Synthesis and biological activities of a new class of heat shock protein 90 inhibitors, designed by energy-based pharmacophore virtual screening

Article abstract of DOI:10.1021/jm4002023

The design through energy-based pharmacophore virtual screening has led to aminocyanopyridine derivatives as efficacious new inhibitors of Hsp90. The synthesized compounds showed a good affinity for the Hsp90 ATP binding site in the competitive binding as

Full text of DOI:10.1021/jm4002023

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Brief Article
Synthesis and Biological Activities of a New Class of Heat Shock Protein
90 Inhibitors, Designed by Energy-Based Pharmacophore Virtual Screening
Antonino Lauria, Ilenia Abbate, Carla Gentile, Francesca
Angileri, Annamaria Martorana, and Anna Maria Almerico
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4002023 • Publication Date (Web): 22 Mar 2013
Downloaded from http://pubs.acs.org on March 23, 2013
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Synthesis and Biological Activities of a New Class of Heat
Shock Protein 90 Inhibitors, Designed by Energy-Based
Pharmacophore Virtual Screening
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Antonino Lauria*, Ilenia Abbate, Carla Gentile, Francesca Angileri, Annamaria Martorana, Anna Maria
Almerico
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Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF”, Sezione di Chimica
Farmaceutica e Biologica - Università di Palermo, Via Archirafi 32, I-90123 Palermo, Italy.
KEYWORDS. Energy-Based Pharmacophore Virtual screening, aminocyanopyridines, Hsp90 inhibitors, competitive binding
assay.
ABSTRACT: The design through Energy-Based Pharmacophore Virtual screening has led to aminocyanopyridine derivatives as
efficacious new inhibitors of Hsp90. The synthesized compounds showed a good affinity for Hsp90 ATP binding site in the com-
petitive binding assay. Moreover, they showed an excellent antiproliferative activity against a large number of human tumor cell
lines. Further biological studies on the derivative with the higher EC50 confirmed its specific influence on the cellular pathways
involving Hsp90.
In the last decade the inhibition of a protein that regulates
multiple signal transduction pathways in tumor cells has be-
come an attractive target for cancer therapy. Heat Shock Pro-
tein 90 (Hsp90) is one of the most promising molecular tar-
gets for such an approach.1 In fact, it is an ubiquitous molec-
ular chaperone protein involved in the folding, reorganiza-
tion and assembling of many key mediators for signal trans-
duction. Hsp90 plays a crucial role in several biological pro-
cesses such as cell proliferation, differentiation, apoptosis or
stress response.2
Herein we present the synthesis and the biological activities
of a new class of Hsp90 inhibitors, designed through Energy-
Based Pharmacophore virtual screening.
DESIGN
The virtual screening protocol was started by selecting four
sets of known Hsp90 inhibitors, belonging to the oxazole,
naphthol, and pyrazole classes (Figure 1).19-21
R4
R2
R3
R3
R1
Inhibition of the ATPase activity of Hsp90 disrupts an ongo-
ing “folding” cycle, including multiple cochaperone proteins,
and in turn leads to the destabilization, ubiquitination, and
ultimately proteasomal degradation of client proteins.3 Many
Hsp90 client proteins are oncogenic proteins, overexpressed
in cancer, often in mutated forms, and are responsible for
unrestricted cancer cell proliferation and survival. The harsh
environmental conditions of the tumor microenvironment,
such as hypoxia and nutrient deficiency, contribute to the
destabilization of proteins and further escalate their depend-
ence on Hsp90, as supported by the higher Hsp90 levels and
the higher ATPase activity found in tumor cells.4,5 On the
other hand, the molecular signature of Hsp90 inhibition, well
established in cancer cell lines, denotes an increased expres-
sion of Hsp72 coupled with the depletion of oncogenic
Hsp90-client proteins.6,7
Many natural-occurring compounds, such as Geldanamycin
or Radicicol, act as Hsp90 inhibitors;8 however, to date, only
few compounds, as the 17-allylaminogeldanamycin (17-
AAG) and the synthetic derivative SNX-5422, exerted a po-
tent antitumor activity in a preclinical model and are current-
ly in clinical trials.9 Thus, it is easy to understand how much
important is the research of new Hsp90 inhibitors with high
selectivity and low toxicity.
R2O
HO
O
NH
O
OR1
HN
N
N
OH
Cl
NH
O2
S
Ar
R1
O
HO
N
R2
OH
N
OH
NH
HO
Figure 1. Pyrazole, naphthol, and oxazole Hsp90 inhibitors clas-
ses.
Each class was submitted to docking studies into the ATPase
binding site of Hsp90 (PDB id: 1UYF). The top scored
docked structure was selected for each class. Thus four ener-
gy-based pharmacophore hypotheses were built through E-
pharmacophore facility, as available in the MAESTRO pack-
age (Version 9.3, Schrödinger, LLC, New York, NY, 2012).
The four hypotheses were built in single mode, with seven
maximum numbers of selected features (Figure 2).
Likewise, a large in house database of heterocyclic com-
pounds (~100.000 structures) was docked with the same pa-
rameters used for the known Hsp90 inhibitor classes. The
best hits (10%) were matched with the four energy based
pharmacophore hypotheses.
In the last years, the in silico methodologies helped to design
compounds with biological activities having different biolog-
ical targets (proteins, DNA, etc.).10-16Also in the studies on
Hsp90, different approaches were used to investigate the
binding mode in the N-terminal domain.17,18
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acids different respect to the other ligands (Phe22 and Ile26).
This suggests a different binding mode if compared to that of
the known ligands.
1
2
3
4
5
Table 1. Analysis of critical amino acids involved in the binding
into Hsp90 ATPase site.
6
7
8
9
1h
ꢀ ꢀ ꢀ
ꢀ ꢀ
ꢀ ꢀ ꢀ ꢀ ꢀ
ꢀ ꢀ ꢀ ꢀ ꢀ
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b
ꢀ
1UYF
1YET
ꢀ
ꢀ
ꢀ ꢀ ꢀ ꢀ ꢀ ꢀ
ꢀ ꢀ ꢀ
ꢀ ꢀ ꢀ ꢀ
ꢀ
In fact, the analysis of the binding mode of 1h (Figure 3b)
evidences the occurrence of several interactions with the ac-
tive site, underlining the central role of some amino acid res-
idues such as Phe22, Ile26, Asn51, Met98, Leu103, Ala111,
Phe138, and Tyr 139, which provide direct non-covalent in-
teractions.
Figure 2. Selected energy-based pharmacophore hypotheses for
naphthol (a, 5 points), pyrazole (b, 7 points; d, 5 points), and
oxazole (c, 7 points) Hsp90 inhibitors classes, docked into Hsp90
ATPase binding site (PDB id: 1UYF).
SYNTHESIS
To optimize the yields of each derivative, the synthetic ap-
proach of the designed compounds was carried out through
two different pathways (Scheme 1).
The structures were ranked on the basis of the fitness scores
and by fixing four as the minimum number of pharmaco-
phore sites to be matched
The best scores were achieved for the class of 2-amino-3-
cyanopyridine 1 (Figure 3a), obtained through the matching
with the energy-based hypothesis built on the top scored
docked structure (Figure 2, b) belonging to pyrazole class.
Scheme 1. Synthetic steps leading to amino-cyanopyridines 1.
O
O
O
HN
R4
R3
CH3SO2Cl
Pyridine
H3CO2
S
+
O
R1
4a-k
R1
2
3
5a-k
NH2
HN
R2
SO2CH3
Method A
NH4OAc
NC
R2
CN
6
HN
R3
R4
NC
H3CO2
S
NH2
O
7
Method B
CN
CN
CN
NH4OAc
R1
R1
9
10
NC
CN
6
HN
R4
R2
R2
SO3CH3
R3
R4
R4
R3
R3
R2
CN
N
CN
H2
N
NH2
a: R1=Cl, R2=R3=R4=H
b: R1=R2=R4=H, R3=NMe2
c: R1=R3=R4=H, R2=F
R4
R1
N
d: R1=OMe, R2=R3=R4=H
e: R1=R4=H, R2=R3=OMe
f: R1=R2=R4=H, R3=OMe
1a-k
R1
R3
g:R1=R2=R4=H, R3=OH
h:R1=OH, R2=OMe, R3=R4=H
i: R1=R3=R4=H, R2=OMe
HN
R2
j: R1=R2=R4=H, R3=NEt2
k: R1=Br, R2=R3=R4=H
SO2CH3
SO2CH3
Figure 3. a: Designed amino-cyanopyridines 1a-k; b: Docking of
the best hit compound 1h into the Hsp90 ATPase site (PDB id:
1UYF).
N
8
H
The first synthetic route (method A), leading to the amino-
cyanopyridines 1, involved the introduction of a sulfamic
moiety on aminoacetophenone 2 to give 3, by reaction with
The analysis of the amino acids, involved in the binding to
Hsp90 ATPase site of 1h, the natural ligand of UYF, the gel-
danamycin (complexed in PDB id: 1YET), and the pyrazole
derivative b used in the selected pharmacophore hypothesis
(Figure 2) is reported in table 1.
It emerges that Asn51, Phe138, and Tyr139 are essential in
the interactions with the Hsp90 ATPase site.
Moreover, the designed ligand 1h, with respect to the other
inhibitors, binds the Hsp90 ATPase site through two amino
methanesulfonyl chloride.22 The
α,β-unsaturated ketones 4
were synthesized through a base catalyzed Claisen-Schmidt
condensation of 3 and aldehydes of type 5. The chalcones 4
were reacted with malononitrile 6, in the presence of ammo-
nium acetate and anhydrous ethanol as solvent, to afford 2-
amino-3-cyanopyridines 1a-k in good yields. The reaction
proceeds through Michael addition of the malononitrile anion
to the α,β-unsaturated ketone to give the adduct 7. The for-
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mation of the Schiff base, which rearranges to the enamine 8, respect to MG_MID values at GI50, TGI and LC50 levels
1
2
3
4
5
6
7
8
can be followed by cyclization and subsequent dehydration,
to give the 2-amino-3-cyanopyridines of type 1.
confirmed that all tested 2-amino-3-cyanopyridines present
both high antiproliferative activity and very low toxicity,
with LC50>10ꢀM for almost all the human tumor cell lines
(supporting information).
The second synthetic approach (method B), bypasses the
chalcone intermediates 4a-k exploiting a one-pot coupling
synthesis of four components. The mixture of aromatic alde-
hydes 5, acetophenone 3, malononitrile 6, and ammonium
acetate is dissolved in dry dioxane and refluxed overnight.
The reaction probably proceeds via intermediate 9, formed
from acetophenone 3 and ammonium acetate, which reacts
with alkylidenemalonitrile 10 (from condensation of aro-
matic aldehyde with malononitrile) to give 8. The above re-
ported cycloaddition, isomerization, and aromatization af-
forded the 2-amino-3-cyanopyridine 1. This approach al-
lowed the isolation of derivatives 1d-f,h,i in higher yields
and in short reaction time with respect to method A.
Table 3. Overview of the GI50, TGI, and LC50 values (
the selected compounds 1a,b,f,h.
ꢀM) for
Cmd
GI50
TGI
LC50
MG_MID
range
0.676
14.454
56.234
9
1a
5.623-0.240 51.286-2.239 >100-23.442
10
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MG_MID
range
0.055
0.955-0.013
0.117
8.710
89.125
>100-8.128
91.201
1b
1f
>100-0.056
11.749
MG_MID
range
1.230-0.016
0.186
>100-0.437
35.481
>100-19.498
93.325
MG_MID
range
1h
0.776-0.024
>100-0.076
>100-14.125
aGI50 is the concentration that inhibits 50% net cell growth. bTGI is
BIOLOGY
c
Hsp90 Competitive Binding Assay: The designed and syn-
thetized compounds were tested to verify the ability to bind
purified human Hsp90. The apparent binding affinities
(EC50) of the selected amino-cyanopyridine derivatives 1a-k
for Hsp90 were determined using a competition-binding as-
say (Table 2).
the concentration giving total growth inhibition. LC50 is the con-
centration leading to 50% net cell death. MG_MID (mean graph
midpoint) is the arithmetic mean value for all tested human cancer
cell lines for each separate experiment.
Table 4. DTP NCI human tumor cell lines panels overview
(GI50).
Panel cell line (ꢀM)
Breast Cancer
CNS Cancer
1a
0.46
0.51
1b
0.10
0.04
1f
0.14
0.08
1h
0.26
0.16
Table 2. Apparent binding affinities (EC50) of selected amino-
cyanopyridine derivatives 1a-k for Hsp90.
Colon Cancer
Leukemia
0.55
0.58
0.66
1.10
0.98
0.78
0.66
0.04
0.04
0.06
0.06
0.08
0.03
0.05
0.10
0.10
0.08
0.13
0.22
0.13
0.13
0.15
0.14
0.12
0.25
0.21
0.13
0.29
Cmd
1a
1b
R1
Cl
H
R2
H
H
F
R3
H
NMe2
H
R4 EC50 (nM)
H
H
H
H
H
H
H
H
H
H
H
950 ± 35
371 ± 28
>104
Melanoma
Non-Small Cell Lung
Ovarian Cancer
Prostate Cancer
Renal Cancer
1c
H
1d
OMe
H
H
H
634 ± 22
867 ± 27
541 ± 29
>104
1e
OMe OMe
1f
H
H
H
OMe
OH
H
1g
H
Analysing further the biological results, it is possible to evi-
dence the excellent activity of 1b against all cell lines of the
melanoma (MG_MID = 0.06 ꢀM), CNS (MG-MID = 0.04
ꢀM), prostate (MG_MID = 0.03 ꢀM) and colon (MG_MID =
0.04 ꢀM) cancer subpanels. Significant results were obtained
in the case of derivative 1f, especially against the cell lines
belonging to CNS, melanoma and colon subpanels, with
GI50 always below 10-1ꢀM. Further, it should be underlined
as the derivative 1h showed the lowest cytotoxicity, with the
largest number of human tumor cell lines (55 out 58) with
LC50>100 ꢀM. Moreover, 1h evidenced an excellent activi-
ty against the full melanoma panel (GI50=0.12 ꢀM) and the
NCI-H522 and SF-295 cell lines with GI50 values of 24 nM
and 43 nM, respectively.
Degradation of Hsp90 client proteins, up-regulation of
Hsp72, and cell cycle distribution: Among the amino-
cyanopyridines, the derivative1h, showing high antiprolifera-
tive activity, low cytotoxicity, and the best EC50 value (252
± 23 nM), was selected for further biological screenings to
elucidate its specific influence on the cellular pathways in-
volving Hsp90. In particular, to link the high binding affinity
of 1h to cellular activity, three in vitro assays were set up
using the human cancer cell line MCF7, an estrogen receptor
positive breast tumor cell line able to express high level of
Hsp90,23 and included in NCI DTP human tumor cell lines
panels.
1h
OH
H
OMe
OMe
H
252 ± 23
>104
1i
H
1j
H
NEt2
H
783 ± 19
>104
1k
Br
H
17-AAG
99 ± 17
The apparent binding affinity (EC50) was evaluated using a compe-
tition-binding assay as described in supporting information. Data are
the mean ± SD of n separate experiments carried out in triplicate.
Statistically significant vs control with p<0.005.
Seven out eleven of the tested compounds showed binding
affinity in the sub-micromolar concentration, with the high-
est activity for 1h.
Antiproliferative screening: The hit compounds 1a-k were
submitted to the Developmental Therapeutics Program of the
National Cancer Institute (DTP NCI) screening on the full
NCI DTP human tumor cell lines panels.
Four out eleven compounds passed the selection criteria of
the DTP NCI protocol and were tested twice to five-dose
concentration screening. All results are reported in support-
ing information and summarized in table 3 and table 4.
All the screened compounds showed anti-proliferative activi-
ty against the tested cell lines in the mid-nanomolar range
and derivative 1b was the most active (MG_MID = 55 nM).
The evaluation of the data for this class of compounds with
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The antiproliferative activity shown by derivative 1h could
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be due to the breakdown of multiple cell survival pathways,
as a consequence of destabilization of Hsp90 client proteins.
To test this hypothesis, we evaluated the effects of 1h on the
degradation of some typical Hsp90 client proteins (Cdk4, c-
Raf, and ErbB2) and on up-regulation of Hsp72 in MCF7
cells, by Western blotting analysis on total extracts following
a 24 h cells exposure to various concentrations of the tested
compound ranges from 2 to ½ of its GI50 value (274 nM,
supporting information). The derivative 1h causes dramatic
depletion of the examined client proteins, inducing a very
strong increase in the expression levels of the chaperone
Hsp72, as shown in figure 4.
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Figure 5. 1h induced cell cycle G2/M arrest in MCF-7 cells.
MCF-7 cells were exposed to 200 nM of 17-AAG or for 1h at 2,
1, and ½ of its GI50 value. Then the cells were collected and
DNA was stained with PI. DNA content was analysed by flow
cytometry for cell cycle distribution. Results shown are mean ±
SD for three independent experiments. * p<0.05; ** p<0.001 as
compared to controls
.
An increase in G2/M phase cells from 24% to 53% (p <
0.0001) was observed as a result of 24 h 1h cells exposure to
2 x GI50 (0.548 ꢀM). The marked accumulation in the G2/M
cell cycle phase was coupled to a decrease in the G0⁄G1
phase cells (65% vs. 40%, p < 0.05) more pronounced than
the decrease in the S phase (11% vs. 7%). When the same
treatment was performed for 48 h, a new population sub-
G0/G1 appeared with a parallel decrease of all the popula-
tions (data not shown).
In light of these findings, the cellular activities of the amino-
cyanopyridine derivative 1h are deeply interesting. The
EC50 values for their apparent binding affinities are compa-
rable to the GI50 value in MCF7 cells and at the similar na-
nomolar concentration 1h is very effective, causing Hsp90-
client protein depletion, Hsp72 up-regulation and G2/M cell
cycle arrest. Collectively our results show good correlation
between Hsp90 binding affinity and cellular activities, sug-
gesting that the effects of 1h on MCF7 cells were conse-
quence of their Hsp90 chaperone inhibitory properties.
Figure 4. Effects of treatment with 1h on Hsp90 client proteins
(Cdk4, ErbB2, c-Raf) levels and Hsp72 expression in MCF-7
cells. MCF-7 cells were exposed to 200 nM 17-AAG or 1h at 2, 1,
or ½ of its GI50 value. Total cellular extracts were obtained 24 h
after treatment.
ing.
β-actin is shown as a control for protein load-
In particular, 1h at 2-fold of its GI50 concentration showed
almost complete knockdown of Hsp90 client proteins. How-
ever, Cdk4 was consistently the most sensitive client protein,
as measured by both rapid disappearances, whereas ErbB-2
levels were less affected. The depletion was dose-dependent
and was achieved with potency similar to that of the refer-
ence compound (17-AAG) when tested in same experimental
conditions.
Antitumor effects can be achieved by cell death and/or cell
cycle arrest. Treatment with derivative 1h for 24 h did not
yield necrosis in MCF7 cells, as demonstrated by negative
response to LDH activity assay (data not shown). Hsp90 in-
hibition induces cell cycle arrest, but the stage of cell cycle
block by Hsp90 inhibitors is cell line- and tumor type-
dependent.6 G2/M arrest is seen in some epithelial cancer cell
lines, including MCF7 cells.24-27 To probe the mechanism of
cell growth inhibition by derivative 1h we examined its ef-
fects on cell-cycle distribution, by DNA staining with pro-
pidium iodide (PI). Cell cycle analysis was performed after
24 h of incubation with 1h derivative in order to detect the
shifts in cell cycle distribution before a significant amount of
cells underwent apoptosis (Figure 5). Untreated MCF7 cells
showed a normal diploid distribution presenting fast prolifer-
ation characteristics, with S + G2/M phase cells accounting
for about 35% of the total cells. Treatment with 1h for 24 h
arrests cell cycle at G2/M phase in a dose-dependent manner.
SUMMARY
The Energy-based pharmacophore virtual screening protocol
was used to identify the amino-cyanopyridine derivatives 1a-
k as a new class of Hsp90 inhibitors. The competitive bind-
ing assay confirmed the Hsp90 binding capability of the syn-
thetized compound at the sub-micromolar concentration.
The antiproliferative activity against a panel of nearly 60 cell
lines was evaluated by NCI in the DTP protocol. The tested
amino-cyanopyridines showed high potency and low toxici-
ty. The hit amino-cyanopyridine1h revealed responses con-
sistent with Hsp90 inhibition, including arrest of cell cycle at
G2/M phase and down-regulation of Hsp90 client protein
levels. In conclusion, the promising biological properties of
these compounds justify further investigation to elucidate the
detailed mechanism of action.
ASSOCIATED CONTENT
Supporting Information. Additional experimental procedures
and biological screening method, the yields, melting point, purity,
1H and 13C NMR data for all the compounds, and the detailed pro-
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says. This material is available free of charge via the Internet at
http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Author
* Phone number: +390916167210; Fax number: +390916230923
Email_Address: antonino.lauria@unipa.it
ABBREVIATIONS
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Hsp90,
Heat
Shock
Protein
90;
17-AAG,
17-
AllylAminoGeldanamycin; PI, Propidium Iodide; DTP, Devel-
opmental Therapeutics Program; NCI, National Cancer Institute;
GI50, Growth Inhibition of 50%; TGI, Total Growth Inhibition;
LC50, Letal Concentration 50%; MG_MID, Mean Graph MID-
point.
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