Inhibiting NF-κB Activation by Small Molecules As a Therapeutic Strategy

Abstract

Because nuclear factor-κB (NF-κB) is a ubiquitously expressed proinflammatory transcription factor that regulates the expression of over 500 genes involved in cellular transformation, survival, proliferation, invasion, angiogenesis, metastasis, and inflammation, the NF-κB signaling pathway has become a potential target for pharmacological intervention. A wide variety of agents can activate NF-κB through canonical and noncanonical pathways. Canonical pathway involves various steps including the phosphorylation, ubiquitnation, and degradation of the inhibitor of NF-κB (IκBα), which leads to the nuclear translocation of the p50- p65 subunits of NF-κB followed by p65 phosphorylation, acetylation and methylation, DNA binding, and gene transcription. Thus, agents that can inhibit protein kinases, protein phosphatases, proteasomes, ubiquitnation, acetylation, methylation, and DNA binding steps have been identified as NF-κB inhibitors. Here, we review the small molecules that suppress NF-κB activation and thus may have therapeutic potential.

1. Introduction

The nuclear factor-κB (NF-κB) signaling pathway plays a major role in the development, maintenance, and progression of most chronic diseases. NF-κB controls the expression of genes involved in a number of physiological responses, including immune inflammatory responses, acute-phase inflammatory responses, oxidative stress responses, cell adhesion, differentiation, and apoptosis [1]. Recent studies have suggested that NF-κB dysregulation is associated with many diseases including AIDS, atherosclerosis, asthma, arthritis, diabetes, inflammatory bowel disease, stroke, muscle wasting and viral infections. Mounting evidence indicates that NF-kB acts as a link between inflammation and cancer progression [2–10], making NF-κB essential to and a potential drug target in hematological malignancies and solid tumors [11, 12]. NF-κB was first identified in 1986 by Sen and Baltimore [5] in the nucleus bound to an enhancer element of the immunoglobulin kappa light chain gene in B cells [5, 13]. It is now known to be ubiquitous in nature present in all the cell types and is evolutionary conserved. It belongs to the family of Rel proteins that includes c-Rel, RelA (p65), RelB, NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100) all of which can form hetero- or homodimers [14–16].

NF-κB activation is tightly regulated mainly through its localization. In resting cells, NF-κB proteins are kept in the cytoplasm in association with inhibitory IkB proteins including IκBα, IκBβ, and IκBε [15] among which IκBα is the most abundant. NF-κB signaling occurs through the canonical (classical) pathway initiated by NF-κB1 (p50/p105) and a noncanonical (alternative) pathway initiated by NF-κB2 (p52/p100) (Fig 1). Before the active NF-κB is translocated into the nucleus, NF-κB1 and NF-κB2 are cleaved to the active p50 and p52 subunits, respectively. While the classical pathway depends on IKK complex consisting of IKKα, IKKβ, IKKγ and the inhibitory subunit IκBs, the alternative pathway depends on IKKα homodimers and NF-κB inducing kinase (NIK) [17–19]. During classical activation, the IKK complex specifically phosphorylates IκBs on two conserved N-terminal serine residues which target them for E2- and E3-ligase-mediated polyubiquitination and subsequent 26S proteasomal mediated degradation. This process releases and activates NF-κB which now translocates to the nucleus. The activation of alternative pathway, which is commonly associated with RelB results in regulated processing of the p100 precursor protein to p52 and subsequent translocation of p52-RelB heterodimers to the nucleus[18]. Although NF-κB activation occurs mainly through canonical and non-canonical pathways, during the past decade a number of pathways for NF-κB activation has been elucidated (Fig 1).

Figure 1.

Schematic representation of major NF-κB activation pathways. In the classical pathway, binding of TNFα to the receptor triggers the sequential recruitment of the adaptors TRADD, TRAF2 and RIP to the membrane. TRAF2 then recruits the IKK complex composed of IKKα, IKKβ and IKKγ (NEMO) through mediation of kinases like TAK1, MEKK1, MEKK3. Activation of the IKK complex leads to the phosphorylation and ubiquitination of IκBα at specific residues followed by its degradation via the proteasome pathway. The p105 subunit of NF-κB then undergoes GSK3β and Tpl2 mediated phosphorylation at S⁹⁰³ and S⁹⁰⁷ and subsequent degradation. The heterodimer p50–p65 is then released and migrates to the nucleus where it undergoes a series of posttranslational modifications including phosphorylation, acetylation and methylation and binds to specific κB sites and activates NF-κB target genes [49, 205, 206]. The alternative pathway is IKKγ independent and is triggered by binding of the CD40, RANK, LTβR, BAFF ligands to their receptor, leading to recruitment of TRAF proteins and the sequential activation of NIK and IKKα. Activation of IKKα then induces the processing of the inhibitory protein p100. p100 proteolysis releases p52 which then translocates to the nucleus and triggers transcription of NF-κB target genes [207]. NF-κB activation in response to UV-C does not depend on IKK activation and relies on sequential recruitment of p38MAPK and CKII. Activated CKII phosphorylates IκBα at C-terminus (S²⁸³-T²⁹⁹). The phosphorylated IκBα undergoes ubiquitination and degradation leading to release of active NF-κB in to the nucleus [208, 209]. EGF induced NF-κB activation proceeds without serine phosphorylation and ubiquitination of IκBα and is IKK independent. It relies on phosphorylation of IκBα at Tyr⁴² through mediation of tyrosine kinases that triggers its proteasome mediated degradation and subsequent release of active NF-κB to the nucleus [210]. NF-κB activation in response to bacterial endotoxin LPS involves Toll like receptor and is mediated through recruitment of MyD88, TRAF6 and ECSIT. Recruitment of these adaptors leads to sequential activation of IRAK1/2 and IKK and eventual release of active NF-κB [211]. NF-κB activation by pervanadate and H₂O₂ induces phosphorylation of IκBα at Tyr⁴² by protein tyrosine kinase like Syk. The Tyr phosphorylation does not lead to IκBα degradation but makes the binding weak thereby dissociating the IκBα and releasing active NF-κB to the nucleus [212, 213]. Antigen receptor viz., T-cell receptor and B-cell receptor mediated signaling to NF-κB activation depends on recruitment of a trimolecular protein complex CARMA1-BCL10-MALT1. In this pathway PKCθ (in T cells) and PKCβ (in B cells) alongwith other kinases act upstream to the trimolecular complex to promote IKKγ polyubiquitination and consequent IKK activation. Activation of IKK through this pathway involves mediation of TRAF2, TRAF6, TAK1 and TAB1 [214, 215]. A novel pathway of NF-κB activation originating from the nucleus is associated with DNA damage. Double-stranded DNA breaks in response to genotoxic agents initiate signals that trigger SUMOylation of nuclear-localized IKKγ, preventing its nuclear export. Concomitantly, these breaks activate ATM which phosphorylates SUMO-modified IKKγ, promoting the removal of SUMO and enhancing IKKγ ubiquitination. Ubiquitinated IKKγ then translocates to the cytoplasm, where it activates IKK in cooperation with ATM and the ELKS protein, leading to IκBα phosphorylation and degradation, p65 nuclear translocation and induction of NF-κB dependent target genes [216–219]. NF-κB can also be regulated by phosphatases. WIP1, a Ser/Thr phosphatase was recently shown to negatively regulate NF-κB activation by dephosphorylating p65 at Ser⁵³⁶ [80].

Abbreviations: AgR, antigen receptor; ATM, ataxia-telangiectasia mutant; BAFF, B-cell activating factor; BCL, B-cell lymphoma; BCR, B cell receptor; CARMA, CARD-containing MAGUK protein; CD40L, CD40 ligand; CK, casein kinase; DSBS, Double-stranded DNA breaks; ECSIT, evolutionary conserved signaling intermediates on Toll pathways; EGF, epidermal growth factor; EGFR, EGF receptor; ELKS, glutamate, leucine, lysine, serine-rich protein; GSK, glycogen synthase kinase; Hsp90, heat shock protein 90; IκB, inhibitor of NF-κB; IKK, IκB kinase; IRAK, IL-1R-associated kinase; LTβ, lymphotoxin β; LPS, lipopolysaccharide; MALT, mucosa-associated lymphoid tissue; MAPK, mitogen activated protein kinase; MAPK/Erk kinase kinase; MyD88, myeloid differentiation factor; NF-κB, nuclear factor-κB; NIK, NF-κB-inducing kinase; NEMO, NF-κB essential modulator; PDK, Phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; RANKL, receptor activator of NF-κB ligand; RIP, receptor-interacting protein; Syk, Spleen tyrosine kinase; TAB, TAK1-binding protein; TAK, transforming growth factor-β-activated kinase; TCR, T cell receptor; TLR, Toll-like receptor; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; Tpl2, tumour progression locus-2; TRADD, TNF-receptor-associated death domain protein; TRAF, TNF-receptor-associated factor.

Once in the nucleus, activated NF-κB undergoes a series of posttranslational modifications, including phosphorylation, acetylation, and methylation. These modifications regulate both the strength and duration of NF-κB activity. RelA/p65 is directly phosphorylated by cAMP- dependent protein kinase (PKA) at Ser²⁷⁶, casein kinase II (CKII) at Ser⁵²⁹, and IKK at Ser⁵³⁶ [20, 21]. RelA dephosphorylation by protein phosphatase 2A (PP2A) has been reported to decrease NF-κB activity [22]. RelA is subject to inducible acetylation by p300/CBP, and acetylated RelA interacts weakly, if at all, with IκBα [23, 24], but maintains its nuclear localization and NF-κB transcriptional response. RelA is also subject to methylation by lysine methyltransferase Set9 (also called Set7 or KMT7) at Lys^314/315 [25].

Activated NF-κB binds to specific DNA sequences in target genes, which are designated as κB elements, and regulates the transcription of over 500 genes involved in immunoregulation, growth regulation, inflammation, carcinogenesis, and apoptosis (Fig 2). NF-κB is frequently constitutively activated in patients with chronic inflammatory conditions such as cancer and pulmonary, cardiovascular, autoimmune, skin, and neurodegenerative diseases [26]. NF-κB’s ability to control multiple genes involved in human diseases makes the NF-κB signaling pathway a novel target for therapy [27, 28].

Figure 2.

A list of gene products regulated by NF-κB.

Due to the various levels of regulation, NF-κB signaling pathway can be potentially targeted at various levels including kinases, phosphatases, ubiquitination, nuclear translocation, DNA binding, protein acetyl transferases and methyl transferases (Fig 3).

Figure 3.

Potential targets for inhibiting the NF-κB activation pathway.

2. Inhibitors of the NF-κB activation pathway

Given NF-κB’s relevance in human diseases and the fact that many drugs interfere with NF-κB signaling, the NF-κB signaling pathway provides a highly attractive target for the therapeutic development. More than 700 inhibitors of the NF-κB activation pathway, including antioxidants, peptides, small RNA/DNA, microbial and viral proteins, small molecules, and engineered dominant-negative or constitutively active polypeptides have been described (Table 1). Several of these molecules act as general inhibitors of NF-κB activation, while other molecules target specific steps; some molecules possibly target multiple steps in the NF-κB pathway (Fig 3).

Table 1.

A list of small molecules as inhibitors of NF-kB pathway

A. Upstream NF-kB	Kahweol	AIDCA derivative	Catalposide	E-73	RelA peptides (P1 & P6)
Natural product	Kava derivatives2	TDZD	Cyclolinteinone	Ecabet sodium	Viral Protein
15d-PGJ(2)	Licorce extracts	TPCA-1	Dihydroarteanniun	Gabexate mesilate	3C protease (EMC virus)
Calagualine	Manumycin A	Pyridine derivatives	Docosahexaenoic acid	Glimepiride	Canine Distemper Virus
Conophylline	Monochloramine	ACHP	Emodin	Hypochlorite	MNF (myxoma virus)
Evodiamine	N-acetylcysteine	Acrolein	Ephedrae herba (Mao) extract	Losartin	Protein
Geldanamycin	Nitric oxide	AGRO100*	Equol	LY294002	C5a
Perrilyl alcohol	Nitrosylcobalamin	Amino-pyrimidine	Erbstatin	Pervanadate	DQ 65-79
PSK	Oleandrin	AS602868	Estrogen	Phenylarsine oxide	Fox1j
Rocaglamides	Omega 3 fatty acids	Aspirin	Ethacrynic acid	Phenytoin	GILZ
Viral protein	ox-LDL	Azidothymidine	Fosfomycin	Ro106-9920**	HSCO
Adenovirus E1A	Panduratin A	BAY-11-7082	Fungal gliotoxin	Sabaeksan	HSP-72
NS5A (Hep-C virus)	PEITC	BAY-11-7083	Gamisanghyulyunbueum***	U0126 (MEK inhibitor)	Interleukin-10
Protein	Petrosaspongiolide M	Benzoimidazole derivative	Genipin	Others	Interleukin -11
Erbin overexpression	Phytic acid	Benzyl isothiocyanate	Genistein	Vagus nerve stimulation	Interleukin -13
Golli BG21	Piceatannol	BMS-345541	Glabridin	Low level laser therapy	MTS-SR-IκBα
KSR	Pinosylvin	Carboplatin	Glucosamine sulfate	Zinc	Onconase
MAST205	Plagius flosculosus extract	CDDO-Me	Glutamine	D. IκB upregulators/	RASSF1A gene
NPM-ALK oncoprotein	Plumbagin	CHS 828*	Gumiganghwaltang***	NF-κB translocation	ROR-alpha
Hep-C virus protease	Pomegranate extract	Compound 5	Isomallotochromanol	Natural product	Surfactant protein A
PEDF	Prostaglandin A1	Compound A	Isomallotochromene	PGG	TAT-SR- IκBα
Rituximab	Quercetin	Cyclopentenones	Kochia scoparla fruit extract	15-deoxyspergualin	ZAS3 protein
TNAP	Rengyolone	CYL-19s	L-ascorbic acid	2',8"-biapigenin	ZUD protein
Synthetic	Rosmarinic acid	CYL-26z	Leflunomide metabolite	5F (from Pteri syeminpinnata)	β-amyloid protein
Betaine	Rottlerin	Diaylpyridine derivative	Melatonin	Agastache rugosa leaf extract	Synthetic
Desloratadine	Saikosaponin-d	DPE	Midazolam	Alginic acid	BMD
LY29 and LY30	Salvia miltiorrhoza extract	Epoxyquinone	Momordin I	Antrodia camphorata extract	Carbaryl
MOL 294 **	Sanguinarine	Gabexate mesilate	Morinda officinalis extract	Apigenin	CGS 25462
Pefabloc	SAm extract	Gleevec	Mosla diantherra extract	Astragaloside IV	DHMEQ
Rhein	Staurosporine	Hydroquinone	Opuntia ficus indica extract	AT514 (serratamolide)	Diltiazem
SMI and FP	Sesquiterpene lactones	Ibuprofen	Platycodin saponins	Atorvastatin	Dioxin
B. IKK activity and IkB	Scoparone	IQCAD	Polymyxin B	Blue honey suckle extract	Dipyridamole
phosphorylation	Silibinin	Indolecarboxamide	Poncirus trifoliata fruit extract	Buthus martensi extract	Disulfiram
Natural product	Silymarin	Isobutyl nitrite	Probiotics	Cantharidin	Enalapril
[6]-gingerol	Sulforaphane	Jesterone dimer	Prostaglandin	Chiisanoside	mEET
1'-Acetoxychavicol acetate	Sulindac	15-deoxyspergualine analog	Resiniferatoxin	Clarithromycin	Fluvastatin
20(S)-Protopanaxatriol	Tetrandine	Methotrexate	Stinging nettle extracts	Cornus officinalis extract	Indole-3-carbinol
4-Hydroxynonenal	Theaflavin	MLB120**	Thiopental	Eriocalyxin B	JSH-23
Acetyl-boswellic acids	Thienopyridine	Monochloramine	Tipifarnib	Gangliosides	KL-1156
Anandamide	Tilianin	MX781 (Retinoid antagonist)	Titanium	Glucocorticoids	Leflunomide
Anethole	Ursolic acid	4-HPR	TNP-470	HP extracts	Levamisole
Apigenin	Vesnarinone	Nafamostat mesilate	Trichomomas vaginalis	Hirsutenone	MEB
Artemisia vestita1	Wedelolactone	NSAIDs	TG-rich lipoproteins	Human breast milk	Moxifloxacin
Baoganning	Withanolides	PS-1145 (MLN1145)	Ursodeoxycholic acid	JM34	Omapatrilat
Betulinic acid	Xanthoangelol D	PQD	Xanthium strumarium extract	KIOM-79	R-etodolac
Black raspberry extracts	Zerumbone	Pyridooxazinone derivative	β- PEITC	Leptomycin B	Rolipram
Buddlejasaponin IV	β-carboline	SC-514**	8-MSO	Neomycin	SC236 (COX-2 inhibitor)
Cacospongionolide B	γ-mangostin	Scytonemin	β-lapachone	Nucling	Triflusal
Calagualine	γ-Tocotrienol	Sodium salicylate	Peptide	Oregonin	Volatile anesthetics
Cardamomin	Peptide	Statins (several)	Penetratin	OXPAPC	E. NF-kB DNA-binding
Cardamonin	IKKβ peptide	Sulfasalazine	VIP	Paeoniflorin	Inorganic Complex
Casparol	NEMO CC2-LZ peptide	Sulfasalazine analogs	Protein	Phallacidin	Metals (chromium, cadmium, gold,
Cobrotoxin	Protein	Survanta	Activated protein C	Piperine	lead, mercury, zinc, arsenic)
Cycloepoxydon	Anti-thrombin III	Thalidomide	HSP-70	Pitavastatin	Natural product
Decursin	Chorionic gonadotropin	THI 52	Interleukin-13	Platycodi radix extract	Actinodaphine
Dehydroascorbic acid	FHIT	YC-1**	Intravenous Ig	Probiotics	Anthocyanins
Dexanabinol	HB-EGF	Others	Murr1 gene product	Rapamycin	Arnica montana extract
Digitoxin	Hepatocyte growth factor	Lead	Neurofibromatosis-2 protein	Rhubarb aqueous extract	Artemisinin
Diosgenin	Interferon-α	Mild hypothermia	PACAP	Salvia miltiorrhoza extract	Baicalein
Diterpenes	Interleukin-10	Saline (low Na+)	SAIF	SH extract	Bambara groundnut
Docosahexaenoic acid	PAN1	C. IkB degradation	ST2 (IL-1-like receptor)	Selenomethionine	β-lapachone
Falcarindol	PTEN	Natural product	α-MSH	Shenfu	Biliverdin
Flavopiridol	SOCS1	5'-methylthioadenosine	γ-glutamylcysteine synthetase	Sophorae radix extract	Brazilian
Furonaphthoquinone	Viral Protein	Artemisia iwayomogi extract	Bacterial/Viral Protein	Sopoongsan	Calcitriol
Garcinone B	Adenovirus	Alachlor	K1L (Vaccinia virus protein)	Sorbus commixta extract	Campthothecin
Glossogyne tenuifolia extract	Core protein (Hep-C virus)	Amentoflavone	Nef (HIV-1)	Sphondin	Sutherlandia frutescens
Glycine chloramine	Cytomegalovirus	Antrodia camphorata#	Vpu protein (HIV-1)	T. polyglycosides	Capsiate
Guggulsterone	E7 (Papillomavirus)	Artemisia capillaries extract	YopJ	Younggaechulgam-tang***	Catalposide
Herbimycin A	MC159	Aucubin	Synthetic	α-pinene	Cat's claw bark
Honokiol	MC160	Baicalein	1-Bromopropane	Peptide	Cheongyeolsaseuptang***
Hypoestoxide	NS5B (Hep-C virus)	Blackberry extract	Acetaminophen	NCPP	Chitosan
Indirubin-3'-oxime	vIRF3 (KSHV)	Buchang-tang***	Diamide	PN50	Chicory root
Isorhapontigenin	Synthetic	Capsaicin	Dobutamine		CSPDP
Clarithromycin	AIM2 overexpression	Raxofelast	F. Proteasome/protease	Mangifera indica bark
Cloricromene	Angiopoietin-1	Ribavirin	Natural product	extract
C-K and Rh(2)	Antithrombin	Rifamides	Cyclosporin A	Mangiferin
Cortex cinnamomi	AvrA protein	Ritonavir	Lactacystine	Melatonin
extract	(Salmonella)	Rosiglitazone	β-lactone	Mn-SOD
Cryptotanshinone	β-catenin	Roxithromycin	Peptide	Mulberry anthocyanins
Cytochalasin D	Bromelain	DAAS	ALLnL	Myricetin
Black rice extract	CaMKK	Serotonin derivative	LLM	N-acetyl-L-cysteine
Danshenshu	CD43 overexpression	Simvastatin	Ubiquitin ligase	Nacyselyn
Diterpenoids	FLN29 overexpression	SM-7368**	Z-LLL	Naringin
Ent-kaurane diterpenoids	FLIP	T-614	Z-LLnV	N-ethyl-maleimide
Epinastine hydrochloride	G-120	Sulfasalazine	Synthetic	Nitrosoglutathione
Epoxyquinol A	Gax (homeobox protein)	SUN C8079	APNE	NDGA
Erythromycin	HIV-1 Resistance Factor	Triclosan plus CPC	Boronic acid peptide	Ochnaflavone
Evodiamine	Interleukin 4	Tobacoo smoke	BTEE	Orthophenanthroline
Fish oil	SspH1 and IpaH9.8	Verapamil	3,4-dichloroisocoumarin	Phenylarsine oxide
Fomes fomentarius	NDPP1 (CARD protein)	Others	Deoxyspergualin	PhIP
extracts	Overexpressed ZIP1	Heat (fever-like)	DFP	Phyllanthus urinaria
Fucoidan	p8	Hypercapnic acidosis	Disulfiram	PMC
Gallic acid	p202a	Hyperosmolarity	FK506 (Tacrolimus)	PTX
Ganoderma lucidum	p21 (Rec)	Hypothermia	Bortezomib	Pyrithione
Garcinol	PIAS1	Alcohol	Salinosporamide A	Pyrrolinedithiocarbamate
Geranylgeraniol	Pro-opiomelanocortin	E. NF-kB transactivation	TLCK	Quercetin
Ginkgolide B	PYPAF1 protein	Natural products	TPCK	Quinozolines
Glycyrrhizin	Raf Kinase inhibitor	4'-DM-6-Mptox	G. Antioxidants	Rebamipide
Halofuginone	protein Rhus verniciflua	4-phenylcoumarins	23-hydroxyursolic acid	Red wine
Hematein	fruits	AHUP	Aged garlic extract	Redox factor 1
Herbal compound 861	SLPI	Adenosine	Anetholdithiolthione	Resveratrol
Hydroxyethyl starch	Siah2	c-AMP	Apocynin	Ginseng derivative
Hydroxyethylpuerarin	SIRT1 Deacetylase	Artemisia sylvatica extract	Apple juice/extracts	Rotenone
Hypericin	overexpression	Bifodobacteria	Arctigenin	Roxithromycin
Kamebakaurin	Siva-1	Blueberry & berry mix	Aretemisa p7F	S-allyl-cysteine
Linoleic acid	Solana nigrum L.	BSASM	Astaxanthin	Sauchinone
Lithospermi radix	Surfactant protein A	BF phenylpropanoids	Benidipine	Spironolactone
Macrolide antibiotics	Tom1 overexpression	cPrG.HC	bis-eugenol	Strawberry extracts
Mediterranean plant	Transdominant p50	Seaweed extract	BG compounds	Taxifolin
extracts	Uteroglobin	Fructus benincasae extract	BHA	Tempol
2-methoxyestradiol	VEGF	Glucocorticoids	CAPE	Tepoxaline
6-MITC	Synthetic	Gypenoside XLIX	Carnosol	tert-butyl hydroquinone
Nicotine	ADP ribosylation inhibitor	Kwei Ling Ko3	Carvedilol	Tetracylic A
Ochna macrocalyx bark	7-amino-4-methylcoumarin	LC root	Catechol derivatives	Vitamin B6
ext.	Amrinone	Luteolin	Celasterol	Vitamin C
Oridonin	Atrovastat	Manassantins A,B	Cepharanthine	Vitamin D
PC-SPES (8 herb mixture)	Benfotiamine	MI bark extract	Chlorogenic acid	Vitamin E derivatives
PGG	Benzamide	Mesuol	Chlorophyllin	Wogonin
Pepluanone	Bisphenol A	Nobiletin	Cocoa polyphenols	xanthohumol
Phyllanthus amarus	Caprofen	Phomol	Curcumin	Yakuchinone A, B
extracts	Carbocisteine	Psychosine	DHEA	α-lipoic acid
Plant compound A	Celecoxib	Qingkailing#	DHEA sulfate	α-tocopherol
Polyozellin	Germcitabine	Saucerneol D & E	Dehydroevodiamine	α-torphryl acetate
Prenylbisabolane 3	Cinnamaldehyde	Shuanghuanglian#	Demethyltraxillagenin	α-torphryl succinate
Prostaglandin E2	2-methoxy CNA	Smilax bockii extract	Diethyldithiocarbamate	β-Carotene
PSK	2-hydroxy CNA	Trilinolein	Diferoxamine
Quinic acid	CDS	Uncaria tomentosum extract	Dihydroisoeugenol
Sanggenon C	CP Compound	WS extracts	Dihydrolipoic acid
Sesamin	Cyanoguanidine	Wortmannin	Dilazep
Shen-Fu#	HMP	α-zearalenol	Fenofibric acid
Silibinin	α-difluoromethylornithine	Viral Protein	DMDTC
Sinomenine	DTD	BZLF1 (EBV protein)	Dimethylsulfoxide
Sword brake fern extract	Evans Blue	SH gene products (PMV)	Disulfiram
Tanacetum larvatum	Evodiamine	Protein	Ebselen
extract	Fenoldopam	Antithrombin	Edaravone
Tansinones	FEX	NF-kappaB-repression factor	EGTA
Taurine + niacine	Fibrates	PIAS3	EPC-K1
TZD MCC-555	FK778	PTX-B	Epigallocatechin-3-gallate
Trichostatin A	Flunixin meglumine	Synthetic	Ergothioneine
Triptolide	Flurbiprofen	17-AAG	Ethyl pyruvate
Tyrphostin AG-126	Hydroquinone	TMFC	Ganoderma lucidum
Ursolic acid	IMD-0354	AQC derivatives	polysaccharides
Withaferin A	JSH-21	9-aminoacridine	Garcinol
Xanthohumol	KT-90	derivatives	γ-glutamylcysteine synthetase
Xylitol	Lovastatin	Chromene derivatives	Ginkgo biloba extract
Yan-gan-wan#	Mercaptopyrazine	D609	Glutathione
Yin-Chen-Hao#	Mevinolin,	Dimethylfumarate	Hematein
Yucca schidigera extract	Monoethylfumarate	EMDPC	Hydroquinone
Peptide	Moxifloxacin	Histidine	Hydroquinone
Ghrelin	Nicorandil	HIV-1 PI	IRFI 042
Peptide YY	Nilvadipine	Mesalamine	Iron tetrakis
Rapamycin	NO-ASA	PEITC	Isovitexin
Viral Protein	Panepoxydone	Pranlukast	Kangen-karyu extract
African Swine Fever virus	Peptide nucleic acids	RO31-8220 (PKC	Ketamine
Sendai Virus-C,V proteins	Perindopril	inhibitor)	Lacidipine
E1B (Adenovirus)	PAD	SB203580 (MAPK inhibitor)	Lazaroids
ICP27 (HSV-1)	α-PBN	Tetrathiomolybdate	L-cysteine
H4/N5 (bracovirus)	Pioglitazone	Tranilast	Ligonberries
NS3/4A (Hep-C)	Pirfenidone	Troglitazone	Lupeol
Protein	PNO derivatives	Others	Magnolol
Adiponectin	Quinadril	Low gravity	Maltol

¹Tibetan medicine;

²Piper methysticum;

³Tortoise shell-Rhizome jelly;

^*Anticancer drugs;

^**small molecules;

^***Oriental medicines;

^#Traditional Chinese Medicine; 15d-PGJ(2), 15-deoxy-prostaglandin J(2); 17-AAG, 17-allylamino-17-demethoxygeldanamycin; 20(S)-PPT, 20(S)-Protopanaxatriol; 4’-DM-6-Mptox, 4’-demethyl-6-methoxypodophyllotoxin; 6-MITC, 6-Methylsulfinyl) hexyl isothiocyanate; α-MSH, α-melanocyte-stimulating hormone; α-PBN, alpha-phenyl-N-tert-butylnitrone; ACHP, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile; AGRO100, G-quadruplex oligodeoxynucleotide; AHUP, 8-acetoxy-5-hydroxyumbelliprenin; AIDCA, (Amino)imidazolyl carboxaldehyde derivative; ALLnL, N-acetyl-leucinyl-leucynil-norleucynal; APNE, N-acetyl-DL-phenylalanine-b-naphthylester; AQC, 6-aminoquinazoline; BAY-11-7082, E3((4-methylphenyl)-sulfonyl)-2-propenenitrile; BAY-11-7083, E3((4-t-butylphenyl)-sulfonyl)-2-propenenitrile; BF, Bupleurum fruticosum; BG, Bruguiera gymnorrhiza; BHA, Butylated hydroxyanisole; BMD, N(1)-Benzyl-4-methylbenzene-1,2-diamine; BMT, o,o’-bismyristoyl thiamine disulfide; BSASM, plant extract mixture; BTEE, N-benzoyl L-tyrosine-ethylester; CaMKK, Calcium/calmodulin-dependent kinase kinase; CAPE, Caffeic Acid Phenethyl Ester; CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDS, Commerical peritoneal dialysis solution; CMP, Chinese medicinal preparations; CNA, Cinnamaldehyde; Compound 5, Uredio-thiophenecarboxamide derivative; CP Compound, 6-Hydroxy-7-methoxychroman-2-carboxylic acid phenylamide); CPC, cetylpyridinium chloride; cPrG.HC, Cycloprodigiosin hycrochloride; CSPDP, Chondrotin sulfate proteoglycan degradation product; CYL-19 s and CYL-26z, two synthetic alpha-methylene-gamma-butyrolactone derivatives; D609, phosphatidylcholine-specific phospholipase C inhibitor; DAAS, diacetoxy acetal derivative of santonin; DFP, diisopropyl fluorophosphates; DHEA, Dehydroepiandrosterone; DHMEQ, Dehydroxymethylepoxyquinomicin; DMDTC, Dimethyldithiocarbamates; DPE, 2-(3,4-dihydroxyphenyl)ethanol; DTD, 4,10-dichloropyrido[5,6:4,5]thieno[3,2-d’:3,2- d]-1, 2, 3-ditriazine; EGTA, Ethylene glycol tetraacetic acid; EMC virus, encephalomyocarditis virus; EMDPC, Ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl)) pyrimidine-5-carboxylate; EPC-K1, phosphodiester compound of vitamin E; FEX, Fexofenadine hydrochloride; FHIT, Fragile histidine triad protein; FLIP, FLICE-Like Inhibitory Protein; G-120, Ulmus davidiana Nakai glycoprotein; GILZ, Glucorticoid-induced leucine zipper protein; HB-EGF, Heparin-binding epidermal growth factor-like growth factor; HMP, 7-(4’-hydroxy-3’-methoxyphenyl)-1-phenylhept-4-en-3-one; HP, Harpagophytum procumbens; HSCO, Hepatoma Subtracted-cDNA library Clone One; IQCAD, Imidazolylquinoline-carboxaldehyde derivative; JSH-23, 4-Methyl- -(3-phenyl-propyl)-benzene-1,2-diamine; KL-1156, 6-Hydroxy-7-methoxychroman-2-carboxylic acid phenylamide; KSHV, Kaposi’s sarcoma-associated herpesvirus; KSR, Kinase suppressor of ras; LC, Ligusticum chuanxiong; LLM, N-acetyl-leucinyl-leucynil-methional; LY294002, [2-(4-morpholinyl)-8-phenylchromone]; MAST205, a serine/threonine kinase; MC160, Mollusum contagiosum virus; MEB, 2-(4-morpholynl) ethyl butyrate hydrochloride; mEET, Mouse estrogen enhanced transcript; Mn-SOD,Manganese superoxide dismutase; MSO, 8-methylsulphinyloctyl; N-(4-hydroxyphenyl) retinamide [4-HPR; NDGA, Nordihydroguaiaritic acid; NCPP, NLS Cell permeable peptides; NFD-37, 2-Methyl-2-(2-methylpropenyl)-2,3-dihydronaphthoquinone [2,3-b]furan-4,9-dione; NH(2)Cl, monochloramine; NO-ASA, Nitric-oxide-donating aspirin; ox-LDL, Oxidized low density lipoprotein; OXPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; PACAP, Pituitary adenylate cyclase-activating polypeptide; PAD, 6(5H)-phenanthridinone; PEDF, pigment epithelium derived factor; PEITC, Phenethyl isothiocyanate; PGG, 1,2,3,4,6-penta-O-galloyl-β-D-glucose; PhIP, 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PI, protease inhibitor; PIAS1, protein inhibitor of activatated STAT1; Plant compound A, plant-derived phenyl aziridine precursor; PMC, (2,2,5,7,8-pentamethyl-6-hydroxychromane); PMV, Paromyxovirus; PNO, Pyridine N-oxide; PQD, Pyrazolo[4,3-c]quinoline derivative; PSK, Protein-bound polysaccharide; PTEN, phosphatase and tensin homolog; PTX, Pentoxyifylline (1-(5’-oxohexyl) 3,7-dimehylxanthine; PTX-B, pertussis toxin binding protein; Pyridine derivatives, 2-amino-3-cyano-4-aryl-6-(2- hydroxy-phenyl)pyridine derivatives; RH(2) & C-K, intestinal bacterial metabolites Rh(2) and compound K (C-K); RORalpha, Retinoic acid receptor-related orphan receptor-alpha; SH, Sargassum hemiphyllum; SAIF, Saccharomyces boulardii anti-inflammatory factor; SLPI, Secretory leucoprotease inhibitor; SMI and FP, Salmeterol and Fluticasone propionate; SOCS, suppressor of cytokine signaling proteins; SspH1 and IpaH9.8, Leucine-rich effector proteins of Salmonella & Shigella; TDZD, 1,2,4-thiadiazolidine derivative; TG, triglyceride; THI 52, 1-naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TLCK, N-α-tosyl-L-lysine chloromethyl ketone; TMFC, 3,4,5-trimethoxy-4’-fluorochalcone; TNAP, TRAFs and NIK-associated protein; ; TP, Tripterygium polyglycosides; TPCA-1, 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thionphenecarboxamides; TPCK, N-α-tosyl-L-phenylalanine chloromethyl ketone; TZD, Thiazolidinedione; VEGF, Vascular endothelial growth factor; VIP, Vasoactive intestinal peptide; VVP, Vaccinia virus protein; WS, Witheringia solanacea; Z-LLL, N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norleucinal; Z-LLnV (carbobenzoxyl-leucinyl-leucynil-norvalinal;

2.1. Inhibition of protein kinases

NF-κB activation requires the phosphorylation, polyubiquitination, and subsequent degradation of its inhibitory subunit, IκBα. Hence, inhibiting IκBα phosphorylation ultimately inhibits NF-κB’s transcriptional activity [29, 30]. IκBα phosphorylation is carried out by IKK, a serine/threonine protein kinase composed of three basic subunits: the kinases IKKα, IKKβ, and the regulatory subunit IKKγ (NEMO). The IKK activation is usually the first common step in the integration of many NF-κB-activating pathways; therefore, one strategy for inhibiting NF-κB activation is to block IKK activation. However, although more than 150 agents have been shown to inhibit NF-κB activation at the IKK step, few studies have investigated the mechanism by which a given agent can inhibit IKK or its activation. The few IKK inhibitors for which a mechanism of action is known can be divided into three general groups: adenosine triphosphate (ATP) analogs, which show some specificity for interacting with IKK; compounds that have allosteric effects on IKK structure; and compounds that interact with a specific cysteine residue (Cys-179) in the activation loop of IKKβ. ATP analogs include natural products such as β-carboline and synthetic compounds such as SC-839, which has an approximately 200-fold preference for IKKβ compared to IKKα [27, 31]. Compounds that have allosteric effects on IKK structure include BMS-345541, a synthetic compound that binds to an allosteric site on both IKKα and IKKβ and has an approximately 10-fold greater inhibitory effect on IKKβ than on IKKα [32]. Compounds that interact with Cys-179 IKKβ include thiol-reactive compounds such as parthenolide, arsenite, and certain epoxyquinoids [33–36]; these compounds’ interactions with Cys-179 are believed to interfere with phosphorylation- induced IKKβ activation because Cys-179 is located between Ser¹⁷⁷ and Ser¹⁸¹, which are required for IKKβ activation in response to upstream signals such as tumor necrosis factor (TNF) and lipopolysaccharide (LPS) [37, 38]. Gene-based inhibitors can also block IKK activation. Specifically, mutations at the ATP-binding site or in the kinase activation loop can create dominant-negative IKKα and IKKβ, which are capable of blocking NF-κB activation [39–43]. Because of their distinct roles in the canonical and non-canonical NF-κB activation pathways, dominant-negative IKK mutants’ can show stimulus-dependent inhibition [44]. Adenoviral-mediated delivery of an IKKβ dominant-negative kinase has been shown to have therapeutic potential for airway inflammatory diseases such as asthma [45, 46]. NEMO can also serve as a target for inhibiting the IKK complex. In particular, introducing a cell-permeable 10 amino-acid peptide that corresponds to the NEMO-binding domain of IKKβ can block the binding of NEMO to IKK in response to TNF in the canonical pathway [47].

While activation of NF-κB by many stimuli depends on the phosphorylation of IκBs at N-terminal sites by the IKK complex, the mechanism of NF-κB activation by ultraviolet (UV) radiation involves the IKK-independent phosphorylation of IκBα at a cluster of C-terminal sites that are recognized by casein kinase II (CKII). CKII activity toward IκBα depends on p38 mitogen-activated protein kinase (MAPK) activation. CKII’s role as a key survival signal that activates NF-κB and protects tumor cells from apoptosis suggests that CKII may be an attractive target for the treatment of diverse cancers. Apigenin, a plant flavonoid, and emodin, a plant anthraquinone, are competitive inhibitors of CKII that directly interact with the nucleotide-binding sites of CKII [48].

Besides phosphorylating and subsequently degrading the molecules that inhibit NF-κB, protein kinases can also target the functional domains of NF-κB proteins themselves to optimally activate NF-κB. NF-κB proteins can be phosphorylated in the cytoplasm or nucleus by such kinases as glycogen synthase kinase 3β (GSK3β) [49], TRAF-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) [50, 51], PKAc [20], mitogen- and stress-activated protein kinase-1 (MSK-1) [52], MAP3K NIK[53], Tpl2, PKC-θ [54], PI3K, Akt [55–57], p38 MAPK [58], protein tyrosine kinase, PKC-δ [59], RHO-kinase 2 [60], Mitogen activated protein kinase kinase 3 (MEKK3) [61], and receptor tyrosine kinases such as epidermal growth factor receptor, human epidermal growth factor receptor 2 [62]. Antagonistic antibodies or kinase inhibitors that target these molecules may decrease NF-κB activation. Some kinase inhibitors that have the potential to inhibit NF-κB activation include SB203580 and PD0980589 (MAPK inhibitors) [58]; denbinobin (TAK1 inhibitor) [63]; tyrosine kinase inhibitors [62]; rhein, (an MEKK inhibitor) [64, 65]; TNAP, betaine (NIK inhibitors) [66–68], epoxyquinol B (a TAK1 crosslinker) [69]; M2L (an extracellular signal-regulated kinase 2 inhibitor) [70, 71]; CCK-8 (a p38 kinase kinase inhibitor) [72], KSR2 (an MEKK3 inhibitor) [73], golli BG21 (a PKC inhibitor) [74].

2.2. Inhibition of NF-κB activation by protein phosphatases

Because protein phosphorylation is a dynamic process whereby phosphatases counterbalance kinase action, phosphatases may be used to inhibit NF-κB activation. Protein phosphatase 2A is a serine/threonine phosphatase that has been reported to dephosphorylate and modulate the activity of IKKβ [75]. Cytosine arabinoside, a pyrimidine analogue used to effectively treat acute leukemia, has been reported to induce apoptosis by activating protein phosphatases 2A and 2B-A and dephosphorylating the p65 subunit of NF-κB [22, 76]. Recently, OspF, a protein phosphatase from Shigella flexneri, was found to dephosphorylate MAPK and prevent histone H3 phosphorylation at Ser¹⁰ in a gene-specific manner to block the activation of a subset of NF-κB responsive genes [77]. Our previous studies have shown that protein tyrosine phosphatase (PTP) inhibitors can suppress NF-κB activation and that phenylarsine oxide, a specific PTP inhibitor, can promote tyrosine 42 phosphorylation of IκBα [78]. While some PTPs stimulate NF-κB activation, other PTPs negatively regulate NF-κB activation. For instance, PTEN, a tumor suppressor with phosphatase activity is known to inhibit NF-κB activation [79]. Recently, Chew et al., [80] found that WIP1, a Ser/Thr PP2C family of phosphatases act as a negative regulator of NF-κB activation. Overexpression of WIP1 was associated with decreased NF-κB activation, whereas WIP1 knockdown resulted in increased NF-κB activation. The group further showed that WIP1 target Ser⁵³⁶ of the p65 subunit of NF-κB.

2.3. Proteasome inhibitors and IκB ubiquitination blockers

The step before NF-κB leaves the cytoplasm involves the ubiquitination of IκB by the SCF-β-TrCP ubiquitin ligase complex followed by the rapid degradation of ubiquitinated IκB by the 26S proteasome [38]. Because IκBα degradation is an important step in the NF-κB activation pathway, inhibiting the proteasomes that degrade IκBα may also serve as a tool for pharmacological intervention. Very specific and potent proteasome inhibitors have been engineered by coupling boronic acid to dipeptides [81]. The dipeptide boronate, bortezomib, the most-studied proteasome inhibitor in clinical development [82], has been shown to inhibit proliferation and induce apoptosis in head and neck [83–85], prostate [86], pancreatic [87], gastric [88], and ovarian [89] cancers. Bortezomib’s antitumor properties correlate in part with its ability to inhibit IκBα degradation [83, 90]. Other well-known proteasome inhibitors include ALLnL, LLM, Z-LLnV, and Z-LLL, lactacystine, N-cbz-Leu-Leu-leucinal (MG132), MG115, and ubiquitin ligase inhibitors [91]. In addition, we recently identified a novel proteasome inhibitor, salinosporamide A (NPI-0052), which can suppress both constitutive and inducible NF-κB activation in a nanomolar range [92].

Several serine protease inhibitors with chymotrypsin-like specificity, including DCIC, TPCK, TLCK, BTEE, APNE, are also able to block proteasome function. However, unlike other protease inhibitors that block only IκB degradation, serine protease inhibitors can block IκB phosphorylation as well as degradation. However, not all serine protease inhibitors can inhibit NF-κB activation [93–95].

Among IκB ubiquitination blockers, the YopJ protein of the bacterial pathogen Yersinia deubiquitinates and stabilizes IκBα to prevent NF-κB nuclear translocation [96]. The small molecule R0196-9920 has been reported to inhibit IκBα ubiquitination and oral inflammation in mouse models [97, 98]. Yaron et al., [98] blocked TNFα-induced IκBα degradation by microinjecting phosphopeptides that corresponded to IκBα’s signal-dependent phosphorylation site. Presumably these phosphopeptides acted as competitive inhibitors for binding to the ubiquitin ligase complex essential to IκBα degradation. Inhibiting β-TrCP (the recognition subunit of the SCF E3 ligase complex) by specific RNAi treatment or by overexpression of dominant-negative β-TrCP mutants blocked NF-κB activity and sensitized breast cancer cells to chemotherapeutic agents [99]. Recently, A20 (TNFAIP3), a cytoplasmic zinc finger protein, was shown to inhibit NF-κB activation in the TNFR and TLR pathways. The ubiquitin editing property of A20 was shown to be essential for NF-κB inhibition [100].

2.4. Blockage of NF-κB nuclear translocation

One approach for inhibiting NF-κB activation is to use small peptides that cross the cell membrane and block the nuclear translocation of the NF-κB complex [101–103]. For example, SN50, a forty-one-residue synthetic peptide that contains a hydrophobic membrane-translocating region and the nuclear localization sequence of NF-κB p50 [101], can enter cells and compete with NF-κB complexes for the machinery responsible for the nuclear translocation of NF-κB. SN50 effectively inhibits the LPS- and TNF-α-induced nuclear translocation of NF-κB in different cell lines [101, 104–107] and mitigates inflammatory responses in vivo [108, 109]. However, SN50 also blocks the nuclear translocation of a number of other transcription factors [102]. Dehydroxymethylepoxyquinomicin, a fungal epoxyquinoid that has anti-inflammatory and antitumor activity in several mouse models, has been reported to be a specific inhibitor of NF-κB nuclear translocation [110].

2.5. Blocking NF-κB activation by inhibitors of p65 acetylation

The activated p65 subunit of NF-κB undergoes acetylation in the nucleus at multiple lysine residues including K¹²², K¹²³, K²¹⁸, K²²¹ and K³¹⁰ [23, 111]. The opposing activities of histone acetyltransferases and histone deacetylases (HDACs) regulate p65 complex acetylation [24]. Acetylation of p65 also depends on coactivators such as p300 and CREB-binding protein (CBP) [112]. The K²²¹ and K³¹⁰ acetylation are associated with increased NF-κB target gene transcription [112] and are required for p65 activation [24], which is supported by the observations that SIRT driven deacetylation at K³¹⁰ inhibits NF-κB target gene transcription [113]. Additionally, K¹²² and K¹²³ acetylation reduces p65 DNA binding affinity and increases IκB interaction and nuclear export [111]. Site-specific p300-mediated p65 acetylation thus regulates the specificity of NF-κB- dependent gene expression [111, 114].

During the last 5 years, a number of compounds have been reported to inhibit NF-κB by inhibiting acetylation. Gallic acid obtained from natural products such as gallnuts, sumac, oak bark, and green tea was recently reported to possess anti-histone acetyltransferase activity, thus showing potential to downregulate NF-κB activation [115]. Daxx, a protein associated with the death domain of Fas receptor, has been reported to suppress NF-κB transcriptional activity by inhibiting p300/CBP-mediated p65 acetylation [116]. Anacardic acid derived from traditional medicinal plants can also inhibit NF-κB activation by inhibiting p65 acetylation [117].

2.6. Blocking NF-κB activation by methyltransferases

The RelA subunits of NF-κB undergo various posttranslational modifications that create specific marks to recruit different effectors to control NF-κB’s temporal and spatial activation [118]. RelA is subject to monomethylation by lysine methyltransferase Set9 (also called Set7 or KMT7) at Lys^314/315 in vitro and in vivo in response to stimuli [25]. RelA methylation at these two residues negatively regulates NF-κB function by triggering the ubiquitination and proteasome-mediated degradation of promoter-associated RelA. RelA methylation also serves as a “death” signal for the destruction of DNA-bound, activated NF-κB [25]. Because RelA subunit methylation negatively impacts NF-κB function, designing a molecule that activates Set9 function could potentiate NF-κB inhibition.

2.7. Blockage of NF-κB to DNA binding

The most direct strategy for blocking NF-κB activation is to block NF-κB from binding to specific κB sites on DNA. Some sesquiterpene lactones (SLs) have been reported to inhibit NF-κB [119] by interacting with Cys-38 in the DNA-binding loop of RelA [120, 121]. Most SLs can also inhibit DNA binding through an analogous Cys residue in the DNA-binding loops of p50 and c-Rel. Recently, a computer-based structural comparison of 103 SLs predicted that a methylene-carbonyl substructure is important for SL-based inhibition of RelA at Cys-38 [122]. Some SLs, including parthenolide, have been shown to inhibit IKKβ through the reactive Cys-179 in the kinase activation loop [34, 121]. Thus, SLs, which target both IKK activity and NF-κB subunit DNA binding [36], have multistep inhibitory activity within the NF-κB signaling pathway.

Blocking specific NF-κB-DNA binding can also be accomplished with decoy oligodeoxynucleotides (ODNs). These ODNs have κB sites and competes for NF-κB dimer binding to specific genomic promoters [123–125]. These oligonucleotides have modifications to increase their stability and their affinity for NF-κB in vivo [126–128]. Decoy ODNs have been reported to have therapeutic potential in a number of animal models of inflammation and cancer; for example, directly injecting NF-κB decoy ODNs into implanted adenocarcinoma colon 26 tumors in mice inhibited cachexia without affecting tumor growth [129].

2.8. Other mechanisms of NF-κB inhibition

2.8.1. By gene transfer

One strategy to block NF-κB activation is through the transfer of genes that code for proteins shown to suppress NF-κB activation. The most direct target is IκBα. IκBα mutation at specific phosphorylation sites (Ser³² and Ser³⁶ replaced to alanine) and ubiquitination sites (Lys²¹ and Lys²² mutated to arginine) results in a nondegradable form of IκBα. This results in a stable cytoplasmic pool of IκBα, thereby preventing NF-κB activation [130–132]. Injecting a nonphosphorylatable form of IκBα into bone marrow macrophages has been shown to inhibit osteoclastogenesis and block bone resorption [133]. Additionally, specific C-terminal serine-to-alanine mutations are sometimes included to reduce the constitutive turnover of IκBα [134]. These super-repressor forms of IκBα can still interact with NF-κB dimers to keep the dimers in the cytoplasm permanently [132, 134, 135]. Such molecules have been used succesfully to inhibit NF-κB activity and to study its role in tumor development [136, 137] and to sensitize tumor cells to apoptosis-inducing agents [134, 135]. Inhibiting NF-κB through the expression of an IκBα super-repressor (IκBαSR) has also been used to sensitize chemoresistant tumors to TNFα- and CPT-11-induced apoptosis, resulting in tumor regression [138], and to inhibit the proliferation of human head and neck carcinoma cells in vitro and in vivo [139]. However, IκBαSRs have also been shown to interact with and affect the activity of non-NF-κB pathway proteins including p53 [140], cyclin-dependent kinase 4 [141], and HDACs [142]. Furthermore, IκBαSR overexpression has been associated with the spontaneous development of squamous cell carcinoma in a murine model [143].

2.8.2. Antioxidants

Antioxidants were suggested as possible NF-κB inhibitors many years ago [144, 145]. Treatment with oxidants such as hydrogen peroxide can activate NF-κB in many cell types. In some cell types, antioxidants can inhibit the induction of NF-κB activity in response to a variety of stimuli (e.g., interleukin-1β, LPS, TNFα) [146, 147]. However, using antioxidants as NF-κB inhibitors is now regarded with increasing scepticism because the NF-κB-inhibiting properties of pyrrolidine dithiocarbamate, a thiol-containing compound, cannot be attributed to its antioxidant function but rather to its effects as an inhibitor of IκB ubiquitin ligase activity [148]. The ways in which antioxidants block NF-κB activation remain unclear, but it is likely that they act at different steps in the NF-κB pathway in different cell types. Antioxidants have been suggested to inhibit NF-κB activation by scavenging reactive oxygen intermediates that act as signaling molecules to activate the NF-κB pathway and by directly inhibiting IKK kinase activity by modifying critical Cys residues in the IKK kinase activation loop [146, 147]. Mitochondrial electron transport inhibitors that suppress reactive oxygen intermediate production (e.g., rotenone) and overexpression of antioxidizing enzymes (e.g., manganese superoxide dismutase and catalase) can block TNFα-induced NF-κB activation [149–151]. Caffeic acid phenethyl ester, a phenolic antioxidant, has been reported to cause direct interference with DNA binding by NF-κB [152] that can be reversed by dithiothreitol [78]. Other antioxidants, viz., N-acetylcysteine, calcium chelators (e.g., EGTA, lacidipine), and vitamin C and E derivatives have been reported to inhibit hydrogen peroxide- or stimulus-induced NF-κB activation.

2.8.3. Bacterial, fungal, and viral proteins

Several microorganisms and viruses encode proteins that can inhibit NF-κB activation. Many viruses have developed a number of mechanisms to inhibit NF-κB signaling [153], and three viruses—African swine fever virus (ASFV) [154], rabbit myxoma virus [155], and insect Microplitis demolitor bracovirus [156]—encode IκB-like NF-κB inhibitors. The ASFV encodes the A238L IκB-like protein, which can stably interact with RelA to inhibit TNFα-, IFN-γ-, and phorbol ester-induced NF-κB-DNA binding [157]. The poliovirus 3C protease cleaves RelA to reduce NF-κB signaling [158]. In addition, several viruses have adaptor-like or small proteins that inhibit IKK activity [153]. For example, the MC160 protein of Molluscum contagiosum [159] and the nonstructural 5B protein of the hepatitis C virus [160] appear to be IKKα-specific and thus may specifically inhibit the noncanonical NF-κB pathway.

The YopJ protein, a Src homology 2 domain protein encoded by Yersinia pseudotuberculosis, inhibits NF-κB activation by preventing the phosphorylation and degradation of IκBα [161]. YopJ has also been shown to bind directly to IKKβ in vitro and in vivo [162]. The Salmonella typhimurium AvrA protein also inhibits NF-κB activation, although its mechanism of action may be different than that of the YopJ protein [163].

Gliotoxin produced by the fungus Aspergillus fumigatus has been reported to inhibit NF-κB activation by preventing IκB degradation [164]. Several other small molecules synthesized by microorganisms or designed derivatives of such compounds that have NF-κB-inhibiting potential include panepoxydone (from Lentinus crinitus) [165], 5,6 epoxycyclohexenone compounds (from Amycolatopsis), and cycloepoxydon [166]. Such compounds may affect distinct parts of the NF-κB pathway including DNA binding, nuclear translocation, and IκBα phosphorylation and degradation.

2.8.4. Anti-inflammatory and immunosuppressive agents

Various anti-inflammatory agents including glucocorticoids, non-steroid anti-inflammatory drugs (NSAIDs), and immunosuppressants have been developed to block NF-κB activation. Glucocorticoids, which are commonly used as anti-inflammatory drugs, strongly inhibit NF-κB activation by mechanisms that are not completely understood but likely include inhibition of DNA binding, IKK activity and transactivation [167]. The glucocorticoids dexamethasone, prednisone and methylprednisolone have been reported to inhibit NF-κB activation. In addition, estrogen and selective estrogen receptor modulators (SERMs) such as raloxifene can act through the estrogen receptor to inhibit NF-κB activation [168, 169].

NSAIDs such as sodium salicylate (aspirin) and sulindac have been reported to inhibit NF-κB activation by inhibiting IκBα phosphorylation [170, 171]. At higher concentrations, aspirin has been shown to block NF-κB activity by directly binding to and inhibiting the kinase activity of IKKβ by reducing its ability to bind ATP [172]. More recently, aspirin was reported to inhibit proteasome activity [173]. As such, high-dose aspirin therapy may have applications in treating diseases in which NF-κB activity is involved, including cancer [174], diabetes [175], and heart disease [176]. Other NSAIDs such as ibuprofen and indomethacin have also been reported to inhibit NF-κB activation in cell culture [177–180].

Several well known immunosuppressants are known to target NF-κB by distinct mechanisms, some precluding NF-κB nuclear translocation [181], some through inhibiting calcineurin [182], some by binding heat-shock proteins [183] and some by modulating the DNA binding or transactivation potential of NF-κB [184–187]. Examples of immunosuppressants having inhibitory effect on NF-κB activation include cyclosporin A (CsA) [181], FK506 [188, 189], PG490 (diterpene triepoxide) [187] and deoxyspergualin [183].

2.8.5. p53 induction

It is known that p53 and NF-κB pathways play opposing roles in human cancer, with p53 acting as a tumor suppressor and NF-κB acting as a tumor activator. The crosstalk between p53 and NF-κB indicates that p53 and NF-κB repress each other’s activities owing to their competition for transcriptional coactivator proteins p300 and CBP [190]. A recent study has proposed an additional mechanism of how CBP phosphorylation by IKKα determines whether CBP binds to p53 or NF-κB [191]. Although a number of studies have focused on identifying p53 activators and NF-κB inhibitors individually, few studies have investigated the molecules that target both the pathways simultaneously. Identifying molecules that simultaneously activate p53 and inhibit NF-κB would have great potential in combination therapy for cancer and various other diseases and could provide helpful tools to better understand the crosstalk between the p53 and NF-κB pathways. Quinacrine, an antimalarial drug, and other derivatives of 9 aminoacridine have been shown to simultaneously repress NF-κB and activate p53 in renal cell carcinoma [192]. Other molecules with similar potential include nutlins [193, 194], cisplatin [195, 196], leptomycin B [197, 198], adenosine-2,3-dialdehyde [199], the NSAID JTE-522 [200], and the cyclin-dependent kinase inhibitors R-roscovitine [201, 202]; and flavopiridol [203, 204].

3. Conclusions and future perspective

NF-κB has been implicated in almost all chronic diseases, and more than 40,000 studies on NF-κB have been published with 9000 on its inhibitors. Although more than 700 different inhibitors (aspirin to IκBα super repressor) of this transcription factor have been reported, yet no NF-κB blocker has been approved for human use. Various steroids and NSAIDs have been found to block NF-κB, but their effects are highly pleiotropic. The molecules that block NF-κB activation lack specificity and thus interfere with NF-κB’s physiological roles in immunity, inflammation, and cellular homeostasis. Additionally, whether the concentrations of inhibitors used in tissue culture experiments can be applied in vivo is often unclear. Therefore, one of the major challenges facing researchers is to develop NF-κB inhibitors aimed at treating different diseases based on their ability to target specific pathways or cells, thereby avoiding the risk of undesired side effects. Future studies should also focus on validating in vitro data in vivo.