Re-engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity

Article abstract of DOI:10.1021/jm4000209

A library of duocarmycin bioprecursors based on the CPI and CBI scaffolds was synthesized and used to probe selective activation by cells expressing CYP1A1 and 2W1, CYPs known to be expressed in high frequency in some tumors. Several CPI-based compounds w

Full text of DOI:10.1021/jm4000209

Brief Article
pubs.acs.org/jmc
Re-engineering of the Duocarmycin Structural Architecture Enables
Bioprecursor Development Targeting CYP1A1 and CYP2W1 for
Biological Activity
Helen M. Sheldrake,†,# Sandra Travica,‡,# Inger Johansson,‡ Paul M. Loadman,† Mark Sutherland,†
Lina Elsalem,† Nicola Illingworth,†,∥ Alexander J. Cresswell,† Tristan Reuillon,† Steven D. Shnyder,†
Souren Mkrtchian,‡ Mark Searcey,§ Magnus Ingelman-Sundberg,‡ Laurence H. Patterson,†
and Klaus Pors*,†
†Institute of Cancer Therapeutics, University of Bradford, Bradford BD7 1DP, U.K.
‡Department of Physiology and Pharmacology, Karolinska Institute, SE-17177 Stockholm, Sweden
§School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.
S
* Supporting Information
ABSTRACT: A library of duocarmycin bioprecursors based on the CPI and CBI scaffolds was synthesized and used to probe
selective activation by cells expressing CYP1A1 and 2W1, CYPs known to be expressed in high frequency in some tumors.
Several CPI-based compounds were pM−nM potent in CYP1A1 expressing cells. CYP2W1 was also shown to sensitize
proliferating cells to several compounds, demonstrating its potential as a target for tumor selective activation of duocarmycin
bioprecursors.
The spirocyclization event can be halted by blocking the OH
group of the seco-duocarmycins (i.e., 2), a strategy that has been
employed in tumor-selective prodrug approaches utilizing a
bioreduction or deglycosylation to restore activity.13 Rather
than masking this phenolic moiety, our aim is to inactivate the
duocarmycins by complete removal of the OH group (i.e., 1)
and to harness the unique capability of selected CYPs to effect
regioselective aryl oxidation in order to generate a potent and
nonreversible active (hydroxyl) metabolite.14 Recently, we have
demonstrated for the first time that a pharmacophore based on
the duocarmycin scaffold is dependent on metabolic hydrox-
ylation by CYP1A1 for biological activity and have shown that
the activation pathway is reminiscent of the natural products
(Figure 1).15,16 Here, we report on a library of duocarmycin
bioprecursors and their propensity for bioactivation by
CYP1A1, 1B1, and 2W1.
INTRODUCTION
■
The cytochromes P450 (CYPs) are responsible for the
oxidation of a diverse range of xenobiotic and endogenous
compounds. Although CYP 1−3 family members operate
mainly to detoxify xenobiotics, isoforms of the CYP1 family are
also known to catalyze one of the first steps in the metabolism
of carcinogens originating from chemical pollutants,1 including
polycyclic aromatic hydrocarbons (PAHs), nitroaromatics, and
arylamines.2 Exposure to such xenobiotics could have a long-
term effect on human health as well as being associated with the
risk of developing cancer. For example, CYP1A1 metabolism of
PAHs often generates more reactive intermediates that are
capable of binding with DNA and causing genetic mutations.1
In addition, CYP1A1 has been associated with estradiol
metabolism, an event correlated with ovarian cancer patho-
genesis.3 The involvement of CYP1A1 in active metabolite
generation and its frequent association with some cancers
indicates this enzyme could be a target for locoregionally
activated cancer therapeutics.4−6 CYP2W1 may also be a
valuable target because we have shown it to be highly expressed
in ∼30% of colon cancers, while the expression in adult
nontransformed tissues remains absent.7 Furthermore, clinical
studies show that the extent of CYP2W1 expression represents
a prognostic marker for malignancy and survival in colon cancer
patients.8,9
RESULTS AND DISCUSSION
■
Chemistry. The bioprecursors based on the CPI scaffold
were synthesized from 5-nitroindole 5 using a version of a
synthetic sequence recently reported by us (Scheme 1).16
Briefly, the indole was protected with Boc and the nitro group
reduced to afford protected amino-indole 7, which was
selectively brominated at C-4 by treatment with NBS. The
resulting bromoindole 8 was transformed to the bis-Boc
protected indole 11 by a high yielding sequence of protecting
group manipulations. Alkylation with 1,3-dichloropropene,
followed by radical cyclization, gave the protected dehydroxy
chloromethylindoline subunit 13. Deprotection with HCl in
We have focused our efforts on the natural product family of
seco-duocarmycins that while showing great promise as
ultrapotent cytotoxins have failed to advance clinically due to
lack of therapeutic index.10−12 Their mechanism of action
involves spirocyclization of a deep-embedded chloromethylin-
doline fragment to trigger production of an N3-adenine
covalent adduct upon binding to the minor groove of DNA.
Received: January 4, 2013
Published: July 11, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
analogue 15. This indicates that the replacement of an indole
with a naphthyl moiety, as in the CBI scaffold, removes a
nitrogen-center possibly critical for coordinating the CYP1A1
catalytic heme toward hydroxylation at the alkylating subunit.
The data obtained from the CHO-1A1 cells revealed an
interesting structure−activity relationship (SAR), with 5-Cl or
−OCH3 monosubstituted analogues possessing sub-nM (15,
21) antiproliferative activity, whereas the 5-OH monosub-
stituted analogue (22) in comparison was significantly less
potent (IC50 = 300 nM). This is unlikely to be due to
differences in cell uptake and the associated availability for
oxidation by CYP1A1 because we previously showed that cell
accumulation of 15 and seco-CPI-MI, which differ only by a
hydroxy group, was similar.16 However, it may be a result of the
diminished lipophilicity of 22 in comparison to 15 or 21 with
reduced DNA affinity as a result. Disubstitution of OCH3 at
positions 5 and 6 (16) did not lead to any loss in activity when
compared with the most potent compound (15) although the
disubstituted fluorine analogue (18) was significantly less
potent (IC50 = 0.23 μM). The natural duocarmycin DNA
recognition motif (DNA-RM) comprising 5,6,7-trimethoxy
substitution and a trimethoxy derivative (17) was also
investigated here, but no significant bioactivation in the
presence of CYP1A1 was evident. Furthermore, compounds
with substitution at positions 5 and 7 (19, 23) were also
inactive in the CYP1A1-transfected cells, suggesting that
functionalization at position 7 may pose steric hindrance and
poor interaction with CYP1A1. The compounds were also
assessed in RT112 bladder cancer cells, which naturally express
moderate levels of CYP1A1.15 Although the compounds were
less potent than in the CHO-1A1 cell line, the SAR pattern was
similar, with analogues 15, 16, and 21 being the most potent.
Next, we investigated CYP2W1 because there is justification
that this enzyme would share a similar substrate affinity profile
with CYP1A1 based on their active site sequence homology.20
CYP2W1 does have a propensity for xenobiotic metabolism20
including indoles21 and may contribute to the pharmacological
activity of the 2-arylbenzothiazole-based Phortress and GW-610
compounds.22,23 Using 2W1-transfected HEK293 cell line in
the chemosensitivity screen, the antiproliferative activity of
three compounds (15, 20, 21) was shown to be potentiated in
the presence of CYP2W1 (Table 1). 16, one of the most potent
CYP1A1-activated bioprecursors, was not a good substrate for
Figure 1. Proposed pathway for CYP activation of duocarmycin
bioprecursors (A = any heterocycle or aromatic ring).
EtOAc followed by EDCI-mediated amide bond formation with
2-carboxylic acid indoles, either commercially obtained or
prepared using previous published procedures,17 afforded seco-
duocarmycin bioprecursor analogues 15−23 in moderate yields
(Scheme 1). The chloromethylbenzoindoline (CBI) analogue
29 was synthesized from 1-bromo-naphthalen-2-ylamine 24
using previously described conditions (Scheme 1).18,19
Chemosensitivity. The duocarmycin bioprecursors were
evaluated for their ability to inhibit the growth of a panel of
CHO and HEK293 cell lines that were naturally devoid of the
CYPs of interest and isogenic variants that were engineered to
be proficient in the target enzymes CYP1A1 or 2W1 as
previously described.7,16 In agreement with the literature, the
authentic seco-CPI-MI and seco-CBI-MI compounds were
shown to possess IC50 values in the pM range, consistent
with the functional requirement of the hydroxyl group for
spirocyclization of the CBI and CPI pharmacophores. In
contrast, all CPI-based bioprecursors displayed low antiprolifer-
ative activity in the 1−25 μM range against CHO cells not
expressing the target CYPs (Table 1). This is consistent with
our previous report16 that showed 15 to possess only a weak
association with the DNA minor groove and poor antiprolifer-
ative activity in ovarian cancer cells. In the CYP1A1-expressing
CHO cells, it was evident that the CBI-based 29 was not
activated to the same extent compared with its direct CPI-based
Scheme 1. Synthesis of Duocarmycin Bioprecursors Based on the CPI and CBI-Based Pharmacophores (See Table 1 for Specific
Structures)
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Table 1. Growth Inhibition of Chloromethylindolines against a Panel of Parental CHO and HEK293 Cell Lines and Their
CYP1A1 and 2W1-Transfected Variants
substitution
R1
CHO
1A1
RT112
1A1
HEK293
2W1
b
compd ID
15
unit
X
R2
R3
wt
PF
wt
PF
a
CPI
CPI
CPI
CPI
CPI
CPI
CPI
CPI
CPI
CPI
CBI
CBI
H
OCH3
OCH3
OCH3
F
H
H
H
3.2 0.3
<0.001
<0.001
>3200
>3000
2.4
0.2 0.0
0.3 0.1
7.0 3.9
1.8 0.1
12.3 0.8
11.1 0.9
0.7 0.5
32.7 12.4
7.7 1.0
125 11.4
8.8 2.6
7.7 1.9
11.8 3.6
9.5 3.2
10.1 2.8
10.5 3.4
12.5 3.6
16.6 4.1
11.5 3.0
453 0.3
0.6 0.6
6.1 2.5
14.7
1.3
1.1
2.6
1.1
21
16
H
OCH3
3.0 0.4
7.5 1.6
18.4 4.8
21 5.2
17
H
OCH3
F
OCH3
3.1 2.9
0.23 0.1
>1
11.1 3.1
3.7 2.8
18
H
H
80
19
H
F
H
F
<21
251
9.1 3.2
20
H
F
H
H
22.6 5.8
23.2 3.3
3.3 0.7
3.2 0.5
167 18.9
0.09 0.0
<0.001
0.5 0.02
0.3 0.02
20.5 3.6
12.2 3.1
376 287.7
21
H
Cl
H
H
>23200
11
41.7
0.8
0.9
1.2
22
H
OH
F
H
H
0.30 0.3
4.1 1.2
624 178.6
0.96 0.7
102 1.9
23
H
H
SO2CH3
0.8
seco-CPI-MI
29
OH
H
OCH3
OCH3
OCH3
H
H
H
H
0.2
c
c
c
c
H
>50
>50
>50
>50
seco-CBI-MI
OH
H
194 30.0
1.9
151 13.9
137 71.4
641 123.3
0.2
a
IC50 values for bioprecursors (μM), CBI (pM), and CPI (pM) are the mean SD of at least three independent assays; dose−response curves for
b
15, 16, 17, CBI, and CPI are shown in SI Figures S1 and S2. Potentiation factor (IC50 in mock-transfected cells/CYP1A1 or 2W1-transfected cells).
c
The compound precipitated above 50 μM.
CYP2W1-activation, while 22, which possessed moderate
CYP1A1-mediated activity, was not active in CYP2W1-
expressing cells. Given that the 5-monosubstituted bioprecur-
sors 15, 20, and 21 were the only analogues potentiated in
CYP2W1-transfected cells, the results suggests that function-
alization at position 6 and 7 is likely to cause steric hindrance
and poor interaction with CYP2W1.
Nonetheless, the results obtained here suggest that selectivity
for targeting CYP2W1 over CYP1A1 can be achieved.
DNA Damage and Cell Death. To obtain a valuable SAR
between DNA damage and cell death, we decided to evaluate
the methoxy-functionalized bioprecursors 15−17. RT112,
HEK293-mock, and 2W1-transfected cells were treated with
15−17 for 48 h, and the appearance of phosphorylated H2A.X
histone (γH2A.X), a positive indicator of DNA damage, was
analyzed by immunodetection, while trypan blue uptake assay
was used as a measure of cell death. The data obtained (Figure
Figure 2. γ-H2AX induction and cell death after 48 h treatment with
15−17. γ-H2AX induction in RT112 (A), HEK/2W1 (C), or HEK-
2) revealed a clear trend showing that 15 was the most potent
bioprecursor of DNA damage and cell death followed by the di-
mock cells (E) as determined by Western blotting. β-Actin and ERp29
and trisubstituted methoxy analogues. Significantly, no
were used as loading control in RT112 (B), HEK/2W1 (D), and
accumulation of γH2A.X was detectable in the HEK293-mock
HEK/mock cells (F), respectively. V is vehicle (DMSO). Cell death
was measured by the trypan blue assay in RT112 (F), HEK/2W1 (G),
transfected cells. To demonstrate the capacity of CYP2W1 in
the bioactivation of 15, the compound was incubated with
recombinant CYP2W1 and cumene hydroperoxide, a com-
monly used constituent employed to reconstitute an electron
donor system.24 As illustrated in Figure 3, CYP2W1 was
capable of oxidizing 15 to a product with m/z 360.1, which is
consistent with the formation of the active spirocyclized CPI-
MI molecule.
or HEK/mock cells (H).
CYP Metabolism of the CBI Pharmacophore. Given that
29 is potently activated by CYP1A1, we further evaluated the
CYP selectivity of this CBI-based member of the duocarmycin
family. Bioprecursor 29 was incubated with several bactosomes
(CYP null, 1A1, 1A2, 1B1, 3A4, and 2D6) for 1 h at 37 °C. Any
metabolites of 29 produced following incubation with specific
CYP bactosomes were extracted and added to EJ-138 bladder
cancer cells (null for CYP1−3 family members).15 This resulted
in 100-fold potentiation by CYP1A1, corroborating the CHO-
1A1 cell data. Neither CYP1A2, 1B1, 2D6, nor 3A4 potentiated
the antiproliferative activity of 29, indicating CYP1A1
selectivity even over CYP1A2, a hepatic CYP1 family member
with over 70% sequence homology (Table 2). Next, we showed
Figure 3. Metabolite profiles following incubation of 15 with
CYP2W1 and CuOOH. (A) 15 (100 μM) and CuOOH (0.5 mM);
(B) 15 (100 μM) and CYP2W1 (20 pmol); (C) 15 (100 μM),
CuOOH (0.5 mM), and CYP2W1 (20 pmol); (D) channel D when
extracted using specific SIR channels at m/z 360.1 (the toxic
spirocyclized metabolite).
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Table 2. Growth Inhibition of Bioprecursor 29 after Incubation with CYP Bactosomes against EJ-138 Bladder Cancer Cell Line
EJ-138
control bactosomes
EJ-138 + 1A1
EJ-138 + 1A2
EJ-138 + 1B1
EJ-138 + 2D6
EJ-138 + 3A4
a
b
a
a
a
a
compd ID
IC50
IC50
1.99
IC50
0.02
PF
100
IC50
2.23
PF
0.9
IC50
1.53
PF
1.30
IC50
2.53
PF
0.79
IC50
1.04
PF
1.91
29
2.88
a
b
IC50 = μM. Potentiation factor (IC50 in EJ-138 + control bactosomes/IC50 in EJ-138 + CYP isoform metabolites).
that the hydroxylated seco-CBI precursor could be observed
directly by LCMS detection after incubation of 29 in the
presence of CYP1A1 bactosomes for 1 h at 37 °C (Supporting
Information (SI) Figure S3). Although the use of a panel of
recombinant CYP proteins was a useful indicator of potential
CYP involvement in activation of 29, it does not fully address
the multiple CYP interactions present in the liver, the major
site of drug metabolism. Clearly the opportunity for tumor
selective activation of duocarmycin bioprecursors must avoid
activation by the liver because this could result in systemic
toxicity. Accordingly, 29 was incubated in the presence of
mouse liver homogenate or microsomal fraction, both
representing a rich source of drug metabolizing CYP enzymes.
Both homogenate and microsomes produced a similar
metabolite profile indicating extensive metabolism (see SI
Figures S4 and S5) although fewer metabolites were observed
in the homogenate, which is likely to be a result of the lower
CYP content per mg of homogenate. Importantly, the active
metabolite of 29 was not produced by either homogenate or
microsomes and provides evidence to support tumor selective
activation.
Discussion. The potential for CYP-selective metabolism of
xenobiotics coupled to their broad substrate specificity provides
a unique opportunity to design drugs whose activity is
dependent on a critical functional group that can be unmasked
or restored by CYP oxidation selectively in tumor tissue.
Several clinically used cancer chemotherapeutics, notably the
DNA alkylating oxazaphosphorines and nitrosoureas, are
known to be metabolized by CYPs to reactive intermediates,
however primarily in the liver.5,25 This systemic CYP
metabolism is associated with profound tissue toxicity and
lack of tumor selectivity. The continued importance of
cytotoxic chemotherapy in management of cancer has identified
a need for more tumor-selective treatments. Furthermore, the
increasing ability to identify the expression of specific targets
associated with patient subgroups provides a basis for
personalized therapy. CYP1A1 and 2W1 are examples of
monooxygenases that are highly expressed in bladder15 and
colon7,9,20 cancer respectively and hence provide a rational basis
for discovery of CYP-selective chemotherapeutic activation and
the ultrapotent duocarmycins are ideal candidates for CYP
mediated bioprecursor drug development.13 In our previous
work,16 we demonstrated CYP1A1-mediated activation of a
duocarmycin analogue capable of generating biological activity
via DNA damage and showed that no hepatic CYP was
contributing to this activity.15 Here, data obtained for the
naphthalene-based analogue 29 revealed that the propensity for
CYP1A1-activation was at least 500-fold less with regard to
antiproliferative potentiation compared with 15. Importantly,
although 29 was shown to undergo hepatic metabolism, no
cytotoxic metabolite was observed to be generated in mouse
liver microsomes or homogenate, demonstrating its potential
for tumor-specific activation. We also prepared CPI analogues
of 15 that differed in their substitution pattern of the DNA-RM
and have shown that subtle changes to the substitution pattern
can result in alteration of the CYP-mediated increase in the
cytotoxicity observed. CYP1A1-mediated bioactivation seemed
to be affected by functionalization at position 7, while
CYP2W1-bioactivation was intolerant to substitution in
positions 6 and 7, indicating that these enzymes do not
metabolize and/or activate these bioprecursors to the same
extent. These results are important in targeting CYP2W1 over
1A1 (or vice versa) and suggest that further re-engineering of
the seco-duocarmycin bioprecursors could lead to even greater
selectivity and ultimately tumor CYP-specific targeting.
EXPERIMENTAL SECTION
■
1
All compounds were analyzed by H NMR. Compounds that had not
previously been reported and the final target compounds (15−23 and
29) were analyzed by 13C NMR, LRMS, and HRMS. The purity of all
compounds was ≥95% as measured by HPLC. Full experimental
protocols can be found in the SI.
Growth Inhibition Assays. The human bladder carcinoma cell
lines, RT112 and EJ-138, were obtained from the European Collection
of Cell Cultures (Salisbury, UK) and were authenticated morpholog-
ically. CHO lines were a gift from the late Dr. T. Friedberg, University
of Dundee. All three cell lines were grown as monolayers in either
RPMI 1640 (RT112 and EJ-138) supplemented with 10% (v/v) fetal
bovine serum, 1 mM sodium pyruvate, and 2 mM of L-glutamine or
DMEM (CHO-1A1) at 37 °C in 5% CO2. The Flp-In system
(Invitrogen) was used for the generation of the human embryonic
kidney HEK293-2W1 cell line, which was transfected as previously
described.7 Compounds were dissolved in DMSO and then diluted in
complete cell culture medium to give a broad range of concentrations
(0.001−100 μM), such that the final DMSO concentration was not
greater than 0.1%. Medium was removed from each well and replaced
with compound or control solutions, and the well plates were then
incubated for a further 96 h before the MTT assay was performed as
previously described.16 Results were expressed in terms of IC50 values
(concentration of compound required to kill 50% of cells), and all
experiments were performed in triplicate.
Role of CYPs in Chemosensitivity of 29. Involvement of specific
CYP isoforms in the activation of 29 was determined by evaluating the
chemosensitivity of CYP-generated metabolites of 29. Metabolites
were created via incubation of 29 (50 μM) in the reaction mixture (2
mM NADPH, 1 mM MgCl2, 50 mM Tris-HCl (pH 7.4), 20 pmol of
CYP1A1, 1A2, 1B1, 2D6, or 3A4 bactosomes (Cypex)). Control
reactions were carried out using CYP-null bactosomes. Following 1 h
incubation at 37 °C, metabolites were extracted using acetonitrile and
centrifugation at 10000g for 10 min. The resultant supernatant was
removed, dried using vacuum evaporation (Genevac), and the
resultant pellet resuspended in DMSO, and the antiproliferative
activity was assessed by the MTT assay following 96 h exposure to EJ-
138 cells as described above.
γH2A.X Phosphorylation and Cell Death. RT112, HEK293-
mock, and 2W1-transfected cells were lysed at 4 °C for 15 min in 1%
Triton X-100 in PBS with addition of a protease inhibitors cocktail
(Roche) or in RIPA buffer with subsequent centrifugation at 13000g.
Cell lysates were centrifuged at 13000g for 10 min. The aliquots of
supernatants were subjected to SDS-PAGE and subsequent immuno-
blotting using CYP2W1, γH2A.X, β-actin, or ERp29 antibodies. Cell
death was measured by use of the trypan blue assay according to
manufacturer’s protocol (Sigma Aldrich).
CYP2W1 Metabolism of 15. CYP2W1 (20 pmol) was incubated
with 15 (25 or 50 μM) in sodium phosphate buffer (pH 7.4) in a total
volume of 400 μL. After 3 min preincubation at 37 °C, the catalytic
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Journal of Medicinal Chemistry
Brief Article
reactions were initiated by the addition of CuOOH (125 μM). After
30 min, the reactions were stopped by snap freezing. Control
incubations included (i) 15, CYP2W1 but no CuOOH, and (ii) 15,
CuOOH but no CYP2W1.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details of synthetic methods used to prepare
target compounds and metabolism data associated with
evaluation of bioprecursor 29. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0)1274 236482. Fax: +44 (0)1274 233234. E-
mail: k.pors1@bradford.ac.uk.
■
Present Address
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M.; Pors, K.; Shnyder, S. D.; Patterson, L. H. Antitumor activity of a
duocarmycin analogue rationalised to be metabolically activated by
cytochrome P450 1a1 in human transitional carcinoma of the bladder.
Mol. Cancer Ther. 2011, 12, 27−37.
∥For N.I.: Clinical Trials Service Unit (CTSU), University of
Oxford, UK.
Author Contributions
#H.M.S. and S.T. contributed equally to this work.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the EPSRC NMSSC for HRMS data. This work was
supported by the EPSRC (HMS) and YCR (grant B207 to
K.P., Programme Grant to L.H.P.) and The Swedish Cancer
Society (MIS). Recombinant CYP2W1 was a kind gift from Dr.
Masaya Tachibana (Karolinska Institute).
ABBREVIATIONS USED
■
CBI, chloromethylbenzoindoline; CHO, Chinese hamster
ovary; CPI, chloromethylpyrroloindoline; CYP, cytochrome
P450; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;
HEK, human embryonic kidney; TTMSS, tris(trimethylsilyl)-
silane
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