Fundamental Role of the Fostriecin Unsaturated Lactone and Implications for Selective Protein Phosphatase Inhibition

Full text of DOI:10.1021/ja038672n

Published on Web 11/26/2003
Fundamental Role of the Fostriecin Unsaturated Lactone and Implications for
Selective Protein Phosphatase Inhibition
Suzanne B. Buck,† Christophe Hardouin,† Satoshi Ichikawa,† Danielle R. Soenen,† C.-M. Gauss,†
Inkyu Hwang,† Mark R. Swingle,‡ Kathy M. Bonness,‡ Richard E. Honkanen,‡ and Dale L. Boger*,†
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and Department of Biochemistry and
Molecular Biology, UniVersity of South Alabama College of Medicine, Mobile, Alabama 36688
Received September 22, 2003; E-mail: boger@scripps.edu
Fostriecin (1), is a novel phosphate monoester produced by
Streptomyces pulVeraceus1 that displays antitumor activity (Figure
1).2 It entered NCI-sponsored clinical trials, but the studies were
discontinued before dose-limiting toxicities or therapeutic plasma
levels were reached when concerns regarding storage stability
surfaced.3 Although early studies demonstrated that 1 inhibits
topoisomerase II (IC50 ) 40 µM),4 its potency and cell cycle effects
were inconsistent with this target of action. Recently, 1 was shown
to inhibit the mitotic entry checkpoint through much more potent
inhibition of protein phosphatase 2A (PP2A).2 Moreover, 1 was
found to be the most selective PP inhibitor disclosed to date (PP2A/
PP4 vs PP1 selectivity g 104).5-7 Consequently, 1 or a more stable
derivative represents an ideal candidate with which to establish the
utility of this novel mechanism of action.
At the time that fostriecin’s therapeutic effects were emerging,
only its two-dimensional structure was known.1,8 Thus, we estab-
lished its stereochemistry9 and thereafter reported its first total
synthesis.10 In a subsequent review of fostriecin,2 we compared its
Figure 1. Fostriecin and key elements of its PP activity.
structure with the pharmacophore11 for nonselective PP1 inhibition
mediates prepared en route to 7 and 14 or generated in efforts to
chronicle the chemical behavior of 1 (Scheme 1).
The partial structure 15 lacking the lactone was found to be 200-
fold less active than 1 against PP2A and exhibited an analogous
and modeled its binding to PP2A enlisting a homology model12
derived from PP1 X-ray structures.13,14 Conserved features of this
pharmacophore present in 1 include (1) a phosphate that binds the
active site metal ions, (2) a methyl group proximal to this acidic
group proposed to mimic the substrate phosphothreonine methyl
group, and (3) an extended hydrophobic segment thought to mimic
the substrate hydrophobic residues (Figure 1). Fostriecin’s most
significant feature that does not correspond to the pharmacophore
is its unsaturated lactone. Our modeled fostriecin-PP2A complex
placed the â12-â13 loop C269 thiol within 2.5 Å of the unsaturated
lactone â-carbon (C3) at a trajectory poised for conjugate addition.
Thus, the unsaturated lactone emerged as a candidate source of
the PP2A/4 inhibition selectivity and potency. Herein, we report a
series of fostriecin derivatives designed to define the importance
of the unsaturated lactone and confirm that of the phosphate
monoester. Their evaluation established a fundamental role for the
unsaturated lactone, and the chemical behavior of 1 implicates its
participation in a reversible, conjugate addition reaction with an
active site nucleophile including C269.
130-fold loss in cytotoxic activity, but it was found to be only ca.
2-fold less active than 1 against PP1/PP5, Table 1. The saturated
lactone 7 exhibited an identical 200-fold decrease in PP2A
inhibition, indicating that this selective loss in activity may be
attributed exclusively to the removal of the conjugated double bond.
Greater 103-104 reductions in PP2A inhibition were observed with
12-14, where a lactone cleavage provided neutral derivatives (ester
or alcohol), whereas the free acids 9-11, like 6, were even less
active (104-105 reduction).
Consistent with the importance of the unsaturated lactone and
its potential alkylation of the PP2A C269 thiol, treatment of 1 with
EtSH (3 equiv) under mild conditions (KHCO3, THF-H2O, 0 °C,
5 h, 77%) provided 8 or 10 (K2CO3, THF-H2O, 0 °C, 4 h, 99%),
Scheme 1.15 Analogous conjugate addition reactions were observed
with MeOH as the nucleophile.
Thus, the unsaturated lactone of fostriecin increases the PP2A
inhibition roughly 200-fold, which potentially may be attributed
to C269 alkylation within the variable PP â12-â13 active site
loop. Supporting this expectation is the observation that PP2A
C269S and C269F mutants are much less sensitive to fostriecin
(>10-fold).16 This behavior of 1 would be analogous to the PP
active site alkylation by microcystin-LR revealed in a cocrystal
X-ray with PP1.13 However, the addition of the PP1 C273 thiol to
the N-methyldehydroalanine residue of microcrystin-LR (which
does not alter the PP inhibition potency) constitutes alkylation of
The derivatives 2-5, including the known dephosphofostriecin
(2)8,9 and cyclic phosphodiester 4,9 were examined to confirm the
importance of the phosphate, whereas 6-15 were prepared to ex-
plore the unsaturated lactone. Of the latter compounds, the saturated
lactone 7, 15 lacking the entire lactone subunit, and diol 14 were
viewed as the key analogues. The remainder (8-13) were inter-
† The Scripps Research Institute.
‡ University of South Alabama College of Medicine.
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J. AM. CHEM. SOC. 2003, 125, 15694-15695
10.1021/ja038672n CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1
two easily generated metabolites, dephosphofostriecin (2) and the
hydrolyzed lactone 6, experience a 105-fold loss in activity, sug-
gesting sites of modification that might enhance the in vivo efficacy.
Interestingly and despite its 200-fold loss in potency, 15 still
represents a remarkably potent and selective PP2A inhibitor. This
suggests that there are additional features of 1 beyond the unsatu-
rated lactone that contribute to its PP inhibition selectivity and that
15 may serve as a lead for further optimization. Studies addressing
such issues and those that exploit the newly recognized origin of
the PP2A potency detailed herein are in progress and will be
reported in due course.
Table 1. Protein Phosphatasea and Cytotoxic Activityb (IC50, µM)
Acknowledgment. We gratefully acknowledge the financial
support of NIH (CA93456, D.L.B.; CA60750 and HL59154,
R.E.H.), the Skaggs Institute for Chemical Biology, and a
NASA fellowship (NTGB-52896, M.R.S.). We are indebted to
Kimberly L. Moring for her assistance with the inhibition stu-
dies and NCI for samples of fostriecin.
Supporting Information Available: Experimental details for the
preparation and characterization of 3 and 7-15 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
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compd
PP2A
PP1
PP5
L1210 L1210/CI-920
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1
2
3
4
5
6
7
8
0.001 ((0.0007) 50 ((10)c 70 ((33)
0.3
20
15
15
>100
>25
>50
3
>50
>25
>25
>25
>25
35
35
>50
35
350 ((100)
2.9 ((1.5)
3.2 ((1.1)
>100
>100
>100c
>100
>100
>100
>100c
>100c
>100
>100c
>100
>100
>100
g100c,d
g100d
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>25
>50
>25
>50
>25
>25
>25
>25
>100
60
73 ((9)
0.21 ((0.05)
0.5 ((0.4)
>50
9
10
11
12
13
14
15
8.8 ((2.5)
>100
1.7 ((0.2)
2.0 ((2.8)
2.1 ((0.6)
0.19 ((0.02)
140 ((50)d >100
g100d
40
a Assays were conducted with native PP2A (rabbit muscle),5b rhPP1R,19
and rhPP5 catalytic subunits19 as detailed.20 b L1210/CI-920 is a L1210
cell line resistant to 1 by virtue of an impaired folate transporter required
to import 1.2b c Also assayed with native PP1 (rabbit muscle) with identical
results.5b d Enzyme inhibition at 100 µM ) 40-50%.
a nearby conserved Cys residue found in all PPs (except PP2B/7),
whereas that of 1 occurs at C269 unique to PP2A/PP4/PP6
contributing to both its potency and selectivity (Figure 1).
As anticipated, but not yet disclosed, the phosphate proved to
be critical to phosphatase inhibition. Its removal with 2 resulted in
a 105-fold loss in PP2A inhibition, whereas its conversion to the
phosphodiesters 3 and 4 resulted in a 103-fold loss in activity.
Acetylation of 4 providing 517 resulted in a further g50-fold loss
in PP2A inhibition, suggesting that the C11 alcohol18 contributes
significantly to fostriecin’s potency. The studies also reveal that
JA038672N
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