Novel analogs of sulfasalazine as system xc ? antiporter inhibitors: Insights from the molecular modeling studies

Received: 17 November 2018

DOI: 10.1002/ddr.21557

Revised: 16 April 2019

Accepted: 27 May 2019

R E S E A R C H A R T I C L E

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Novel analogs of sulfasalazine as system x_cantiporter

inhibitors: Insights from the molecular modeling studies

Dhavalkumar Patel¹| Prashant S. Kharkar¹| Neha S. Gandhi²| Ekjot Kaur³

Shilpee Dutt³| Mukesh Nandave^1,4

|

¹Shobhaben Pratapbhai Patel School of

Abstract

Pharmacy and Technology Management,

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System x_c(Sx_c⁻), a cystine-glutamate antiporter, is established as an interesting tar-

get for the treatment of several pathologies including epileptic seizures, glioma, neu-

rodegenerative diseases, and multiple sclerosis. Erastin, sorafenib, and sulfasalazine

(SSZ) are a few of the established inhibitors of Sx_c⁻. However, its pharmacological

inhibition with novel and potent agents is still very much required due to potential

issues, for example, potency, bioavailability, and blood–brain barrier (BBB) permeabil-

ity, with the current lead molecules such as SSZ. Therefore, in this study, we report

the synthesis and structure–activity relationships (SAR) of SSZ derivatives along with

molecular docking and dynamics simulations using the developed homology model of

SVKM's NMIMS (Deemed to be University),

Vile Parle (West), Mumbai, Maharashtra, India

²School of Mathematical Sciences and

Institute for Health and Biomedical Innovation,

Queensland University of Technology,

Gardens Point Campus, Brisbane, Queensland,

Australia

³Shilpee Dutt Laboratory, Advanced Centre

for Treatment, Research and Education in

Cancer (ACTREC), Tata Memorial Centre,

Navi Mumbai, Maharashtra, India

⁴Department of Pharmacology, Delhi

Pharmaceutical Sciences and Research

University, New Delhi, India

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xCT chain of Sx_cantiporter. The generated homology model attempted to address

the limitations of previously reported comparative protein models, thereby increasing

the confidence in the computational modeling studies. The main objective of the pre-

sent study was to derive a suitable lead structure from SSZ eliminating its potential

issues for the treatment of glioblastoma multiforme (GBM), a deadly and malignant

grade IV astrocytoma. The designed compounds with favorable Sx_c⁻inhibitory activ-

Correspondence

Mukesh Nandave, Department of

Pharmacology, Delhi Pharmaceutical Sciences

and Research University, New Delhi, India.

Email: mukeshnandave@gmail.com

Funding information

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ity following in vitro Sx_cinhibition studies, showed moderately potent cytotoxicity

Department of Biotechnology , Ministry of

Science and Technology, Grant/Award

Number: BT/PR4605/MED/30/719/2012

in patient-derived human glioblastoma cells, thereby generating potential interest in

these compounds. The xCT-ligand model can be further optimized in search of potent

lead molecules for novel drug discovery and development studies.

K E Y W O R D S

cystine-glutamate antiporter, erastin, GBM, glioblastoma multiforme, sorafenib, sulfasalazine,

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system x_c

conquer cancer (Cassetta & Pollard, 2018; Kharkar, 2017; Shah &

Kharkar, 2018). In order to survive, the cancer cells have to combat

1

| INTRODUCTION

Cancer is a generic term used to describe numerous types of malig-

nancies of the body. Despite the time and efforts worth countless dol-

lars, cancer remains undefeated. The overall survival rate has

improved by only 3.4 months in 2013 compared to the treatments

available in 2003 (Salas-Vega, Iliopoulos, & Mossialos, 2017). Newer

targets and treatment modalities have been and are being explored to

oxidative stress produced as a result of aerobic metabolism (Sosa

et al., 2013). In this context, cancer cells adapt by a mechanism involv-

ing manipulation of intracellular signaling through aberrant gene

expression and thus maintain intracellular redox status (Balendiran,

Dabur, & Fraser, 2004). To neutralize the higher concentration of

reactive oxygen species (ROS) produced by the aerobic metabolism,

Drug Dev Res. 2019;1–20.

wileyonlinelibrary.com/journal/ddr

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PATEL ET AL.

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the cancer cells may synthesize a key antioxidant, glutathione (GSH),

thereby maintaining DNA synthesis, growth, and multidrug/radiation

resistance (Balendiran et al., 2004). The continuous supply of cystine

is mandatory for the production of adequate levels of GSH in tumor

cells (Habib, Linher-Melville, Lin, & Singh, 2015). Many studies have

reported that a variety of cancer cell types like glial (Sontheimer,

2008), head and neck (Chintala et al., 2010), lung (Guan et al., 2009),

breast (Balendiran et al., 2004), gastrointestinal (Ishimoto et al., 2011),

pancreatic (Lo, Ling, Wang, & Gout, 2008), ovarian (Okuno et al.,

2003), colon (Verrey et al., 2004), and many others maintain the intra-

cellular redox balance by importing one molecule of cystine (which

gets converted to cysteine intracellularly) with the simultaneous

release of one molecule of glutamate (Figure 1).

Sontheimer, 2008). Overexpression of Sx_cwas found to be associ-

ated with increased GSH synthesis in cancer cells in vitro, ultimately

compensating the antioxidant requirement of cancer cells and helping

in their survival. Sx_c⁻, basically a hetero(di)meric amino acid trans-

porter (HAT), consists of two subunits, xCT and 4F2hc. The xCT sub-

unit is known to perform the antiporter activity while 4F2hc subunit

is involved in trafficking of xCT (Verrey et al., 2004; Wagner, Lang, &

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Bröer, 2001). Inhibitors of Sx_cantiporter are likely to throw cancer

cells off-balance in their redox environment, thereby killing them due

to excessive ROS production. Additionally, these inhibitors are also

useful in several other therapeutic areas such as glutamate-induced

excitotoxicity, and epileptogenesis.

In a recent review, Patel, Kharkar, and Nandave (2015) have

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extensively reviewed emerging roles of Sx_cantiporter and its inhibi-

The equimolar exchange of cystine and glutamate is mediated by

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system x_c(Sx_c⁻) antiporter present in the cell membrane (Balendiran

tors in variety of CNS disorders such as Alzheimer's disease, psycho-

sis, drug addiction, depressive disorders, multiple sclerosis,

hypoglycemic neuronal cell death, glioma, and excitotoxicity. Over the

et al., 2004; Chintala et al., 2010; Guan et al., 2009; Habib et al.,

2015; Ishimoto et al., 2011; Lo et al., 2008; Okuno et al., 2003;

(a)

(b)

FIGURE 1 (a) Glutathione

synthesis through intracellular

movement of cystine by Sx_c⁻in

cancer cells to neutralize ROS;

(b) molecular structures of well-

known Sx_c⁻inhibitor drugs along

with their cellular potency data.

ROS, reactive oxygen species

PATEL ET AL.

3

years, Sx_c⁻antiporter always intrigued researchers for its involvement

in many pathies outlined previously. In their recent work, McCormick

et al. (2019) assessed the tumor redox status with the help of positron

emission tomography (PET) imaging of the antiporter activity using

radiotracer (S)-4-(3-[¹⁸F]fluoropropyl)-L-glutamic acid ([¹⁸F]FSPG).

The authors have clearly demonstrated reduced retention of the

radiotracer with increased levels of oxidative stress. In summary, the

authors proposed the utility of [¹⁸F]FSPG PET as an early redox indi-

cator of tumor response following treatment. Working on the similar

of the study was in identification of a promising strategy involving

inactivation of ATF4 for abrogating chemoresistance and enhanced

TMZ efficacy in human gliomas.

SSZ,

a mutual prodrug of 5-aminosalicylic acid (5-ASA) and

sulfapyridine, has been in therapy for ulcerative colitis due to its enzy-

matic breakdown in the gut. The highly polar and hydrophilc nature of

1 makes it poorly bioavailable (Robe et al., 2009). The randomized phase

1/2 trial of 1 for treating gliomas was an utter failure. Despite these facts,

the interest of the scientific community in SSZ continued over all these

years, Sehm et al. (2016) demonstrated that SSZ did not affect cell viabil-

ity below 200 μM and above that it exhibited gliomatoxicity. It has been

shown to (a) induce ferroptotic cell death in glioma cells, (b) potentiate

the chemotherapeutic and autophagy-inducing activities of TMZ, and

(c) reduced glioma-derived edema. Overall, SSZ has been viewed as a

tumor microenvironment-normalizing drug.

lines, Čolovic et al. (2018) synthesized and evaluated ¹⁸F-labeled

ꢀ

boramino acid analog of aminosuberic acid for PET imaging of the

antiporter. The PC3 tumor xenografts in mice were visualized using

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the radiotracer because the tumor cells could take it up via Sx_canti-

porter. Both these studies unequivocally supported the importance of

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Sx_cin tumor imaging in particular. In an interesting study, Mil-

Several researchers devoted substantial amount of time and

efforts for developing potent, drug-like small-molecule Sx_c⁻inhibitors.

Patel et al. (2010) synthesized derivatives of α-amino-3-hydroxy-

5-methyl-4-isoxazolepropionic acid (AMPA) and α-amino-3-carboxy-

5-methyl-isoxazole propionic acid (ACPA) and analogs of 1 to evaluate

adinovic, Ungard, Linher-Melville, Popovic, and Singh (2018) demon-

strated the functional effect of the actions of neurotrophin nerve

growth factor on its cognate receptor tyrosine kinase TrkA on xCT,

culminating in reduced cancer-induced bone pain. The TrkA inhibitor

AG879 inhibited functional system x_c⁻activity. The study emphasized

the importance of Sx_c⁻-mediated TrkA activation as a means of thera-

peutic intervention in cancer-related pain management.

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their Sx_cinhibitory activity. Of all the synthesized molecules, amino

acid naphthyl-ACPA and hydrazine carboxylic acid demonstrated simi-

lar potency as endogenous substrate, L-cystine, with K_ivalues 52, 64,

and 59 μM, respectively, while corresponding isoxazolo[3,4-d] pyri-

dazinones were far less potent. The molecules were designed essen-

tially to mimic important structural features of 1, including azo

linkage. In yet another interesting study, Shukla et al. (2011) synthe-

Two FDA-approved drugs, sulfasalazine (SSZ) (1, Figure 1) and

sorafenib (2, Figure 1) are few of the potent inhibitors of Sx_c⁻. Addi-

tionally, erastin (3, Figure 1) was shown to have the highest potency

among all the Sx_c⁻inhibitors investigated till date (Dixon et al., 2014).

Sorafenib, a conventional molecularly targeted anticancer drug, is

associated with a lot of adverse effects (Dahlmanns et al., 2017; Gran-

ito et al., 2016), while erastin lacks promising in vivo anticancer data.

In a recent study, Sato et al. (2018) showed that erastin, a ferroptosis

(regulated necrotic cell death) inducer, synergized with cisplatin to

increase its cytotoxicity in cancer cells via irreversible inhibition of

Sx_c⁻. In yet another interesting study, Liu et al. (2017) and Clemons,

Liu, Duong, and Phillips (2017) discussed the importance of therapeu-

tic strategies for targeting mutant p53 protein. One such strategy

involved suppressing function of xCT along with APR-246, a small-

molecule known to restore sequence-specific DNA binding of mutant

p53 resulting in wild-type p53 transcriptional activity and tumor-

suppressor function. This, beyond doubt, delineates the importance of

xCT inhibitors in cancer treatment. Wang et al. (2017) studied the

effects of propofol, a general anesthetic on proliferation and invasive-

ness of C6 glioma cells. The study successfully demonstrated the

inhibitory effects (reduced cell viability, invasiveness, and migration

coupled with decreased glutamate release) of propofol via Ca2+

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sized several analogs of 1 as potential Sx_cinhibitors. The individual

drugs making up 1, that is, 5-ASA and sulfapyridine exhibited half

maximal inhibitory concentration (IC₅₀₎values for Sx_c⁻-mediated cys-

tine uptake >1,000 μM, while the corresponding value for 1 was

30 μM. In an attempt to replace the problematic azo linkage, several

saturated and unsaturated linkers such as alkene, alkyne or the

corresponding alkane, with the distal sulfapyridine moiety constant,

yielded one equipotent (IC₅₀= 30 μM) and other less potent SSZ ana-

logs, including 1 minus the pyridine sulfonamido compounds. Overall,

the study mainly focused on replacing the azo linkage with other

linkers. No attempt was made to explore the isosteres of the salicylic

acid as well as the sulfapyridine moieties. Among these inhibitors, 1,

an approved anti-inflammatory drug, turned out to be a promising

lead for further chemical optimization of potency and pharmacoki-

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netic properties because it inhibited Sx_cwith moderate potency and

it lacked the adverse effects associated with approved anticancer

drugs. However, SSZ is problematic due to its very poor systemic bio-

availability (approximately 12%), rapid cleavage by colonic bacteria

into inactive constituents and short half-life (~80 min). Its blood–

brain-barrier (BBB) permeability is not known. It is likely to be

prevented from crossing the BBB because it is so very hydrophilic

and most importantly acidic. Acids are unlikely to cross BBB

(Kharkar, 2014).

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permeable AMPA receptor (CPAR)—Sx_cpathway. Chen, Rauh,

Buchfelder, Eyupoglu, and Savaskan (2017) and Chen et al. (2017)

investigated the role of activating transcription factor 4 (ATF4) which

critically regulates the oxido-metabolic state in gliomas in the patho-

genesis of temozolomide (TMZ) (first-line treatment for malignant gli-

omas)-resistance. The authors found out that the TMZ treatment led

to increased expression of ATF4, xCT among others and the elevation

of xCT as a consequential event of ATF4 activation. The significance

Careful examination of the molecular structure of 1 (Figure 1)

reveals a lot of problematic features such as azo linkage (─N N─),

the pyridine ring, and salicylic acid moiety. Reduction of the azo

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PATEL ET AL.

linkage by the intestinal bacteria yields potentially mutagenic struc-

tures (with anilino groups, sulfapyridine and 5-ASA). The salicylic acid

moiety can undergo glucuronidation quickly during conjugation reac-

tion. Even the highly anionic nature of the salicylic acid is likely to

hamper membrane permeability during random-walk process, includ-

ing BBB permeation. The literature reports indicated few relatively

closer analogs of 1 being less potent. (Shukla et al., 2011). We thought

of systematically maneuvering the SSZ structure in an attempt to

arrive at a hit structure(s) which is tailor-made and preferably devoid

of issues of 1. In systematizing our design and synthesis efforts, we

initiated our work with building a homology model of the xCT chain of

secondary structure elements, we started from the xCT and the tem-

plates alignment derived by the HHpred server. Hence, initially, back-

bone atom coordinates for transmembrane segments only were

obtained from the relative coordinates of AdiC or ApcT. Subsequently,

insertions and deletions were included and their backbones modeled.

Loops were modeled on the basis of the structure of compatible frag-

ments found in the PDB, and side chains were picked from a rotamer

library using SCWRL4 (Krivov, Shapovalov, & Dunbrack Jr., 2009).

Models were submitted to stepwise energy minimizations to reduce

steric contacts (r.m.s. gradient = 0.01). Initially, hydrogens were mini-

mized retaining the backbone and side chains fixed. The charges of

side-chains were assigned at physiological pH. Minimizations were

further carried by gradually realizing harmonic restraints on the side

chains and keeping backbone fixed. In next step, harmonic restraints

were only applied to the backbone. In the final step of minimization,

the entire protein was treated as flexible. In this way, 50 initial models

based on the CHARMM27 force field were generated, all sharing the

same transmembrane topology but differing in local conformations.

The quality of the models was evaluated by various diagnostics tools

available on MolProbity webserver (Davis et al., 2007) and the Struc-

tural Analysis and Verification Server (SAVES, http://nihserver. mbi.

ucla.edu/SAVES/) before and after the refinement step. The energeti-

cally most favorable models in apo (inward facing), open-occluded and

open-out (Figure 3) conformations were selected for docking of the

inhibitors.

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Sx_cto rationally aid our design process. In the absence of the 3D

structural information, the main emphasis was placed on building a

comparative model of the antiporter which would overcome issues

with similar, previously reported homology models (Matti et al., 2013).

The developed homology model, after extended molecular dynamics

simulations, was further used for molecular docking studies of the

designed analogs of 1.

In the present study, we report the homology model development

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of the Sx_cantiporter and provide an atomic description of SSZ-

binding site. We also report the use of this model to guide the design

of novel SSZ analogs along with their synthesis and in vitro biological

evaluation in target-specific inhibition assays. Furthermore, we evalu-

ated their cytotoxicity in patient-derived glial cell lines. Overall, the

present investigation is a rational attempt to advance the design and

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development of novel Sx_cantiporter inhibitors as potential antican-

cer agents.

2.2 | Docking studies

2.2.1 | Software

2

| MATERIALS AND METHODS

2.1 | Homology modeling of system x_c⁻antiporter

All the molecular modeling studies described herein were performed

using Small-Molecule Drug Discovery Suite 2015–4 (Schrödinger

Release 2015-4, 2015) and the modules included therein for per-

forming various molecular modeling operations as described below.

To identify appropriate templates, the sequence of human cys-

tine/glutamate transporter (Uniprot accession No. Q9UPY5) was sub-

mitted to homology recognition algorithms (threading servers) such as

Homology detection & structure prediction (HHpred) by HMM-HMM

comparison (http://toolkit.tuebingen.mpg.de/hhpred) (Söding, Biegert, &

Lupas, 2005) AND LOMETS (Local MEta-Threading-Server) (http://

zhanglab.ccmb.med.umich.edu/LO-METS/) (Wu & Zhang, 2007) for

detecting remote homologies (Figure 2 and Figure S1S). We then used

the structures of bacterial L-arginine/agmatine antiporter, AdiC open-

to-out Arg⁺bound conformation (PDB code 3OB6) (Kowalczyk et al.,

2011) and the outward-facing Arg⁺-bound occluded (PDB 3L1L) (Gao

et al., 2010) as templates for modeling the transmembrane domains.

The substrate-free structures (apo) of ApcT, a broad-specificity amino

acid transporter, and AdiC closely resembled each other with RMSD

~1.25 Å and, therefore, we modeled xCT in the fully occluded/inward

facing conformation using apo-ApcT (PDB code 3GIA) (Shaffer,

Goehring, Shankaranarayanan, & Gouaux, 2009) as reported previously

(Gao et al., 2010).

2.2.2 | Protein structures

The developed homology models of xCT—apo, open-occluded and

open-out—were subjected to Protein Preparation wizard using default

settings as implemented in Schrödinger suite 2015–4 (Sastry,

Adzhigirey, Day, Annabhimoju, & Sherman, 2013). The termini were

capped. The H-bonds were optimized and, in the end, restrained mini-

mization was performed wherein the heavy atoms were converged to

root mean square deviation (RMSD) 0.3 Å. The prepared protein was

then subjected to further minimization using Prime 4.2 (default set-

tings) (Jacobson et al., 2004; Jacobson, Friesner, Xiang, & Honig,

2002; Halgren et al., 2004; Sastry et al., 2013). Glide version 6.9

(Friesner et al., 2004), as implemented in Schrödinger suite 2015–4,

was used for performing docking studies of SSZ (1, Figure 1) and the

designed molecules (Table 1).

Homology modeling was performed using MODELER-9v11 (Sali &

Blundell, 1993) tool implemented in the UCSF Chimera (Pettersen

et al., 2004). For the correct identification and positioning of the

PATEL ET AL.

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FIGURE 2 Sequence alignment of the xCT and apo ApcT, used for homology modeling. Sequences were aligned based on HMM profile

alignment techniques such as HHpred. HHpred, Homology detection and structure prediction

2.2.3 | Ligand structures

2.2.4 | Receptor grid generation

The ligand structures were built in Maestro 10.4. All the structures

were then optimized using the LigPrep version 2.6, as implemented in

Schrödinger suite 2015–4 with default settings. Ligand preparation

was followed by the docking studies. The study was aimed at investi-

gating the binding modes of 1 (Figure 1) and few moderately potent

inhibitors identified in this study.

The refined protein structures were used to define the ligand-binding

pocket using Receptor Grid Generation module. The centroid of residue

Arg396 was used for placing the enclosing box. The grid was then

used for docking the ligands in the ligand-binding site. For docking

studies, Extra Precision (XP) (Friesner et al., 2006) mode was used.

The docked poses were minimized and RMSD to input ligand

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PATEL ET AL.

FIGURE 3 The conformation of the xCT ligand-binding site in (a) open-out conformation, (b) open-occluded conformation, and (c) inward-

occluded conformation. His110 in the helix lies toward the cytosolic region

geometries were calculated. Table 1 lists the docking scores for all the

molecules used in the docking studies along with interacting amino

residues. Figure 4 shows the docking poses of representative mole-

cules (1, 15, and 12) into the ligand-binding pocket of xCT. After

docking studies, the pose viewer file was used for studying the bind-

ing modes of the ligands with the xCT chain of the antiporter. The

docked posed of all other ligands in Table 1 are shown in Figures S4S

(4), S5 (5), S6 (7), S7 (11), S8 (14), S9 (18), and S10 (19) (see Supporting

Information section).

solvated with TIP3 water molecules. We have not taken into account

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the heterodimeric structure of Sx_c(Sato, Tamba, Ishii, & Bannai,

1999) because of the experimental data on interactions of xCT with

the heavy chain, 4F2 is sparsely available (Palacín, Errasti-Mur-

ugarren, & Rosell, 2016). The activation, inactivation, and ion indepen-

dent mechanism of the receptor are beyond the scope of this study.

All protein residues were set to the standard CHARMM protonation

states at neutral pH. The system was neutralized by adding NaCl as

counterions and also adding a concentration of 150 mM. The simula-

tion system had a dimension of approximately 93.1 Å × 93.1 Å × 120 Å

and contained 96,593 atoms, including 208 POPC molecules, 20,477

CHARMM TIP3P water molecules, 79 Na⁺and 88 Cl⁻ions (Figure 5).

The optimized protocol for running CHARMM simulations in

GROMACS was used in this work (Lee et al., 2016). The structure was

first energy-minimized for 5,000 steps using a steepest-descent algo-

rithm with a tolerance of 1,000 kJ mol⁻¹nm⁻². The systems were

then equilibrated by applying position restraints on the complex and

NVT ensemble simulations for 400 ps at a temperature of 303 K. Sub-

sequently, the built systems were further equilibrated without any

restraints under semi-isotropic Berendsen pressure coupling. After

the standard input builder minimization and equilibration steps, the

production run of each simulation was performed or 600 ns. The van

der Waal interactions were calculated with a force-based switch cut-

off of 1.2 nm. Electrostatic corrections were made by Particle Mesh

Ewald (PME) algorithm (Essmann et al., 1995), with 1.2 nm cut-off for

Columbic interactions. All atom bond lengths were linked using the

LINCS algorithm (Hess, Bekker, Herman, & Johannes, 1997) in con-

junction with the simulation time step of 2 fs. In addition, the pressure

was maintained at 1 bar. To maintain the temperature, a Nosé–

Hoover temperature coupling method (Hoover, 1985; Nosé,

1984; Parrinello & Rahman, 1981) with a tau-t of 1 ps was used, and

for pressure coupling, a semi-isotropic Parrinello–Rahman method

2.3 | Computational prediction of physicochemical

and pharmacokinetic properties

The molecular/physicochemical properties for the designed molecules

were calculated/predicted with the help of Canvas and QikProp mod-

ules of Schrödinger Small Molecule Drug Discovery Suite 2015–4.

The compounds were first drawn in 2D, prepared using LigPrep mod-

ule as per standard protocols. All the minimized structures were

loaded into QikProp module and the physicochemical and pharmacoki-

netic properties were predicted.

2.4 | Molecular dynamics simulations

The molecular interactions were computed by Gromacs v5.0.4

(Abraham et al., 2015) and using the CHARMM36 force field as

parameter (Pettersen et al., 2004). The CHARMM-GUI website

(http://www.charmm-gui.org) was used to automate the system setup

for MD simulation (Wu et al., 2014). Lipid bilayers models were built

using a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC) phospholipids using CHARMM-GUI membrane builder (Jo,

Kim, & Im, 2007). The docked xCT-SSZ complex was placed in the

rectangular simulation box and the preassembled bilayers were

PATEL ET AL.

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PATEL ET AL.

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FIGURE 4 Binding mode of 1, 15, and 12 in the ligand-binding site of Sx_c⁻antiporter; (a) 2D representation of the drug (1)—transporter

interactions depicting H-bonds, π–π stacking and hydrophobic interactions; (b) 2D representation of ligand (15)—antiporter interaction, and (c) 2D

representation of ligand (12)— antiporter interaction. 2D, two-dimensional

(Nosé, 1984; Nosé & Klein, 1983) with a tau-p of 5 ps and a com-

2.4.1 | Cavity-volume calculator

pressibility of 4.5 × 10⁻⁵bar⁻1 was used.

The trajectory was subjected for analyses of secondary structure,

root mean square deviation (RMSD) on the backbone atoms of protein,

all atoms of the binding site and heavy atoms of ligands using

AMBERTools16 (Case et al., 2016). Hydrogen bonds, native (any atoms

that are closer than 7 Å in the specified reference docked pose) and

nonnative contacts of the protein within 7 Å from the ligand and clus-

tering analyses were also performed. A hydrogen bond between a

donor (D–H) and a heavy acceptor atom (A) was considered to be

formed when the A to D distance was <3.0 Å and the A-H-D angle is

greater than the angle cut-off of 135^ꢀ. These geometric criteria for

defining hydrogen bonds are consistent with those used in prior stud-

ies. Secondary structure content was assessed with the DSSP algo-

rithm. Visualization was carried out using UCSF Chimera (Pettersen

et al., 2004) and Discovery Studio 4.1.

The volume of the SSZ cavity was calculated by POVME (Durrant,

Votapka, Sørensen, & Amaro, 2014). The xCT snapshots were extracted

from the trajectory every frame (60,000 frames in total from 600 ns)

and superimposed onto the reference structure with a pregenerated

3D-grid representing the SSZ cavity. The volume was calculated by

counting the grid points located within 10 Å in the SSZ-cavity.

Clustering analysis of the trajectory obtained from MD simulation

was performed using CPPTRAJ of Amber Tools 16 with the hierarchi-

cal agglomerative algorithm and average-linkage approach and cluster

size set to 10, to determine various populations of interactions of SSZ

with the receptor, water, and ions in the cavity. Cluster analysis of

30,000 frames saved at 20-ps intervals during the 0–600 ns period

was done by aligning the first root mean square coordinate deviation

on the backbone of xCT residues and then by clustering based on the

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PATEL ET AL.

FIGURE 5 Schematic representation of protein–ligand complex embedded in the POPC bilayer. Water molecules and lipids are shown as

sticks, the protein in ribbon colored from N- (blue color) to C-terminal (red) and the ions as spheres. Sulfasalazine is shown in van der Waals

spheres with the receptor shown in a representative snapshot taken from the top eight clusters

movement of the ligand with respect to the last RMS-fit (in this case

the backbone atoms of residues xCT).

with salicylic acid resulted in 7. Amides were synthesized according to

Scheme 3. The ─COOH group of 8 was protected as an ester to give

9

which on further reaction with different aromatic acids gave

corresponding amides 15a and 15 h along with the acylated phenolic

impurities. The ester derivatives (15a and 15 h) and their acylated phe-

nolic impurities were further subjected to hydrolysis using LiOH.H₂O to

give the corresponding acids 11 and 12. In another diazotization reaction

(Scheme 4), 4-aminobenzensulfonic acid (13) on diazotization followed

by coupling with α- and β-naphthols yielded 14 and 15. Sulfonamides

(18 and 19) were synthesized by reaction of p-nitrobenzenesulfonyl

chloride (16) with aromatic amines (17a and 17b) (Scheme 5).

2.5 | Chemistry

2.5.1 | General

All the solvents (including dry solvents) and chemicals were procured

from commercial suppliers such as Sigma-Aldrich Chemical Corporation

(St. Louis, MO), Fischer Scientific (Hampton, NH), and VWR Interna-

tional (Radnor, PA), and were used as received unless otherwise indi-

cated. All reactions were performed under an inert atmosphere (N₂)

unless otherwise noted. Analytical silica gel 60 F₂₅₄-coated TLC plates

were purchased from Merck Millipore (Billerica, MA) and were visual-

ized with UV light or by treatment with TLC reagents such as ninhydrin,

Dragandorff's or phosphomolybdic acid (PMA). Flash-column chroma-

tography was carried out on Combiflash R_f(Lincoln, NE) using silica gel

(230–400 mesh). ¹H-NMR spectra were routinely recorded on Bruker

400 MHz FT-NMR (Billerica), with tetramethylsilane (TMS) as an inter-

nal standard. Mass spectra were obtained using Agilent LCMS, 6110AA

model (Santa Clara, CA). Reversed-phase HPLC (RP-HPLC) analyses

were performed using Agilent HPLC instrument (Santa Clara) equipped

with PDA absorption detector. Melting points were recorded using

Veego Instruments, VMP-DS model, capillary melting point apparatus

(Mumbai, Maharashtra, India) and are uncorrected. Radioactivity was

measured by Hidex-Triathler Liquid Scintillation Counter (Brandon, FL).

2.5.3 | Synthesis of ethyl 2-hydroxy-5-[(E)-2–4

{4-[(pyridine-2-yl)sulfamoyl]phenyl}diazen-1-yl]-

benzoate (4)

A mixture of 1 (0.5 g, 10 mmol), absolute EtOH (10 mL) and conc.

H₂SO₄(1 mL) was boiled under reflux with stirring for 8.5 hr followed

by evaporation to dryness. Water (5 mL) was added to the residue

and the mixture filtered. The filtrate was basified with saturated

NaHCO₃solution and the precipitated solid was collected by filtra-

tion. The filtrate was extracted with DCM and the solvent evaporated

to dryness. The residue was triturated with petroleum ether

(40–60 ^ꢀC) and filtered to yield 4 as yellow solid (0.46 g, 87%). R_f:

0.68 (CHCl₃/MeOH 9:1); IR (KBr): ν 3,417 (m; ν[O─H]), 3,203 (s;

ν[N─H]), 2,926 (m; ν[C─H_Alipha]), 2,385 (m; ν[N N]),1,685 (s; ν[C O]),

[

³⁵S]L-cystine was purchased from BRIT (Mumbai).

1,295 (s; ν[S─O] cm⁻^{1 1}H-NMR (400 MHz, DMSO-d₆): δ (ppm) 11.1

;

(s, 1H), 8.32 (d, 1H), 8.0–8.1 (m, 3H), 7.92–7.99 (m, 3H), 7.75 (t, 1H),

7.17–7.25 (m, 2H), 6.85 (t, 1H); 4.41, (q, 2H), 1.40 (t, 3H); LC–MS

(ESI): 427.1 [M + H]⁺; Melting point: 213 ^ꢀC; Elemental analysis calcu-

lated (%) for C₂₀H₁₈N₄O₅S: C 56.33, H 4.25, N 13.14; Found: C

56.72, H 4.11, N 12.92.

2.5.2 | Synthetic schemes

The synthetic route leading to compounds 4 and 5 is shown in Scheme 1.

Esterification of 1 resulted in 4, which upon further amidation led to 5.

In Scheme 2, diazotization of sulphanilamide (6) followed by coupling

PATEL ET AL.

11

SCHEME 1 Reagents and conditions: (a) EtOH, H₂SO_4,RT; (b) EtOH, NH₃, RT

SCHEME 2 Reagents and conditions: (a) conc. HCl, NaNO₂, 0–5 ^ꢀC; (b) salicylic acid, NaOH, 0–5 ^ꢀC

SCHEME 3 Reagents and conditions: (a) EtOH, H₂SO₄, reflux; (b) (i) 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-Oxide

Tetrafluoroborate (TBTU), di-isopropyl ethylamine (DIPEA), (ii) benzoic acid, (iii) hydrocinnamic acid, DCM; (c) LiOH.H₂O, tetrahydrofuran-

methanol (THF-MeOH)

2.5.4 | Synthesis of 2-Hydroxy-5-[(E)-2-{4-[(pyridine-

2-yl)sulfamoyl]phenyl}diazen-1-yl]benza-mide (5)

reaction mixture was filtered to collect yellow solid (0.15 g, 58%). R_f:

0.51 (CHCl₃); IR (KBr): ν 3,425 (m; ν[O─H]), 3,199 (s; ν[N─H]), 2,993

(m; ν[C─H_Alipha]), 2,361 (m; ν[N N]), 1,685 (s;ν[C O]), 1,388 (s;ν

Compound 4 (0.3 g, 0.7 mmol) was dissolved in EtOH (20 mL). The

reaction mixture was saturated with NH₃gas and further kept for

24 hr. The reaction was monitored with TLC for completion. The

;

[S─O]) cm⁻^{1 1}H-NMR (400 MHz, DMSO-d₆): δ (ppm) 13.8 (brs, 1H),

8.72 (brs, 1H), 8.6 (d, 1H), 8.12 (s, 1H), 8.05–8.09 (m, 2H), 7.95–8.0

12

PATEL ET AL.

SCHEME 4 Reagents and conditions: (a) (i) conc. HCl, NaNO₂, 0–5 ^ꢀC; (ii) aq. NaOH, (1) α-naphthol or (2) β-naphthol, 0–5 ^ꢀC

SCHEME 5 Reagents and

Conditions: (a) pyridine, 80 ^ꢀC

(m, 2H), 7.9–7.94 (m, 2H), 7.74–7.8 (t, 1H), 7.2 (d, 1H), 7.05–7.09 (d,

1H), 6.85–6.9 (t, 1H); LC–MS (ESI): 397.1 [M + H]⁺; Melting point:

190–192 ^ꢀC. Elemental analysis calculated (%) for C₁₈H₁₅N₅O₄S: C

54.40, H 3.80, N 17.62; Found: C 54.18, H 3.39, N 17.28.

(ppm) 8.38 (d, 1H), 8.14–8.20 (dd, 1H), 8.0 (m, 4H), 7.5 (m, 2H),

7.16–7.18 (d, 1H); LC–MS (ESI): 322.14 [M + H]⁺; Melting point:

264–266 ^ꢀC; Elemental analysis calculated (%) for C₁₃H₁₁N₃O₅S: C

48.49, H 3.45, N 13.08; Found: C 48.13, H 3.05, N 13.35.

2.5.5 | General procedure for synthesis of 7, 14–15

2.5.7 | Synthesis of 4-[(E)-2-(4-Hydroxynaphthalen-

(Method A)

1-yl)diazen-1-yl]benzene-1-sulfonic acid (14)

Respective amines along with NaNO₂(0.13 g, 15 mmol) were dis-

solved in of water (1.5 mL) and maintained at 0–5^ꢀC. Conc. HCl

(1.5 mL) diluted with water (1.5 mL) maintained at 0–5 ^ꢀC was then

added to the above mixture. After 10 min. Stirring (0–5 ^ꢀC), the solids

in the above mixture were filtered and added to respective phenols

(5.4 mmol) dissolved in 2.5 M NaOH (10 mL) and stirred at 0–5 ^ꢀC.

After 10 min., conc. HCl (1.5 mL) was added and allowed to stir for

45 min at 0–5^ꢀC. NaCl (1 g) was then added to the reaction mixture

and heated until all the solids dissolved. The reaction was monitored

by TLC for completion. The reaction mixture was cooled at RT and

then on an ice bath for 15 min. Solids were filtered and air dried. The

crude product was purified by recrystallization from EtOH to give title

compound.

Reaction of 13 (0.49 g, 2.8 mmol) following diazotization with

α-naphthol (0.38 g, 2.8 mmol) as described in method A resulted in

compound 14 as brown solid (0.4 g, 47%). Rf: 0.33 (CHCl₃); IR (KBr): ν

=3,430 (s; ν[O − H]), 2,362 (m; ν[N=N]), 1,652 (w;ν[C-H Ar]), 1,173 (s;

ν[S − O]) cm⁻^{1 1}H-NMR (400 MHz, DMSO-d₆): δ (ppm) 8.7 (d, 2H),

;

8.15 (d, 1H), 8.01 (d, 1H), 7.6–7.8 (m, 4H), 7.5 (m, 2H), 7.3 (t, 1H), 6.42

(d, 1H); LC–MS (ESI): 329.1 [M + H]+. Melting point >300^ꢀC; Elemen-

tal analysis calculated (%) for C₁₆H₁₂N₂O₄S: C 58.53, H 3.68, N 8.53;

Found: C 58.42, H 3.60, N 8.24.

2.5.8 | Synthesis of 4-[(E)-2-(2-Hydroxynaphthalen-

1-yl)diazen-1-yl]benzene-1-sulfonic acid (15)

Compound 15 was obtained as orange solid (0.72 g, 81%) by reaction

of diazotised 13 (0.49 g, 2.8 mmol) with β-naphthol (0.38 g, 2.8 mmol)

as described in method A. R_f: 0.39 (CHCl₃); IR (KBr): ν = 3,429 (s; ν[O─H]),

2.5.6 | Synthesis of 2-Hydroxy-5-[(E)-

2-[4-sulfamoylphenyl]diazen-1-yl]benzoic acid (7)

2,356 (m; ν[N N]), 1,661 (w;ν[C─H_Ar]), 1,195 (s; ν[S─O]) cm^{−1 1}H-

;

Compound 7 was obtained as red solid (1.1 g, 63%) by diazotization

reaction of 6 (1 g, 5 mmol) with salicylic acid (0.76 g, 5 mmol) as

described in Method A. R_f: 0.4 (CHCl₃/MeOH 8:2); IR (KBr):

NMR (400 MHz, DMSO-d₆): δ (ppm) 15.8 (s, 1H), 8.55 (d, 1H), 7.92 (d,

1H), 7.79 (d, 2H), 7.75 (s, 1H), 7.71 (d, 2H), 7.60 (t, 1H), 7.41 (t, 1H),

6.46 (d, 1H); LC–MS (ESI): 327.05 [M–H]⁻; Melting point >300 ^ꢀC;

Elemental analysis calculated (%) for C₁₆H₁₂N₂O₄S: C 58.53, H 3.68,

N 8.53; Found: C 58.16, H 3.62, N 8.29.

ν

3,367 (w; ν[O─H]), 3,199 (m; ν[N─H]), 2,369 (w; ν[N N]), 1,685

(s; ν[C O]), 1,299 (s; ν[S─O]) cm^{−1 1}H-NMR (400 MHz, DMSO-d₆): δ

;

PATEL ET AL.

13

and the precipitated solids were filtered, washed with cold water and

air dried.

2.5.9 | General procedure for the synthesis of 18–19

(Method B)

4-Nitrobenzenesulfonyl chloride was dissolved in pyridine (15 mL).

Respective amines (4 mmol) were added to above solution and stirred

for 2 hr at 80 ^ꢀC. The reaction was monitored by TLC. The excess of

pyridine was removed by washing with water (30 mL) yielding crude

solid product.

2.5.13 | Synthesis of 5-Benzamido-

2-hydroxybenzoic acid (11)

Reaction of 9 (0.5 g, 2.76 mmol) and benzoic acid (0.33 g, 2.76 mmol)

formed 10a which was further hydrolysed to obtain 11 as brown solid

(0.054 g, 27%) as described in general method C. R_f: 0.4 (CHCl₃/

MeOH = 9:1); IR (KBr): ν = 3,287 (s; ν[N─H]), 1,664 (s; ν[C O Acid]),

2.5.10 | Synthesis of 4-nitro-N-(pyridine-2-yl)

benzene-1-sulfonamide (18)

1,528 (s; ν[C O Amide]) cm^{−1 1}H-NMR (400 MHz, DMSO-d₆): δ

;

(ppm) 14.1 (brs, 1H), 11.1 (brs, 1H), 10.2 (s, 1H), 8.25 (d, 1H), 7.91 (d,

2H), 7.82 (dd, 1H), 7.52 (m, 2H), 7.45 (m, 1H), 6.93 (d, 1H); LC–MS

(ESI): 316.1 [M + H]⁺; Melting point: 197 ^ꢀC; Elemental analysis calcu-

lated (%) for C₁₄H₁₁NO₄: C 65.37, H 4.31, N 5.44; Found: C 64.96, H

4.21, N 5.39.

Compound 18 was obtained as creamy white solid (0.76 g, 60%) from

16 (1 g, 4 mmol) and 17a (0.38 g, 4 mmol) as described in Method

B. R_f: 0.52 (CHCl₃/MeOH = 9:1); IR (KBr): ν =2,930 (m; ν[N─H]),

1,514 (w; ν[N─O]), 1,130 (s;ν[S─O]) cm⁻^{1 1}H-NMR (400 MHz,

;

DMSO-d₆): δ (ppm) 11.17 (s, 1H), 8.42 (d, 1H), 8.33 (d, 2H), 8.15–8.25

(m, 3H), 7.85 (t, 1H), 7.16 (t, 1H); LC–MS (ESI): 278 [M–H]⁻; Melting

point: 192 ^ꢀC; Elemental analysis calculated (%) for C₁₁H₉N₃O₄: C

47.31, H 3.25, N 15.05; Found: C 47.20, H 3.05, N 14.85.

2.5.14 | Synthesis of 2-Hydroxy-

5-(3-phenylpropanamido)benzoic acid (12)

Reaction of 9 (0.5 g, 2 mmol) and hydrocinnamic acid (0.5 g,

2.76 mmol) formed 10b which was further hydrolysed to obtain 12 as

white solid (0.066 g, 33%) as described in general method C. R_f: 0.21

(CHCl₃/MeOH = 9:1); IR (KBr): ν = 3,265 (s; ν[N─H]),1,640 cm−¹(s;

2.5.11 | Synthesis of 4-nitro-N-phenylbenzene-

1-sulfonamide (19)

ν[C OAcid]), 1,569 (s;ν[C OAmide]) cm⁻¹

;

¹H-NMR (400 MHz,

Compound 19 was obtained as creamy white solid (0.84 g, 65%) from

16 (1 g, 4 mmol) and 17b (0.37 g, 4 mmol) as described in Method

B. R_f: 0.62 (CHCl₃/MeOH = 95:5); IR (KBr): ν = 3,127 (m; ν[N─H]),

1,511 (s; ν[N─O]), 1,147 (s;ν(S─O)) cm⁻¹; ¹H-NMR (400 MHz, DMSO-

d₆): δ (ppm) 10.6 (s, 1H), 8.33 (d, 2H), 7.94 (d, 2H), 7.21–7.28 (t, 2H),

7.05–7.12 (m, 3H); LC–MS (ESI): 277 [M–H]⁻; Melting point: 153 ^ꢀC;

Elemental analysis calculated (%) for C₁₂H₁₀N₂O₄S: C 51.79, H 3.62,

N 10.07; Found: C 51.42, H 3.58, N 10.40.

DMSO-d₆): δ (ppm) 14.0 (brs, 1H), 11.1 (brs, 1H), 9.85 (s, 1H), 8.10 (d,

1H), 7.62 (dd, 2H), 7.24 (m, 4H), 7.15 (t, 1H), 6.85 (d, 1H), 2.90 (t, 2H),

2.50 (t, 2H); LC–MS (ESI): 286.2 [M + H]⁺; Melting point: 211 ^ꢀC;

Elemental analysis calculated (%) for C₁₆H₁₅NO₄: C 67.36, H 5.30, N

4.91; Found: C 67.21, H5.21, N 4.69.

2.6 | Biology

2.6.1 | In vitro Sx_c⁻inhibition assay ([³⁵S]L-Cystine

uptake assay)

2.5.12 | General procedure for synthesis of 11–12

(Method C)

Uptake assay was performed as reported earlier by Liu, Rush, Zapata,

and Lobner (2009) with a few modifications. Briefly, 12 × 10⁴cel-

ls/mL were seeded in 96-well plates and incubated (5% CO₂, 37 ^ꢀC)

for 24 hr. Media were removed and cells were washed with uptake

buffer. Immediately after washing, [³⁵S] L-cystine (5 μCi/mL) was

exposed to cells along with title compounds and unlabeled cystine for

3 min. Duration. [³⁵S]L-cystine present in the extracellular environ-

ment was washed with ice-cold PBS. Radioactivity was measured

after dissolving the cells using 0.3 N NaOH. Values were normalized

to positive blank which contained exposure of [³⁵S]L-cystine to cells

in the absence of synthesized compounds.

5-Aminosalicylic acid (15 g, 97 mmol), absolute EtOH (250 mL) and

7.5 mL conc. H₂SO₄were refluxed with stirring for 8.5 hr followed by

evaporation to dryness. Water (20 mL) was added to the residue and

the mixture was filtered. The filtrate was basified with saturated bicar-

bonate solution and solids were collected by filtration. The filtrate

was extracted with DCM and evaporated to dryness. The residues

were triturated with petroleum ether (40–60 ^ꢀC) and filtered to give

ethyl 5-amino-salicylate. This compound (0.5 g, 2.76 mmol) was dis-

solved in DMF (10 mL) along with aromatic acids (1 eq) and TBTU

(1 eq) followed by stirring at 0 ^ꢀC. Diisopropylethyl amine (5 eq) was

then added dropwise and kept at RT overnight. The reaction was

monitored by TLC. The reaction mixture was partitioned between

EtOAc and water. The dried organic layers were evaporated to get

crude product, which was further dissolved in THF:MeOH (1:1) and

LiOH.H₂O (1 eq) was added to the reaction flask. The reaction was

monitored by TLC. The reaction mixture was acidified with 1 N HCl

2.6.2 | In vitro anticancer assay

−

Cytotoxicity of moderately active Sx_cinhibitors (4, 5, 12, 15 and 19)

was evaluated using MTT assay on three of the glioblastoma cell lines:

U87 MG (human), SF268 (human) and C6 (mouse) (van de Loosdrecht,

14

PATEL ET AL.

Beelen, Ossenkoppele, Broekhoven, & Langenhuijsen, 1994). Briefly,

3,000 cells were seeded in each of the 96-well plates for different

concentration of six compounds ranging from 10 μM to 1,000 μM.

After 18 hr of seeding, different drug concentrations were added into

the cells along with DMSO (vehicle control). Following 48 hr of incu-

bation with the compounds, cells were incubated with MTT reagent

(5 mg/mL) for 4 hr in incubation and the crystals were solubilized

using DMSO. The absorbance was then measured at 570 nm using

SPECTROstar Nano Microplate Reader. The IC₅₀values for the title

compounds were determined using GraphPad prism software using

nonlinear regression analysis.

3.1.2 | Threading

In this study, three homology models of the human xCT were con-

structed in outward-open, outward-occluded and inward-occluded con-

formations based on AdiC and ApcT crystal structures (PDB ID 3OB6,

3L1L, and 3GIA, respectively). The stereochemical quality of the

threaded models, both before and after energy refinements, were eval-

uated using the Molprobity and SAVES server which uses PROCHECK,

ERRAT and VERIFY 3D programs (Table S1S). Molprobity analyses

showed the absence of poor rotamers, cβ deviations, and bad bonds

and angles in all three models. The outlier residues shown in Ram-

achandran plot were not refined because the residues did not consti-

tute part of the active site. The VERIFY 3D scores were lower for the

refined structures than the templates, but acceptable.

3

| RESULTS AND DISCUSSION

3.1 | Homology modeling

3.1.3 | Main structural features

3.1.1 | Selection of suitable templates for model

building

The homology models derived from AdiC and ApcT conform to the char-

acteristic fold of the SLC7 families of amino acid transporters: the model

comprises of 12 transmembrane all-helical segments with the N- and C-

termini located inside the cell (Fotiadis, Kanai, & Palacin, 2013). The

substrate-binding site is quite hydrophobic. Residues His110 and Thr112

are at the surface and are close to the substrate-binding/permeation

pathway of xCT. Cys327 seats right near the extracellular hydrophobic

cavity whereas His110 is at the top of Arg396. Structural alignment of

the models based on substrate-free and substrate-bound templates rev-

ealed major conformational changes in TM2, TM6, TM8, and TM10.

Transmembrane domain 8 is straight in the outward-open structure and

kinked in the inward-open model and this is in agreement with the previ-

ous result for AdiC and ApcT (Jeschke, 2013).

Threading methods returned AdiC and ApcT as the best and the only

reliable templates. Metrics indicating the reliability of structural rela-

tion were unambiguous: both servers yielded the highest possible

confidence value despite sequence identity of less than 21% between

xCT and AdiC/ApcT (Figure 2 and Figure 1S). Furthermore, both pro-

teins have antiporter activity, are ion independent, and are L-amino

acid transporters. Thus, HHpred server estimated the probability

(i.e., the degree of confidence) of homology between the entire trans-

membrane domains (TMD) of AdiC and ApcT as 100%. This remark-

ably high score was statistically supported by notable E-value (1E⁻⁴⁵

for AdiC and 2.7E⁻⁴¹for ApcT) and p-value (2.7E⁻⁵⁰for AdiC and

7.3E⁻⁴⁶for ApcT) scores. LOMETS, which takes predictions from nine

different servers that represent a diverse set of state-of-the-art

threading algorithms, also ranked AdiC and ApcT as top-two tem-

plates. These servers also predicted glutamate/γ-aminobutyrate

(GABA) antiporter; LEUT, glutamate-GABA antiporter as a suitable

template (based on E and p values), but we did not use this template

to model xCT because the sequence identity dropped below 15%.

The researchers at the University of Montana used ClustalW

alignment method to thread xCT model using the apo structure of

ApcT (Matti et al., 2013). We expect significant differences in the con-

formation of active site owing to the differences in the alignment

methods and the availability of newer templates in holo- and apo-con-

formations. Profile HMMs make allowance for amino acid and inse-

rtion/deletion frequencies, and thus, for the importance of each

position for defining protein homologies. Pair-wise comparison of pro-

file HMMs is currently considered the most powerful and accurate

method for detecting and aligning remotely related sequences and a

method like HHPRED also takes into account secondary structure

prediction (Söding, 2005). In conclusion, two quantitative estimates

(HHpred and LOMETS metaserver) converge to the same conclusion,

indicating that AdiC and ApcT are the best available and highly useful

templates for homology modeling of xCT.

The Computed Atlas of Surface Topography of proteins (CASTp)

web server (Dundas et al., 2006) was used to locate, measure, and

characterize the pockets on the protein surfaces for all three models

and the voids in the interior of proteins. The molecular surface volume

and area calculated by CASTp for all models are reported in Table S2

The binding pocket of the models based on open-out and open-

occluded conformations is widely open toward the extracellular side

(Figure 3), reflected by the larger mouth volume compared to that of

the model based on the apo structure.

3.2 | Predicted physicochemical and

pharmacokinetic properties

As represented in Table 2, most of the designed compounds pos-

sessed acceptable molecular, physicochemical, and pharmacokinetic

properties such as logD, logS@ pH 7.4, #H-bond donors and accep-

tors as well as TPSA and #rotatable bonds. Most of the designed mol-

ecules had TPSA of ~100 Å²and few exhibited logD_7.4> 2.5,

indicating that these molecules (including masked acid, 4) were likely

to be orally absorbed and may potentially gain entry through BBB.

The calculation/predictions of molecular properties was performed to

eliminate designs likely to fail down the line due to unacceptable

properties.

PATEL ET AL.

15

TABLE 2 ^aPhysicochemical and pharmacokinetic properties of the synthesized molecules^a

Molecule

logS @ pH 7.4

3.796

logP

logD_7.4

−0.13

MW

HBD

3

HBA

9

TPSA

141.3

130.3

147.1

142.4

86.63

99.32

104.9

91.99

Rotatable bonds

1

2.838

3.587

2.339

1.948

2.678

2.927

3.533

3.449

1.506

2.624

398.4

426.4

397.4

321.3

257.2

285.3

328.3

279.3

278.3

6

8

6

4

6

3

4

−0.06904

0.3461

4.745

3.587

2

9

5

2.339

3

9

7

−1.41

3

8

11

12

14

15

18

19

3.697

−0.8747

−0.4391

0.7908

0.8726

1.506

3

5

3.541

3

5

3.557

2

6

3.485

2

6

2.447

1

7

2.252

2.624

1

6

Abbreviations: HBD, H-bond donors; HBA, #H-bond acceptors; logP, Predicted partition coefficient; logD_7.4, distribution coefficient at pH 7.4;

MW, molecular weight in daltons; TPSA, topological polar surface area (Å²); Rotatable Bond, # Rotatable bonds.

^alogS @ pH 7.4: Predicted aqueous solubility at pH 7.4, log S. S in mol dm⁻³

.

We have compared the secondary structures of the xCT-SSZ com-

plex from the docked state and during the MD simulations. The DSSP

algorithm (Kabsch & Sander, 1983) was used to monitor the evolution

of secondary structures over the entire 600 ns trajectory. While there

were some transient turn and bend content in the expected locations,

there were no pronounced changes in the helical content (66%).

The RMSDs were calculated for the xCT chain (backbone atoms

only), residues in the binding pocket and ligand from the 600 ns tra-

jectory with reference to the docked complex. The moving averages

of the rmsds were smooth with an interval of 30. The moving average

for the protein backbone at 600 ns converged at ~4 Å whereas the

active site residues RMSD was ~3.3 Å (Figure S2). In case of the

ligand, the plot showed that the ligand diffused from the original bind-

ing site during the production phase with 2–12 Å minimum RMSD rel-

ative to a top-ranked docking pose. The movement of the ligand could

be attributed to various interactions as discussed further below.

The volume of the ligand cavity diverged from the docked com-

plex during the simulations (Figure S3). Some divergence from the ini-

tial conformation was not unexpected when going from the docked

structure environment to aqueous solution in the simulations and pro-

vided that there were various conformational changes reported for

membrane receptors upon binding of the substrate or ligand. The

change in volume of the ligand/substrate cavity could be explained by

the rotation of the aromatic cluster consisting of three tyrosines (Tyr

240, 241 and 244) and the water occupancy in the cavity. Figure 6

shows the average side-chain torsion (chi1) rotation during simulation

wherein Tyr241 and Tyr244 flip to alternate conformations. These

residues are localized near the lipid/water interface and affect the

binding of the ligand in the pocket. Grid and water H-bond analyses

using CPPTRAJ tool in AMBER suggested the location of internal

waters near the two Arginines (135 and 396) and Ser330.

3.3 | Molecular docking studies and molecular

dynamics simulations

The open-out conformation of xCT was used for the docking studies.

Initial docking runs with the open-occluded and inward-occluded con-

formations did not yield reasonable binding poses (data not shown).

Hence, we explored further docking runs with the open-out confor-

mation. Trial runs with SSZ (1) yielded only one binding pose using XP

mode of Glide (Figure 4a). The ─COO⁻group of 1 interacted with

Arg396 via a salt-bridge and a strong H-bonding interaction with

Thr56 (Figure 4A). In addition, the other end of 1 showed edge-to-

face π–π stacking interaction with Tyr241. The sulphonamide ─NH

formed a H-bond with Asn390. The hydrophobic central portion was

found to interact with the hydrophobic amino acid residues such as

Leu389, Ile142, Ala245 and others. Previously, isoxazole hydrazide

inhibitors were shown to interact with Arg135 (cation-π interaction),

Tyr251 (H-bonding), Ile134 (hydrophobic), Tyr244 (hydrophobic),

Ser330 (H-bonding), Thr56 (H-bonding), etc. (Matti et al., 2013). Over-

all, similar ligand-antiporter interactions were found in our docking

analyses as reported previously.

Trial runs with 1 to optimize docking run settings were followed

by similar runs with the designed analogs of 1 (Table 1). Figure 4

shows the binding mode of 15 (IC₅₀= 116.74 μM) in the ligand-

binding pocket of xCT. Here, the ─COOH group in 1 was substituted

with ─SO₃H group. In addition to dominant salt-bridge interactions

with Arg396, the acidic functionality in 15 also formed strong elec-

trostatic interactions with Arg135. Arg396 also exhibited cation–π

interaction with the aromatic ring bearing ─SO₃H group. The hydro-

phobic naphthyl ring formed several interactions with the hydropho-

bic residues in the ligand-binding pocket of the antiporter. Similarly,

12, a secondary amide (IC₅₀= 138 μM), exhibited similar binding

mode with salt-bridge interaction with Arg135 and cation–π interac-

tion of the acid-bearing aromatic ring with Arg369 (Figure 4c).

Table 1 lists the major ligand-binding pocket residues interacting

with the ligands.

Details of key H-bonding interactions and contacts between 1 and

binding site within the xCT over the course of the MD simulations are

presented in the Supporting Information (Table S3). From the data, it

was clear that the intramolecular H-bond between the 2-hydroxyl

16

PATEL ET AL.

FIGURE 6 The conformation of ligand in the cavity obtained from clustering analysis on 30,000 frames based on the ligand fit. (frames were

taken every 20 ps). The trajectory was divided into 10 clusters. Clusters 9 and 10 had <1% occupancy and therefore were not shown. Key

residues Arg135, Arg396, Tyr240, Tyr241, and Tyr244 interacting with SSZ are shown for eight clusters. Water molecules are shown as lines,

protein as ribbon colored from N (blue) to C-terminal (red) and residues as sticks. SSZ, sulfasalazine

group and the carbonyl O atom of the carboxylic acid group played a

significant role in the stabilization of the molecule as highlighted in the

monoclinic polymorphic crystal structure and quantum chemical studies

(Patel & Gandhi, 2011). We believed this was one of the key features

required for activity of SSZ analogs. The O atoms of the carboxylic acid

moieties formed electrostatic interactions with the side chains of

Arg135 and Arg136. Due to the conformational changes in the binding

site, at around 500 ns, the carbonyl O atom of the ─COOH group

showed transient H-bonding with the side-chains of Ser330 or water-

mediated H-bonds with Ser330, Arg135 and Arg136. Similar H-bonds

were observed with the ─COOH or its isosteric ─SO₃H groups from

ligands with either Arg135, Arg396 or both (Figure 4b,c).

site, the native contacts with the aromatic triad of tyrosines were

absent. Cluster 1 displayed role of π–sulfur interactions arising from the

Cys327 residue of helix 8. This result indicated slight conformational

changes were occurring upon ligand binding and was compatible with

the previous study highlighting the role of Cys327 in substrate binding

site (Jiménez-Vidal et al., 2004). Besides the top two clusters, other clus-

ters showed transient interactions with the side chain of Ser330 and

close contacts with Ile142 and Ala145.

A recent study based on the mutagenesis experiment and superposi-

tion of C. glabrata cystine transporter (Deshpande et al., 2017), CgCYN1

model onto mammalian cystine transporter xCT identified residues

Cys327, Phe146, Ala247, Gly333, Leu389, and Val404 of xCT as critical

for substrate binding. The SSZ-binding site in our model indicated an

important role of Cys327, Ala247, Arg135, and 136, and other aromatic

residues as discussed previously, whereas residues Phe146, Gly333,

Leu389, and Val404 were within 5 Å from the SSZ-binding site. Our

modeling reasonably was in agreement with their study on CgCYN1

despite <40% sequence similarity between the mammalian and yeast

cystine transporter.

Next, we performed RMSD-based structural clustering to identify

distinct poses of the SSZ bound to the xCT. For SSZ, the clusters with

occupancy >1% identified from the atomistic simulations are shown in

Figure 6. The three most populated clusters reflected the ligand mobility

with differences originating from interactions with residues in particular

from helix-1 and helix-8. The interactions of the ligand with residues in

the binding pocket are shown in Figure 7. The results indicated that in

cluster 2, SSZ retains all native interactions with the protein as observed

in the docked complex and at the start of the simulation. The carboxylic

oxygens were involved in H-bonding with both arginines and the aro-

matic rings of SSZ were in close contacts with Tyr240, Tyr241, Tyr244,

and Ala247 thus, forming π–π and π–alkyl interactions. The most popu-

lated cluster 1 showed reorientation of SSZ in the pocket. While the car-

boxylic group still retained interactions with the arginines in the active

3.4 | In vitro Sx_c⁻antiporter inhibition

A total of five series of title compounds were synthesized and tested

−

for in vitro Sx_cinhibition. The potency data along with the molecu-

lar structures, docking scores, and interacting residues are given in

−

Table 1. The Sx_cantiporter inhibition potential of the title

PATEL ET AL.

17

FIGURE 7 Details of various interactions of sulfasalazine with the receptor shown in a representative snapshot taken from the top eight clusters

compounds was evaluated using uptake assay in C6 glioma cell lines.

A total of 13 out of 28 compounds were able to inhibit the anti-

porter activity in the IC₅₀range of 110–492 μM (Compound 1,

IC₅₀= 70 μM) (Table 1). These potency ranges (i.e., higher μM) are in

accordance with the previous reports for 1 and its analogs (Matti

et al., 2013; Shukla et al., 2011).

suitable replacement for the salicylic acid moiety, α- and β-naphthol

substructures were tried. To our surprise, one of the two compounds,

15 picked up the moderate activity (IC₅₀= 116 μM), compared to 14,

which was completely inactive (IC₅₀> 500 μM). As seen from the

binding mode (Figure 4), 15 formed salt-bridge interactions between

−

the highly acidic SO₃group and the two arginines (135 and 396).

The structure–activity relationship (SAR) for Sx_c⁻antiporter inhibi-

tion can be highlighted as follows: (a) conversion of 1 to its ester (4)

led to improved cellular activity without much loss (~30%) in potency

(76 μM vs 111 μM, Table 1). Masking one of the polar functionalities

of 1 led to improved cellular activity, as expected. Further conversion

of the ester to the amide (5) led to reduced in vitro potency. This

could potentially be due to the neutral nature of the primary amide

(─CONH₂in 5) compared to the highly acidic ─COOH group, thereby

compromising the salt-bridge interaction with either Arg135 or

Arg396. 2) Removal of the terminal pyridine ring in 1 (compound 7)

resulted in complete loss of antiporter activity. This activity loss was

surprising despite the presence of the proximal salicylic acid function-

ality. The absence of the pyridine ring might have compromised the

anchoring of the molecule in the ligand-binding pocket, thereby

potentially interfering with the favorable interactions between the

ligands and the corresponding residues that mattered. Moreover, the

removal of pyridine ring is likely to reduce logD_7.4compared to

1 (Table 2). (c) Replacement of the azo linkage with the secondary

amide (─CONH─) group along with the pyridine sulfonamido group

completely abolished the antiporter activity (11, Table 1). Further

extension of the ─CONH─ group by ethylene linker regained the

potency, albeit less than 1 (12, Table 1). (d) In an attempt to identify

The presence of 2-OH group favorably led to H-bonding interaction

with the backbone ─C O of Ile57. 5) Other modifications involving

deletion of the azo linkage with simultaneous boisosteric replacement

of the salicylic acid moiety with neutral –NO₂group identified 18 with

two-fold less potency compared to 1 (IC₅₀= 170 μM). Further

replacing the pyridine heterocycle in 18 with the corresponding car-

bocycle (19) reduced potency further, delineating the important role

of pyridine in biding of 1 with the antiporter. Overall, the study identi-

fied around five moderately potent (within two-fold potency on the

lower side from 1) inhibitors of Sx_c⁻. The SAR was found to be

realtively steep. This is in accordance with the earlier reports (Shukla

et al., 2011).

3.5 | Sx_c⁻inhibitors show anticancer potential in

human glioblastoma cells

Because there could be potential differences between mouse (C6 cell

−

line) and human glioblastoma cells in terms of sensitivity towards Sx_c

inhibitors, we analyzed the anticancer activity of the five identified

−

Sx_cinhibitors (hits) (4, 5, 12, 15 and 19) along with 1 on the human

glioblastoma cells (U87 MG and SF268) and compared it with their

activity (potency) against C6 cell line. The cytotoxic activity was

18

PATEL ET AL.

determined by MTT assay. All the five inhibitors showed cytotoxicity

for U87 cell lines with an IC₅₀range of 38.24–169.1 μM (Table 1).

However, only two Sx_c⁻inhibitors (4 and 19) were able to inhibit pro-

liferation of SF268 cell line, albeit at quite high concentration. Being a

highly malignant cell line, SF268 might express efflux transporters

leading to lower effective cellular concentration. We could not under-

stand this low potency by the hit molecules. The two glioma cell lines

used although are both glioma grade IV but have different genetic

background. These data suggest that compounds 4 and 19 are the

most promising in terms of potentiating cell death of glioma cells

irrespective of their genetic background. Since C6 cell line was used

for the primary screening, we evaluated all the hits in MTT assay using

C6 cell line. Compound 5 was the most potent hit with IC50 48.71

6.9 μM while 4 was a close competitor (IC50 = 53.04 6.2 μM). On

the similar lines, 19 was slightly (1.five-fold) less potent than 1.

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SUPPORTING INFORMATION

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How to cite this article: Patel D, Kharkar PS, Gandhi NS,

Kaur E, Dutt S, Nandave M. Novel analogs of sulfasalazine as

system x_c⁻antiporter inhibitors: Insights from the molecular

modeling studies. Drug Dev Res. 2019;1–20. https://doi.org/

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oncotarget.8651

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Structure and mechanism of

a Na+−independent amino acid

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