Gossypol with Hydrophobic Linear Esters Exhibits Enhanced Antitumor Activity as an Inhibitor of Antiapoptotic Proteins

Article abstract of DOI:10.1021/acsmedchemlett.6b00302

A series of gossypol Schiff bases that were derived from unnatural linear amino acid methyl esters were identified and found to be much more potent than gossypol and ABT-199 in terms of anticancer activity. This is the first example of gossypol Schiff bases with increased activity. The investigation of the Schiff base side chain of gossypol revealed that the unique anticancer effect was achieved by the introduction of hydrophobic ester groups. The optimized products showed low micromolar pan antitumor activities against NCI-60 tumor cell lines, which is promising for further drug development. Studies on the preliminary mechanism of action for their cellular activities was also carried out with antiapoptotic protein (Bcl-2 and Mcl-1) inhibition FP assays. The molecular modeling analysis demonstrated a possible binding mode for these compounds with Bcl-2, which could explain the binding affinity of the novel gossypol Schiff bases with these proteins.

Full text of DOI:10.1021/acsmedchemlett.6b00302

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Letter
Gossypol with Hydrophobic Linear Esters Exhibits Enhanced
Anti-Tumor Activity as an Inhibitor of Antiapoptotic Proteins
Yuzhi Lu, Shuangchan Wu, Yuan Yue, Si He, Jun Li, Jun Tang, Wei Wang, and Hai-Bing Zhou
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00302 • Publication Date (Web): 19 Oct 2016
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Gossypol with Hydrophobic Linear Esters Exhibits Enhanced
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Yuzhi Lu,† Shuangchan Wu,† Yuan Yue,† Si He,† Jun Li,† Jun Tang,† Wei Wang†,* and HaiꢀBing Zhou†,*
†Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University School of
Pharmaceutical Sciences, Wuhan, 430071, China
KEYWORDS: Gossypol Schiff bases, linear amino acid esters, antiapoptotic proteins
ABSTRACT: A series of gossypol Schiff bases that were derived from unnatural linear amino acid methyl esters were identified
and found to be much more potent than gossypol and ABTꢀ199 in terms of anticancer activity. This is the first example of gossypol
Schiff bases with increased activity. The investigation of the Schiff base side chain of gossypol revealed that the unique anticancer
effect was achieved by the introduction of hydrophobic ester groups. The optimized products showed low micromolar pan antiꢀ
tumor activities against NCIꢀ60 tumor cell lines, which is promising for further drug development. Studies on the preliminary
mechanism of action for their cellular activities was also carried out with antiꢀapoptotic protein (Bclꢀ2 and Mclꢀ1) inhibition FP
assays. The molecular modeling analysis demonstrated a possible binding mode for these compounds with Bclꢀ2, which could exꢀ
plain the binding affinity of the novel gossypol Schiff bases with these proteins.
Natural polyphenols have received considerable attention
because of their biological activity.1 Gossypol, a polyphenol
that is isolated from cotton seeds, has long been investigated
for antiꢀtumor activity in addition to the reported antiviral
3
(such as antiꢀHIV2 and antiꢀH5N1 ), antifungal4, and antiꢀ
malarial5 activity as well as the inhibition of trypanosome T.
brucei activity6. (ꢀ)ꢀGossypol is currently in Phase II clinical
trials and displays singleꢀagent antiꢀtumor activity in patients
with advanced malignancies.7 The removal or mask of aldeꢀ
hyde groups has been shown to significantly reduce the toxiciꢀ
ty of gossypol in humans.8 As such, a variety of gossypol deꢀ
rivatives have been developed, including gossypol lactones9,
gossypol nitriles5, gossypolone10, and the modification of the
main structure of naphthalene7, 11, etc. Among these, gossypol
Schiff bases displayed widespread biological activities. The
purpose of the introduction of the imine through the aldehyde
was to reduce the toxicity of gossypol without a loss in the
activity since the hydroxyl group appears to be essential for
the activity in many cases.12 However, from known results, the
reported activities of aldehyde derived compounds, including
Figure 1. A comparison of our newly synthesized Schiff bases
gossypol Schiff bases, decreased dramatically compared with
with compounds reported to have anticancer activity.
gossypol itself in terms of antiꢀtumor activity (Figure 1A).12ꢀ15
The antiꢀtumor activities of gossypol Schiff bases were imꢀ
proved slightly only when the framework of gossypol was
changed into gossypolone (Figure 1B).15 The effects of the
introduced substituents of gossypol Schiff bases on the antiꢀ
tumor activity were still ambiguous. As a matter of fact, an
investigation of the mechanism of the gossypol Schiff bases
was not pursued in these previous studies. On the other hand,
studies have shown that gossypol may inhibit tumor cells by
interacting with Bclꢀ2 family proteins, which are the central
regulators of apoptosis (programmed cellꢀdeath).16 The moduꢀ
lation of the antiapoptotic Bclꢀ2 family (Bclꢀ2 and Mclꢀ1) is
necessary for protein-protein interactions (PPIs) during apopꢀ
tosis. Thus, an ideal inhibitor of antiapoptotic proteins should
bind to both Bclꢀ2 and Mclꢀ1.17
Therefore, to address the role of substituents on gossypol
Schiff bases, to explore the SAR, and ultimately to discover
agents that increase the broad biological activities with the
reduced toxicity of gossypol, we systematically investigated
many types of gossypol Schiff bases that targeted both Bclꢀ2
and Mclꢀ1 (including a small number of previous synthetic
compounds that lacked antiꢀtumor activity information, see
details in Supplemental Materials).18 The results demonstrated
that gossypol with hydrophobic linear esters increased antiꢀ
tumor activity and reduced cytotoxicity. This finding is differꢀ
ent from the conclusion of increased activity that is induced by
hydrophilic effects in the literature.14ꢀ15
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>100
>100
>100
>100
>100
>100
>100
>100
The results indicated that compound 16 with a hydrophobic
1
2
3
4
5
6
7
8
linear ester was the most active, and the compound showed
pan antiꢀtumor activity with GI50 values of 0.1 to 1.7 ꢁM in
NCIꢀ60 human cancer cell lines (Figure 1C). The therapeutic
index of compound 16 was over 30ꢀfold higher than gossypol,
and showed 10ꢀfold better antiꢀtumor activity and 10ꢀfold
lower cytotoxicity than gossypol. The competitive FP assays
demonstrated that the active compounds had high binding
affinity to Bclꢀ2 and Mclꢀ1. The results of the molecular modꢀ
eling study indicated that hydrophobic groups that were introꢀ
duced by linear amino acid methyl esters were responsible for
the extra affinity for the potent compounds.
6
7
20.93 ± 0.66 24.7 ± 1.13 37.3 ± 1.56 21.40 ± 0.54
8
21.92 ± 0.43
>100
>100
>100
>100
>100
83.13 ± 1.45
>100
>100
12.80 ± 0.63
>100
9
10
11
12
13
14
29.15 ± 5.76
15.47 ± 0.70
8.30 ± 0.65
42.36 ± 0.77
9
10
11
12
13
14
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16
17
18
19
20
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60
>100
Cell viability of human and mouse cancer cell lines. The
SAR information on the reported substrates of gossypol Schiff
bases is limited and includes few discussions about the effect
of the substituents. It was reported that the natural product
cryptosphaerolide, with an aliphatic chain, exhibited HCTꢀ116
activity through the Mclꢀ1 pathway.19 Thus, we proposed that
the introduction of the hydrophobic moiety into gossypol
could be beneficial for the activity of the compound. Thereꢀ
fore, we synthesized a set of different types of amines from
gossypol that included aromatic amines, aliphatic amines, and
amino acid derivatives, etc., described in detail in Suppleꢀ
mental Materials. The antiproliferative activity of the target
gossypol Schiff bases was initially studied with an MTT cell
viability assay for human and mouse cancer cell lines. The
results are shown in Table 1.
12.14 ± 1.75 77.34 ± 2.27 77.10 ± 1.05 5.85 ± 0.17
17.68 ± 1.11
6.23 ± 0.35
>100
0.52 ± 0.78
>100
8.24 ± 0.57 7.75 ± 0.99 29.70 ± 1.23
a
IC50 values were the means of three independent experiꢀ
ments.
With compound 14 in hand, we next investigated gossypol
Schiff bases that were derived from unnatural amino acꢀ
ids/esters with different chain lengths (15ꢀ26). As shown in
Table 2 and Figure 2, in human cancer cell lines MCFꢀ7, DUꢀ
145, and HCTꢀ116, gossypol Schiff bases that were conjugatꢀ
ed with unnatural amino acid esters (14ꢀ19) were more potent
than the corresponding amino acids 20ꢀ25. For the latter comꢀ
pounds, the chain length had little effect on the cell growth
inhibitory activities (Figure 2). For the amino acid methyl
esters 14ꢀ19, compound 16 (n = 3) exhibited the most potent
inhibitory activity (Figure 2). After the replacement of COOH
or COOMe groups by a larger group, such as CONHCH2Ph in
compound 26, a decreased activity was observed. Compounds
27 and 28, analogues of the gossypol derivative megosin with
different chain lengths, were generated to increase water soluꢀ
bility but displayed modest inhibitory activity in the four canꢀ
cer cell lines.20 Almost all the compounds exhibited good inꢀ
hibitory activity in the mouse colon cell line CTꢀ26 (Tables 1
and 2). For example, compounds 2 (IC50 = 5.4 ꢁM) and 9 (IC50
= 12.8 ꢁM) exhibited good results that were absent in the othꢀ
er three cancer cell lines (IC50 > 100 ꢁM).
First, compounds 1ꢀ6 with different sizes and lengths of aliꢀ
phatic hydrophobic groups (such as adamantyl and tertꢀoctyl)
were attached, but only compounds 1 and 3 with medium size
groups (tertꢀbutyl and cyclohexyl) showed moderate inhibitoꢀ
ry activity; others did not exhibit activity in cellular assays.
We also checked the anticancer activity of Schiff bases with
aromatic hydrophobic groups (compounds 7ꢀ11), which were
more potent than aliphatic groups (compounds 1ꢀ6). Comꢀ
pound 7, with a benzoic acid methyl ester, exhibited micromoꢀ
lar inhibitory activity in all of the four cellular assays. We then
investigated several natural amino acid methyl ester groups.
Gossypol Schiff bases that were derived from natural amino
acids have been demonstrated to exhibit good antiviral activity
against HIVꢀ1 and H5N1,2ꢀ3 but their antiꢀtumor activity was
not prominent. In this regard, compounds 12, 13, and 14 were
prepared from Valine, Methionine and Glycine methyl esters,
respectively. These ester compounds exhibited better activities
than aliphatic (compounds 1ꢀ6) and aromatic (8ꢀ11) ones. Noꢀ
tably, compound 14 is the first modified imine compound with
similar inhibitory activity compared to gossypol in all the asꢀ
says.
Table 2. Further evaluation of the optimized gossypol Schiff
bases with different linker lengths using cell viability assays.
DU-145
MCF-7
HCT-116
CT-26
Comp.
15
R =
IC50a (µM) IC50a (µM) IC50a (µM) IC50a (µM)
9.47 ± 0.87 5.64 ± 0.89 5.24 ± 1.21 4.25 ± 0.25
16
17
18
19
20
21
22
23
2.39 ± 0.21 4.39 ± 0.46 2.75 ± 1.01 8.39 ± 0.98
7.36 ± 2.05 8.22 ± 1.20 3.38 ± 2.32 16.59 ± 3.01
8.56 ± 2.44 11.46 ± 0.52 0.64 ± 0.35 13.75 ± 1.27
11.42 ± 1.27 8.95 ± 0.21 13.12 ± 2.58 5.59 ± 0.49
34.80 ± 1.33 14.43 ± 2.33 26.78 ± 0.51 48.91 ± 2.52
21.61 ± 3.09 19.37 ± 1.19 21.01 ± 3.42 67.28 ± 4.32
18.14 ± 0.14 20.03 ± 0.44 32.33 ± 4.36 28.82 ± 5.64
20.68 ± 0.75 15.94 ± 1.67 22.16 ± 1.87 4.46 ± 0.58
Table 1. Systematic evaluation of gossypol Schiff bases using
cell viability assays.
DU-145
MCF-7
HCT-116
CT-26
Comp.
R =
IC50a (µM)
IC50a (µM) IC50a (µM)
IC50a (µM)
17.16 ± 4.17
>100
>100
>100
>100
>100
>100
>100
12.76 ± 1.21
5.40 ± 0.33
1
2
3
4
20.65 ± 1.24
>100
80.30 ± 1.24 25.25 ± 1.43
>100 21.7 ± 0.62
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26
27
28
27.48 ± 1.56 17.65 ± 0.65 23.12 ± 0.71 4.54 ± 0.79
19.06 ± 0.56 11.48 ± 0.81 17.31 ± 1.92 2.08 ± 1.07
22.90 ± 3.97 16.10 ± 3.17 29.80 ± 3.21 47.93 ± 2.01
28.87 ± 1.54 15.9 ± 1.22 48.91 ± 1.19 3.19 ± 0.39
36.03 ± 1.11 17.72 ± 0.69 43.10 ± 2.65 35.58 ± 0.46
7.93 ± 0.32 60.10 ± 0.18 3.03 ± 1.43 46.78 ± 0.77
19.17 ± 3.66 75.00 ± 0.55 5.01 ± 0.27 61.90 ± 1.14
7.97 ± 1.13 54.00 ± 1.09 3.59 ± 0.76 23.20 ± 0.59
bases with different chain lengths (compounds 14, 16ꢀ20)
1
2
3
4
5
6
7
8
showed significant cytotoxicity against human cancer cells. It
should be noted that the newly synthesized gossypol Schiff
bases that were derived from unnatural amino acid esters were
more potent than all unnatural amino acids, similar to the reꢀ
sults that are presented in Table 2. Notably, the GI50 value for
the most potent gossypol Schiff base, compound 16 which was
derived from a γꢀaminobutyric acid methyl ester, was less than
1.0 ꢁM for 50 tumor cell lines out of the 60 cell lines that were
tested, which was approximately 10ꢀfold more potent than the
parent (±)ꢀgossypol (average GI50 = 3.1 ꢁM) and the known
Bclꢀ2 inhibitor drug ABTꢀ199 (GI50 = 2.77 ꢁM) (Table 3 and
Supplemental Table S2). In addition, in most cases, methyl
esters of glycine and 6ꢀaminocaproic acid derived gossypol
Schiff bases 14 and 18 showed the same level of cytotoxicity
as (±)ꢀgossypol. However, compared to the (±)ꢀgossypol 5ꢀ
amino valeric acid methyl ester that was derived from gossyꢀ
pol, Schiff base 17 significantly affected the growth of several
cancer cells, including a leukemia cell line, the nonꢀsmall cell
lung cancer HOPꢀ92 cell line, the CNS cancer SNBꢀ19 and
U251 cell lines, and the renal cancer RXF 393 cell line (see
Supplemental Table S2), etc. Based on the data obtained from
NIH and our MTT assays, we proposed that the introduction
of a linear amino acid methyl esters side chain could benefit
the antiꢀtumor activity of gossypol Schiff bases. Cytotoxicity
studies that were performed with HEK (human embryonic
kidney) 293T and Vero cells showed that most compounds
were less toxic than gossypol (see Supplemental Table S5). In
addition, in a separate study compound 16 was found to be
stable enough at pH 1ꢀ10 and to microsomes (Supplemental
Tables S3 and S4).
(±)-Gc
(+)-G
(-)-G
ꢀb
ꢀ
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
26
27
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60
ꢀ
a
IC50 values were the means of three independent experiꢀ
ments.
b “ꢀ” Means no substitution. c “G” means gossypol.
Table 3. Average cell growth inhibition data for compounds
14 and 16ꢀ18 (GI50, ꢁM) against a panel of 60 human cancer
cells.
Figure 2. The cell viability assays of the unnatural amino acid
methyl esters (14-19), their corresponding acids (20-25), and
amide 26 in selected tumor cell lines.
Average cell growth inhibition (GI50, ꢁM)a
Cell type (number
of cell lines)
ABTꢀ
199
(±)ꢀ
Gossyꢀ
14
1.88
1.65
1.63
1.63
2.84
1.74
2.19
1.92
2.44
16
17
18
To examine whether these novel gossypol Schiff bases were
superior to the parent (±)ꢀgossypols in terms of inhibitory acꢀ
tivity, we also prepared (+)ꢀgossypol and (ꢀ)ꢀgossypol comꢀ
pounds (see the Supplemental Materials Chemistry section).
Antiproliferative data revealed that the linear chain methyl
esters of gossypol Schiff bases 14ꢀ19 were more active than
the parent (±)ꢀgossypols and performed better than the correꢀ
sponding enantiomers (Table 2). Notably, the newly syntheꢀ
sized compounds significantly affected the growth of MCFꢀ7
and CTꢀ26 cancer cells, in which (±)ꢀgossypol and even (ꢀ)ꢀ
gossypol were ineffective. Compound 16, which was the most
potent compound (IC50 = 4.39 ꢁM) that inhibited the growth
of MCFꢀ7 cells, was 12ꢀfold more potent than (ꢀ)ꢀgossypol
(IC50 = 54.0 ꢁM). Similarly, compound 25, with an IC50 value
Leukemia (6)
2.11
2.47
2.85
3.19
2.97
3.22
3.50
3.16
NDb
ND
0.56
0.48
0.46
0.60
0.99
0.59
0.82
0.41
0.74
0.81
1.05
1.00
1.27
1.42
1.26
1.14
1.09
1.08
1.03
1.51
1.44
1.46
2.35
2.65
2.08
1.41
1.72
NonꢀSmall Cell
Lung Cancer (9)
2.7
Colon Cancer (7)
2.85
3.35
1.75
3.33
2.6
CNS cancer (6)
Melanoma (9)
Ovarian Cancer (7)
Renal Cancer (8)
Prostate Cancer (2)
Breast Cancer (6)
3.51
2.77
of 2.08 ꢁM, was 11ꢀfold more potent than (ꢀ)ꢀgossypol (IC50
23.2 ꢁM) in CTꢀ26 cells.
=
a GI50: concentration required for 50% cell growth inhibition. b
ND: not determined.
In vitro NCI-60 human cancer cell lines assay. Most of the
gossypol Schiff bases in this study (compounds 1ꢀ3, 8, 9, 11,
14, 16ꢀ20, and 23ꢀ28) were also selected for cytotoxicity
screening against 60 human tumor cell lines. As anticipated,
the data obtained by this cellꢀbased assay correlated well with
our previous MTT observations (Tables 1 and 2). Namely,
compounds with aliphatic (compounds 1ꢀ3) and aromatic subꢀ
stituents (compounds 8, 9, 11) generally showed poor or modꢀ
est inhibitory activities at concentrations up to 10 ꢁM in both
experimental approaches (see Supplemental Table S1). On the
contrary, unnatural amino acids/esters derived gossypol Schiff
Binding affinity to Bcl-2 and Mcl-1 proteins. Gossypol is a
potent inhibitor of Bclꢀ2 and Mclꢀ1.16, 21 It binds to Bclꢀ2 and
Mclꢀ1 with Ki values of 0.32 and 0.18 ꢁM, respectively, as
determined by competitive fluorescence polarizationꢀbased
(FPꢀbased) binding assays.7 The NIH data and our MTT assay
results revealed that our newly synthesized gossypol Schiff
bases may have a similar mechanism as the parent compound.
To confirm this point, we evaluated the binding affinity of
the selected active compounds for Bclꢀ2 using FP assays, in
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possible aggregation of hydrophobic compounds (Table 4 and
Figure 3A). The βꢀalanine methyl ester derived gossypol
Schiff base 15 displayed a high binding affinity for Bclꢀ2, with
a Ki value of 0.090 ꢁM, and the glycine methyl analogue 14
showed an increased binding affinity, with a Ki value of 0.005
ꢁM. The other ester compounds 16ꢀ18 and amino acid derivaꢀ
tives 20, 22, and 23 exhibited similar binding affinities with Ki
values that ranged from 0.171 to 0.348 ꢁM, and all were better
than (±)ꢀgossypol (Ki = 0.511 ꢁM) (Figure 3A). As mentioned
above in the MTT assay, unnatural amino acid methyl esters
that were derived from the gossypol Schiff bases 14ꢀ18 disꢀ
played potent efficacy in the inhibition of cell growth in the
MCFꢀ7 cell line, which expresses moderate levels of Bclꢀ2
and Mclꢀ1 (Table 2).22ꢀ23 This observation correlated with the
binding data of compounds 14ꢀ18 for Bclꢀ2, which indicated
that Bclꢀ2 was the potential target of these compounds. For the
enatiomers of the optimized compound 16 (Supplemental Maꢀ
terials), both of the binding affinity of (ꢀ)ꢀ16 (Ki = 0.131 ꢁM)
and (+)ꢀ16 (Ki = 0.167 ꢁM) were higher than (±)ꢀ16 (Ki =
0.341 ꢁM) for Bclꢀ2. Although gossypol Schiff bases 20, 22,
and 23 that were derived from the amino acids displayed
strong binding affinity (Ki = 0.224, 0.235, 0.171 ꢁM, respecꢀ
tively) to Bclꢀ2 in the FP assays, they showed a 2.1ꢀfold lower
efficacy in the inhibition of the growth of the MCFꢀ7 cell line,
with an average IC50 of 16.8 ꢁM, compared to the correspondꢀ
ing ester analogues 14ꢀ19 with an average IC50 value of 7.82
ꢁM. The ester analogues 14ꢀ19 are also 7.7 times more potent
than gossypol (IC50 = 60.10 ꢁM). This discrepancy is likely
due to the high hydrophilicity of these unnatural amino acids,
so they always exhibit poor cell membrane permeability comꢀ
pared to the corresponding esters. Therefore, the gossypol
unnatural amino acids 20ꢀ23 displayed good binding affinity
to Mclꢀ1 and Bclꢀ2 proteins, and they always showed relativeꢀ
ly weaker efficacy in the inhibition of cancer cells. It was also
reported the high hydrophilicity of apogossypolone derivatives
could result in a low cell permeability and would therefore
exhibit moderate antiꢀtumor activity, although those comꢀ
pounds displayed good binding affinity to Bclꢀ2 family proꢀ
teins.24
2
3
4
5
6
7
8
9
Figure 3. Competitive binding curves of 14, 16, 20, 22, and
(±)ꢀgossypol to (A) Bclꢀ2 and (B) Mclꢀ1, as determined with
the fluorescenceꢀpolarizationꢀbased binding assay.
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Next, we evaluated the binding properties and specificity of
selected gossypol Schiff bases against Mclꢀ1 using FP assays
(Table 4); Mclꢀ1 was expressed at high levels in many cancer
cell lines such as the colon cancer HCTꢀ11625 and prostate
cancer DUꢀ14526ꢀ27 cell lines. The cell growth inhibition of
these compounds in these two cell lines is shown in Tables 1
and 2. All of the tested compounds displayed superior affinity
against Mclꢀ1 in the FP assays, similar to that of gossypol (Ki
= 0.162 ꢁM) (Figure 3B). Consistent with binding data, (±)ꢀ
gossypol exhibited potent efficacy in the inhibition of cell
growth in HCTꢀ116 and DUꢀ145 cell lines according to the
results of the MTT assay, with IC50 values of 3.03 and 7.93
ꢁM, respectively (Table 2). Other unnatural amino acid methyl
esters that were derived gossypol Schiff bases 14ꢀ18 also disꢀ
played strong inhibitory properties against the Mclꢀ1 protein.
The most potent compound, compound 17, displaced BH3
binding to Mclꢀ1, with IC50 values of 7.36 and 3.38 ꢁM to
DUꢀ145 and HCTꢀ116, respectively (Table 2). However, the
compounds 20, 22, and 23 displayed very high binding affinity
to Mclꢀ1 in the FP assays, with average IC50 values of 0.10
ꢁM; the compounds showed a relatively weak efficacy in inꢀ
hibiting the growth of DUꢀ145 and HCTꢀ116 cell lines, with
average IC50 values of approximately 24.54 and 27.09 ꢁM,
respectively. The lower IC50 of cellular data of compounds 20ꢀ
23 with higher Mclꢀ1 binding affinity may be due to a permeꢀ
ability issue, as observed in Bclꢀ2.
To investigate the activities and mechanisms of gossypol
imines, ABTꢀ199 was selected as a control compound. ABTꢀ
199 is known as a highly Bclꢀ2 selective antagonist. Our data
revealed that compound 16 had lower Bclꢀ2 binding affinity
than ABTꢀ199 but had higher Mclꢀ1 affinity. According to the
NCI cell line data, ABTꢀ199 exhibited the highest activity in
the leukemia cancer cell line HLꢀ60, which expresses high
levels of Bclꢀ2. In addition, compound 16 exhibited an equal
potency to ABTꢀ199 in the HLꢀ60 cell line (Supplemental
Table S2). Furthermore, compound 16 was more potent in
most human cancer cell lines than ABTꢀ199 (Supplemental
Table S2). Mclꢀ1, but not Bclꢀ2, is expressed in most human
cancer cell lines.26 Thus, it was possible to infer that the gossꢀ
ypol Schiff bases were superior to ABTꢀ199 via the inhibition
of both Bclꢀ2 and Mclꢀ1 mechanisms.
Table 4. Crossꢀactivity of gossypol Schiff bases with selected
linker length against Bclꢀ2 and Mclꢀ1 in fluorescence polarizaꢀ
tion assays.
IC50a (ꢁM) FPA
Ki (ꢁM)
Compound
Bclꢀ2
Mclꢀ1
Bclꢀ2
Mclꢀ1
14
15
16
17
18
20
22
23
0.103±0.019
1.433±0.33
0.005±0.004
0.090±0.018 0.579±0.166
0.341±0.05
0.182±0.052
0.348±0.035 0.195±0.032
0.224±0.042 0.121±0.01
0.235±0.044 0.094±0.014
0.171±0.024 0.085±0.007
0.167±0.008 0.240±0.061
0.131±0.014 0.178±0.033
0.511±0.312 0.162±0.065
0.276±0.06
0.286±0.039 2.959±0.847
0.825±0.106 0.947±0.364
0.483±0.111 0.740±0.154
0.840±0.075 1.021±0.166
0.573±0.091 0.521±0.053
0.596±0.095 0.661±0.071
0.458±0.053 0.478±0.036
0.452±0.016
0.375±0.031 0.937±0.175
1.191±0.667 0.856±0.332
0.180±0.071
0.139±0.03
(+)-16
(-)-16
gossypol
1.249±0.32
ABTꢀ199
<0.001
>4
<0.001
>0.78
With the combination of binding affinities and all the in
vitro cell viability data, one can conclude that gossypol Schiff
bases that were derived from unnatural linear amino acid meꢀ
thyl esters could enhance the antiꢀtumor activity of gossypol.
Furthermore, the chain length is also important for Bclꢀ2 famiꢀ
ly protein binding; the data revealed that the threeꢀcarbon
linker was the best in the series. Studies have indicated that
small molecular inhibitors of the Bclꢀ2 family with longer,
a
IC50 values were the means of three independent experiꢀ
ments.
flexible hydrophobic groups have displayed higher potencies
4
than small, short, and rigid hydrophobic groups.28ꢀ29 Thereꢀ
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ACS Medicinal Chemistry Letters
fore, computational docking studies were carried out to underꢀ been shown to be one of the most precise MD scoring tools for
docking studies.32ꢀ33 The first model had a much lower score, ꢀ
24, than the second model, which had a score of ꢀ14 based on
the AmberScore. In contrast, the score of gossypol in the form
of (ꢀ)ꢀ16 was ꢀ14, as shown in Figure 4D; this score was lower
than the form that is presented in Figure 4C. The amino acid
ester chains that are shown in Figure 4D did not contribute
extra binding energy. The hydrophobic interaction could
therefore improve the interaction between the receptor and the
ligand, even when the H boning interaction was sacrificed.
These results are also consistent with the following findings:
from BIM to different types of peptides (stapled or unnatural),
the four hydrophobic residues h1 to h4 (ILIF) were widely
applied to design novel peptide inhibitors.34 The activity of
gossypol imines was improved by the hydrophobic interaction
that resulted from the introduction of the aliphatic side chains.
1
2
3
4
5
6
7
8
stand the binding model of the selected compounds to Bclꢀ2
family proteins and the SAR of the different substituents.
B
A
P3
P2
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D
C
In summary, a series of gossypol Schiff bases with linear
unnatural amino acids/methyl esters were demonstrated to be
potent inhibitors of antiapoptotic proteins. Compound 16 was
found to bind to Bclꢀ2 and Mclꢀ1 with IC50 values of 0.341
and 0.180 ꢁM, respectively. The in vitro cellular assay indiꢀ
cated that compound 16 potently inhibited the growth of canꢀ
cer cells. The cell growth inhibition data from the NIH were
consistent with our results, and the GI50 values of compound
16, the most potent compound, ranged from 0.1 to 1.7 ꢁM,
which were better than the values that were obtained with
gossypol and ABTꢀ199.
Figure 4. (A, B) The interaction models of compound (ꢀ)ꢀ16
with Bclꢀ2. A The binding site and orientation of the comꢀ
pound (represented by compound (ꢀ)ꢀ16) in the hydrophobic
groove P2 and P3 of Bclꢀ2. B The protein is rendered in a
surface model, while the compound is rendered in a stick
model and a van der Waals sphere (CPK) model. (C, D) Two
possible poses were assessed in the molecular docking study,
C with no ligandꢀprotein hydrogen bonding interaction and D
with a ligandꢀprotein hydrogen bonding interaction; Bclꢀ2 is
rendered as a cartoon, while the compound and the residues
around it are rendered as sticks (enlarged pictures, see Supꢀ
plemental Figure S11).
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and chemical data for final compounds,
molecular modeling, biochemical assays, and stability assays.
NCIꢀ60 one dose (10 ꢁM) screening data for compounds 1ꢀ3, 8, 9,
11, 14, 16ꢀ20, 23ꢀ26, and 28. Details of NCIꢀ60 information for
compounds ABTꢀ199, 14, 16, 17, and 18. (PDF). The Supporting
Information is available free of charge on the ACS Publications
website.
Molecular modeling. The enamine form of the gossypol
Schiff base analogue was confirmed by Xꢀray crystal strucꢀ
ture,4 so we used this form in our molecular model study. One
advantage of the gossypol enamine tautomer is that it is stabiꢀ
lized by intramolecular hydrogen bonding. Compound (ꢀ)ꢀ16
was chosen to be studied by molecular docking since the (ꢀ)ꢀ
gossypol conformation exhibits a better binding affinity than
the corresponding (+)ꢀconformation.
AUTHOR INFORMATION
Corresponding Author
As observed in Figures 4A and B, the aliphatic chain could
occupy the P2 and P3 pockets of the Bclꢀ2 protein without the
flexible loop domain in the model.30 The size and orientation
could fit the pocket quite well in the ligand CPK model. We
then analyzed the details of the compound (ꢀ)ꢀ16 and the proꢀ
tein interaction from the molecular modeling point of view.
*Eꢀmail: waw6@whu.edu.cn. Phone: 86ꢀ2768759586
*Eꢀmail: zhouhb@whu.edu.cn. Phone: 86ꢀ2768759586
ACKNOWLEDGMENTS
This project was supported by the National Natural Science
Foundation of China (21202125, 31300060, 81573279), the Huꢀ
bei Province Natural Science Foundation (2014CFB241), the
Innovation Seed Fund of Wuhan University School of Medicine,
and the Hubei Province Engineering and Technology Research
Center for Fluorinated Pharmaceuticals.
The major surface of the binding groove is a hydrophobic
region that includes A97, F101, M112, N149, and F150 and
the polar residue rich region that contains R104, D108, R143
and Y199. The helixes, which contain Y199 and D100, were
observed to have strong H bonding according to Xꢀray strucꢀ
ture data and formed the wide hydrophobic pocket P3 for the
peptide or ligand interaction. R143 was found to interact with
D of the BIM peptide through H bonding. However, in the
reported small molecule and Bclꢀ2 complex, hydrogen bond
interactions with the receptor were observed less often, except
in one example in which the ABTꢀ199 indole analogue interꢀ
acted with D100.31 In our cases, two possible binding models
are presented in Figures 4C and D; one model included the
most observed hydrophobic interaction without the proteinꢀ
ligand hydrogen bonding interaction, and the other model used
the hydrogen bonds between the hydroxyl group of gossypol
and the nitrogen of R143 as well as the oxygen of the carbonyl
group of gossypol and the nitrogen of R104. We used Amberꢀ
Score to evaluate the binding energy because AmberScore has
ABBREVIATIONS
SAR, structureꢀactivity relationships; PPIs, proteinꢀprotein interꢀ
actions; MTT, 3ꢀ(4,5ꢀdimethylꢀ2ꢀthiazolyl)ꢀ2,5ꢀdiphenylꢀ2ꢀHꢀ
tetrazolium bromide; CNS, central nervous system; Bclꢀ2, Bꢀcell
lymphoma 2; Mclꢀ1, myeloid cell leukemia 1; FP, fluorescence
polarization; CPK, CoreyꢀPaulingꢀKoltun.
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Gossypol with Hydrophobic Linear Esters Exhibits Enhanced Antiꢀtumor Activity as an Inhibitor of Antiapoptotic Proteins
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