Molecular basis for nonspecificity of nonsteroidal anti-inflammatory drugs (NSAIDs

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Drug Discovery Today � Volume 00, Number 00 �March 2015 REVIEWS

Molecular basis for nonspecificity ofnonsteroidal anti-inflammatory drugs(NSAIDs)Avaneesh K. Dwivedi1,3, Vaishali Gurjar1,3, Sanjit Kumar2 andNagendra Singh1

1 School of Biotechnology, Gautam Buddha University, Greater Noida, Uttar Pradesh 201308, India2Center for Bioseparation Technology, VIT University, Vellore, Tamil Nadu 632014, India

Inhibition of the production of inflammatory mediators by the action of nonsteroidal anti-

inflammatory drugs (NSAIDs) is highly accredited to their recognition of cyclooxygenase enzymes.

Along with inflammation relief, however, NSAIDs also cause adverse effects. Although NSAIDs strongly

inhibit enzymes of the prostaglandin synthesis pathways, several other proteins also serve as fairly

potent targets for these drugs. Based on their recognition pattern, these receptors are categorised as

enzymes modifying NSAIDs, noncatalytic proteins binding to NSAIDs and enzymes with catalytic

functions that are inhibited by NSAIDs. The extensive binding of NSAIDs is responsible for their limited

in vivo efficacy as well as the large spectrum of their effects. The biochemical nature of drugs binding to

multiple protein targets and its implications on physiology are discussed.

IntroductionNonsteroidal anti-inflammatory drugs (NSAIDS) are one of the

most prominent categories of compounds occupying the drug

market. NSAIDs have been prescribed by physicians for over a

hundred years. These drugs provide anti-inflammatory, antipy-

retic and analgesic effects in various pathological conditions

[1,2]. NSAIDS are so-called because they are not steroids in nature

but cause the same effects as steroids by inhibiting synthesis of

prostaglandins (PGs). NSAIDs are recommended for treatment of

acute or chronic conditions of pain and inflammation. In the USA

over 30 billion doses were sold and there were seven million

prescriptions of NSAIDs in 2001 [3]. The well-known members

of this category are naproxen, aspirin and ibuprofen, which are

available worldwide for the treatment of pain and inflammation

[4].

NSAIDs act by suppressing cyclooxygenase (COX)-1 and COX-2

enzymes or tumour necrosis factor (TNF)-a and inducible nitric

oxide synthase (iNOS). Phospholipase A2 (PLA2) or phospholipase

C (PLC) catalyses liberation of arachidonic acid from membrane

Please cite this article in press as: Dwivedi, A.K. et al. Molecular basis for nonspecificity of non10.1016/j.drudis.2015.03.004

Corresponding author: Singh, N. ([email protected])3 These authors contributed equally to this review.

1359-6446/� 2015 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.drudis.2015.03.004

phospholipids, which is further oxygenated into PGs by the action

of lipoxygenase (LOX) and COX enzymes. By blocking the action

of COX, NSAIDs can relieve inflammation by reducing vasodilata-

tion and pain, which is produced mainly by prostaglandin E

(PGE)2 and prostacyclin (PGI2). Most of the NSAIDs block synthe-

sis of PGs by inhibiting COX enzymes nonselectively [5]. The

inhibition of COX is also responsible for damage of the gastroin-

testinal mucosa, because the PGs are also involved in mucosal

defence and repair mechanisms [6,7]. A most commonly pre-

scribed NSAID, aspirin has been found to cause gastrointestinal

bleeding in half of the cases in the USA [8–10] and 12,000 cases of

intestinal bleeding and 1200 deaths every year in the UK [11].

Long-term use of NSAIDs increases the risk of having acute cardiac

disease and gastrointestinal-related side-effects [5]. Depending on

the selectivity for either of the COX isoforms, NSAIDs have been

divided into conventional and COX-2-selective categories. Con-

ventional NSAIDs such as phenoprofen, ibuprofen, indomethacin,

ketoprofen, keterolac, naproxen, paracetamol, piroxicam and as-

pirin are nonselective COX inhibitors, whereas etodolac, melox-

icam, diclofenac, celecoxib, nimesulide, valdecoxib, parecoxib,

rofecoxib and etoricoxib are selective COX-2 inhibitors and are

also known as coxibs.

steroidal anti-inflammatory drugs (NSAIDs), Drug Discov Today (2015), http://dx.doi.org/

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REVIEWS Drug Discovery Today � Volume 00, Number 00 �March 2015

DRUDIS-1593; No of Pages 11

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Most of the nonselective and selective NSAIDs have mild-to-

severe side-effects with long-term usage. NSAIDs are also associat-

ed with a broad spectrum of adverse effects on the functioning of

the liver, kidney, cardiovascular system, skin and gut. Gastroin-

testinal side-effects are the most common and constitute a wide

clinical spectrum ranging from dyspepsia, heartburn and abdomi-

nal discomfort to more serious events such as peptic ulcer with life-

threatening complications of bleeding and perforation [12–16].

Besides COX enzymes, NSAIDs have been observed recognising

various other physiological targets as well.

In this review, we report the comparative details of the atomic

interactions of NSAIDs with their target and off-target protein

receptors available in the Protein Data Bank (PDB). The atomic

coordinates of the complexes of NSAIDs and receptors were down-

loaded from the PDB website (http://www.rcsb.org/pdb/home/

home.do) and the intermolecular interactions were studied using

COOT [17], PyMOL [18] and CCP4 [19].

Protein targets binding to NSAIDsCOX-1COX enzymes catalyse the rate-limiting steps of synthesis of PGs,

prostacyclins and thromboxanes, collectively known as prosta-

noids. COX enzymes are also known as prostaglandin-endo per-

oxide synthases (PTGS/PGHS, EC: 1.14.99.1). COX enzymes

catalyse the oxidation of arachidonic acid (a v-6 polyunsaturated

fatty acid; PUFA) to PGH2, which is further converted into series 2

prostanoids.

Currently, three forms of COX are known: COX-1, COX-2 and

COX-3. COX-1 and COX-2 share 81% homology and COX-3 is a

splice variant of COX-1 [20]. COX-1 and COX-2 are also known as

PGHS1 and PGHS2, respectively. COX-1 and COX-2 function in

very similar ways but selective inhibition can make a difference to

their side effects. The expression of COX-1 is constitutive, whereas

COX-2 is induced in macrophages at the site of inflammation

[21,22]. COX-1 is also supposed to be additionally responsible for

maintaining gastrointestinal mucosa, therefore a new category of

NSAIDs specific only for COX-2 were designed. Rofecoxib and

etoricoxib are the most selective COX-2 inhibitors [23]. Recently,

rofecoxib (produced by Merck) was not found to be associated with

gastrointestinal risk but increases the chances of acute myocardial

infarction in high-risk patients, which led to the withdrawal of the

drug from the market. Other COX-2-selective drugs were also

found to be associated with myocardial infarction and thrombo-

sis-related issues [24–28].

The 3D structure of COX-1 was found to have three indepen-

dent folding units, one epidermal growth factor (EGF) domain, a

membrane-binding motif and a catalytic domain [29]. The sub-

strate- and NSAID-binding site on COX-1 is present in the form of a

long hydrophobic channel. The structure of COX-1 comprised a

homodimer. Most of the NSAIDs have been found to bind to one of

the monomers and block total activity of the dimer through

crosstalk between both monomers. However, it has also been

observed that a COX-2-selective inhibitor binding to one mono-

mer does not affect the activity of the other one [30]. The drug-

binding hydrophobic cavity is formed predominantly by hydro-

phobic amino acids Val116, Tyr348, Val349, Leu352, Leu359,

Phe381, Leu384, Tyr385, Trp387, Phe518, Ile523, Ala527 and

Leu531. Three polar amino acids Arg120, Tyr355 and Ser530 are


2 www.drugdiscoverytoday.com

also present in the binding site. The crystal structure of COX-1 has

also been solved in complex with various NSAIDs such as flurbi-

profen, diclofenac, celecoxib, ibuprofen, alclofenac and nimesu-

lide [29–33]. The basic chemical structure of NSAIDs is generally

composed of a hydrophobic aromatic nucleus and an attached

acidic group. The aromatic portion lies in the hydrophobic chan-

nel of COX-1, whereas the acidic group is H-bonded to the side-

chain hydroxyl group of Tyr355 and guanidinium group of Arg120

(Fig. 1a). All the NSAIDs except aspirin inhibit COX-1 in a similar

way by working as competitive inhibitors, whereas aspirin and its

analogues acetylate the active-site Ser530 to act as suicide inhibi-

tors of COX enzymes.

COX-2Three dimensional structures of native and complexed states of

COX-2 have been determined [34]. The structure of COX-1 and

COX-2 is similar with a root-mean-square deviation (RMSD) of

0.9 A of their Ca atoms. The drug-binding sites in both enzymes are

identical except for the presence of the smaller Val509 instead of

Ile523 and Arg499 in place of His513 in COX-1, which causes a

slight increase in the volume of the drug-binding cavity and

creates a side-pocket in COX-2. The crystal structures of COX-2

have been reported in complex with naproxen, aspirin, flurbipro-

fen, indomethacin, diclofenac and celecoxib [31,34–37]. Various

modes of inhibitor binding to the COX active-site have been

reported including ionic interaction of carboxylic-acid-containing

NSAIDs with Arg120 of COX-1, COX-2 and insertion of arylsulfo-

namides and sulfones into the side-pocket of COX-2. Most of the

NSAIDs bind COX-2 by introducing an ionic interaction with

Arg108 and H-bond with Tyr341 in a similar way as for COX-1

(Fig. 1b), although diclofenac has been found to interact with the

COX-2 site in an inverted manner. The carboxylate group of

diclofenac makes H-bonds with Tyr385 and Ser530 instead of

Arg120 and Tyr355 [36]. COX-2-selective NSAIDs such as etodolac,

meloxicam, diclofenac, celecoxib, nimesulide, valdecoxib, pare-

coxib, rofecoxib and etoricoxib have a different mode of binding

to COX-2 than nonselective drugs. The sulfonamide group of

coxibs lies in the space created in the side pocket owing to the

presence of Val523 in COX-2. Celecoxib makes ionic interactions

with side-groups of His75 and Arg499 along with H-bonded inter-

actions with main-chain CO groups of Leu338 and Ser339 residues

of COX-2. The sulfonamide group of celecoxib is additionally H-

bonded to Gln178 as well [36,37]. In this manner, coxibs gain one

extra salt bridge and three extra H-bonded interactions compared

with nonselective COX inhibitors (Fig. 1c). The biochemical se-

lectivity of several NSAIDs was evaluated using the COX–isozyme

activity. It was observed that valdecoxib, etoricoxib, 5,5-dimethyl-

3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone

(DFU) and 5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonyl-

phenyl)-2(5H)-furanone (DFP) inhibited COX-1 and COX-2 with

the following COX-1:COX-2 IC50 ratios: 61.5, 344, 660 and 1918,

respectively; whereas indomethacin, celecoxib and rofecoxib had

the corresponding values of 30, 29.6 and 272 [38], indicating the

role of the sulfonamide group in providing specificity to coxibs.

PLA2Although inhibition of PG synthesis by NSAIDs has been attribut-

ed to their binding to COX enzymes, another mechanism of


http://www.rcsb.org/pdb/home/home.do

http://www.rcsb.org/pdb/home/home.do





Arg 120

(a) (b)

(c) (d)

Arg 499

His 75

Ala 513

Phe 367

Leu 370

Tyr 371

Trp 31

Phe 105Asp 49

His 48

OW Ile 9

Ile 19

Leu 17

Leu 10

Leu 3

Leu 2

Lys 69

Phe 5

Tyr 52

Tyr 22

Ser 23

Ala 18

Trp 373

Tyr 341

Ser 516

Ser 339

Leu 338

Gln 178

Val 509

Arg 108

Ala 513

Val 509

Ser 516

Phe 367

Leu 370

Ile 523

Phe 381

Leu 384

Ser 530Tyr 385

Tyr 355

Tyr 348

Tyr 341

Tyr 371

Trp 373

Leu 338Ser 339

Trp 387Val 349

Leu 352Leu 359

Drug Discovery Today

FIGURE 1

Binding of nonsteroidal anti-inflammatory drugs (NSAIDs) to prostaglandin synthesis pathway enzymes. (a) Ribbon diagram of cyclooxygenase (COX)-1 (blue) in

complex with flurbiprofen is shown. (b) Structure of COX-2 (blue) in complex with indomethacin, a nonselective COX inhibitor, and (c) COX-2 complexed withcelecoxib, a selective COX-2 inhibitor, are drawn. The figure indicates a deeper binding of coxib. (d) Diclofenac is shown in bound state to the active site of

phospholipase A2 (PLA2). The carboxyl group of the drug makes H-bonded interactions with catalytic residues His48 and Asp49 through the conserved water

molecule (OW). The drug is completely surrounded by the hydrophobic residues of the binding site of PLA2. Residues contributing to the formation of the drug-binding site and interacting with the drug are represented as ball and stick models. Drugs are shown by magenta ball and stick models. H-bonded interactions are

shown as dotted lines. The figure was drawn using PyMOL [18].


inhibition of PG synthesis is to stop liberation of free arachidonic

acid from phospholipids by the action of PLA2. PLA2 is a key

enzyme involved in the production of proinflammatory media-

tors. It is responsible for the liberation of arachidonic acid

from membrane phospholipids. This arachidonic acid serves as

a substrate for COX and LOX enzymes for synthesis of PGs and


leukotrienes. The substrate binding to PLA2 occurs through a

hydrophobic channel. Many of the NSAIDs have been found to

be strong inhibitors of PLA2 enzymes. To determine the viability of

PLA2 as a target molecule for the structure-based drug design

against inflammation, arthritis and rheumatism, the crystal struc-

ture of the complex of PLA2 with various NSAIDs such as aspirin,







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nimesulide, oxyphenbutazone, indomethacin, diclofenac and

paracetamol have been determined [39–42].

The drug-binding site in PLA2 includes mainly hydrophobic

residues such as Leu2, Leu3, Phe5, Ile9, Ala18, Ile19, Tyr22, Ser23,

Trp31, Lys69 and Phe105. The active site of PLA2 comprises His48,

Asp49 and Tyr52 residues, located at the interior end of the

substrate-binding hydrophobic channel. The stereochemical en-

vironment of the hydrophobic channel is very compatible for

binding to NSAIDs. Most of the NSAIDs have been found to block

the phospholipid-binding site and act as competitive inhibitors of

PLA2 enzymes. The hydrophobic aromatic rings of NSAIDs make

several van der Waals contacts with the substrate-binding site

residues; whereas the charged or polar functional group of the

drugs makes ionic- or H-bonds with catalytically essential residues

His48 and Asp49 directly or through a catalytically conserved

water molecule (Fig. 1d). Although most of the NSAIDs act as

competitive inhibitors of PLA2, indomethacin has been found

binding at an alternate site near to the substrate-binding hydro-

phobic channel and categorised as a noncompetitive inhibitor of

the enzyme with a Ki of 12 mM [40,43]. It has been shown that the

cumulative anti-inflammatory effects of NSAIDs are based on not

only their binding to COXs but also their strong affinity for PLA2

[39–41].

Prostaglandin keto reductase (PTGR)PTGR2 belongs to the superfamily of dehydrogenase and reduc-

tase enzymes. These enzymes catalyse NADPH-dependent reduc-

tion of unsaturated bonds in PGs and are responsible for the

eventual inactivation of PGs. Selective inhibition of PTGR2 also

enhances insulin sensitivity. Indomethacin has been shown to

interact strongly with the PTGR2, located at the site closer to the

NADP-binding site [44], carboxylic acid making H-bonds with the

sugar OH group of NADP. The hydrophobic rings of the drug

makes many van der Waals interactions with the residues Tyr51,

Cys54, Thr60, Ile65, Phe99, Tyr100, Met135, Leu288, Val289 and

Leu290.

Several NSAIDs have also been observed inhibiting other mem-

bers of the aldoketo-reductase (AKR) family. AKR1C3 acts to

suppress cell differentiation and promote proliferation in myeloid

tissues and represents a novel target for COX-independent anti-

neoplastic actions of NSAIDs. Indomethacin and flufenamic acid

have been found forming complexes with AKR1C3 [45]. Indo-

methacin binds to the active site, whereas flufenamic acid bound

additionally to a beta hairpin loop. Indomethacin and flufenamic

acid bind in a similar manner at the active site of AKR1C3. The

carboxylic acid groups of indomethacin and flufenamic acid make

two H-bonds with the hydroxyl group of Tyr55 and one salt bridge

with His117. Additionally, indomethacin makes several van der

Waals contacts with Tyr24, Leu54, Trp86, Met120, Tyr216,

Trp227, Phe306, Phe311, Pro318 and Tyr319 residues. The binding

of drugs has caused drastic readjustments of the orientation of

side-chains of several residues of the drug-binding site including

Phe306, Phe311 and Trp227, indicating an induced fit mechanism

of drug binding.

Cytochrome P450 (CYP450)The CYP family comprises a diverse group of catabolic enzymes,

responsible for oxidation of drugs and toxic chemicals and for drug



biotransformation. The enzymes are located in the smooth endo-

plasmic reticulum of cells and are highly concentrated in intesti-

nal and liver cells [46]. Various metabolic intermediates also serve

as substrates to these enzyme complexes, accounting for �75% of

the total number of different metabolic reactions catalysed by over

11 500 CYP enzymes [47,48]. Binding and structural studies of

NSAIDs to the various components of CYP have been studied.

CYP4502C5/3LVdH and related enzyme 2C5dH catalyse hydrox-

ylation of diclofenac with binding affinities of 80 mM and 57 mM,

respectively. The crystal structure of human CYP4502C9 com-

plexed with flurbiprofen was determined by X-ray crystallography

[49]. The hydrophobic rings of NSAIDs oriented towards the heme

group for the addition of an oxygen atom. The acid group inter-

acted with polar side-chains of Asn204 and Ser289 directly or via

water molecules. Other hydrophobic residues of CYP stabilising

the binding of NSAIDs are Val100, Leu103, Ala113, Val205,

Ala294, Leu359, Leu363 and Phe474 (Fig. 2a). Relatively few polar

interactions are present between CYP and NSAIDs in comparison

with COX enzymes and the drug-binding cavity is also much wider

in the case of CYP to ensure recognition of a wide variety of

xenobiotic organic compounds. CYP450-mediated oxidised inter-

mediate forms of diclofenac have been found to be implicated in

NSAID-induced intestinal and idiosyncratic hepatotoxic reactions

[50,51]. As a consequence of recognition of CYP450 enzymes by

NSAIDs, the oxidised intermediates of these drugs could also be

having further roles in contributing to other adverse effects on

human physiology.

Lactoperoxidase (LPO)LPO (EC 1.11.1.7) is a heme-containing peroxidase enzyme secret-

ed from mammary glands. LPO catalyses hydrogen-peroxide-me-

diated oxidation of thiocyanate and works as an antimicrobial

agent. NSAIDs have been found to inhibit peroxidase activity by

acting as competitive substrates. The peroxidases being mainly

provided by the invading eosinophils, NSAIDs can affect the host

defence mechanism by inhibiting the activity of the peroxidase

enzymes [52,53]. NSAID molecules such as aspirin, indomethacin,

nimesulide and paracetamol have been observed to be interacting

with LPO in structural studies [54]. Nimesulide and indomethacin

bind LPO as inhibitors by replacing the conserved water molecule

required for catalysis, whereas aspirin and paracetamol work as

substrates for the enzyme. All the drugs bind at the substrate-

binding distal heme cavity of LPO. The main residues involved in

formation of the drug-binding site include the heme group,

His109, Phe113, Phe254, Arg255, Glu258, Gln423, Pro424 and

Phe381 (Fig. 2b,c). The polar groups of paracetamol (4GN6.pdb)

and aspirin (3GCL.pdb) interact with the heme iron through the

conserved water molecule, whereas indomethacin (3OGW.pdb)

and nimesulide (3QL6.pdb) interact directly with the heme iron by

replacing the catalytically essential water molecule in the distal

heme cavity of LPO (Fig. 2c).

Transthyretin (TTR)TTR is a transporter protein for thyroxin and retinol, present in

serum and cerebrospinal fluid. It is a 55 kDa tetramer of a 127

amino-acid-long polypeptide chain. The oligomerisation of TTR

creates two thyroxin-binding sites per tetrameric structure [55].

Dissociation, misfolding and aggregation of TTR have been






Leu 103

Leu 363

Leu 359

HEME

Asn 204

His 109

Arg 255

Phe 254

Phe 381

Pro 424

Gln 423His 351

His 109Arg 255

Phe 254

Phe 381

Pro 424

Gln 423

His 351

OW 856OW

OW 857

Val 205Val 100Phe 473

(a) (b)

(c)

Phe 114

Ala 113

Ala 294

Ser 289


FIGURE 2

Recognition of nonsteroidal anti-inflammatory drugs (NSAIDs) by oxidising enzymes. (a) The binding of diclofenac (magenta) is shown in the active site of

cytochrome P450 (CYP450) enzyme (blue). The heme group of the enzyme is also shown as a green ball and stick model. (b) Aspirin (yellow) binding as substrate

to the distal heme cavity of lactoperoxidase (LPO), by interacting with the heme group through the catalytically conserved water molecule (OW). (c) Indomethacin

(magenta) bound to the distal heme cavity of LPO by replacing the conserved water molecule and binding in inhibitory mode to the enzyme. Heme group of LPOis also shown in ball and stick model in green.


reported to be associated with the amyloid diseases senile systemic

amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and

familial amyloid cardiomyopathy (FAC) [56–59]. Aggregated

TTR deposited on peripheral nerves and heart tissues is responsible

for amyloidosis. Several NSAIDs including diclofenac, flufenamic

acid and flurbiprofen have been found inhibiting TTR fibril


formation in vitro. To design drugs against amyloid diseases, crystal

structures of these NSAIDs have also been reported in complex

with TTR [60].

The structure of TTR consists of a homodimer, where each dimer

binds NSAIDs in a similar fashion. The drug-binding site present

on TTR is comparatively shallow and less-specific to NSAIDs. The







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site is formed by contribution of two parallel and one antiparallel

b-strand. Drugs bind to the concave surface of this mixed topology

b-sheet structure in random orientations, unlike COX enzymes.

The carboxylic group of diclofenac makes two H-bonds with the

polar side-chains of Ser117 and Thr119, whereas flufenamic acid

and flurbiprofen bind differently with their carboxylate group


Val 121Thr 106

Thr 119 Ala 108Lys 15

Leu 17Ser 117

Ile 110

Thr 663

(a) (b)

(c)

G

Tyr 155

Tyr 113

Leu 108

Val 103

Leu 110

FIGURE 3

Nonsteroidal anti-inflammatory drugs (NSAIDs) binding to nonenzymatic targets. (ab-strands of transthyretin (TTR). Chemical interactions between TTR (blue) and two Nlactoferrin in complex with diclofenac (magenta) showing a shallow binding site.

bromodomain (BRD)2 protein complex with paracetamol is shown. The drug-bind

makes a single H-bond along with several van der Waals contacts in the drug-bi


facing towards Lys15. The hydrophobic portions of the drugs make

limited van der Waals interactions with Leu17, Thr106, Ala108,

Leu110 and Val121 located on the surface of the b-sheet (Fig. 3a).

The binding of drugs on the surface of TTR prevents intermolecu-

lar interaction of TTR molecules and inhibits aggregation, essential

for fibril formation.



Glu 664

Glu 431lu 659

Val 591

Pro 591

Pro 429Tyr 660

Asn 156 Cys 152

Ile 162

Phe 99

Gly 652

) NSAIDs in two opposite orientations on the concave surfaces of antiparallel

SAIDs, diclofenac (magenta) and flurbiprofen (cyan), are shown. (b) C-lobe of NSAIDs bind lactoferrin with relatively weaker affinities. (c) The human

ing site in BRD2 is contributed to from five a-helices. Paracetamol (magenta)

nding site.






Lactoferrin (LF)LF is an iron transport protein from mammary gland secretions.

Four NSAIDs: indomethacin, aspirin, diclofenac and ibuprofen,

have been shown to induce gastropathy in animal models [61].

The C-terminal half of the bovine milk LF prevented the NSAID-

induced gastropathy by binding to drugs with affinities ranging

from 2.6 to 4.8 � 10�4M. The crystal structures of the C-lobe of LF

with NSAIDs have been determined [61]. The structure of the

complexes showed that drugs bind to the C-lobe at a site formed

by two a-helices: a10 and a11. The drug-binding site on the C-lobe

represents a deep cavity that can accommodate NSAIDs in a

nonselective manner. The drug-binding site is formed by

Pro429, Glu431, Val591, Pro593, Gly652, Glu659, Thr663 and

Glu664 residues. The carboxylate group of aspirin and diclofenac

is H-bonded to Thr663. Drugs also make several van der Waals

contacts with Pro593 and Tyr660 residues (Fig. 3b). NSAIDs are

found to bind nonspecifically and in variable orientations to the C-

lobe of LF. LF and transferrin, being very similar in structure and

functions, work as transporting proteins for NSAIDs and reduce

the effective biological concentration of the drugs in body fluids.

Moreover, oral recombinant human LF supplementation during a

short-term indomethacin challenge has been observed reducing

the NSAID-mediated increase in small intestinal permeability and,

hence, provides a nutritional tool in the treatment of hyperper-

meability-associated disorders [62].

AlbuminHuman serum albumin (HSA) is abundantly present in plasma and

restricts free concentration of a wide variety of molecules in the

blood. HSA is known to have an important role in drug transport

and metabolism. At least two distinct drug-binding sites have been

observed in HSA by crystallographic studies [63]. Drug-binding

sites on HSA show flexible binding of a large variety of compounds.

Various NSAIDs such as aspirin, ibuprofen, oxyphenbutazone,

indomethacin, among others, have been shown to interact with

HSA [63,64]. Aspirin is hydrolysed into salicylic acid and the acetyl

group is transferred to Lys199 by HSA using its esterase activity.

Oxyphenbutazone and salicylic acid bind at the same site, whereas

ibuprofen and indomethacin recognise multiple and distinct sites

located deeper on HSA. All the drug-binding sites have polar and

hydrophobic amino acids to make attractive interactions with

NSAIDs. The drug-binding sites on HSA are nonspecific in nature

because other compounds also interact in similar ways to the

protein. Presence of multiple drug-binding sites on HSA contrib-

utes to its higher efficiency of drug transport and drug distribution.

Peroxisome proliferator activator receptor (PPAR)gPPARg is a member of a family of nuclear receptors. It works as a

transcription factor for genes involved in glucose homeostasis and

insulin sensitisation [65–67]. It has been implicated in the pathol-

ogy of many diseases such as obesity, diabetes, atherosclerosis and

cancer. PPARg decreases the inflammatory response of many

cardiovascular cells, particularly endothelial cells [68]. Agonists

of PPARg have been used in the treatment of hyperlipidaemia and

hyperglycaemia states [69]. PPARg is a potential therapeutic target

for metabolic syndrome and inflammatory diseases [70,71]. Indole

acetate derivatives are known to be activators of PPARg receptor,

hence indomethacin has also been reported to be an agonist of


PPARg and beneficial in the treatment of hyperlipidaemia and

hyperglycaemia [72]. The 3D crystal structures of PPARg in com-

plexes with indomethacin and other indole derivatives have been

reported [73]. Indomethacin binds more strongly than 5-methoxy-

indole acetate (MIA) and 5-hydroxy-indole acetate (HIA) to PPARg

with a dissociation constant of 9.73 mM [72]. Indomethacin shares

the same space with other indole derivatives and potent agonists

in the ligand-binding site of PPARg [73]. Two molecules of indo-

methacin were found strongly held between a-helices of the

activation function (AF)2 site in the ligand-binding domain.

The carboxylic acid group of indomethacin makes one H-bond

with Tyr473 and two salt bridge interactions with His323 and

His449 residues. The ligand-binding site is highly hydrophobic in

chemical nature and constituted by residues Ile281, Phe282,

Cys285, Arg288, Ile326, Tyr327, Leu330, Ile341, Met348,

Leu353, Leu356, Phe360, Phe363, Met364, Leu453, Leu465 and

Leu469. Indomethacin makes extensive van der Waals interac-

tions with these hydrophobic amino acids and is strongly held in

the binding site.

Human bromodomain (BRD)2BRD is known to bind acetylated lysine. It has a four-helical-

bundle structural fold [74,75]. BRD2 is encoded by the BRD2 gene

[76]. Human BRD2 has been suggested to be involved in signal

transduction for growth control. The therapeutic benefits of mod-

ulating this target class are largely unexplored owing to the lack of

suitable chemical probes [77]. Paracetamol has been shown to

interact consistently with human BRD2. The crystal structure of

the complex of paracetamol and BRD2 has been reported [77].

Paracetamol binds in the cavity surrounded by helices. The drug

makes a single direct H-bond with Asn156 and the drug-binding

cavity is formed by Phe99, Val103, Leu108, Leu110, Tyr113,

Cys152, Tyr155 and Ile162 residues (Fig. 3c). BRD-containing

receptor proteins regulate expression of multiple genes such as

those involved in inflammatory reactions and tumour cell growth.

The inhibitors of BRD have profound antiproliferative and anti-

inflammatory effects [78]. Similarly, paracetamol can also be

implicated in antiproliferative effects along with its anti-inflam-

matory activities.

Mechanisms of NSAID-induced reactionsNSAIDs are the most widely prescribed category of drugs used

against inflammatory disorders in society. Continuing usage of

NSAIDs for longer timeframes leads to a number of lethal side-

effects such as heart attack, stroke and toxicity, as well as some

severe side-effects including gastrointestinal injury and peptic

ulceration along with a broad spectrum of adverse effects in the

liver, kidney and cardiovascular systems. The mechanisms of

adverse as well as beneficial effects of NSAIDs are COX-dependent

and COX-independent in nature. Although the precise mecha-

nism is not well understood, NSAIDs have also been found in-

creasing the survival of colon and breast cancer patients [78].

The chemo-preventive activity of NSAIDs is thought to be the

result of their binding to the aryl hydrocarbon (ArH) receptor to

modulate carcinogen metabolic enzymes [79,80]. It has also been

reported that, in several cancer cell lines, NSAIDs induced knock-

down of specificity protein transcription factors (Sp1, Sp3 and Sp4)

resulting in the decreased expression of growth-promoting and







Review

s�P

OSTSCREEN

proinflammatory genes [81–85], which, along with reducing the

risk of Alzheimer’s disease [86], adds to the beneficial effects of

NSAIDs. Although gastrointestinal problems are known to be

associated with inhibition of COX-1, a recent report suggested

that nonspecific simultaneous inhibition of COX-1 and COX-2 is

responsible for the damage of gastric mucosa in rats [87]. The drug-

induced liver injury (DILI) is caused by NSAID antagonism of

fernasoid-X-receptors (FXR) present on the liver cells [88]. Several

clinical studies have also reported NSAIDs inhibiting uridine 50-

diphospho-glucuronosyltransferase (UGT) enzymes, involved in

xenobiotic metabolism in hepatocytes [89], which could further

add to the COX-independent side-effects of NSAIDs. Similarly,


TABLE 1

Interaction features of the NSAIDs and protein targets.

Target protein NSAIDs PDB code No. of polar interaction

including solvent

(2.8–3.5 A)a

COX-1 Flurbiprofen 3N8Z 3

Diclofenac 3N8Y 3

Celecoxib 3KK6 3

Ibuprofen 1EQG 3

Alclofenac 1HT8 3

Nimesulide 3N8X 7

Aspirin 1PTH 2

COX-2 Naproxen 3NT1 3

Flurbiprofen 3PGH 3

Indomethacin 4COX 3

Diclofenac 1PXX 4

Celecoxib 3LN1 4

PLA2 Aspirin 1TGM 2

Nimesulide 1ZWP 12

Oxyphenbutazone 1Q7A 1

Indomethacin 4EIX 3

Diclofenac 2B17 4

Paracetamol 2DPZ 6

CYP450 Diclofenac 1NR6 4

Flurbiprofen 1R9O 3

TTR Diclofenac 1DVX 5

Flufenamic acid 3OZL 0

Flurbiprofen 1DVT 1

Lectoferrin Indomethacin 3IB1 5

Aspirin 3IAZ 4

Diclofenac 3IB0 4

Ibuprofen 3IB2 1

HSA Aspirin 2I2Z 0

Ibuprofen 2BXG 6

Oxyphenbutazone 2BXB 6

Indomethacin 2BXM 4

PTGR Indomethacin 1S2A 6

Flufenamic acid 1S2C 6

PPARg Indomethacin 3ADS 3

BRD2 Paracetamol 4IOO 4

LPO Aspirin 2QQT 3

Indomethacin 3OGW 1

Nimesulide 3QL6 8

Paracetamol 3PY4 2

Abbreviations: BRD, bromodomain; COX, cyclooxygenase; CYP450, cytochrome P450; HSA, huma

Protein Data Bank; PLA, phospholipase A; PPAR, peroxisome proliferator-activated receptor; Pa The numbers of interactions between the drugs and receptor proteins are calculated using


coxibs have also been found interacting with ion channels on

heart muscles in Drosophila, which could additionally be respon-

sible for their adverse effects on the heart [90]. Recently, new and

more-specific coxibs without cardiovascular deleterious effects

have been developed [91], indicating a possibility of more-specific

COX inhibitors having reduced adverse reactions.

Concluding remarksCOX enzymes catalyse the synthesis of the proinflammatory

agents from membrane-liberated arachidonic acid and are hence

used as targets for synthesis of anti-inflammatory drugs. Besides

inhibiting the activity of COX enzymes, NSAIDs have also been


s No. of nonpolar

interactions

(distance up to 4.0 A)a

Binding affinity

of the NSAID

References

63 Ki = 1.0 mM [33,95]

61 Ki = 0.15 mM [33,96]

387 Ki = 0.30 mM [30]35 Ki = 9.0 mM [32,97]

42 Not reported [32]

81 Ki = 0.12 mM [33,98]25 IC50 = 126 mM [31]

58 Ki > 10.0 mM [35]

50 Ki = 0.77 mM [99]

82 Ki = 0.60 mM [34]

51 Ki = 50 nM [36,98]84 Ki = 0.87 mM [37,98]

15 KD = 6.4 mM [41]

58 – To be published

57 KD = 64 nM [39]24 KD = 1.3 mM [40]

53 Ki = 48 nM [42]


52 KD = 50 mM [100]56 Ki = 9.6 mM [49]

89 56.06% inhibition [60]



31 KD = 260 mM [61]28 KD = 330 mM [61]

46 Not reported [61]

22 KD = 480 mM [61]

11 KD = 70 mM [64,101]54 KA = 1.0 � 105/M [63,102]

102 KA = 2.97 � 106/M and

7.07 � 104/M

[63]

105 KA = 2.5 � 105/M [63,103]

106 Ki = 2.1 mM [45]

154 Ki = 3.1 mM [45]

127 KD = 9.73 mM [73]

46 KA = 50 mM [31,77]

60 Not reported [54]110 – To be published



n serum albumin; LPO, lactoperoxidase; NSAID, nonsteroidal anti-inflammatory drug; PDB,

TGR, prostaglandin keto reductase; TTR, transthyretin.

the CCP4 package [19].






found to interact with several other physiological receptor pro-

teins and enzymes, such as PLA2, HSA, LF, CYP450, TTR, LPO, PG

reductases, BRD2, transcription factors, FXR, UGT and PPARg,

which could additionally be responsible for their physiological

effects. Structural analysis has also revealed that, although NSAIDs

primarily inhibit catalytic functions of COX and PLA2 enzymes,

which are responsible for production of eicosanoids, the binding

affinities of drugs targeting CYP450, TTR, AKR and PTGR are also

fairly strong in nature (Table 1). On the basis of current analysis

and reported values, it can be stated that, besides inhibiting COX

enzymes, indomethacin binds strongly to PTGR and LPO whereas

diclofenac is the most potent inhibitor of PLA2 and flurbiprofen

has very high affinity for CYP450 and TTR receptors. NSAIDs

inhibit PLA2, PTGR and PPARg with micromolar affinity with a

large number of protein–ligand interactions, which are compara-

ble to their binding to COX enzymes, indicating a highly nonspe-

cific recognition of COX enzymes by these drugs (Table 1). Based

on their interactions with the drugs, the receptor protein targets

can be divided into three groups. Group I: CYP450 and LPO

enzymes, which catabolise NSAIDs. Group II: NSAIDs bind to

nonenzymatic receptors such as LF, TTR, transcription factors

and albumin, to prevent surface–surface interactions with other


molecules or transportation. Group III: NSAIDs work as inhibi-

tors for COX, PLA2, AKR, PTGR, PPARg, FXR and UGT enzymes.

LPO can be categorised in group I and III because drugs bind as

substrates as well as in inhibitors. These nonspecific bindings of

NSAIDs to diverse groups of enzymes are additionally responsible

for their beneficial and/or adversarial physiological effects on the

human body. To develop more-specific anti-inflammatory drugs,

it is essential to avoid their binding to additional physiological

receptor molecules responsible for pathological effects that will

further increase the effective bioavailability of the drugs. In the

preview of these observations, new drugs inhibiting catalytic

activity exclusively of enzymes responsible for production of

eicosanoids might have diminished adverse effects on the hu-

man physiology. In fact, to support this hypothesis, new cate-

gories of highly selective COX-2 inhibitors are being designed to

avoid nonspecific receptors binding to reduce adverse reactions

[91–94].

AcknowledgementsThe authors acknowledge the financial assistance from the Indian

Council of Medical Research (ICMR) and Department of Science &

Technology (DST), Ministry of Science and Technology, India.

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Molecular basis for nonspecificity of nonsteroidal anti-inflammatory drugs (NSAIDs

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