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

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 II^ndResearch 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.^2,3In 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 isotype³⁻⁵is 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.^6,7Crucial 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.¹In 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

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large-GTPase GBP1, whose incorporation into microtubules is

facilitated by the presence of βIII-tubulin.^6,7For 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.⁸⁻¹⁰Natural 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.^11,12Although

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).¹³To 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.^14,15Motivated 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.¹⁷Activity of each compound

measured as GI₅₀is 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 GI₅₀in a given cell line

for a drug, μ is the average of the GI₅₀of 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-(CH₂)₃

6,7-O(CH₂)₂O

6-OCH₃

OCH₃

H

OCH₃

H

OCH₃

H

OCH₃

H

OH

OCH₃

H

OCH₃

H

Cl

H

OCH₃

H

OCH₃

H

OCH₃

H

OCH₃

H

OH

OCH₃

H

OCH₃

H

Br

H

Cl

H

Cl

H

OCH₃

H

OCH₃

H

OCH₃

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

Cl

H

OCH₃

H

OCH₃

H

OCH₃

H

6-OCH₃

H

6-OCH₃

H

6-OCH₃

H

OCH₃

H

Within the mechanistic set, the most represented drugs are

known MTAs such as quinazolinone,¹⁹maytansine,²⁰colchi-

cines, and its analogue benzoyl-deacetyl-colchicine,²¹vinblas-

tine,²²and baccatin III.²³In 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

H

OCH₃

H

OCH₃

H

6,7-OCH₃

H

6,7-OCH₃

H

6,7-OCH₃

H

OCH₃

H

6,7-OCH₃

Br

H

6,7-OCH₃

Cl

H

6,7-OCH₃

Cl

H

6-CH₂CH₃

OCH₃

H

OCH₃

H

OCH₃

H

Br

H

Cl

H

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.⁵βIII-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.⁶GBP1 then is capable

to bind a panel of prosurvival kinases like PIM1, thus recruiting

them into cytoskeleton and prolonging their activity.⁶At

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 GI₅₀value. 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

R₅substituent 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−R₄are 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 R₅substituent (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−R₄as

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 (sp²like geometry). On the contrary, the ethyl chain

projects out of the ring plane (sp³geometry). 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.²⁹These

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.³⁰In other GTPases (e.g., Ras, Rho) this regulation is

performed by partner proteins (e.g., GAP and GEF).³¹Analysis

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.³²The

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.^13,14We found that the synthesis

of products where R₁is 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 ([C₂₀H₁₆Cl NO₅

+ H]⁺calcd 386.0790).

protein/

mutants

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 ([C₂₂H₂₀Br 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-d₆as 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

([C₂₂H₂₀Cl NO₃+ H]⁺calcd 382.1204).

General Synthesis of 4-Aza-2,3-didehydropodophyllotoxin

Derivatives. These derivatives were synthesized by following

previously reported method.^13,14An 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

([C₂₁H₁₇Cl₂NO₅+ 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 ([C₂₂H₁₉Cl₂NO₃+ 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 ([C₂₃H₂₅NO₇+ 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 ([C₂₁H₁₈Br 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 ([C₂₂H₂₃NO₆+ 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 ([C₂₁H₁₈Cl 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 ([C₂₁H₂₁NO₅+

H]⁺calcd 368.1492).

4-(2-Hydroxyethyl)-6-methoxy-9-phenyl-4,9-dihydrofuro[3,4-b]-

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 ([C₂₀H₁₉NO₄+

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

([C₂₃H₂₅NO₇+ 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 ([C₂₁H₂₁NO₅+ 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 ([C₂₂H₂₃NO₆+ 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 ([C₂₀H₁₈Br 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 ([C₂₁H₂₁NO₅+ 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

([C₂₄H₂₇NO₈+ 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 ([C₂₂H₂₃NO₆+ 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 ([C₂₄H₂₇NO₆+

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 ([C₂₁H₂₀Br 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 ([C₂₁H₂₁NO₃+

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 ([C₂₁H₂₀Cl 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

([C₂₁H₂₀Br 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 ([C₂₁H₁₉Cl₂NO₅+ 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

([C₂₁H₂₀Cl 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.¹⁸The

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.⁶Cells harvested in

cold PBS were extracted in lysis buﬀer, and coimmunoprecipitation

and Western blots were done as described previously.³³Antibodies

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.⁶Additional 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.³⁴The 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

Cl₂NO₃+ 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 10⁻⁴M 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 0.¹⁷Percentage 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 analysis¹⁷was 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 GI₅₀was 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 GI₅₀in a

given cell line for a drug, μ is the average of the GI₅₀of 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.⁶For 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/),²⁸a 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 (hGBP1^FL; 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 hGBP1^FLdownloaded 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 hGBP1^FLwas

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 hGBP1^FLwere 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 pK_avalues of azapodophyllotoxin

derivatives were calculated by using the ACD/pK_aDB, 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.³⁵The

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 PM7³⁶in the MOPAC2012 package³⁷and EF

(eigenvector following routine)³⁸as 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 hGBP1^FL.

The obtained homology model of hGBP1^FLwas 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 hGBP1^FLhomology 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;⁴⁰Insight2005, 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/Å²as 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 10⁶kcal/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 hGBP1^FLsequence (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 procedure⁴⁰and 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 10⁴minimization 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 Procheck³⁹structure 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

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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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Article Doi

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

DOI: 10.1021/jm5009902

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