Identification of the first inhibitor of the GBP1:PIM1 interaction. Implications for the development of a new class of anticancer agents against paclitaxel resistant cancer cells

Article abstract of DOI:10.1021/jm5009902

Class III β-tubulin plays a prominent role in the development of drug resistance to paclitaxel by allowing the incorporation of the GBP1 GTPase into microtubules. Once in the cytoskeleton, GBP1 binds to prosurvival kinases such as PIM1 and initiates a sig

Full text of DOI:10.1021/jm5009902

Article
pubs.acs.org/jmc
Identification of the First Inhibitor of the GBP1:PIM1 Interaction.
Implications for the Development of a New Class of Anticancer
Agents against Paclitaxel Resistant Cancer Cells
∥ ,◆
Mirko Andreoli,†,◆ Marco Persico,‡,◆ Ajay Kumar,
Nausicaa Orteca,‡ Vineet Kumar,§
Antonella Pepe,§ Sakkarapalayam Mahalingam,○ Antonio E. Alegria,⊥ Lella Petrella,# Laima Sevciunaite,#
Alessia Camperchioli,# Marisa Mariani,† Antonio Di Dato,‡ Ettore Novellino,‡ Giovanni Scambia,▽
Sanjay V. Malhotra,*,§ Cristiano Ferlini,*,† and Caterina Fattorusso*,‡
†Danbury Hospital Research Institute, 24 Hospital Avenue, Danbury, Connecticut 06810, United States
‡Department of Pharmacy, University of Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
§Laboratory of Synthetic Chemistry, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, 1050
Boyles Street, Frederick, Maryland 21702, United States
∥
̀
School of Environmental Affairs, Universidad Metropolitana, Avenue Ana G. Mendez, San Juan, Puerto Rico PR 00928, United
States
⊥Department of Chemistry, University of Puerto Rico at Humacao, 100 Road 908, Humacao, Puerto Rico PR 00791, United States
#Laboratory of Molecular Oncology, Jean Paul IInd Research Foundation, Largo A. Gemelli 1, 86100 Campobasso, Italy
▽Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Roma, Italy
○Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States
S
* Supporting Information
ABSTRACT: Class III β-tubulin plays a prominent role in the
development of drug resistance to paclitaxel by allowing the
incorporation of the GBP1 GTPase into microtubules. Once in
the cytoskeleton, GBP1 binds to prosurvival kinases such as
PIM1 and initiates a signaling pathway that induces resistance
to paclitaxel. Therefore, the inhibition of the GBP1:PIM1
interaction could potentially revert resistance to paclitaxel. A
panel of 44 4-azapodophyllotoxin derivatives was screened in
the NCI-60 cell panel. The result is that 31 are active and the
comparative analysis demonstrated specific activity in paclitax-
el-resistant cells. Using surface plasmon resonance, we were able to prove that NSC756093 is a potent in vitro inhibitor of the
GBP1:PIM1 interaction and that this property is maintained in vivo in ovarian cancer cells resistant to paclitaxel. Through
bioinformatics, molecular modeling, and mutagenesis studies, we identified the putative NSC756093 binding site at the interface
between the helical and the LG domain of GBP1. According to our results by binding to this site, the NSC756093 compound is
able to stabilize a conformation of GBP1 not suitable for binding to PIM1.
tissue dependent and can be modified in response to
microenvironmental stimuli.2,3 In fact, the microenvironment
surrounding cancer cells can trigger the expression of specific
tubulin subtypes, such as βIII-tubulin, able to induce the
emergence of drug resistance. The overexpression of βIII-
tubulin isotype3−5 is an example of this survival mechanism,
which is not limited to this protein acting as a single driver of
the resistant phenotype but it involves a multimolecular
complex that is able to activate a cytoskeletal gateway for the
incorporation into microtubules of pro-survival kinases such as
PIM1 and NEK6.6,7 Crucial element of this gateway is the
INTRODUCTION
■
Drug resistance is the most relevant clinical problem in the
management of solid malignancies. In many cases, after an
initial response to treatment, cancer cells develop a resistant
phenotype which is ultimately responsible for the fatal
progression of the disease. Mechanisms that induce drug
resistance are complex and rely on multiple functional
pathways. Microtubule targeted agents (MTAs) are the
chemotherapeutics most commonly used for the management
of solid malignancies. Unfortunately, treatment with MTAs
eventually induces drug resistance. Microtubules are formed by
heterodimers of α/β tubulin isotypes.1 In mammals, multiple
genes encode for at least seven α and six β tubulin genes.
Microtubule composition of the different tubulin isotypes is
Received: April 9, 2014
Published: September 11, 2014
© 2014 American Chemical Society
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large-GTPase GBP1, whose incorporation into microtubules is
facilitated by the presence of βIII-tubulin.6,7 For this reason, it
would be desirable to control βIII-tubulin role by switching off
such a gateway of drug resistance and possibly restores
sensitivity to MTAs. The present study was aimed at identifying
specific inhibitors of such functional gateway, that is,
compounds able to interfere with the involved protein−protein
interaction network.
Scheme 1. Representative Example of the Synthesis of
Azapodophyllotoxin Derivatives
Over the last two decades, there has been significant interest
in developing therapeutics and chemical probes that inhibit
specific protein:protein interactions. Although developing small
molecules that are capable of occluding the large, often
relatively featureless protein:protein interaction interface has
been challenging, there are increasing numbers of examples of
small molecules that function in this manner with reasonable
potency.8−10 Natural products and their derivatives have
historically been invaluable as a source of therapeutic agents.
Among these podophyllotoxins are the first and perhaps the
best known example of the use of a lignan as a lead compound,
a cytotoxic aryltetralin lactone originally obtained from
Podophyllum peltatum L. and related species.11,12 Although
the semisynthetic derivatives etoposide, etoposide phosphate,
and teniposide are currently used in clinic for the treatment of a
variety of malignancies, there are side effects associated with the
use of these agents in clinic (including myelosuppression,
neutropenia, and nausea).13 To overcome these limitations, we
modified the original structure and prepared a small set of
azapodophyllotoxins (APTs) which showed reduced toxicity
and high cancer inhibitory activity.14,15 Motivated by these
results, we have now prepared a larger set of APTs and tested
for their potential to inhibit the activity of GBP1 function.
The data presented herein demonstrate that some APTs are
selectively more active in the cell lines most resistant to MTAs
like paclitaxel. The ability of the identified analogues in
preventing the formation of the GBP1:PIM1 complex was
tested, and compound NSC756093 was found active. The
molecular bases of this inhibition were investigated using
bioinformatics, molecular modeling, and mutagenesis studies,
and a putative binding site and mechanism of action was
hypothesized. Results obtained could potentially allow the
development of a new generation of compounds active on
MTA-resistant tumors.
in the NCI-60 panel of cell lines15 (see Experimental Section
for details; the methodology for NCI-60 cell line screening is
also described at http://dtp.nci.nih.gov/branches/btb/ivclsp.
html).
Briefly, the panel is organized into nine subpanels
representing diverse histologies: leukemia, melanoma, lung,
colon, kidney, ovary, breast, prostate, and central nervous
system. The results of the reference drugs carboplatin
(NSC241240), cisplatin (NSC119875), and paclitaxel
(NSC125793) are publicly available at http://dtp.nci.nih.gov.
Sensitivity of NCI-60 cells to paclitaxel is summarized in
Supporting Information, Figure 1SI. The screening was a two-
stage process, beginning with the evaluation of all compounds
against the 60 cell lines at a single dose of 10 μM. There were
31 compounds that exhibited significant growth inhibition and
they were evaluated against the NCI-60 at five concentration
levels to a final concentration of 0.1 nM (for more active
compounds, see Supporting Information, Figures 2SI−5SI).
The results were then analyzed according to the principles of
the COMPARE analysis.17 Activity of each compound
measured as GI50 is ranked with a Z-score within the cell
lines of the NCI-60 panel. Each Z-score is calculated with the
formula z = (x − μ/σ), where x is the GI50 in a given cell line
for a drug, μ is the average of the GI50 of the same drug within
the NCI-60 panel, and σ is the standard deviation. To perform
the COMPARE analysis, all these values were correlated with a
Spearman test with the Z-scores of another reference drug,
resulting in a ρ coefficient. A positive ρ value indicates an
overlapping mechanism of action with significant cross-
resistance/sensitivity as compared with the reference drug.
On the contrary, a negative ρ value signifies an increased
activity in the cells which are resistant to the reference drug.
As a first analysis, we analyzed the 31 active 4-APTs using as
references cisplatin (NSC119875) (Figure 1A), carboplatin
(NSC241240) (Figure 1B), and paclitaxel (NSC125973)
(Figure 1C). As expected, there was a strong correlation (ρ >
0.8) in the NCI-60 panel for carboplatin and cisplatin (Figure
1A,B) because the two drugs have a similar mechanism of
action.
RESULTS AND DISCUSSION
■
Chemistry. The OH-functionalized derivatives of 4-aza-2,3-
didehydropodophyllotoxin at the N atom in ring “‘C’”, i.e., the
N-(2-hydroxy-ethyl)-2,3-didehydroazapodophyllotoxins, were
synthesized in one step by simple refluxing in ethanol as
reported previously (Scheme 1).16
Hydroxy-functionalized 2,3-didehydroazapodophyllotoxin
derivatives were prepared, in two simple steps, by reacting
commercially available substituted anilines with 2-chloroethyl-
chloroformate in dry dichloromethane in the presence of
pyridine followed by reacting with KOH in ethanol. These
arylamino alcohols are not stable for long periods of time at
room temperature and, therefore, they were synthesized freshly
before use. The overall yields of products through our protocol
were in the 50−70% range. Structures were corroborated with
the help of 1H, COSY, 13C, NMR, as well as 1H NMR coupled
with deuterium exchange experiments, FTIR spectroscopy, and
HRMS.14
On the contrary, the 31 active 4-APT compounds exhibited a
significant negative correlation as compared with paclitaxel
(Figure 1C) but not with either cisplatin or carboplatin (Figure
1A,B). This finding suggests that 4-APTs are more active in
paclitaxel-resistant cells.
To better characterize these compounds we also ran a
COMPARE analysis for the 21 most active 4-APTs using the
mechanistic set of NCI-reference compounds18 (Supporting
Information, Figure 6SI). All the compounds capable of
Cell Based Assays. A panel of 44 4-azapodophyllotoxins
(4-APTs) whose structure is reported in Table 1 was screened
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Table 1. Structures of 4-Azapodophyllotoxins (4-APTs)
compd
R1
R2
R3
R4
NSC750210
NSC750716
NSC750717
NSC750211
NSC750718
NSC751499
NSC756083
NSC750212
NSC751500
NSC750719
NSC750213
NSC751501
NSC751502
NSC756084
NSC756085
NSC756086
NSC750720
NSC750721
NSC750722
NSC750723
NSC751503
NSC751504
NSC756087
NSC756088
NSC756089
NSC756090
NSC756091
NSC756092
NSC756093
NSC756094
NSC756095
NSC756097
NSC756098
NSC756099
NSC756100
NSC756102
NSC756103
NSC756104
NSC756105
NSC756106
NSC756108
NSC756110
NSC756111
NSC756112
6,7-OCH2O
6,7-OCH2O
6,7-OCH2O
6,7-OCH2O
6,7-OCH2O
6,7-OCH2O
6,7-OCH2O
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-(CH2)3
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6,7-O(CH2)2O
6-OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
H
H
H
H
OCH3
H
OH
OCH3
H
OCH3
H
Cl
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
H
H
H
H
OCH3
H
OH
OCH3
H
OCH3
H
Br
H
Cl
H
H
Cl
Cl
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
H
H
H
H
OCH3
H
OH
OCH3
H
OCH3
H
Br
H
Figure 1. Plot chart showing the results of Spearman correlation test
between the Z-score of the tested active 4-APTs with carboplatin (A),
cisplatin (B), and paclitaxel (C) in the NCI-60 cell lines. In x- and y-
axis, the p value of the Spearman correlation and ρ values are plotted,
respectively. Positive and negative ρ values indicate cross-sensitivity
and cross-resistance, respectively.
Cl
H
H
Cl
Cl
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
H
6-OCH3
6-OCH3
H
6-OCH3
H
H
6-OCH3
H
OCH3
H
H
Within the mechanistic set, the most represented drugs are
known MTAs such as quinazolinone,19 maytansine,20 colchi-
cines, and its analogue benzoyl-deacetyl-colchicine,21 vinblas-
tine,22 and baccatin III.23 In synthesis, although there was no a
single agent in the mechanistic set which exhibited significant
homology with all the 4-APTs, this analysis provided evidence
that at least some of the 4-APTs could interact with
cytoskeleton, with the compounds NSC756093 and
NSC756090 as representative of two clusters with the most
divergent activity (Supporting Information, Figure 6SI).
For the activities in paclitaxel-resistant cells we reported
above (Figure 1C), we chose to test 4-APTs for their ability to
inhibit the cytoskeletal gateway of drug resistance mediated by
GBP1.
We are aware that drug resistance in general, and paclitaxel-
resistance in particular, may be driven by multiple mechanisms.
In this study, we focused on a specific single mechanism of
resistance linked to the cytoskeletal gateway of drug-resistance
and GBP1 expression without assessing directly the antitubulin
activity of the whole set of compounds. Also no metabolic
analysis were conducted and we do not know if some of these
compounds may act in vivo as pro-drugs of other members of
6-OCH3
Br
H
6,7-OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
H
6,7-OCH3
6,7-OCH3
H
6,7-OCH3
H
H
6,7-OCH3
H
OCH3
H
H
6,7-OCH3
Br
H
6,7-OCH3
Cl
H
H
6,7-OCH3
Cl
Cl
H
6-CH2CH3
6-CH2CH3
6-CH2CH3
6-CH2CH3
6-CH2CH3
OCH3
H
OCH3
H
OCH3
H
Br
H
H
Cl
H
H
Cl
Cl
H
yielding a ρ value higher than 0.55 were selected to identify
drugs sharing similar mechanisms of action. Either the parent
podophyllotoxin (NSC24818) or etoposide (NSC141540) did
not have a ρ value higher than 0.55. Cluster analysis was applied
to identify groups of 4-APT compounds sharing most similar
mechanisms of action (Supporting Information, Figure 6SI).
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the same family. According to these limits, we cannot conclude
that the increased effect we noticed in paclitaxel-resistant cells
for 4-APT is driven by a single mechanism of action.
GBP1:PIM1 Interaction. One of the mechanisms of
paclitaxel resistance with strong support in clinical studies is
represented by class III β-tubulin overexpression.5 βIII-Tubulin
is not suitable for in vitro screening because its active
conformation require posttranscriptional changes not obtain-
able with the current technology of production of recombinant
protein. Recently, we discovered that βIII-tubulin is capable
enhancing incorporation of the GBP1 GTPase into the
cytoskeleton in stressing conditions.6 GBP1 then is capable
to bind a panel of prosurvival kinases like PIM1, thus recruiting
them into cytoskeleton and prolonging their activity.6 At
variance of βIII-tubulin, GBP1 and PIM1 can be expressed in
vitro and used to screen compounds capable of disrupting such
a protein:protein interaction. To develop a quantitative system
of screening, the kinetics of the GBP1:PIM1 interaction was
monitored with surface plasmon resonance technology (SPR).
PIM1 was immobilized on the biochip as ligand (50 μg/mL).
In a parallel flow path, carbonic anhydrase (CA) was
immobilized on the chip as negative control. The binding of
GBP1 was tested on both targets, flowing the protein on the
chip surface at different concentrations (8.25−140 nM). CA
was also used as analyte control in the same range of [GBP1],
thereby demonstrating that GBP1:PIM1 interaction is not
dependent on any aspecific binding of PIM1 (Supporting
Information, Figure 7SI). We repeated the experiment in
duplicate, and we tested the interaction also in the presence of
GDP and the GTP nonhydrolyzable analogue GppNHp (1 μM
in PBST). The signal generated from the protein:protein
interaction was dose-dependent in the range 8.25−140 nM, and
the kinetic analysis revealed a KD value of 38 14 nM. No
binding was detectable using CA as ligand or analyte. These
findings show that SPR technology is able to detect the specific
GBP1:PIM1 interaction. We used the SPR method to test a
potential inhibition of the GBP1:PIM1 interaction. All the 44 4-
APTs were screened in PBST and dimethyl sulfoxide (DMSO)
0.2% v/v with GBP1 280 nM in PBST solution in duplicate
experiments. The negative control of each experiment was
represented by CA that was flowed on the chip surface in
parallel with the test compounds. All the analyses were
performed in two independent channels of the biochip.
There were 32 compounds that were completely inactive as
inhibitor of the GBP1:PIM1 interaction. There were 11
compounds that were capable of producing an inhibition of
the binding around 10−20%, while only NSC756093 was able
to inhibit 65% of the GBP1:PIM1 interaction (Figure 2A).
The inhibition of the GBP1:PIM1 interaction by
NSC756093 was dose-dependent (Figure 2B) and statistically
significant as compared with the control without drug (p <
0.001, Anova). Representative biosensograms are shown in
Figure 2C (NSC756093) and D (NSC756090). In all the
experiments, CA was used as negative control and the
maximum signal was calculated with GBP1 without inhibitors.
This analysis demonstrated that NSC756093 is a specific
inhibitor of the GBP1:PIM1 interaction in a pure in vitro
system using recombinant proteins. Importantly, we can
exclude that PIM1 is the target of the tested compounds
because we did not notice any change of the baseline for any of
the tested compound up to the maximum tested concentration
in the absence of GBP1(Supporting Information, Figure 8SI).
Figure 2. (A) Bar chart showing the % of inhibition of the 12
compounds capable of producing an inhibition of the GBP1:PIM1
interaction >10% at a drug concentration of 100 nM. The maximum
signal (100%) was obtained in the absence of any compound. Bar and
error bars refer to mean and SD of duplicated experiments. (B) Line
chart reporting the dose dependent growth inhibition of NSC756093
and NSC756090. Each dot and bar refer to mean and SD of duplicated
experiments. (C,D) Representative biosensograms for NSC756093
(C) and NSC756090 (D). CA (dotted line) represented the negative
control while GBP1 (red line) the maximum signal without inhibitor.
To confirm the activity of the drug in cell lines, we assessed
the ability of NSC756093 to inhibit the GBP1:PIM1 interaction
in SKOV3 cells. The cells were treated for 3 h using 100 nM of
the drug, then the cells were scraped and the pellet was used for
coimmunoprecipitation of PIM1 (bait) with GBP1. The results
demonstrated that treatment with NSC756093 inhibits the
interaction also in vitro, while treatment with the vehicle
DMSO or the inactive compound (NSC756090) did not yield
any modulation of the GBP1:PIM1 interaction (Figure 3).
To further substantiate the link between activity of
NSC756093 and the expression of GBP1 and PIM1, we
downloaded the gene expression data of the NCI-60 cells which
are publicly available at http://dtp.nci.nih.gov. Therefore, we
sorted the cell lines according to the GI50 value. The first and
the third tertile were classified as NSC756093-sensitive and
NSC756093-resistant cells. A significant difference was noticed
between NSC756093-sensitive and NSC756093-resistant cells.
In fact expression of GBP1 and PIM1 conferred increase
sensitivity to NSC756093 (Figure 4).
Identification of NSC756093 Putative Binding Site. To
identify a putative binding site for NSC756093, first, a
structural and bioinformatic analysis was performed on the X-
ray structures of: (i) etoposide in complex with topoisomerase
II (TopoIIβ) and DNA (PDB ID: 3QX3) and (ii)
podophyllotoxin in complex with tubulin (PDB ID: 1SA1).
Indeed, podophyllotoxin (1), etoposide (2), and NSC756093
share a common structural skeleton, composed by the
polycyclic system B−D and the phenyl ring E, but presenting
different substituents (Table 2).
The rationale applied to our analysis is that similar ligand
substructures recognize homologous structural elements of the
protein target, thus, if two binding pockets of different proteins
share a common motif, it is likely that ligands or ligand
fragments that bind within one binding pocket will also be
recognized in the respective part of the other binding
pocket.24−27
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domain (WHD) and the topoisomerase-primase domain
(TOPRIM) of TopoIIβ for 2. In the case of 2, DNA is also
involved (Figure 6A and Supporting Information, Figure 9SI).
On the other hand, the results obtained by the eukaryotic
linear motifs resource for functional sites in proteins28 (http://
elm.eu.org) showed that the binding sites of the two complexes
include several consensus sequences of functional motifs
(Figures 6B, 7B, and Supporting Information, Figures 9SI,
and 10SI), and that the E ring of the two ligands interacts with
the consensus sequence of the same protein motifs in both
proteins (colored in yellow in Figures 6B and 7B).
The bioinformatics analysis applied on the structures of
TopoIIβ and tubulin, was successively performed on PIM1 and
GBP1 X-ray structures. In agreement with the results of the
SPR experiments, it resulted that only GBP1 contained a three-
dimensional combination of consensus sequence of functional
motifs, similar to those found in the active sites of TopoIIβ and
tubulin (Supporting Information, Figure 11SI). Importantly,
the identified sequences located at the interface of the two main
GBP1 structural domains (namely LG and helical). Thus, a
putative binding site of NSC756093 on GBP1 has emerged
from our analysis (Figure 8A).
Docking Studies. By using the identified putative binding
site as the NSC756093 starting position, a dynamic docking
study was performed on the NSC756093/GBP1 complex,
defined as binding domain the whole GBP1 structure. To test
the thermodynamic stability of the resulting docked complexes,
these latter were also subjected to a molecular dynamics
simulated annealing protocol (see Experimental Section for
details). The resulting complexes were ranked by their
conformational energy values, and the lowest energy complex
was chosen as the more representative one (Supporting
Information, Figure 11SI, Table 1SI). The quality of the
obtained docked complex was assessed using Procheck (http://
www.ebi.ac.uk/thornton-srv/software/PROCHECK/) and re-
sulted comparable to that obtained for human GBP1 X-ray
structure (PDB ID 1DG3; Ramachandran plots, Chi1−Chi2
plots, main chain parameters, side chain parameters are
reported in the Supporting Information).
Figure 3. (A) Representative coimmunoprecipitation (IP) of PIM1
and GBP1 in SKOV3 cell line treated with NSC756093 (3 h at 100
nM). The signal was revealed using anti-GBP1 antibody detected as a
specific band of 67 kD. Lane 1: input of SKOV3 lysate. Lane 2: flow
through co-IP. Lane 3: flow through control. Lane 4: co-IP.
PIM1:GBP1 (antibody anti-PIM1). Lane 5: co-IP. Negative control
(antibody anti-IGg). The presence of the signal in lane 4 means no
interference in the GBP1:PIM1 binding in DMSO and the inactive
NSC756090; the absence of detectable signal in the presence of
NSC756093 means that the compound is able of inhibiting the
GBP1:PIM1 interaction. (B) Bar chart showing the densitometric
analysis of the experiment shown in A, performed in two independent
experiments. A significant (double asterisks = p < 0.001, Anova)
suppression of the co-IP was noticed in both experiments only with
the compound NSC756093.
Docking results supported our binding hypothesis. In the
calculated complex, NSC756093 is bound at the interface
between the LG domain and the helical domain (Figure 8).
It is noteworthy that the subset constituted by all GBP1
residues within 5 Å from any given NSC756093 atom (namely
the binding site) contained the consensus sequences of protein
motifs also found in podophyllotoxin/tubulin binding site, one
of which is also present in the etoposide/TopoIIβ binding site
(Figure 9 vs Figures 6 and 7).
In particular, the E ring of the three ligands interacts with the
consensus sequence of the same functional motif in all binding
sites, colored in yellow in Figures 6, 7, and 9. Similar
interactions are established by the polycyclic systems of 1
and NSC756093 with tubulin and GBP1, respectively; indeed,
the conserved D ring targets the consensus sequence of same
functional motif in both binding sites, colored in blue in Figures
7 and 9. In addition, also the smaller and quite similar R5s of 1
and NSC756093 (Table 2) target a consensus sequence motif
present in both binding sites, colored in magenta in Figures 7
and 9. On the contrary specific ligand substructures such as the
R5 substituent of 2 (Table 2), represented by a bulky glycosidic
moiety, as well as, the A ring of 1, replaced by a 6-OCH3 group
in NSC756093, target protein motifs which do not present
shared consensus sequences with the other two complexes.
Figure 4. Analysis of PIM1/GBP1 gene expression and NSC756093
sensitivity. Median gene expression values (Z-score) for GBP1 and
PIM1 were downloaded and combined in an index of expression.
Green line corresponds to the average of each group. This index was
significantly lower in the cells resistant to NSC756093, as compared
with the NSC756093-sensitive cells (p = 0.01, t test).
Thus, we superimposed the two X-ray complexes by fitting
the common polycyclic system of the ligands (Figure 5).
The first observation resulting from the overall structural
comparison of 1 and 2 in complex with their molecular targets
is that, in both cases, the small ligand is engaged in molecular
interactions at the interface between two protein domains, such
as the α and β monomers of tubulin for 1 and the winged helix
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Table 2. Structures of Podophyllotoxin (1), Etoposide (2), and NSC756093
Structure−Activity Relationships. NSC756093 is the steri-
cally less hindered structure of the series, all other analogues
presenting bulkier substituents at B and E rings (Table 1), and
it is also the only tested 4-APT which resulted in being able to
significantly inhibit GBP1:PIM1 interaction (Figure 2). This
indicates that bulkier substituents at R1−R4 are not tolerated by
the GBP1 binding pocket. Accordingly, in the calculated
NSC756093/GBP1 complex, NSC756093 is almost completely
embedded in a binding pocket within GBP1 structure, with
only the D ring and the “northern” part of the structure,
including the R5 substituent (Table 2), partially exposed to the
solvent (Figure 10).
To rationalize the inactivity of the other tested 4-APTs and
at the same time validate our NSC756093/GBP1 interaction
model, compounds bearing minimal extra-volume at R1−R4 as
compared with NSC756093 (i.e., NSC756094, NSC756095,
NSC756100, and NSC756108; Table 1) were subjected to an
in-depth conformational analysis and the conformers within 5
kcal/mol from the global energy minimum (GM) were placed
in the putative GBP1 binding site by superimposing their B−D
rings on those of NSC756093 in the calculated complex with
GBP1 (see Experimental Section for details). First, all possible
orientations of the substituents were taken in to account and all
possible steric clashes with the protein were analyzed
(Supporting Information, Figure 12SI). It resulted that the
substitution of the E ring at meta or para positions,
(NSC756095 and NSC756094), or the introduction of an
additional methoxy group at position 7 of the B ring
(NSC756100), were not tolerated due to steric clashes with
specific residues present in the corresponding GBP1 binding
cleft (Supporting Information, Figure 12SIA,B). Also, the
replacement of the methoxy group at position 6 with an ethyl
chain, as in compound NSC756108, produced a steric clash
with the protein (Supporting Information, Figure 12SIC).
Indeed, even if at a first look the ethyl chain and the methoxy
group could occupy a similar molecular volume, nevertheless,
they are differently oriented with respect to the polycyclic
system plane. The methoxy group, due to oxygen conjugation
with the aromatic ring, lays on the same plane of the polycyclic
system (sp2 like geometry). On the contrary, the ethyl chain
projects out of the ring plane (sp3 geometry). In agreement
with the observed steric clashes, the subsequent full energy
minimization of all possible complexes of GBP1 with the
conformers of NSC756094, NSC756095, NSC756100, and
NSC756108, did not provide any solution with acceptable
ligand conformation (i.e., ΔE from the global energy minimum
Figure 5. Superimposition of the X-ray structure of the etoposide/
TopoIIβ complex (orange; PDB ID: 3QX3) on the X-ray structure of
podophyllotoxin/tubulin complex (green; PDB ID: 1SA1) by fitting
the A−D rings of the ligands. The yellow circle highlights the position
of the fitted ligands. Proteins are displayed as ribbons, ligands are
displayed as CPK. DNA is omitted, for clarity of presentation.
Taken together, these results indicate that the molecular
skeleton composed by the fused rings B−D and the pendant
phenyl ring E bind at the interface of two protein domains. This
interface includes the consensus sequences of protein func-
tional motifs, and the different selectivity toward a specific
target depends on the introduced substituents. In this view,
even minor modifications in the structure of the ligand may
drive the binding to a different combination of protein motifs,
i.e., to a different protein target. On the other hand, the same
ligand could recognize a similar pattern of protein motifs at
different protein targets. In this regard, it is noteworthy that
etoposide is reported to be able to inhibit TopoIIβ also by
binding at the ATPase domain of the protein.29 These
observations are in agreement with the results of the biological
investigation reported in the previous section. Indeed, although
it resulted that some APTs could interact with cytoskeleton,
only NSC756093 inhibited the GBP1:PIM1 interaction in vitro
as well as in paclitaxel resistant cells. The structural features
responsible for such a selectivity are discussed in the next
paragraph.
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Figure 6. (A) Overview of the etoposide binding site in TopoIIβ/DNA complex (PDB ID: 3QX3). TopoIIβ (white) and DNA (violet) are displayed
as ribbons. Etoposide is displayed in stick and colored by atom type (C = green, O = red, H = white). (B) Detailed view of protein motifs involved in
etoposide binding site. Key interacting residues are displayed in stick and underlined in the sequence; key consensus sequence residues are in bold.
Etoposide is displayed as ball and stick and colored by atom type. Protein motifs involved in the binding site are displayed as ribbons and colored:
DOC_WW_Pin1_4 motif (orange); MOD_GlcNHglycan and MOD_GSK3_1 motifs (yellow). Hydrogens are omitted for sake of clarity.
Figure 7. (A) Overview of podophyllotoxin binding site on α/β tubulin heterodimer (PDB ID 1SA1). Tubulin is displayed as ribbons and colored in
white. Podophyllotoxin and α tubulin GTP nucleotide are displayed in stick and colored by atom type (C = green, O = red, N = blue, P = magenta,
H = white). (B) Detailed view of protein motifs involved in podophyllotoxin binding site. Key interacting residues are displayed in stick and
underlined in the sequence; key consensus sequence residues are in bold. Podophyllotoxin is displayed as ball and stick and colored by atom type.
Protein motifs involved in this binding site are displayed as ribbons and colored: LIG_FHA_1 motif (magenta); CLV_NDR_NDR_1 motif (blue);
CLV_PCSK_SKI1_1 motif (cyan); MOD_GlcNHglycan and MOD_GSK3_1 motifs (yellow). Hydrogens are omitted for sake of clarity.
<10 kcal/mol). These results account for the observed SARs,
thus supporting the binding of NSC756093 to the hypothesized
site of GBP1.
(helical domain), was observed. Thus, by affecting the
interactions between the LG and the helical domains of
GBP1, NSC756093 could drive the formation of GBP1
conformations not compatible with PIM1 binding. Although
results obtained with the GBP1 mutants support our allosteric
hypothesis (see next paragraph), a limitation of our study
consists in the fact that the calculated GBP1 conformational
change is not proved by any experimental data. Future studies
will address this issue with the use of appropriate experimental
techniques (e.g., circular dichroism).
GBP1Mutants. On the basis of the results obtained by our
molecular modeling studies, to gain insights into the structure
of the NSC756093 inhibition of GBP1:PIM1 interaction, a
series of mutants was designed. A panel of mutants with single
or double deletion of the coding sequence of human GBP1 was
obtained. Eight mutants were prepared as described in Table 3.
In conclusion, according to our molecular modeling studies,
NSC756093 binds at the interface between the helical domain
and the LG domain of GBP1 (Figure 8). This binding could
affect all key protein domain interactions for the allosteric
modulation of GBP1 conformations/functions. Indeed, the
conformational equilibrium between the helical and the LG
domain regulates GBP1 catalytic activity and oligomerization
ability.30 In other GTPases (e.g., Ras, Rho) this regulation is
performed by partner proteins (e.g., GAP and GEF).31 Analysis
of the overall GBP1 conformational changes caused by the
dynamic docking procedure (Figure 8), due to the disruption of
key ionic interactions, showing a significant movement of the
α4′ helix (LG domain) with respect to the α12 and α13 helices
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Figure 8. Overall view of NSC756093/GBP1 starting (A), docked (B), and annealed (C) complexes. GBP1 structure is displayed as ribbons, where
the LG domain is in cyan, the connecting region in blue, the helical domain in red and α12/α13 in orange. NSC756093 is displayed as CPK and
colored by atom type (C = green, O = red, N = blue, H = white). Key interacting residues between LG domain and α12/α13 are displayed in stick,
colored by atom type and labeled.
Figure 9. (A) Overview of NSC756093 binding site in GBP1 (white) in the final annealed complex. GBP1 is displayed as ribbons. NSC756093 is
displayed in stick and colored by atom type (C = green, O = red, N = blue, H = white). (B) Detailed view of protein motifs involved in NSC756093
binding site. Key interacting residues are displayed in stick and underlined in the sequence; key consensus sequence residues are in bold.
NSC756093 is displayed as ball and stick and colored by atom type. Protein motifs involved in this binding site are displayed as ribbons and colored:
LIG_FHA_1 motif (magenta); CLV_NDR_NDR_1 motif (blue); MOD_GlcNHglycan motif (yellow). Hydrogens are omitted for sake of clarity.
The spatial positioning of mutated residues within GBP1
structure is depicted in Supporting Information, Figure 13SI.
The mutants were designed to mutate residues of the LG
domain by playing a key role in the allosteric regulation of
GBP1 structure/function, and binding experiments were
performed using GDP and the GTP nonhydrolyzable analogue
GppNHp (1 μM in PBST).
A complete absence of interaction was noticed only for the
mutant R227E/K228E in both GTP and GDP conditions.
R227 and K228 residues were reported to play a crucial role in
GBP1 catalytic activity and homo-oligomerization ability.32 The
positively charged side chains of R227 and K228 (α4′ helix, LG
domain) are coupled with four negatively charged glutamate
residues present on α12 and α13 helices (i.e., E556, E563,
E568, and E575) (Figure 8A). As a consequence, when R227
and K228, are replaced with two negatively charged glutamate
residues, as in the R227E/K228E GBP1 mutant, a complete
decoupling of the LG and the helical domains is induced. A
double mutation of the same residues (R227A/K228A), but
with the hydrophobic residue alanine, is not able to generate
the same disruptive effect on the protein:protein interaction.
Importantly, according to our docking results, NSC756093
also affected the ionic interactions involving K227 and R228
(Figure 8) and induced a dramatic change in the positioning of
the α4′ helix (LG domain) and the α12 and α13 helices (helical
domain) (Figure 8). Thus, these findings support the
hypothesis that the cleft in which NSC756093 binds is an
allosteric site capable of controlling the active conformation of
GBP1 and its ability to interact with partners such as PIM1.
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acid, substituted aniline, and aromatic aldehyde was dissolved in the
minimum volume of ethanol. The reaction mixture was refluxed for
30−90 min. After cooling, the precipitate was filtered off, washed with
minimal cold ethanol, and then recrystallized from ethanol to afford
the desired compound. Characterization data (NMR, HRMS etc.) of
compounds NSC750210−750213, 750716−750723, and 751499−
751504 have been published earlier.13,14 We found that the synthesis
of products where R1 is methoxy at the meta position produced
regioisomeric products with some aromatic aldehydes as observed
from NMR data, while all remaining aryl amino alcohols produced
only one regioisomeric expected product.
5-(2-Hydroxyethyl)-9-(3-chlorophenyl)-6,9-dihydro-[1,3]dioxolo-
[4,5-g]furo[3,4-b]quinolin-8(5H)-one (NSC756083). Yield: 78%. 1H
Figure 10. (A) NSC756093 bound to GBP1. The ligand is displayed
as CPK and colored by atom type (C = green, O = red, N = blue, H =
white), the solvent accessible surface of the protein is displayed as solid
(transparency = 50%) and colored in white, protein backbone is
displayed as white ribbon. (B) solvent accessible surface of
NSC756093 in complex with GBP1.
Table 3. Kinetic Analysis of a Panel of GBP1 Mutants for the
GBP1:PIM1 Interaction Assessed with SPR Technology
NMR (DMSO-d6, 400 MHz): δ 3.56−3.86 (m, 4H), 4.96 (t, 1H),
5.03−5.05 (d, J = 8 Hz, 2H), 5.11 (s, 1H), 5.86−5.92 (m, 2H), 6.69 (s,
1H), 6.94 (s, 1H), 7.45−7.53 (m, 1H), 7.58−7.63 (m, 1H), 7.95−7.80
(m, 1H), 8.03−8.05 (m, 1H). 13C NMR (DMSO-d6, 100 MHz): δ
39.36, 48.22, 57.89, 65.95, 94.07, 96.51, 101.50, 110.12, 117.83,
121.48, 121.90, 129.93, 131.25, 134.52, 143.47, 147.27, 147.79, 148.84,
160.81, 172.07. LC-MS (ESI-TOF): m/z 386.0770 ([C20H16 Cl NO5
+ H]+ calcd 386.0790).
protein/
mutants
KD (nM) in PBST (1 μM
KD (nM) in PBST (1 μM
a
GppNHp )
GDP)
GBP1
67 20
>10000
30 10
>10000
R227E/
K228E
R48A
290 21
50 10
21 0.1
26 11
4-(2-Hydroxyethyl)-10-(3-bromophenyl)-3,4,6,7,8,10-hexahydro-
1H-cyclopenta[g]furo[3,4-b]quinolin-1-one (NSC756084). Yield:
K51A
D184A
Q72A
D103N
80
62
21
5
2
1
77
3
161 17
15 0.1
R227A/
R228A
137 14
61
4
a
Guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate, non-
hydrolyzable analogue of GTP.
1
CONCLUSION
70%. H NMR (DMSO-d6, 400 MHz): δ 1.90−2.00 (m, 2H), 2.61−
■
2.77 (m, 2H), 2.80−2.84 (t, 2H), 3.65−3.78 (m, 3H), 3.80−3.92 (m,
1H), 4.98−5.06 (m, 2H), 5.10−5.17 (m, 2H), 6.92 (s, 1H), 7.11 (s,
1H), 7.17−7.25 (m, 2H), 7.30−7.38 (m, 1H), 7.42 (s, 1H). 13C NMR
(DMSO-d6, 100 MHz): δ 25.13, 31.46, 32.33, 39.41, 48.23, 57.70,
65.84, 94.81, 110.17, 121.71, 123.84, 126.58, 126.87, 129.13, 130.19,
130.51, 134.81, 139.03, 143.73, 149.91, 160.82. LC-MS (ESI-TOF):
m/z 426.0665 ([C22H20 Br NO3 + H]+ calcd 426.0699).
We have demonstrated that 4-APTs are more active in cancer
cell lines less responsive to paclitaxel. In particular, compound
NSC756093 is able to modulate the GBP1:PIM1 interaction,
and this is the first study reporting the inhibition of such a
system. The results of our investigation on the NSC756093
mechanism of action provide useful insights on the design of
novel inhibitors of the GBP1:PIM1 interaction with higher
specificity. After this additional step of optimization, it will be
possible to move the experimentation to preclinical models of
taxane resistance.
4-(2-Hydroxyethyl)-10-(3-chlorophenyl)-3,4,6,7,8,10-hexahydro-
1H-cyclopenta[g]furo[3,4-b]quinolin-1-one (NSC756085). Yield:
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a MEL-TEMP
instrument and are uncorrected. IR spectra were recorded on a
PerkinElmer Spectrum 100 FTIR spectrometer on ATS mode. H,
■
1
COSY, 13C, DEPT45, DEPT90, DEPT135, and HETCOR NMR
spectra were measured on a Bruker 400 Ultra Shield spectrometer
using DMSO-d6 as solvent. All chemical shifts are reported in parts per
million relative to tetramethylsilane. Coupling constants (J) are
reported in Hz. The LC-MS data was taken on an Agilent 1200 series
system with Agilent 6210 time-of-flight mass detector. Absorption
spectra were obtained in DMSO, using DMSO as blank, with an
Agilent 8453 absorption spectrometer. The purities of all of the tested
compounds were >95% as estimated by HPLC.
1
79%. H NMR (DMSO-d6, 400 MHz): δ 1.90−2.00 (m, 2H), 2.61−
2.77 (m, 2H), 2.80−2.84 (t, 2H), 3.65−3.80 (m, 3H), 3.82−3.92 (m,
1H), 4.98−5.08 (m, 2H), 5.09−5.19 (m, 2H), 6.93 (s, 1H), 7.11 (s,
1H), 7.17−7.23 (m, 2H), 7.25−7.31 (m, 2H). 13C NMR (DMSO-d6,
100 MHz): δ 25.13, 31.46, 32.33, 39.43, 47.89, 57.70, 65.85, 94.80,
110.16, 123.84, 126.23, 126.44, 126.57, 127.37, 130.16, 132.96, 134.81,
139.02, 143.72, 149.65, 160.82. LC-MS (ESI-TOF): m/z 382.1206
([C22H20 Cl NO3 + H]+ calcd 382.1204).
General Synthesis of 4-Aza-2,3-didehydropodophyllotoxin
Derivatives. These derivatives were synthesized by following
previously reported method.13,14 An equimolar mixture of tetronic
4-(2-Hydroxyethyl)-10-(3,4-dichlorophenyl)-3,4,6,7,8,10-hexahy-
dro-1H-cyclopenta[g]furo[3,4-b]quinolin-1-one (NSC756086). Yield:
1
58%. H NMR (DMSO-d6, 400 MHz): δ 1.90−2.01 (m, 2H), 2.65−
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118.44, 118.78, 127.98, 128.96, 129.41, 130.31, 130.50, 130.90, 139.57,
142.73, 147.83, 160.75, 172.12. LC-MS (ESI-TOF): m/z 456.0373
([C21H17 Cl2 NO5 + Na]+ calcd 456.0376).
4-(2-Hydroxyethyl)-6-methoxy-9-(3,4,5-trimethoxyphenyl)-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756090). Yield: 54%. H
2.75 (m, 2H), 2.80−2.84 (t, 2H), 3.65−3.78 (m, 3H), 3.85−3.90 (m,
1H), 5.00−5.06 (m, 2H), 5.10−5.16 (m, 2H), 6.92 (s, 1H), 7.12 (s,
1H), 7.19−7.22 (m, 1H), 7.49−7.51 (m, 2H). 13C NMR (DMSO-d6,
100 MHz): δ 25.13, 31.44, 32.33, 39.43, 47.91, 57.67, 65.92, 94.46,
110.27, 123.46, 126.57, 128.10, 128.91, 129.49, 130.51, 130.87, 134.77,
139.14, 143.89, 148.16, 160.95, 172.09. LC-MS (ESI-TOF): m/z
416.0803 ([C22H19 Cl2 NO3 + H]+ calcd 416.0815).
NMR (DMSO-d6, 400 MHz): δ 3.58 (s, 3H), 3.65−3.72 (m, 10H),
3.74 (s, 3H), 4.85 (s, 1H), 4.98−4.52 (m, 1H), 5.06−5.19 (q, 2H),
6.51 (s, 2H), 6.59−6.62 (m, 1H), 6.71−6.72 (d, J = 4 Hz, 1H), 7.11−
7.13 (d, J = 8 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 38.86,
47.70, 55.23, 55.73, 57.85, 59.79, 65.83, 96.13, 100.35, 104.53, 108.64,
118.97, 131.82, 135.85, 137.07, 143.09, 152.72, 158.68, 160.83, 172.19.
LC-MS (ESI-TOF): m/z 428.1699 ([C23H25 NO7 + H]+ calcd
428.1704).
6-(2-Hydroxyethyl)-10-(bromophenyl)-2,3,7,10-tetrahydro-[1,4]-
dioxino[2,3-g]furo[3,4-b]quinolin-9(6H)-one (NSC756087). Yield:
9-(3,4-Dimethoxyphenyl)-4-(2-hydroxyethyl)-6-methoxy-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756091). Yield: 48%. H
1
70%. H NMR (DMSO-d6, 400 MHz): δ 3.62−3.75 (m, 3H), 3.76−
3.85 (m, 1H), 4.13−4.18 (m, 2H), 4.18−4.26 (m, 2H), 4.94 (s, 1H),
5.02−5.18 (m, 3H), 6.57 (s, 1H), 6.76 (s, 1H), 7.18−7.25 (m, 2H),
7.31−7.38 (m, 1H), 7.43 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ
39.11, 48.03, 57.70, 63.95, 64.24, 65.84, 94.15, 103.11, 118.80, 118.87,
121.72, 126.76, 129.17, 130.13, 130.32, 130.51, 139.48, 142.62, 149.60,
160.65, 172.15. LC-MS (ESI-TOF): m/z 444.0404 ([C21H18 Br NO5
+ H]+ calcd. 444.0441).
NMR (DMSO-d6, 400 MHz): δ 3.67−3.72 (m, 9H), 3.73 (s, 3H),
3.83−3.92 (m, 1H), 4.57−5.16 (m, 4H), 6.57−6.60 (m, 1H), 6.64−
6.67 (m, 1H), 6.69−6.70 (d, J =J = 4 Hz, 1H), 6.78−6.80 (d, J = 8 Hz,
1H), 6.83−6.84 (d, J = 4 Hz, 1H), 7.02−7.05 (m, 1H). 13C NMR
(DMSO-d6, 100 MHz): δ 38.45, 47.76, 55.23, 55.38, 55.49, 57.81,
65.75, 96.41, 100.25, 108.57, 111.43, 111.81, 119.17, 119.28, 131.94,
137.20, 140.18, 147.21, 148.54, 158.60, 160.45, 172.18. LC-MS (ESI-
TOF): m/z 398.1592 ([C22H23 NO6 + H]+ calcd 398.1598).
6-(2-Hydroxyethyl)-10-(3-chlorophenyl)-2,3,7,10-tetrahydro-
[1,4]dioxino[2,3-g]furo[3,4-b]quinolin-9(6H)-one (NSC756088).
4-(2-Hydroxyethyl)-6-methoxy-9-(3-methoxyphenyl)-4,9-
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756092). Yield: 52%
1
Yield: 61%. H NMR (DMSO-d6, 400 MHz): δ 3.62−3.75 (m, 3H),
3.76−3.85 (m, 1H), 4.13−4.18 (m, 2H), 4.18−4.26 (m, 2H), 4.95 (s,
1H), 5.00−5.17 (m, 3H), 6.57 (s, 1H), 6.76 (s, 1H), 7.16−7.24 (m,
2H), 7.25−7.32 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 39.14,
48.02, 57.70, 63.95, 64.24, 65.84, 94.14, 103.10, 118.79, 118.86,
126.26, 126.34, 127.29, 130.17, 130.33, 132.97, 139.48, 142.61, 149.34,
160.64, 172.14. LC-MS (ESI-TOF): m/z 400.0931 ([C21H18 Cl NO5
+ H]+ calcd 400.0946).
6-(2-Hydroxyethyl)-10-(3,4-dichlorophenyl)-2,3,7,10-tetrahydro-
[1,4]dioxino[2,3-g]furo[3,4-b]quinolin-9(6H)-one (NSC756089).
[regio-isomeric mixture]. 1H NMR (400 MHz, acetonitrile-d3) δ
3.74−3.67 (m, 1H), 3.75 (s, 3H), 3.79 (s, 3H), 3.91−3.79 (m, 4H),
4.95 (s, 1H), 5.10−4.97 (m, 2H), 6.59 (dd, J = 8.5, 2.5 Hz, 1H), 6.65
(d, J = 2.5 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.81 (dd, J =
2.6, 1.7 Hz, 1H), 6.85 (dt, J = 7.6, 1.2 Hz, 1H), 7.04 (dd, J = 8.4, 0.8
Hz, 1H), 7.21 (t, J = 7.9 Hz, 1H). 13C NMR (101 MHz, CD3CN) δ
40.44, 48.86, 55.71, 56.03, 59.37, 67.08, 101.29, 109.46, 112.32,
114.58, 120.03, 120.91, 130.38, 133.01, 138.59, 150.00, 160.17, 160.77,
161.48, 173.56. LC-MS (ESI-TOF): m/z 368.1491 ([C21H21 NO5 +
H]+ calcd 368.1492).
4-(2-Hydroxyethyl)-6-methoxy-9-phenyl-4,9-dihydrofuro[3,4-b]-
1
1
Yield: 70%. H NMR (DMSO-d6, 400 MHz): δ 3.62−3.83 (m, 4H),
quinolin-1(3H)-one (NSC756093). Yield: 50%. H NMR (DMSO-d6,
4.08−4.30 (m, 4H), 4.93−5.20 (m, 4H), 6.58 (s, 1H), 6.78 (s, 1H),
7.19−7.21 (d, J = 8 Hz, 1H), 7.50 (s, 2H). 13C NMR (DMSO-d6, 100
MHz): δ 38.15, 48.05, 57.69, 63.95, 64.25, 65.91, 93.84, 103.21,
400 MHz): δ 3.67−3.71 (m, 3H), 3.73 (s, 3H), 3.81−3.92 (m, 1H),
4.93−5.16 (m, 4H), 6.56−6.59 (m, 1H), 6.70−6.71 (d, J = 4 Hz, 1H),
6.96−7.01 (m, 1H), 7.12−7.18 (m, 1H), 7.20−7.28 (m, 4H). 13C
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NMR (DMSO-d6, 100 MHz): δ 38.95, 47.85, 55.24, 57.77, 65.81,
96.27, 100.38, 108.62, 126.13, 127.54, 128.22, 132.05, 137.40, 158.68,
160.56, 172.0. LC-MS (ESI-TOF): m/z 338.1382 ([C20H19 NO4 +
H]+ calcd 338.1387).
4-(2-Hydroxyethyl)-6-methoxy-9-(4-methoxyphenyl)-4,9-
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756094). Yield: 52%
9-(3,4-Dimethoxyphenyl)-4-(2-hydroxyethyl)-6,7-dimethoxy-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756098). Yield: 64%. H
NMR (DMSO-d6, 400 MHz): δ 3.61 (s, 3H), 3.68−3.69 (2s, 9H),
3.78 (s, 3H), 3.88−3.40 (m, 1H), 4.85 (s, 1H), 4.99−5.14 (m, 3H),
6.66−6.69 (m, 1H), 6.72 (s, 1H), 6.74 (s, 1H), 6.78−6.80 (d, J = 8 Hz,
1H), 6.87−6.88 (d, J = 4 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ
38.90, 47.87, 55.40, 55.43, 55.86, 55.95, 58.11, 65.66, 95.03, 99.44,
111.38, 111.68, 114.42, 118.34, 119.20, 129.33, 139.88, 145.08, 147.20,
148.00, 148.53, 160.29, 172.36. LC-MS (ESI-TOF): m/z 428.1704
([C23H25 NO7 + H]+ calcd 428.1704).
4-(2-Hydroxyethyl)-6,7-dimethoxy-9-(3-methoxyphenyl)-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756099). Yield: 68%. H
[regio-isomeric mixture]. 1H NMR (400 MHz, acetonitrile-d3) δ
7.25−7.09 (m, 2H), 7.00 (dd, J = 8.5, 0.8 Hz, 1H), 6.90−6.77 (m,
2H), 6.64 (d, J = 2.4 Hz, 1H), 6.59 (dd, J = 8.5, 2.5 Hz, 1H), 5.11−
4.95 (m, 2H), 4.93 (s, 1H), 3.88−3.80 (m, 3H), 3.78 (s, 3H), 3.76 (s,
3H), 3.75−3.62 (m, 2H). 13C NMR (101 MHz, CD3CN): δ 172.71,
160.28, 159.17, 158.31, 139.88, 137.72, 132.18, 128.72, 119.63, 113.66,
108.59, 100.28, 97.40, 66.09, 58.50, 55.11, 54.83, 47.93, 38.69. LC-MS
(ESI-TOF): m/z 368.1485 ([C21H21 NO5 + H]+ calcd 368.1492).
9-(3-Bromophenyl)-4-(2-hydroxyethyl)-6-methoxy-4,9-
NMR (DMSO-d6, 400 MHz): δ 3.60 (s, 3H), 3.69−3.73 (m, 5H), 3.79
(s, 3H), 3.87−3.98 (m, 1H), 4.90 (s, 1H), 5.00−5.15 (m, 3H), 6.68−
6.73 (m, 2H), 6.76 (s, 1H), 6.78−6.81 (m, 2H), 7.13−7.17 (m, 1H).
13C NMR (DMSO-d6, 100 MHz): δ 39.38, 47.91, 54.84, 55.89, 55.95,
58.10, 59.72, 65.70, 94.84, 99.54, 111.19, 113.53, 114.48, 117.85,
119.77, 129.22, 130.11, 145.11, 148.12, 148.54, 159.20, 160.40, 172.28.
LC-MS (ESI-TOF): m/z 398.1596 ([C22H23 NO6 + H]+ calcd
398.1598).
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756095). Yield: 51%. H
4-(2-Hydroxyethyl)-6,7-dimethoxy-9-phenyl-4,9-dihydrofuro[3,4-
1
b]quinolin-1(3H)-one (NSC756100). Yield: 60%. H NMR (DMSO-
NMR (CD3CN, 400 MHz): δ 3.79 (s, 3H), 3.80−3.82 (m, 4H), 4.82−
5.03 (m, 4H), 6.59−6.62 (m, 1H), 6.55−6.66 (d, J = 4 Hz, 1H), 6.98−
7.00 (d, J = 8 Hz, 1H), 7.22−7.27 (m, 2H), 7.34−7.38 (m, 1H), 7.42−
7.43 (m, 1H). 13C NMR (CD3CN, 100 MHz): δ 38.95, 47.68, 54.83,
58.10, 65.93, 100.24, 108.46, 118.07, 121.74, 126.54, 129.14, 129.94,
130.02, 131.93, 137.50, 149.51, 159.13, 160.39, 172.23. LC-MS (ESI-
TOF): m/z 438.0291 ([C20H18 Br NO4 + Na]+ calcd 438.0311).
4-(2-Hydroxyethyl)-6,7-dimethoxy-9-(3,4,5-trimethoxyphenyl)-
4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756097). Yield: 45%.
d6, 400 MHz): δ 3.51 (s, 3H), 3.60−3.73 (m, 6H), 3.78−3.90 (m, 1H),
4.85 (s, 1H), 4.95−5.06 (m, 3H), 6.57 (s, 1H), 6.68 (s, 1H), 7.03−
7.10 (m, 1H), 7.13−7.20 (m, 4H). 13C NMR (DMSO-d6, 100 MHz):
δ 39.38, 47.91, 55.87, 55.95, 58.08, 65.71, 94.94, 99.55, 114.53, 117.96,
126.16, 127.50, 128.20, 130.17, 145.12, 146.98, 148.09, 160.36, 172.28.
LC-MS (ESI-TOF): m/z 368.1490 ([C21H21 NO5 + H]+ calcd
368.1492).
4-(2-Hydroxyethyl)-6,7-dimethoxy-9-(4-methoxyphenyl)-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756102). Yield: 74%. H
1H NMR (DMSO-d6, 400 MHz): δ 3.59 (s, 3H), 3.62 (s, 4H), 3.70 (s,
8H), 3.78 (s, 3H), 4.85 (s, 1H), 4.96−4.98 (t, 1H), 5.04−5.19 (m,
2H), 6.55 (s, 2H), 6.76 (s, 1H), 6.82 (s, 1H). 13C NMR (DMSO-d6,
100 MHz): δ 39.54, 47.78, 55.75, 55.91, 55.96, 58.14, 59.80, 65.73,
94.70, 99.49, 104.49, 114.33, 118.15, 129.77, 135.84, 142.79, 145.12,
148.08, 152.72, 160.69, 172.39. LC-MS (ESI-TOF): m/z 458.1805
([C24H27 NO8 + H]+ calcd 458.1809).
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NMR (DMSO-d6, 400 MHz): δ 3.59 (s, 3H), 3.67−3.83 (m, 9H),
3.84−3.95 (m, 1H), 4.87 (s, 1H), 5.02−5.15 (m, 3H), 6.63 (s, 1H),
6.74 (s, 1H), 6.77−6.83 (m, 2H), 7.11−7.15 (m, 2H). 13C NMR
(DMSO-d6, 100 MHz): δ 38.60, 47.89, 54.91, 55.88, 55.96, 58.08,
65.64, 95.19, 99.50, 113.55, 114.56, 118.35, 128.45, 130.12, 139.41,
145.11, 148.03, 157.61, 160.10, 172.30. LC-MS (ESI-TOF): m/z
398.1597 ([C22H23 NO6 + H]+ calcd 398.1598).
9-(3-Bromophenyl)-4-(2-hydroxyethyl)-6,7-dimethoxy-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756103). Yield: 70%. H
160.98, 172.25. LC-MS (ESI-TOF): m/z 426.1915 ([C24H27 NO6 +
H]+ calcd 426.1911).
6-Ethyl-4-(2-hydroxyethyl)-9-phenyl-4,9-dihydrofuro[3,4-b]-
1
quinolin-1(3H)-one (NSC756108). Yield: 56%. H NMR (DMSO-d6,
NMR (DMSO-d6, 400 MHz): δ 3.60 (s, 3H), 3.68−3.78 (m, 2H), 3.79
(s, 4H), 3.85−3.95 (m, 1H), 4.98 (s, 1H), 5.02−5.16 (m, 3H), 6.67 (s,
1H), 6.77 (s, 1H), 7.18−7.25 (m, 2H), 7.32−7.38 (m, 1H), 7.41−7.45
(m, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 39.26, 47.97, 55.93,
58.02, 65.82, 94.38, 99.64, 114.48, 117.12, 121.68, 126.76, 129.14,
130.14, 130.19, 130.45, 145.22, 148.29, 149.48, 160.61, 172.20. LC-MS
(ESI-TOF): m/z 446.0528 ([C21H20 Br NO5 + H]+ calcd 446.0598).
9-(3-Chlorophenyl)-4-(2-hydroxyethyl)-6,7-dimethoxy-4,9-
400 MHz): δ 1.06−1.10 (t, 3H), 2.45−2.51 (q, 2H), 3.58−3.70 (m,
3H), 3.75−3.3 (m, 1H), 4.88 (s, 1H), 4.96−4.50 (m, 1H), 5.02−5.09
(m, 2H), 6.73−6.75 (m, 1H), 6.90−6.92 (d, J = 8 Hz, 1H), 6.95 (s,
1H), 7.04−7.08 (m, 1H), 7.12−7.17 (m, 4H). 13C NMR (DMSO-d6,
100 MHz): δ 15.58, 27.98, 38.94, 47.69, 57.74, 65.81, 95.67, 113.42,
122.92, 123.90, 126.17, 127.58, 128.25, 131.14, 136.21, 143.32, 147.14,
160.70, 172.16. LC-MS (ESI-TOF): m/z 336.1599 ([C21H21 NO3 +
H]+ calcd 336.1594).
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756104). Yield: 72%. H
9-(3-Bromophenyl)-6-ethyl-4-(2-hydroxyethyl)-4,9-dihydrofuro-
[3,4-b]quinolin-1(3H)-one (NSC756110). Yield: 55%. 1H NMR
NMR (DMSO-d6, 400 MHz): δ 3.60 (s, 3H), 3.68−3.77 (m, 2H), 3.79
(s, 4H), 3.87−3.95 (m, 1H), 4.99 (s, 1H), 5.02−5.16 (m, 3H), 6.67 (s,
1H), 6.77 (s, 1H), 7.18−7.25 (m, 2H), 7.26−7.30 (m, 2H). 13C NMR
(DMSO-d6, 100 MHz): δ 39.26, 47.97, 55.92, 58.03, 65.83, 94.37,
99.64, 114.45, 117.13, 126.25, 126.35, 127.30, 130.11, 130.18, 132.93,
145.22, 148.28, 149.24, 160.62, 172.21. LC-MS (ESI-TOF): m/z
402.1080 ([C21H20 Cl NO5 + H]+ calcd 402.1103).
(DMSO-d6, 400 MHz): δ 1.14−1.18 (t, 3H), 2.54−2.59 (q, 2H),
3.65−3.82 (m, 3H), 3.84−3.92 (m, 1H), 5.01−5.19 (m, 4H), 6.83−
6.85 (m, 1H), 6.98−7.00 (d, J = 8 Hz, 1H), 7.05 (s, 1H), 7.21−7.25
(m, 2H), 7.33−7.36 (m, 1H), 7.43 (s, 1H). 13C NMR (DMSO-d6, 100
MHz): δ 15.53, 27.98, 38.90, 47.76, 57.70, 65.93, 95.08, 113.63,
121.71, 123.09, 123.15, 126.89, 129.17, 130.22, 130.51, 131.17, 136.19,
143.64, 149.62, 160.98, 172.09. LC-MS (ESI-TOF): m/z 414.0626
([C21H20 Br NO3 + H]+ calcd 414.0699).
9-(3,4-Dichlorophenyl)-4-(2-hydroxyethyl)-6,7-dimethoxy-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756105). Yield: 71%. H
9-(3-Chlorophenyl)-6-ethyl-4-(2-hydroxyethyl)-4,9-dihydrofuro-
[3,4-b]quinolin-1(3H)-one (NSC756111). Yield: 53%. 1H NMR
NMR (DMSO-d6, 400 MHz): δ 3.60 (s, 3H), 3.68−3.77 (m, 2H), 3.79
(s, 4H), 3.87−3.95 (m, 1H), 4.99 (s, 1H), 5.02−5.06 (m, 2H), 5.08−
5.15 (m, 2H), 6.67 (s, 1H), 6.77 (s, 1H), 7.19−7.22 (m, 1H), 7.48−
7.53 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 38.55, 47.98, 55.92,
58.00, 65.90, 94.06, 99.69, 114.37, 116.70, 128.00, 128.88, 129.42,
130.14, 130.46, 130.83, 145.29, 147.74, 148.37, 160.73, 172.18. LC-MS
(ESI-TOF): m/z 436.0646 ([C21H19 Cl2 NO5 + H]+ calcd 436.0713).
6-Ethyl-4-(2-hydroxyethyl)-9-(3,4,5-trimethoxyphenyl)-4,9-
(DMSO-d6, 400 MHz): δ 1.14−1.18 (t, 3H), 2.54−2.58 (q, 2H),
3.68−3.80 (m, 3H), 3.80−3.91 (m, 1H), 5.03−5.19 (m, 4H), 6.83−
6.85 (d, J = 8 Hz, 1H), 6.99−7.01 (d, J = 8 Hz, 1H), 7.05 (s, 1H),
7.18−7.23 (m, 2H), 7.25−7.30 (m, 2H). 13C NMR (DMSO-d6, 100
MHz): δ 16.02, 28.49, 39.44, 48.26, 58.20, 66.43, 95.58, 114.12,
123.57, 123.65, 126.76, 126.97, 127.90, 130.66, 131.65, 133.48, 136.69,
144.14, 149.86, 161.48, 172.60. LC-MS (ESI-TOF): m/z 370.1170
([C21H20 Cl NO3 + H]+ calcd 370.1204).
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756106). Yield: 54%. H
NMR (DMSO-d6, 400 MHz): δ 1.14−1.18 (t, 3H), 2.53−2.59 (q,
2H), 3.59−3.72 (m, 12H), 3.95−4.02 (m, 1H), 4.88 (s, 1H), 4.99 (bs,
1H), 5.06−5.21 (q, 2H), 6.53 (s, 2H), 6.83−6.85 (d, J = 8 Hz, 1H),
7.04 (s, 1H), 7.12−7.14 (d, J = 8 Hz, 1H). 13C NMR (DMSO-d6, 100
MHz): δ 15.60, 28.00, 38.86, 47.55, 55.73, 57.83, 59.79, 65.82, 95.52,
104.56, 113.40, 122.96, 124.02, 130.90, 135.88, 142.96, 143.29, 152.74,
9-(3,4-Dichlorophenyl)-6-ethyl-4-(2-hydroxyethyl)-4,9-
1
dihydrofuro[3,4-b]quinolin-1(3H)-one (NSC756112). Yield: 70%. H
NMR (DMSO-d6, 400 MHz): δ 1.14−1.18 (t, 3H), 2.54−2.59 (q,
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the whole mechanistic set, and similarities were selected if the ρ value
was >0.55 with a p-value <0.0001. To identify scaffold with divergent
activities as compared to the mechanistic set, the results were then
analyzed with hierarchical cluster analysis. All the statistical analyses
were performed with the JMP 9 software (SAS). GBP1 and PIM1 gene
expression Z-scores for the NCI-60 panel were downloaded from
http://dtp.nci.nih.gov/. An index of GBP1 and PIM1 expression was
calculated by multiplying the two values. Cells were then sorted for the
sensitivity to NSC756093 measured as GI50. The first and the third
tertile were categorized as NSC756093-sensitive and NSC756093-
resistant, respectively.
Cell Lines and Coimmunoprecipitation Experiments. The
NCI-60 assay was performed at NCI as previously described.18 The
cell line SKOV3 was purchased from ECACC and cultured as directed.
This cell line was chosen for the detectable expression of both GBP1
and PIM1 and was cultured as previously described.6 Cells harvested in
cold PBS were extracted in lysis buffer, and coimmunoprecipitation
and Western blots were done as described previously.33 Antibodies
were anti-GBP1 and anti-PIM1 (1:200 in 5% milk−TBST, Santacruz).
Gel images were quantified from three independent experiments using
an Image Station (Carestream) and densitometric analysis.
Biosensor Experiments and Proteins. Interaction analyses were
done using the ProteON Xpr 36 (Biorad). Carbonic anidrase (CA)
protein utilized as negative control was purchased from Sigma. The
GLM sensor chip (Biorad) was equilibrated at room temperature for
45 min before use. During the binding procedure, TBST buffer (tween
0.005%, Biorad) was utilized. After the air initializing procedure, the
sensor chip was conditioned on the horizontal side with first injection,
sodium dodecyl sulfate (SDS, Bio Rad) 0.5%, 30 μL/min for 60 s;
second injection, sodium hydroxide (NaOH, Bio Rad) 50 mM, 30 μL/
min for 60 s; third injection, hydrogen chloride (HCl, Bio Rad) 100
mM, 30 μL/min for 60 s. The injections set was repeated for the
vertical side of the chip to complete the conditioning protocol. The
surface of the chip was activated using a 1:1 solution of 1-ethyl-3-[3-
(dimethylamino)propyl] carbodiimide hydrochloride (EDC) and N-
hydroxysulfosuccinimide (S-NHS), with single injections of 30 μL/
min for 300 s. Once the chip surface was activated, the ligand protein
PIM1 was captured on the chip (50 μg/mL) in at least two of the six
available channels to have a duplicate of every protein interaction
experiment. One channel was treated with acetate buffer pH 5.5 as
negative control surface. PIM1 was diluted in acetate buffer pH 5.5 and
then injected in the instrument 30 μL/min for 300 s. A signal of 2000
response units (RU) was obtained. The surface of the chip was then
deactivated with an injection of 1 M ethanolamine−HCl solution, 30
μL/min, for 300 s. Two injections of TBST (30 μL/min for 60 s) were
utilized to remove from the chip surface the excess of ligand, then the
surface was flushed overnight with ultra pure water filtered with Milli
Q system (Millipore, Billerica, MA). The day after the analyte was
injected from lowest to highest concentrations, using a solution of 280
nM GBP1 in TBST, injected 30 μL/min for 120 s (Tween 0.05%),
and allowing 15 min of dissociation time. The kinetic of the binding
was measured using the Langmuir kinetic model with the ProteON
software. After the immobilization, the biochip surfaces was
regenerated with 30 μL/min for 60 s injections of 4 M guanidinium
chloride (GuHCl). Production of GBP1 and PIM1 recombinant
proteins have been described elsewhere.6 Additional controls were
carried out to ensure specificity and quality of the binding. In
particular, quality of the binding was confirmed in each chip with a
simultaneous run of negative and positive controls (shown in
Supporting Information, Figures 7SI and 8SI). In particular, in each
run in which PIM1 was used as analyte, we used GBP1 and CA as
positive and negative controls of the interaction. Preliminary
experiments were also conducted using CA instead of PIM1 as ligand.
In these experiments, GBP1 did not show any signal on the chip, thus
excluding aspecific binding of GBP1 to the chip. Additional controls
included also the use of denaturated GBP1 (heated at 55 °C for 20
min). Denaturated GBP1 did not produce any binding, while in the
parallel lane the not denaturated protein produced the expected signal.
Mutants of GBP1 were prepared using SLIM-PCR as previously
described.34 The sequence of each mutant was confirmed with Sanger
2H), 3.69−3.81 (m, 3H), 3.83−3.91 (m, 1H), 5.03−5.19 (m, 4H),
6.83−6.85 (m, 1H), 6.99−7.01 (d, J = 8 Hz, 1H), 7.05 (s, 1H), 7.18−
7.22 (m, 2H), 7.28−7.30 (m, 1H). 13C NMR (DMSO-d6, 100 MHz):
δ 15.53, 27.99, 38.94, 47.76, 57.70, 65.93, 95.08, 113.63, 123.07,
123.15, 126.26, 126.47, 127.40, 130.16, 131.15, 132.98, 136.19, 143.63,
149.36, 160.98, 172.10. LC-MS (ESI-TOF): m/z 404.0711 ([C21H19
Cl2 NO3 + H]+ calcd 404.0815).
NCI-60 Panel Assay and COMPARE Analysis. Growth
inhibition experiments were performed at NCI with the use of the
NCI-60 panel of cell lines.
Details of the methodology are described at http://dtp.nci.nih.gov/
branches/btb/ivclsp.html. Briefly, the panel was organized into nine
subpanels representing diverse histologies: leukemia, melanoma, and
cancers of lung, colon, kidney, ovary, breast, prostate, and central
nervous system. The cells were grown in supplemented RPM1 1640
medium for 24 h. The test compounds were dissolved in DMSO and
incubated with cells at five concentrations with 10-fold dilutions, the
highest being 10−4 M and the others being 10−5, 10−6, 10−7, and 10−8
M. The assay was terminated by addition of cold trichloroacetic acid,
and the cells were fixed and stained with sulforhodamine B. Bound
stain was solubilized, and the absorbance was read on an automated
plate reader. Growth inhibition effects of all the compounds were
calculated in terms of GI50, which is the concentration of the drug that
causes 50% of growth inhibition, after correction for the cell count at
time 0.17 Percentage growth inhibition (GI50) was calculated from time
zero, control growth, and the five concentration level absorbance. The
inhibitory concentrations (LC50) represent the average of two
independent experiments. The one-dose data of all the compounds
is reported as a mean graph of the percent growth of treated. The
number reported for the one-dose assay is growth relative to the no-
drug control and relative to the time zero number of cells. This allows
detection of both growth inhibition (values between 0 and 100) and
lethality (values less than 0). For example, a value of 100 means no
growth inhibition. A value of 40 would mean 60% growth inhibition. A
value of 0 means no net growth over the course of the experiment. A
value of −40 would mean 40% lethality. A value of −100 means all
cells are dead. The drug response curves from the five-dose data on the
NCI-60 panel for NSC756090, NSC756092, 756093, and 756095 are
given in Supporting Information, Figures 2SI−5SI.
The principle of COMPARE analysis17 was used to analyze the
activity of 4-APTs. The Z-score of the reference compounds paclitaxel,
carboplatin, and cisplatin were downloaded from http://dtp.nci.nih.
gov/. Using the data of all the NCI-60 cell lines, a Spearman
correlation assay was performed with the Z-score of all the 31 active 4-
APTs.
Activity of each compound measured as GI50 was ranked with a Z-
score within the cell lines of the NCI-60 panel. Each Z-score was
calculated with the formula z = (x − μ/σ), where x is the GI50 in a
given cell line for a drug, μ is the average of the GI50 of the same drug
within the NCI-60 panel, and σ is the standard deviation. To perform
the COMPARE analysis, all these values are correlated with a
Spearman test with the Z-scores of another reference drug, resulting in
a ρ coefficient.
This approach allowed us to identify pattern of cross-resistance/
sensitivity to the reference drugs. The NCI COMPARE mechanistic-
set served to test the hypothesis that 4-APTs belong to a group of
compounds with a given mechanism of action. The mechanistic
diversity set consists of 879 compounds and represent a broad range of
growth inhibition patterns in the NCI-60 cell line. Similarity with a
member of the mechanistic set is featured by a positive and significant
ρ value. The most active 21 4-APTs were analyzed for similarity with
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sequencing and the recombinant protein produced as previously
described.6 For the experiments of inhibition with the compounds, the
following protocol was adopted. First, the compounds were flowed on
the chip in the absence of GBP1 using the following concentrations:
0.1, 1, 10, and 100 nM. Thereafter, we monitored the inhibition of the
GBP1:PIM1 interaction by injecting concomitantly the tested
compounds and GBP1. Each run was performed at least three times
at the four concentrations indicated above plus CA (280 nM) and
GBP1 (280 nM) as negative and positive control. The % of inhibition
was calculated by analyzing the signal of the interaction (RU) in the
presence of the compound over the maximum interaction signal with
GBP1. A value of 100% indicated no inhibition. Analysis of variance
(Anova) was used to identify compounds with significant inhibitory
activity.
the PDB structures assuming a pH of 7.2. These structures were
analyzed using Biopolymer and Homology module of Insight 2005
(Accelrys, San Diego).
Linear functional motifs present in (i) tubulin podophyllotoxin
binding site (i.e., within a 5 Å radius from any given ligand atom), (ii)
topoisomeraseIIβ etoposide binding site, (iii) GBP1, and (iv) PIM1
were identified by using the Eukaryotic Linear Motif server (http://
elm.eu.org/),28 a resource for predicting small functional sites in
eukaryotic proteins. The identified motifs in GBP1 and PIM1 were
logically intersected with the union of those present in the binding
sites of tubulin and topoisomerase. This allowed us to identify the
putative binding site of NSC756093 in GBP1, characterized by three
linear functional motifs present in tubulin and/or topoisomeraseIIβ
binding sites.
Modeling of GBP1. The molecular model of full length human
GBP1 in free form was built starting from the experimentally
determined structure of free form GBP1 (hGBP1FL; PDB ID: 1DG3),
which lacks four loops (amino acids 63−73, 157−166, 190−193, 244−
256), the N-terminal region (aa1−5), and the C-terminal region
(aa584−592). The sequence of 1DG3 was aligned with the sequence
of hGBP1FL downloaded from the UniProtKB/Swiss-Prot Data Bank
(http://www.uniprot.org; entry P32455) by using the Multiple_Align-
ment algorithm (Homology module, Accelrys, San Diego). Sub-
sequently, the secondary structural prediction of the hGBP1FL was
performed using the Structure Prediction and Sequence Analysis
server PredictProtein (http://www.predictprotein.org/). The coor-
dinates of the structurally conserved regions aa6−62, aa74−156,
aa167−189, aa194−243, and aa257−583 of hGBP1FL were accordingly
assigned by the SCR-AssignCoords procedure (Homology module)
using 1DG3 as template structure. The lacking loop segments aa63−
73, aa157−166, aa190−193, and aa244−256 were inserted by using
the Generate Loops procedure. With the Generate Loops procedure, a
peptide backbone chain was built between two conserved peptide
segments using randomly generated values for all the loops’ φs and ψs.
The chain was defined starting from the N-terminal end of the loop
being built; the Start and Stop Residues were defined as the SCR
residues of the model protein at either end of the loop itself. The
geometry about the base was described by the four distances between
Cα and N-termini of the Start residue and the Cα and C-termini of the
Stop residues. In the process of closing the loop, the values for the
generated φs and ψs were adjusted until the four distance criteria are
met. Specifically, a function was defined for the distances in terms of
the dihedral angles (Scale Torsions: 60). The differences between the
desired distances and their current values were minimized using a
linearized Lagrange multiplier method. After a series of 1000 iterations,
the loop was closed, except in the case where the distances between
the ends of the loop were not respected (Convergence = 0.05). The
geometry at the base of the loop is then checked for proper chirality.
Finally, the loops were screened on the basis of steric overlap
violations. All loops that are found to have unacceptable contacts were
rejected. Because successive calculations can correct some bad
contacts, a fairly large overlap factor was used (Internal and External
overlap = 0.6). A bump check of the 10 generated loops together with
the evaluation of their conformational energy were used as selection
criteria. The lowest conformational energy loop presenting no steric
overlap with the rest of the protein was selected.
Molecular Modeling Studies. Molecular modeling calculations
were performed on SGI Origin 200 8XR12000 and E4 Server Twin 2×
Dual Xeon-5520, equipped with two nodes. Each node: 2× Intel Xeon
QuadCore E5520-2.26 Ghz, 36 GB RAM. The molecular modeling
graphics were carried out on SGI Octane 2 workstations.
Conformational Analysis of the New Azapodophyllotoxin
Derivatives. The apparent pKa values of azapodophyllotoxin
derivatives were calculated by using the ACD/pKa DB, version
12.00, software (Advanced Chemistry Development Inc., Toronto,
Canada). All compounds were considered neutral in all calculations
performed as a consequence of the estimation of percentage of
neutral/ionized forms computed at pH 7.4 (physiological value) and
pH 7.2 (cytoplasmic value) using the Handerson−Hasselbalch
equation. The compounds were built using the Insight 2005 Builder
module (Accelrys Software Inc., San Diego, CA). Atomic potentials
and charges were assigned using the CVFF force field.35 The
conformational space of compounds was sampled through 200 cycles
of simulated annealing (ε = 1). In simulated annealing, the
temperature is altered in time increments from an initial temperature
to a final temperature by adjusting the kinetic energy of the structure
(by rescaling the velocities of the atoms). The following protocol was
applied: the system was heated to 1000 K over 2000 fs (time step of
3.0 fs); a temperature of 1000 K was applied to the system for 2000 fs
(time step of 3.0 fs) to surmount torsional barriers; successively,
temperature was linearly reduced to 300 K in 1000 fs (time step of 1.0
fs). Resulting conformations were then subjected to Molecular
Mechanics (MM) energy minimization within the Insight 2005
Discover module (CVFF force field (ε = 1) until the maximum rmsd
was less than 0.001 kcal/Å, using conjugate gradient as the
minimization algorithm. Resulting conformers were grouped into
families on the basis of their torsional angles and ranked by their
potential energy values (i.e., ΔE from the global energy minimum).
The MM conformers were then subjected to a full geometry
optimization by semiempirical calculations, using the quantum
mechanical method PM736 in the MOPAC2012 package37 and EF
(eigenvector following routine)38 as geometry optimization algorithm.
GNORM value was set to 0.01. To reach a full geometry optimization,
the criterion for terminating all optimizations was increased by a factor
of 100, using the keyword PRECISE. Resulting conformers were
grouped into families on the basis of their torsional angles and ranked
by their potential energy values (i.e., ΔE from the global energy
minimum).
Bioinformatics Analysis. The experimentally determined structures
of (i) tubulin in complex with podophyllotoxin (PDB ID: 1SA1), (ii)
TopoisomeraseIIβ in complex with etoposide (PDB ID: 3QX3), (iii)
GBP1 (PDB IDs: 2B8W, 2B92, 2BC9, 2D4H, 1DG3, 1F5N), and (iv)
PIM1 (PDB IDs: 4MBL, 4K0Y, 4K18, 4K1B, 4JX7, 4JX3, 4GW8,
4ENX, 4ENY, 4DTK, 4AS0, 4ALW, 4ALV, 4ALU, 4A7C, 3VC4,
3VBW, 3VBX, 3VBY, 3VBV, 3VBT, 3VBQ, 3UIX, 3UMW, 3UMX,
3T9I, 3R00, 3R01, 3R02, 3R04, 3QF9, 3MA3, 3JXW, 3JY0, 3JYA,
3JPV, 3F2A, 3DCV, 3CXW, 3CY2, 3CY3, 3C4E, 3BWF, 3BGP,
3BGQ, 3BGZ, 3A99, 2XIZ, 2XIY, 2XIX, 2XJ0, 2XJ2, 2XJ1, 2J2I, 2OI4,
2O3P, 2O63, 2O64, 2O65, 2OBJ, 2C3I, 2BZH, 2BZI, 2BZK, 2BZJ,
2BIK, 2BIL, 1YWV, 1YXS, 1YXT, 1YXU, 1YXV, 1YXX, 1YHS, 1YI3,
1YI4, 1XWS, 1XQZ, 1XR1) were downloaded from the Protein Data
Bank (PDB; http://www.rcsb.org/pdb/). Hydrogens were added to all
Finally, the coordinates of the N-terminal and C-terminal amino
acids (1−5 and 584−592, respectively) were assigned using the
EndRepair command in Homology module of Insight 2005. In
particular, according to results obtained from the secondary structural
prediction, the residues 584−585 (with the standard geometry of an α-
helix) and the residues 1−5 and 584−592 (with an extended chain
conformation) were appended to the molecular model of hGBP1FL.
The obtained homology model of hGBP1FL was completed
inserting the water molecules of GBP1 experimentally determined
structure (PDB ID: 1DG3) through the UnMerge and Merge
commands (Biopolymer module, Accelrys, San Diego).
The entire GBP1 model was then subjected to a full energy
minimization within Insight 2005 Discover_3 module (Steepest
Descent algorithm, maximum RMS derivative = 10 kcal/Å; Conjugate
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Gradient algorithm, maximum RMS derivative = 1 kcal/Å; ε = 1).
During the minimization, only the whole disordered N−C terminal
and loop backbone and side chains were left free to move, whereas the
structurally conserved regions (SCRs) of GBP1 were fixed to avoid
unrealistic results.
Each step of refining procedure was followed by a structural check
by using the Struct_Check command of the ProStat pulldown in the
Homology module to verify the correctness of the geometry
optimization procedure before moving to the next step. Checks
included φ, ψ, χ1, χ2, χ3, and ω dihedral angles, Cα virtual torsions,
and Kabsch and Sander main chain H-bond energy evaluation. The
final generated model was checked for quality using Procheck39
structure evaluator software. The obtained hGBP1FL homology model
was used for successive dynamic docking studies.
Docking Studies on GBP1 in Complex with the New
Azapodophyllotoxin Derivatives. The putative NSC756093/GBP1
complex was subjected to dynamic docking studies (Affinity,
SA_Docking;40 Insight2005, Accelrys, San Diego). A docking
methodology (Affinity, SA_Docking; Insight2005, Accelrys, San
Diego) which considers all the systems flexible (i.e., ligand and
protein) was used. Although in the subsequent dynamic docking
protocol all the systems were perturbed by means of Monte Carlo and
simulated annealing procedures, nevertheless, the dynamic docking
procedure formally requires a reasonable starting structure. Accord-
ingly, the starting model of GBP1 was subjected to a preliminary
energy minimization to generate roughly docked starting structure
(Steepest Descent algorithm, maximum RMS derivative = 10 kcal/Å;
Conjugate Gradient algorithm, maximum RMS derivative = 1 kcal/Å;
ε = 1).
calculations were performed: one, where all water molecules were left
free to move, another, where the water molecules within the active site
of GBP1, were tethered with a force constant of 100 kcal/Å2 as for the
preliminary energy minimization.
A Monte Carlo/minimization approach for the random generation
of a maximum of 20 acceptable complexes was used. During the first
step, starting from the previously obtained roughly docked structures,
the ligand was moved by a random combination of translation,
rotation, and torsional changes to sample both the conformational
space of the ligand and its orientation with respect to the protein
(MxRChange = 3 Å; MxAngChange = 180°). During this step, van der
Waals (vdW) term was scaled to a factor of 0.1 to avoid severe
divergences in the vdW energies. If the energy of a complex structure
resulting from random moves of the ligand was higher by the energy
tolerance parameter than the energy of the last accepted structure, it
was not accepted for minimization. To ensure a wide variance of the
input structures to be successively minimized, an energy tolerance
value of 106 kcal/mol from the previous structure was used. After the
energy minimization step (conjugate gradient; 2500 iterations; ε = 1),
the energy test, with an energy range of 50 kcal/mol, and a structure
similarity check (rms tolerance = 0.3 kcal/Å) was applied to select the
20 acceptable structures. Each subsequent structure was generated
from the last accepted structure. Following this procedure, the
resulting docked structures were ranked by their conformational
energy and were analyzed by a structural check by using the
Struct_Check command of the ProStat pulldown in the Homology
module to verify the correctness of their dihedral angles values.
Structures characterized by unrealistic backbone geometry were
discarded.
During the minimization, the whole system was left free to move,
whereas a tethering restraint was applied on structurally conserved
regions (SCRs) to avoid unrealistic results. To identify SCRs, we
analyzed the hGBP1FL sequence (UniProtKB/Swiss-Prot Data Bank;
entry code GBP1: P32455) using the Structure Prediction and
Sequence Analysis server PredictProtein (http://www.predictprotein.
org/). In GBP1, 16 α helix and four β-sheet secondary structures were
predicted to be highly conserved (α1, aa26−32; α2, aa113−121; α3,
aa139−151; α4, aa199−205; α5, aa221−227; α6, aa261−272; α7,
aa292−303; α8, aa315−322; α9, aa326−334; α10, aa350−370; α11,
aa376−397; α12, aa404−423; α13, aa433−451; α14, aa457−465; α15,
aa469−563; α16, aa567−585; β1, aa12−14; β2, aa39−43; β3, aa79−
80; β4, aa92−95). Accordingly, for the α-helices, the distance between
hydrogen bond donors and acceptors was constrained within 2.5 Å
using a force constant of 100 kcal/mol/Å (Restrain command;
Discover_3 module, Accelrys, San Diego). On the other hand, for the
β-sheets, the φ and ψ torsional angle were constrained within −130°
and +125°, respectively, using a force constant of 100 kcal/mol/Å
(Restrain command; Discover_3 module, Accelrys, San Diego).
Moreover, during these calculations, also the water molecules within
the active site of GBP1 were tethered with a force constant of 100
kcal/Å2. In particular, all the water molecules within a 6 Å radius from
any given nucleotide atom (i.e., GppNHp for GBP1 (PDB ID: 1F5N))
were considered.
Flexible docking was achieved using the Affinity module in the
Insight 2005 suite, setting the SA_Docking procedure40 and using
three different methods for the calculation of the nonbond
interactions: (i) Quartic_vdW_no_Coul (vdW CUT_OFF: 5), (ii)
Cell_Multipole, and (iii) Group Based (vdW and Coul CUT_OFF:
15).
The docking protocol included a Monte Carlo based conforma-
tional search of the ligand (NSC756093) within the obtained
homology model of GBP1. The binding domain area was defined as
a subset including all residues of GBP1 protein (aa1−592). All atoms
included in the binding domain area were left free to move during the
entire course of docking calculations, whereas, in order to avoid
unrealistic results, a tethering restraint was applied on the structurally
conserved regions (SCRs) of protein. The set of restraints applied was
the same as for the preliminary energy minimization. On the other
hand, in order to analyze the role of the water molecules within the
binding site of GBP1, for each set of docking, two typologies of
To test the thermodynamic stability of the resulting docked
complexes, these latter were subjected to a molecular dynamics
simulated annealing protocol. Two typologies of calculations were
performed using or the Cell_Multipole or the Group Based method
(CUT_OFF: 50) for the calculation of the nonbond interactions. A
tethering restraint was applied on the structurally conserved regions
(SCRs) of the complex and on the water molecules within the
nucleotide binding sites. The set of structural restraints applied was the
same as for previous docking calculations. The protocol included 5 ps
of a dynamic run divided in 50 stages (100 fs each), during which the
temperature of the system was linearly decreased from 500 to 300 K
(Verlet velocity integrator; time step = 1.0 fs). In simulated annealing,
the temperature was altered in time increments from an initial
temperature to a final temperature. The temperature was changed by
adjusting the kinetic energy of the structure (by rescaling the velocities
of the atoms). Molecular dynamics calculations were performed using
a constant temperature and constant volume (NVT) statistical
ensemble, and the direct velocity scaling as temperature control
method (temp window = 10 K). In the first stage, initial velocities were
randomly generated from the Boltzmann distribution, according to the
desired temperature, while during the subsequent stages initial
velocities were generated from dynamics restart data. The temperature
of 500 K was applied with the aim of surmounting torsional barriers,
thus allowing an unconstrained rearrangement of the “ligand” and the
“protein” active site (initial vdW and Coulombic scale factors = 0.1).
Successively temperature was linearly reduced to 300 K in 5 ps, and,
concurrently, the vdW and Coulombic scale factors have been similarly
increased from their initial values (0.1) to their final values (1.0). A
final round of 104 minimization steps (conjugate gradient, ε = 1)
followed the last dynamics steps, and the minimized structures were
saved in a trajectory file. After this procedure, the resulting structures
were analyzed by a structural check by using the Struct_Check
command of the ProStat pulldown in the Homology module to verify
the correctness of the geometry. The resulting complexes were ranked
by their conformational energy and analyzed for the linear functional
motifs present in their ligand binding sites. The complex characterized
by the lowest conformational energy and a ligand binding site
presenting at least three linear functional motifs in common with
tubulin and/or topoisomeraseIIβ binding sites, was selected. The
structure was further minimized (CVFF force field; Group Based
method for nonbond interaction (CUTOFF: 20)) by a combination of
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Steepest Descent (maximum rms derivative less than 0.1 kcal/Å) and
Conjugate Gradient algorithms (maximum rms derivative less than
0.01 kcal/Å) to allow the relaxation of the whole protein and
resubjected to the above-reported structural evaluation. Obtained
complex was checked for quality using Procheck39 structure evaluator
software.
The PM7 lowest energy conformers of each conformational family
of compounds NSC756094, NSC756095, NSC759100, and
NSC756108 within 5 kcal/mol from the global energy minimum
were superimposed on NSC756093 in complex with GBP1, and the
obtained complexes were energy minimized using the above-described
procedure. Finally, all ligand/enzyme complexes were ranked by
considering the potential energy values of the ligands (i.e., ΔE from
the global energy minimum).
of Health through grant no. 8 P20 GM 103475 (A.K.), by the
Connecticut State Grant 2014-0908 (C.Fe.), by a liberal
donation from Rudy and Sally Ruggles (C.Fe.), by the
Associazione Oppo e le sue stanze ONLUS (G.S.), by the
EU project Bluegenics grant 311848 (C.F.), and by the Italian
MIUR financial FIRB RBFR12WB3W_003 (M.P). Sponsors of
the research were not involved in the design and interpretation
of the experimental activities.
ABBREVIATIONS USED
■
MTAs, microtubule targeted agents; APTs, azapodophyllotox-
ins; CA, carbonic anhydrase; TopoIIβ, topoisomerase IIβ
ASSOCIATED CONTENT
* Supporting Information
REFERENCES
■
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Dot chart showing the sensitivity to paclitaxel in the NCI-60
panel; five dose data on the NCI-60 panel for NSC756090,
NSC756092, NSC756093, and NSC756095; heat-map showing
the results of two-way hierarchical clustering of the COMPARE
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NSC756093/GBP1 complexes obtained by docking studies;
protein functional motifs indentified in the binding sites of
etoposide, podophyllotoxin, and NSC756093; Ramachandran
plots, Chi1−Chi2 plots, main chain and side chain parameters
of human GBP1 X-ray structure (PDB ID 1DG3) and
molecular model of docked NSC756093/GBP1 complex.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*For C.F.: phone, +39081678544; fax, +39081678552; E-mail,
caterina.fattorusso@unina.it.
*For S.V.M.: phone, 301-846-5141; fax, 301-846-5206; E-mail,
malhotrasa@mail.nih.gov.
*For C.Fe.: phone, +1 203 739 8390; fax, +1 203073908739; E-
mail, cristiano.ferlini@danhosp.org.
■
Author Contributions
◆M.A., M.P., and A.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NCI Developmental Therapeutics Program for
60 cell line screening. We also thank Robert Bedoukian for his
advice. The study was partially supported by the National
Cancer Institute, National Institutes of Health, under contract
no. HHSN261200800001E (S.V.M., A.P., and V.K.), by the
National Center for Research Resources and the National
Institute of General Medical Sciences of the National Institutes
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