Strictosidine Synthase Triggered Enantioselective Synthesis of N-Substituted (S)-3,14,18,19-Tetrahydroangustines as Novel Topoisomerase i Inhibitors

Article abstract of DOI:10.1021/acschembio.7b00740

Monoterpenoid indole alkaloids (MIAs) comprise an important class of molecules for drug discovery, and they have variant carbon skeletons with prominent bioactivities. For instance, in spite of limitations to their use, camptothecins are the only clinically approved topoisomerase I (Top1) inhibitors. The enzyme strictosidine synthase, which is key for MIA biosynthesis, was applied to the enantioselective preparation of three N-substituted (S)-3,14,18,19-tetrahydroangustine (THA) derivatives. These non-camptothecin MIAs were shown to have moderate in vitro HepG2 cytotoxicity and Top1 inhibition activities. The (S)-configured MIAs had stronger cytotoxicity and Top1 inhibition than their chemically synthesized (R)-enantiomers, which aligned with the results of molecular dynamics simulations. A series of N-substituted (S)-THAs were then chemoenzymatically synthesized to investigate structure-activity relationships. The most active analogue observed was the N-(2-Cl benzoyl)-substituted derivative (7i). Insight into the binding mode of 7i and a Top1-DNA covalent complex was investigated by molecular dynamics simulations, which will facilitate future efforts to optimize the Top1 inhibitory activities of non-camptothecin MIAs.

Full text of DOI:10.1021/acschembio.7b00740

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Article
Strictosidine Synthase Triggered Enantioselective Synthesis of N-substituted
(S)-3,14,18,19-Tetrahydroangustines as novel Topoisomerase I Inhibitors
Yunrui Cai, Huajian Zhu, Zaccary Alperstein, Wenjun Yu, Artem Cherkasov, and Hongbin Zou
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00740 • Publication Date (Web): 15 Nov 2017
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Strictosidine Synthase Triggered Enantioselective Synthesis
of N-substituted (S)-3,14,18,19-Tetrahydroangustines as novel
Topoisomerase I Inhibitors
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†,||
‡,†,||
§,||
†
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Yunrui Cai, Huajian Zhu,
Zou
Zaccary Alperstein, Wenjun Yu, Artem Cherkasov, Hongbin
†
,*
†
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Zhejiang University City College, Hangzhou 310015, Zhejiang, China
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Vancouver Prostate Centre, University of British Columbia, Vancouver, British Columbia V6H 3Z6, Canada
ABSTRACT: Monoterpenoid indole alkaloids (MIAs) comprise an important class of molecules for drug discovery, and
these have variant carbon skeletons with prominent bioactivities. For instance, in spite of limitations to their use, camptothe-
cins are the only clinically approved Topoisomerase I (Top1) inhibitors. The enzyme STR1, which is key for MIA biosyn-
thesis, was applied to the enantioselective preparation of three N-substituted (S)-3,14,18,19-tetrahydroangustine (THA) de-
rivatives. These non-camptothecin MIAs were shown to have moderate in vitro HepG2 cytotoxicity and Top1 inhibition ac-
tivities. The (S)-configured MIAs had stronger cytotoxicity and Top1 inhibition than their chemically synthesized (R)-
enantiomers, which aligned with the results of molecular dynamics simulations. A series of N-substituted (S)-THAs were
then chemo-enzymatically synthesized to investigate structure-activity relationships. The most active analogue observed was
the N-(2-Cl benzoyl) substituted derivative (7i). Insight into the binding mode of 7i and topoisomerase I-DNA covalent com-
plex was investigated by molecular dynamics simulations, which will facilitate future efforts to optimize the Top1 inhibitory
activities of non-camptothecin MIAs.
Monoterpenoid indole alkaloids (MIAs) comprise an im-
portant group of natural secondary metabolites [including
camptothecin (CPT), ajmaline, vindoline, quinine, and oth-
ers; Figure 1], and these are famous for their diverse struc-
MIAs by one-step syntheses starting from the STR1 reac-
12
tion product strictosidine.
13
Since CPT, a natural product MIA , was established as a
Topoisomerase I (Top1) inhibitor with excellent antitumor
activity, Top1 has become a hot-target in cancer chemo-
therapy due to its role in DNA replication, transcription and
1,2
tural skeleton and prominent pharmacological activities.
Strictosidine synthase (STR1, Figure 1) is a gateway en-
zyme which conducts asymmetric Pictet-Spengler (P-S)
condensation between tryptamine and secologanin to Stric-
1
4,15
other processes through relaxation of supercoiled DNA.
Although the camptothecins such as Topotecan, Irinotecan
and Belotecan were developed as the only three clinically
3,4
tosidine for the biosynthesis of some 2000 MIAs. Owing
to the critical function of STR1 in the biosynthetic pathway
of MIAs, it has been comprehensively studied in chemical
16,17
approved Top1 inhibitors,
they have several limitations
to their use specifically including poor chemical stability
4
–10
15,17
biology following the disclosure of its 3D structure.
The
due to the spontaneous opening of the lactone.
There-
P-S condensation catalyzed by STR1 is very attractive due
to its advantages in high stereoselectivity and efficiency as
well as mild reaction conditions. However, there are few
examples demonstrating the chemo-enzymatic use of STR1
to synthesize new alkaloids in the search for bioactive sub-
fore, discovery of novel non-CPT Top1 inhibitors has be-
come a promising research field, and several novel Top1
18,19
inhibitors have been reported.
These diversified inhibi-
tors include compounds in clinical trial such as indeno-
20
quinolines
(BMS-247615,
Phase
II) ,
5,11
stances.
Previously, we reported preparation of several
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Figure 1. STR1 as gateway enzyme for natural MIAs biosynthesis
2
1,
indenoisoquinolines (NSC-724998, Phase 2I)
diben-
hydrogenated vincosamide, and the selective 3R configura-
tion originated from the spontaneously lactamized product
of vincoside, isolated from Adina rubescens.
2
zonaphthyridinones (Genz-644282, Phase I) as well as
some molecules under preliminary investigations such as
30
2
3
the evodiamines (Figure 2A) .
,14,18,19-Tetrahydroangustine (THA, Figure 1) and
angustine are proposed biogenetically as belonging to
Interestingly, the structural arrangement of THA is simi-
lar to CPT and the evodiamines (Figure 2A), which sug-
gests that compounds with this skeleton may also be poten-
tial Top1 inhibitors. A structure-activity relationship (SAR)
study of evodiamines accordingly inspired the synthesis of
the N-acylated THA derivatives. In order to also identify
which configuration might promote the desired bioactivity,
3(S)-THAs (7a–c) were prepared by the STR1 chemo-
enzymatic approach, and their R-enantiomers (8a–c) were
chemically synthesized from vincosamide. Preliminary
inhibitory screening results showed that the 3(S)-THA de-
rivatives (7a–c) were superior for both in vitro HepG2 cy-
totoxicity and Top1 inhibition than their (R)-enantiomer
counterparts (8a–c), which aligned with the results of mo-
lecular dynamics simulations. Initiated by immobilized
STR1 as a biocatalyst, a series of 3(S)-THAs was stereose-
lectively generated (Figure 2B) and tested in vitro for Top1
inhibitory activity and HepG2 cytotoxicity.
3
STR1 catalyzed MIAs, and showed in vitro antiprolifera-
2
4,25
tive and anti-inflammatory activities.
When first isolat-
2
6
ed from Strychnos angustflpra , the carbon skeleton of
angustine aroused significant interest from chemists to at-
tempt total synthesis, and several successful strategies were
27–29
reported.
However, these strategies include many steps
and low yields, or lack the flexibility for facile analogue
production. Notably, the enantiomer 3(R)-THA was also
generated from
RESULTS AND DISCUSSION
Chemistry. The synthesis of strictosidine can be
achieved via the condensation of tryptamine and secolo-
ganin in water by reflux in the presence of acetic acid, lead-
ing to a mixture of strictosidine and the spontaneously lac-
tamized product of vincoside, vincosamide. Pure vincosa-
mide can be obtained by filtration due to its lower water
solubility, leaving a strictosidine mixture that is challeng-
31
Figure 2. STR1 as biocatalyst for potential Top1 inhibitor THAs
ing to purify.
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a
Scheme 1. Synthesis of N-substituted THA derivatives
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a
Reagents and conditions: a) STR1, KPi buffer, pH 7.0, 5C; b) 5% Na CO , 70 C; c) H , Pd, r.t; d) glucosidase, acetate buffer, pH 5.0,
37 C; e) NH OAc, EtOH, reflux; f) p-TsOH, toluene, 90 C; g) acyl or alkyl halides, t-BuOK, THF, r.t.; h) acetic acid, water, reflux
2
3
2
4
In the novel approach presented, strictosidine (1) was syn-
thesized by the STR1 catalyzed enantioselective P-S reac-
tion of tryptamine and secologanin in mild reaction condi-
tions, and then converted into strictosidine lactam (2) in the
presence of sodium carbonate. The synthetic routes of N-
substituted (S)-THA derivatives 7a–r and their three R-
enantiomers 8a–c are depicted in Scheme 1. The hydrogen-
ated product (3) of 2 was gently incubated with β -
glucosidase, extracted from almonds, to give its aglycone
4), which reacted with ammonium acetate in refluxing
(
ethanol to afford its E ring aza- product (5). Reaction of
compound 5 with p-toluenesulfonic acid (p-TsOH) in re-
fluxing toluene led to dehydration and oxidation to provide
the hetero-aromatized (S)-THA (6). In the presence of po-
tassium tert-butoxide (t-BuOK), compound 6 was treated
with acyl or alkyl halides to give the N-substituted (S)-THA
derivatives 7a–r. With vincosamide in hand, the same
strategy was applied to prepare the corresponding N-
substituted (R)-3,14,18,19-THA derivatives (8a–c).
Figure 3. Top1-mediated DNA cleavage assay gel. (A) Inhibition
of Top1 relaxation activity at 200 and 500 μM: lane 1, DNA
alone; lane 2, DNA + Top1; lane 3‒16, DNA + Top1 + CPT or
test compounds (7a, 8a, 7b, 8b, 7c, 8c). (B) Inhibition of Top1
relaxation activity at 200 μM: lane 1, DNA alone; lane 2, DNA +
Top1; lanes 3‒21, DNA + Top1 + CPT or test compounds (7a‒7r,
respectively). (C) Inhibition of Top1 relaxation activity at 50, 100,
and 200 μM: lane 1, DNA alone; lane 2, DNA + Top1; lane 3‒11,
DNA + Top1 + CPT or test compounds (7a, 7i).
The study of the favorable configuration of C-3.
In order to identify the favorable configuration of C-3, six
N-substituted THA derivatives (7a–c, 8a–c) were synthe-
sized and screened for in vitro Top1 inhibitory activity and
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Figure 4. Molecular dynamics (MD) simulations of 7a and 8a in Top1 (PDB ID: 1k4t). . (A) Three evenly spaced snapshots taken from
the simulations of 8a in the color-coded order of Green, red, and purple. (B) Three evenly spaced snapshots taken of the simulation with
compound 7a, in the color-coded order of Green, red, and purple. (C) Final snapshot of the simulations of 8a. (D) Final snapshot of the
simulation of 7a. In all of the picture the protein and most of the DNA has been removed for clarity.
Table 1. In vitro cytotoxic activity of the THAs enantio-
mers against HepG2
ly more preventive of supercoiled DNA relaxation than its
enantiomer (8a). These results suggest that N-substituted
THA derivatives with the S configuration possess better
Top1 inhibitory activities than their R-enantiomers.
Cytotoxicity
Cytotoxicity
Compound
Compound
(IC50, μM)
(IC50, μM)
To further test the hypothesis of preferred C-3 configura-
tion, compounds 7a–c and 8a–c were also assayed for their
in vitro cytotoxic activities against HepG2 cell line using
the MTT assay. The results listed in Table 1 also showed
that N-substituted THA derivatives with S configuration
7
7
a
8.5 ± 0.06
20.9 ± 0.09
23.4 ± 0.17
0.68 ± 0.01
8a
8b
8c
29.2 ± 0.19
65.2 ± 0.24
57.8 ± 0.11
b
7
c
CPT
(
7a–c) are each about three-fold more potent than their R-
antiproliferative activity using HepG2 cells. Many Top1
inhibitors act by stabilizing a covalent Top1-DNA, so-
called “cleavable complex”, to prevent the relaxation of
supercoiled DNA. To investigate the inhibitory activity,
Top1 and supercoiled DNA pBR322 were incubated in the
presence of the test compounds or CPT. Inhibition was
observed when the supercoiled complexes were prevented
from being relaxed. As shown in Figure 3A, compounds
enantiomers. In addition, compound 7a, having a 4-
chlorobenzoyl substituent, exhibited the most potent cyto-
toxic activity of the analogues tested, which was consistent
with the observed Top1 inhibitory activity.
In order to rationalize the differences between the S- and
R- enantiomers of the parent compounds (7a–c and 8a–c),
molecular dynamics (MD) simulations were performed
with the OPLS3 force field implemented in Maestro 2016-3
7
a–c and 8a–c are all active against Top1-mediated relaxa-
32-37
software (Figure 4).
The docked structures of the S-
tion of supercoiled DNA at a high concentration (500 μM),
indicating that N-substituted THA derivatives were novel
non-CPT inhibitors of Top1. At lower concentration (200
μM), 7a–c and 8a maintained inhibition, while none was
observed for 8b and 8c. Meanwhile, compound 7a is slight-
enantiomer (7a) and R-enantiomer (8a) in topoisomerase I
pdb: 1k4t) were simulated for 156 ns and 100 ns, respec-
tively, and visualized with the MOE suite software. After
the first ~60 ns of the simulation of compound 8a, most of
the compound was observed to be pushed out of the DNA
(
4
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Figure 5. (A) Superimposed docked complexes of 7i (green) and CPT (purple). (B) Comparison of the final snapshot of 7i (green) with
CPT (purple) in Top1-DNA complex. (C) Binding mode of compounds 7i and 3D superimposed structure of the final simulated snapshot
(gray). In the PDB entry 14kt, Met 230 and Lys 334 correspond to Met 428, and Lys 751.
leaving only part of the pyridine ring left between the bases.
The system was simulated for another 100 ns to see if the
compound would find its way back between the nucleobas-
es. This was not observed, however, after a 100 ns simula-
tion of compound 7a, an induced fit was observed with pi-
stacking interactions that remained strong throughout the
simulation (Figure 4B). To accommodate compound 7a,
the pocket between the DNA increased vertically by around
1 Angstrom (Figure 5B, green). Furthermore, it can be seen
that adding large substituents on the R enantiomer results in
an even more unfavorable geometry, due to the steric inter-
action resulting from the large out of plane angle of the
stereocenter (Figure 4C). Therefore, it appears that the lack
of potency of this set in the R-enantiomers was largely cor-
related to the out of plane between the indole ring and the
rest of the ring system. The S-enantiomer is much more
planar, facing more empty room in the pocket, allowing for
free substitution on the indole nitrogen (Figure 4D. This
explains the greater flexibility in modifications permitted to
the S-enantiomer, with the 4-chlorobenzoyl analogue hav-
ing exhibited the most potent cytotoxic activity of the com-
pounds tested.
Structure-activity relationship study. Substituted
benzoyl, acyl, cyano, and alkyl groups were introduced to
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prepare N-substituted (S)-THA derivatives 7d–r for SAR
ing the size of the gap between the DNA bases relative to
study (Scheme 1). As shown in Figure 3B, at the concentra-
tion of 200 μM, compounds 7a–c, 7h, 7i were found to be
active against Top1-mediated relaxation of supercoiled
DNA, while compounds 7d, 7f, 7o showed moderate Top1
inhibitory activity, and the rest were almost inactive. Over-
all, N-benzoyl compounds exhibited the most potent Top1
inhibitory activity, especially those with the Cl-substituted
benzoyl group (7a, 7h‒i). This can be understood by the
results of the simulation, where it is apparent that the ben-
zoyl ring makes a close interaction with a methionine resi-
due from Top1 (Figure 5C). Compound 7i was then select-
ed to test the in vitro Top1 inhibitory activity at three dif-
ferent concentrations with 7a and CPT as control (Figure
CPT (Figure 5B), in which 7i is stabilized by additional
amino acid contacts that predominantly include methionine.
This interaction appears to be driven by a strong aromatic-
dipole interaction at the ortho-substituted benzoyl ring with
methionine 428 (Figure 5C). Interestingly, during the simu-
lation of compound 7a a strong water mediated contact
with lysine 751 of Top1 was observed for more than half of
the simulation (Supplementary Figure S1, S2).
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Conclusion. In summary, immobilized STR1 was uti-
lized to enantioselectively synthesize a series of N-
substituted (S)-THA derivatives and successfully provide a
short and efficient chemo-enzymatic approach to prepare
polycyclic MIAs. These N-substituted (S)-THAs were iden-
tified as novel Top1 inhibitors. Interestingly, the Top1 and
HepG2 inhibitory activities observed in vitro showed that
the C-3(S) configuration of N-substituted THAs are pre-
ferred for bioactivity. This was supported by MD simula-
tion models that the R-enantiomer may be pushed out of
pocket of Top1 while the corresponding S-enantiomer may
remain between the bases. Moreover, the SAR study of N-
substituted (S)-THAs was conducted, and the Top1 inhibi-
tor 7i was identified as the most potent analogue tested.
The formation a novel methionine-aromatic contact be-
tween 7i and Top1 was proposed to explain the observed
activity. Indeed, this type of strong interaction is only be-
ginning to be recognized as a stabilizing force in protein
3
C). Furthermore, the antiproliferative activity of 7a–r
against HepG2 cell lines was investigated using CPT as a
reference drug. As shown in Table 2, compound 7i was the
most potent in vitro antiproliferative agent against HepG2,
with an IC50 value of 1.8 μM. Overall, IC50 values of most
N-benzoyl compounds and some N-acylated compounds
were < 10 μM, but most N-alkylated compounds exhibited
weak cytotoxicity. Taken together, the results of cytotoxici-
ty assay are consistent with Top1 inhibitory activity.
Table 2. Antiproliferative activity of the N-Substituted (S)-
THA derivatives against HepG2
Cytotoxicity
IC50, μM)
Cytotoxicity
Compound
Compound
38
(
(IC50, μM)
structures. The results described here provide further in-
sight into the biocatalytic character of STR1, and demon-
strate an excellent opportunity for its use as a biocatalyst
for the enantioselective construction of diversified MIAs
with potentially meaningful bioactivity.
7
7
a
9.1 ± 0.02
16.6 ± 0.08
23.5 ±0.09
7.5 ± 0.01
9,7 ± 0.03
5.6 ± 0.01
8.3 ± 0.02
12.3 ± 0.02
1.8 ± 0.01
5.2 ± 0.03
7k
7l
18.2 ± 0.03
9.6 ± 0.02
24.6 ± 0.15
b
7
c
7m
7n
a
7
d
na
7
e
7o
33.5 ± 0.24
a
METHODS
7
f
7p
na
a
7g
7h
7i
7q
na
Materials. Secologanin was isolated from Lonicera Tatarica.
Topoisomerase I, purified from calf thymus was purchased from
Thermo Fisher Scientific. All of the other chemicals were ob-
tained from commercial sources.
a
7r
na
CPT
0.70 ± 0.004
7j
Synthesis of N-substituted THAs. Synthetic methods are
summarized in the legends of Schemes 1. Fully detailed protocols,
along with the characterization of synthetic products by NMR and
MS, are described in detail in the Supporting Information.
a
na= not active
To further understand the contribution toward Top1 inhibi-
tion of the novel THA pharmacophore, the most active de-
rivative, compound 7i, was compared with CPT by docking
into the final snapshot of the 7a simulated structure, and
superimposing this with the CPT crystal structure (Figure
5). The overlap of A, B, and C rings between these mole-
cules suggested their similar binding modes (Figure 5A).
Interestingly, both internal amides of the two compounds
overlapped in their nitrogen and carbonyl oxygen atoms in
the same region, perhaps satisfying an electrostatic need. In
addition, compound 7i was observed to induce its fit into
the cleaved DNA complex with topoisomerase I by increas-
Topoisomerase I Inhibition Assay. The enzyme activity
was measured by assessing relaxation of supercoiled pBR322
plasmid DNA. Test compounds were dissolved in DMSO and
were tested at final concentrations as shown. The reaction mixture
contained 2 × DNA Top1 buffer (10 μL), Top1 (0.5 U), the test
compounds (various concentrations), pBR322 plasmid DNA (0.25
μg), and distilled water in a final volume of 20 μL. Reactions
were carried out for 30 min at 37 °C and then stopped by adding
SDS [0.5% (w/v) final concentration]. After that, 3.5μL of 6 ×
loading buffer [0.1 mM EDTA, 7% (v/v) glycerol, 0.01% (w/v)
xylene cyanol FF, 0.01% (w/v) bromophenol blue] was added.
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Reaction products were electrophoresed on a 0.8% (w/v) agarose
gel in TAE (Tris-acetate-EDTA) running buffer at 60 V for 1.5 h.
the Zhejiang Provincial Fund for Distinguished Young
Scholars (LR15B020001), the National High-tech Research
and Development Program (863 Program, 2015AA020922),
and the Fundamental Research Funds for the Central Uni-
versities (2017FZA7016). We appreciate C. B. Naman for
critical editing of the manuscript.
To visualize the reaction products, the gel was stained with 0.5 μ
–
1
g mL ethidium bromide for 10 min. DNA bands were visualized
using a UV transilluminator.
In Vitro Cytotoxicity Assay. Cells were plated in 96-well
3
microtiter plates at a density of 5 × 10 cells/well and incubated in
a humidified atmosphere with 5% CO at 37 °C for 24 h. Test
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on
the ACS Publications website at DOI:
2
compounds were added to wells at different concentrations, and
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.1% (v/v) DMSO was used as a control. After samples were
incubated for 72 h, 20 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-
–1
2
,5-diphenyl-tetrazolium-bromide) solution (5 mg mL ) was
added to each well, and the plate was incubated for an additional 4
h. The formazan was dissolved in 100 μL of DMSO. The ab-
sorbance (OD) was quantitated with microplate spectrophotome-
ter at 570 nm. Wells containing no drugs were used as blanks for
the spectrophotometer. The survival of the cells was expressed as
percentage of untreated control wells. All experiments were per-
formed in triplicate.
Detailed synthetic procedures, supplementary figures,
quantificaiton of Topoisomerase I inhibitory activity
and NMR spectra
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AUTHOR INFORMATION
Corresponding Author
(8) Loris, E. A., Panjikar, S., Ruppert, M., Barleben, L., Unger,
*
M., Schübel, H., Stꢀckigt, J. (2007) Structure-based engineering
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Biol. 14, 979–985.
E-mail: zouhb@zju.edu.cn
Author Contributions
(9) Maresh, J. J., Giddings, L. A., Friedrich, A., Loris, E. A.,
|
|
These authors contributed equally to this work.
Panjikar, S., Trout, B. L., Stꢀckigt, J., Peters, B., O'Connor, S. E.
(2008) Strictosidine synthase:ꢁ mechanism of a Pictet−Spengler
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Notes
The authors declare no competing financial interest.
THA in this paper is the abbreviation of 3,14,18,19-
Tetrahydroangustine.
ACKNOWLEDGMENTS
(11) Zhu, H., Kerčmar, P., Wu, F., Rajendran, C., Sun, L.,
This work was supported by the National Natural Science
Foundation of China (No. 21472170, 21272207, 21702183),
Wang, M., Stöckigt, J. (2015) Using strictosidine synthase to
prepare novel alkaloids. Curr. Med. Chem. 22, 1880–1888.
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