time after injection. (A) Apoptosis after injection with
Nod (O), PGx-Nod (b), Nod-AM2 (4), or Nod-PM2 (2). (B)
Apoptosis after injection of MC-LR (open circles) or MC-LA-
AM2 (filled circles). The data represent average of three experiments
and SEM.
Nod-PP2A complex in which the two carboxyl groups of either
Nod or MC form tight hydrogen bonds with hydrophilic
moieties in the PP2A inhibitor binding site.13,14 Thus, the
introduction of the acyloxymethyl moieties will not only disrupt
the hydrogen bonds but also introduce unfavorable bulk and
hydrophobicity in the tight, hydrophilic binding region.
Because the diacyloxymethyl-esters had severely de-
creased ability to interact with phosphatase and thus were
nontoxic, they had to be de-esterified to restore the toxin
activity. The ability of Nod-AM2 and -PM2 to be enzyma-
tically processed to Nod was confirmed by HPLC analysis
after in vitro incubation with pig liver esterase (not shown).
More importantly, Nod-AM2, Nod-PM2 (Figure 3A), and
MC-LA-AM2 (Figure 3B) all induced apoptosis when
injected into intact cells, albeit more slowly than Nod
(Figure 3A) and MC-LR themselves (Figure 3B). This is
expected if the compounds had to be converted by esterases
before becoming biologically active. We conclude that the
acyloxymethyl-esters of MC-LA and Nod could be activated
by esterases and therefore become functional when intro-
duced into intact cells. The PM2 analogue of Nod was at least
as efficient as the AM2 analogue in inducing apoptosis
(Figure 3A). This indicated that the extra bulk of the PM
moiety did not interfere with esterase cleavage, indicating
that esterase-sensitive attachment of Nod or MC to cell-
targeting macromolecules destined for endocytosis could be
achieved via longer acylesters than AM.
Concluding Remarks
In conclusion, we have demonstrated the inactivation of
the apoptogenic toxins Nod and MC-LA through esterification
of their essential carboxyl groups. The diacyloxymethyl-esters
were readily reactivated upon exposure to esterases in vitro.
When injected into cells, the ester linkage was cleaved and the
liberated active toxin produced apoptosis (Figure 3). We
believe therefore that cancer cells with specific receptors linked
to endocytosis can be targeted by ester-linking Nod or MC to
carriers, like cell-specific receptor ligands or antibodies destined
for endocytosis.11,20 We have also demonstrated that hydro-
phobic toxin derivatives, like phenylglyoxal-Nod (Table 1), can
The phenylglyoxal-substituted Nod is biologically active,
inducing apoptosis rapidly after microinjection into cells

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5761
Nod of >99.5% purity was obtained in >90% yield by
semipreparative HPLC (stationary phase: Knauer Vertex col-
umn 250 mm ꢀ 16 mm packed with Kromasil 100, C-8, 5 μm;
mobile phase: 18% CH3CN, 0.1 M NaClO4, 0.001 M phos-
phate, pH=6.8). Desalting was on a Sep-Pak C-18 cartridge
(Waters Corp. Milford, MA). After washing with water, Nod
was eluted with 70% aq CH3CN.
MC-LA was extracted from freeze-dried cells of the Micro-
cystis aeruginosa strain IZANCYA122 using 1 mM aq triethyl-
amine. The supernatant was applied on Sep-Pak C-18 cartridge
and the activity eluted with acetonitrile before further purifica-
tion by ion-exchange and reversed-phase chromatography. The
final fractions were subjected to MS-analysis on a Bruker-
Daltonic Esquire-LC-ESI-MS system.
Synthesis of AM- and PM-Esters of Nod and MC-LA. Nod or
MC-LA (1.2 and 1.0 μmol, respectively) was dissolved in 7 μL of
dry DMSO under argon protection. To the resulting solution
DIEA (2.5 μmol) and AM-Br (2.4 μmol) were added and the
mixture kept for 1.5-2 min at room temperature. The reaction
was then stopped rapidly by freezing in liquid nitrogen. All
volatile components were removed in vacuum. Purification of
the AM-esters was performed on a semipreparative HPLC
column (Knauer Kromasil RP-8, 5 μm, 250 mm ꢀ 10 mm) using
water and acetonitrile (both added 0.05% TFA) as mobile
phases. Purity of compounds was determined by analyses of
the DAD-spectra of rechromatographed fractions. Synthesis of
the PM-esters was performed similarly, but PM-Br was used for
esterification.
The conditions for modification of the guanidine group of
nodularin with phenylglyoxal were optimized using the model
compound Z-Arg-Leu-Val-Gly-OH. Even under optimal con-
ditions (aq THF pH 8.5-9), we achieved only a 1:1 stoichio-
metry of modification both for the model compound and for
Nod itself. Synthesis of Nod modified at the guanidine group
of arginine with phenylglyoxal (PGx-Nod; Figure 1A, R2,
IIa-c) was performed in aq THF pH 8.8. Purification to homo-
geneity and analyses of reaction products were as for the ester
analogues.
Cell Handling and Experimental Conditions. Hepatocytes
were isolated from male Wistar rats (100-150 g) by in vitro
collagenase perfusion23 as previously described.24 The hepato-
cytes were resuspended (8.0 ꢀ 105 cells/mL) in pregassed (5%
CO2/95% O2) buffer (120 mM NaCl, 5.3 mM KCl, 0.01 mM
KH2PO4, 1.2 mM MgSO4, 1.0 mM CaCl2, 10 mM Hepes (pH
7.4)) supplemented with lactate (5 mM) and pyruvate (5 mM).
The cells were incubated with toxins in 48-well tissue culture
plates in a 5% CO2 atmosphere at 37 ꢀC for 90 min, fixed in
buffered (pH 7.4) formaldehyde to a final concentration of 2%,
and percent apoptotic cells determined by differential inter-
ference contrast (DIC) microscopy.
Figure 4. Acyloxymethyl esters of Nod and MC-LA, and PGx-
Nod added to cell medium failed to induce fibroblast apoptosis but
did induce hepatocyte apoptosis. (A,B) Freshly isolated rat hepa-
tocytes were incubated in suspension culture for 90 min in the
presence of various concentrations of the toxins. The percentage
of apoptotic cells was determined by differential interference con-
trast microscopy. (A) Nod (O), PGx-Nod (b), Nod-AM2 (4), and
Nod-PM2 (2). (B) MC-LR (O), MC-LA-AM2 (b). The insets of (A)
show DIC-micrographs of cells treated with vehicle or with 80 nM
Nod, 4 μM PGx-Nod, 60 μM Nod-AM2, or 60 μM Nod-PM2. (B)
shows cells treated with 0.2 μM MC-LR or 35 μM MC-LA-AM2.
Bar is 15 μm. (C,D) Swiss 3T3 fibroblasts were incubated for 3 h in
serum-free RPMI-medium in the presence of various concentra-
tions of Nod, MC, or their protoxins. Apoptosis was assessed based
on fluorescent microscopy of cells whose chromatin was stained
(Hoechst 33342) and differential interference contrast microscopy.
(C) Nod (O), PGx-Nod (b), Nod-AM2 (4), and Nod-PM2 (2). (D)
MC-LR (O) and MC-LA-AM2 (b). The data represent the average
and SEM from three experiments.
Swiss 3T3 mouse fibroblasts were cultured in RPMI-medium
with 10% fetal calf serum at 37 ꢀC in a 5% CO2 atmosphere. For
testing of toxin analogues in the medium, about 7000 cells were
plated in 0.25 mL 48-well tissue culture plate wells and incubated
for 3 h after changing to fresh medium without added serum.
The incubation was stopped by adding 0.25 mL of 4% buffered
formaldehyde solution (pH 7.4) containing the fluorescent
DNA-stain Hoechst 33342. The percentage of apoptotic cells
was determined as described previously.7 For microinjection,
the fibroblasts were plated in 3.5 cm tissue culture dishes at a
density of 60000/dish. The microinjection was performed using
an Eppendorf 5170 micromanipulator mounted on a Zeiss
Axiovert 35 M inverted microscope. Microcapillaries (type
BF100-10, outer and inner diameter 1.0 and 0.78 mm, re-
spectively) and puller (model P-87) were from Sutter instru-
ments Co (Novato, CA). For each toxin, between 50 and 60 cells
were injected, and the percentage of apoptotic cells were deter-
mined by differential interference microscopy.7 Each experi-
ment was performed in triplicate, and the average and SEM was
calculated.
retain biological activity (Figure 3A). Such derivatives can
allow more toxin to be loaded in the lipid core of nanoparticles,
which emerge as a vehicle for the selective delivery of cytotoxic
substances to cancer cells.21
Materials and Methods
Materials. DMSO and di-isopropylethylamine (DIEA) of
highest purity available were stored over preactivated molecular
˚
sieves (3 A). AM-Br was from Sigma-Aldrich (St. Louis, MO)
and PM-Br a generous gift from Dr. C. Schultz (EMBL,
Heidelberg, Germany).
Purification of Nodularin-R and Microcystin-LA from Cyano-
bacteria. Nodularin-R was extracted from 130 g (dry weight) of
Nodularia spumigena strain AV1 cells, first with a 1:1:1 mixture
of methanol:butanol:water and next with 1 mM aq ammonia.
After ion exchange chromatography (Macro-Prep Q system,
Bio-Rad Laboratories, Hercules, CA) and preparative reversed
phase chromatography, 220 mg of 94% pure Nod was obtained.

5762 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Herfindal et al.
Determination of ability of MC, Nod and their analogues to
bind to the active site of protein phosphatase 2A was done with a
MC-YR-[125I] displacement assay as described.17,25 Protein
phosphatase 2A (PP2A) was obtained from rabbit muscle
following the procedure of ref 26.
In Silico Predications of Physiochemical Properties. Estima-
tions of logD, pI, and ionization conditions were done with
the Calculator Plug-in in Marvin version 5.0.1 (2008) for Apple
(ChemAxon Ltd., www.chemaxon.com). The molecules were
drawn in 2D mode and converted to 3D structures with the fine
build clean 3D mode in Marvin. Standard ionic condition (0.1 M
Cl- and 0.1 M Naþ/Kþ) was used for estimation of logD at pI
and physiological pH (7.4). pKa was estimated in macro mode
under the following conditions: min basic pK1, -10; max acidic
pKa, 20; temperature 310 K.
(11) Singh, R.; Erickson, H. K. Antibody-cytotoxic agent conjugates:
preparation and characterization. Methods Mol. Biol. 2009, 525,
1–23.
(12) Zheng, Y.; Cai, Z.; Song, X.; Chen, Q.; Bi, Y.; Li, Y.; Hou, S.
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Strack, S.; Stock, J. B.; Shi, Y. Structure of protein phosphatase
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(14) Kelker, M. S.; Page, R.; Peti, W. Crystal structures of protein
phosphatase-1 bound to nodularin-R and tautomycin: a novel
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(project MAST3-CT97-0156), the Norwegian Cancer Society,
and the Norwegian Research Council. Nina Lied Larsen
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