The selective inhibition of phosphatases by natural toxins: The anhydride domain of tautomycin is not a primary factor in controlling PP1/PP2A selectivity

Article abstract of DOI:10.1016/S0960-894X(03)00105-7

Analogues of the potent and moderately selective PP1/PP2A inhibitor tautomycin (TM) were prepared with modifications in the C1′-C7′ anhydride moiety. While all retain varying degrees of activity within a 3000-fold range of potencies, they also show remarkable constancy in their IC₅₀ ratios, suggesting that the anhydride moiety is not critical in controlling the selectivity of inhibition.

Full text of DOI:10.1016/S0960-894X(03)00105-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1597–1600
The Selective Inhibition of Phosphatases by Natural Toxins:
The Anhydride Domain of Tautomycin Is Not a Primary
Factor in Controlling PP1/PP2A Selectivity
Wen Liu,a James E. Sheppeck, II,a David A. Colby,a Hsien-Bin Huang,b
Angus C. Nairnc and A. Richard Chamberlina,*
aDepartment of Chemistry, University of California at Irvine, Irvine, CA 92697, USA
bInstitute of Biochemistry, Chu-Tzi College of Medicine, Hualien 970, Taiwan
cThe Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
Received 30 September 2002; accepted 20 November 2002
Abstract—Analogues of the potent and moderately selective PP1/PP2A inhibitor tautomycin (TM) were prepared with modifi-
cations in the C10–C70 anhydride moiety. While all retain varying degrees of activity within a 3000-fold range of potencies, they also
show remarkable constancy in their IC50 ratios, suggesting that the anhydride moiety is not critical in controlling the selectivity of
inhibition.
# 2003 Elsevier Science Ltd. All rights reserved.
The dephosphorylation of phosphoserine and phospho-
threonine residues of protein substrates is a vital part of
many cell-signalingcascades, playinga crucial role in
cell proliferation and many other processes.1 Protein
phosphatases 1 and 2A (PP1 and PP2A) represent two
of the four major classes of serine-threonine phospha-
tases, which also include PP2B (calcineurin), PP2C, and
others.2 The more abundant PP1 and PP2A can be
pharmacologically differentiated from all other phos-
phatases with a number of naturally-occurringtoxins,
includingokadaic acid (OA), the microcystins, tauto-
mycin (TM), cantharidin, and calyculin, that much
more strongly inhibit PP1 and PP2A than any of the
others.3
10-fold selective for PP1 over PP2A7) beingthe only
available small molecule PP1 inhibitor.3,8
Because of the lack of highly PP1-selective inhibitors,
the underlyingstructural origins of inhibition selectivity
by TM and related toxins are of interest, as a first step
in designing new selective inhibitors. A number of
studies have clearly illustrated that PP1/PP2A selectivity
can be affected dramatically by structural modifications
of the inhibitors.9 For example, Forsyth recently repor-
ted that modification of OA resulted in a 17-fold
increase in PP2A selectivity.9a In addition, our group
has shown that the non-selective microcystin-LA
becomes moderately selective for PP1 (7:1) when the
leucine residue is replaced by a sterically more demand-
9b
The inhibition of PP1 and PP2A by these toxins is
known to occur competitively at a single site on each
enzyme,4 but because of the considerable sequence
homology between PP1 and PP2A,5 most of the toxins
do not inhibit this pair with good selectivity. The main
exception to this generalization is the ꢀ100-fold selec-
tive inhibition of PP2A by okadaic acid.3 On the other
hand, highly selective inhibitors of PP1 have proven to
be rather elusive, with tautomycin6 (which is only 5- to
ingcyclohexylalanine.
Significant differences in selec-
tivity within a series of cantharidin analogues have also
been reported recently.9c Unfortunately, the data from
these individual studies do not yet suggest a unified
predictive paradigm for discriminating between the two
enzymes, and further studies are clearly warranted.
The interestingstructural complexity and bioloigcal
activity of TM have spurred a number of syntheses10
and structure–activity investigations.11 The latter stud-
ies focused on modifications of the C1–C26 ‘main
chain;’ therefore there is little correspondingSAR data
*Correspondingauthor. Tel.: +1-949-824-7089; fax: +1-949-824-
8571; e-mail: archambe@uci.edu
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00105-7

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W. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1597–1600
for the C10–C70 anhydride domain. Indeed, this anhy-
dride-containingsegment of TM is a critical structural
element for bindingto PP1 and PP2A; however, its role
in controllingthe selectivity of inhibition has not been
evaluated. This is a particularly interestingissue because
all active PP1/PP2A inhibitors, natural or synthetic,
contain a homologous moiety that can be viewed as
mimickingthe phosphate group of the phosphoprotein
substrates of these enzymes.12 We have therefore asses-
sed whether changing the identity of the C10–C70
domain of tautomycin affects the selectivity of inhibi-
tion of PP1 and PP2A.
In order to address this issue, we envisioned a series of
tautomycin derivatives in which the C10–C70 region of
TM (1) is substituted with different phosphate mimics
(Fig. 1), all of which can be accessed by minor variation
of our published route to the parent toxin.10d The ana-
logue 2, 30-epi-tautomycin, has been reported pre-
viously, but biological activity has not been
published.11c Compounds 3 and 4 have been designed to
incorporate the phosphate mimic of okadaic acid, an
a-hydroxy carboxylic acid that in direct contrast to TM
is highly selective for PP2A. The analogue 5 was derived
to examine the effect of removingthe C7 0 carboxylate of
the parent compound while 6 was designed to substitute
a simple phosphonic acid for the C30–C70 region of TM.
Figure 2. Conformations of the C10–C70 subunits of tautomycin and
analogues. The C24 main chain attachment is represented as CPK.
The subunit 2 can assume a conformation similar to 1
despite beingepimeric, but there are energetic penalties to
be paid. In one rotamer of 2 (A), the C30-OH maintains its
position as in TM, but 1,3-allylic strain13 develops
between the cis-methyl and the allylic methylene groups.
Alternatively, 2 may avoid this A-strain by bindingas in
B, but the position of the hydroxyl group, which may be
important for tight binding, differs from that in TM (1).
Some reassurance that each of these modified phosphate
mimics might be reasonable structural surrogates for the
anhydride domain of TM was obtained by conforma-
tional analysis and comparison to the parent compound
by minimization and dynamics usingthe Discover_3
module of Insight II. The anhydride moiety of 1 and 2
was assumed to exist as the diacid, as proposed by Iso-
be11c and the modelingwas carried out as described
previously.12 The calculated structures are shown as the
excised C10–C70 subunits for direct comparison (Fig. 2).
The lowest energy conformer of 3 has a similar place-
ment of the correspondinghydroxy group to 1 while 4
must rotate into a higher energy conformation with the
carboxylate and OH interchanging positions. The ana-
logue 5 can clearly assume a conformation identical to 1
(but lackingone carboxylate rgoup) and the lowest
energy conformer of 6 aligns well with the correspond-
ing groups in the other analogues, but obviously lacks
several of the structural elements shared by the other
analogues (OH and one acid group). Based on these
considerations, one might reasonably expect reduced
potencies for these analogues towards PP1 and PP2A
compared to TM itself. However, it is the question of
differential effects in the phosphate mimic domain, that
is potential changes in PP1/PP2A selectivity, that this
study sought to clarify.
The syntheses of analogues 2–614 were closely based on
our published synthesis of tautomycin.10d Preparation
of the epimer 2 was carried out as described, with the
acid 7 (Fig. 3) in place of the natural enantiomer of this
fragment. The syntheses of 3 and 4 began with an
Evans’ Aldol reaction of the ketone 8 (Fig. 4) to g ive a
mixture of alcohols 9 and 10, which were easily sepa-
rated and individually carried to the respective targets.
Each alcohol was protected as a silyl ether under stan-
dard conditions, and the oxazolidinone was removed
with base to give the corresponding acids 11 and 12.
After conversion to the acid chloride, addition to the
previously prepared Weinreb intermediate 1310d gave 14
Figure 1. Structures of tautomycin and derivatives.

W. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1597–1600
1599
Figure 3. Synthesis of 30-epi-tautomycin 2.
Figure 5. Synthesis of analogue 5.
Figure 6. Synthesis of analogue 6.
and 2A were determined in the standard phosphorylase-
a inhibition assay17 (Table 1). Synthetic tautomycin (1)
gave IC50 values of 0.19 nM for PP1 and 0.94 nM for
PP2A, similar to published values for natural tautomy-
cin7 and correspondingto a selectivity favoringPP1 by
4.9 to 1. The totally inactive bis-TMSE ester of tauto-
mycin (previously reported10d) served as a negative
control.
Figure 4. Synthesis of analogues 3 and 4.
and 15 in good yields. The Weinreb amides were then
reduced with DIBAL-H to give the aldehydes 16 and 17.
The syntheses of 3 and 4 were completed usingthe
reported Mukaiyama Aldol strategy.10a,d
The mono-acid 5 was synthesized by a similar strategy
startingfrom ( R)-malic acid (Fig. 5). Benzylation and
selective reduction gave the diol 18, which was con-
verted into the bis-(triethylsilyl) ether and reductively
deprotected with palladium on carbon to give the
requisite acid 19. This acid was coupled to the main
chain C17-OH precursor, as described, to give the
Weinreb intermediate 20. Swern oxidation15 of the pri-
All of the analogues show the expected decrease in
potency, with the most potent of the group, 30-epi-TM
(2), beingapproximately 1000-fold less active than the
parent compound. Still, at approximately IC50=100
nM, the analogue 2 not only retains a significant level of
inhibitory potency, but it also maintains a level of
selectivity for PP1 over PP2A (3.8:1) that is comparable
to tautomycin itself. More significantly, both of the
okadaic acid-like analogues (3 and 4) also selectively
inhibit PP1, although not quite at the same level as
mary triethylsilyl ether in this intermediate followed by
16
Wittigolefination
with functionalized ylide 21 pro-
vided the Weinreb intermediate 22, which converted
into the target analogue 5 via the reported protocol.
Table 1. Phosphatase inhibition by tautomycin and analoguesa
Compd
IC50 (mM)
Selectivity
(PP1 : PP2A)
Finally, the phosphonate analogue 6 was synthesized
from readily available carboxy phosphonic acid 23 (Fig.
6). Conversion into the tris-acid chloride, followed by
perbenzylation and selective saponification of the car-
boxylate ester gave the acid 24. Once again following
the general strategy employed to prepare the analogues
2–5, the acid 24 was converted into 6.
PP1
PP2A
1
2
3
4
1.9ꢁ10ꢂ4
0.13
80
9.4ꢁ10ꢂ4
0.50
100
4.9:1
3.8:1
1.3:1
1.5:1
1.9:1
2.0:1
n/a
19
2.1
150
>500
29
4.0
300
>500
5
6
bis-TMSE ester
With the tautomycin derivatives in hand, the respective
IC50 values for the inhibition of protein phosphatase 1
aValues from assay are based on phosphorylase-a as substrate.

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W. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1597–1600
tautomycin itself and with further reduced potencies.
Interestingly, it is the analogue 4 with the unnatural
okadaic acid stereochemistry that is the (somewhat)
more potent inhibitor of the two. Nonetheless, because
okadaic acid itself is highly selective for PP2A it is par-
ticularly noteworthy that both of the okadaic acid-tauto-
mycin hybrids 3 and 4 selectively inhibit PP1. Similarly,
the analogue 5, which was the second most active inhi-
bitor in this group of five analogues, retained the PP1
selectivity of the parent tautomycin. Finally, the analogue
6, although the least potent inhibitor of the group,
shows a selectivity profile essentially identical to the
other analogues.
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Taken as a whole, these results indicate that the selec-
tivity of PP1/PP2A inhibition is not a function of the
0
structure of the anhydride-containingC1 –C70 domain
that constitutes the ‘phosphate mimic’ of tautomycin.
Indeed, all of these analogues exhibit nearly identical
selectivity patterns over a wide range of potencies. The
resultant generalized hypothesis is that modification of
the phosphate mimic domains of other toxins such as
okadaic acid is not likely to significantly alter PP1/
PP2A selectivity, and that the structural elements that
control selectivity reside elsewhere in the toxins. Further
experiments based on this generalization are in progress.
Acknowledgements
We are grateful to the National Institutes of Health
(NS57550) for providingfinancial support.
12. Gauss, C.-M.; Sheppeck, J. E.; Nairn, A. C.; Chamberlin,
A. R. Bioorg. Med. Chem. 1997, 5, 1751.
13. Broeker, J. L.; Hoffman, R. W.; Houk, K. N. J. Am.
Chem. Soc. 1991, 113, 5006.
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