Nucleotide-Binding Oligomerization Domain (NOD) - American

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Nucleotide-Binding Oligomerization Domain (NOD) Inhibitors: A Rational Approach toward Inhibition of NOD Signaling Pathway Ž iga Jakopin*

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Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia ABSTRACT: Dysregulation of nucleotide-binding oligomerization domains 1 and 2 (NOD1 and NOD2) has been implicated in the pathology of various inflammatory disorders, rendering them and their downstream signaling proteins potential therapeutic targets. Selective inhibition of NOD1 and NOD2 signaling could be advantageous in treating many acute and chronic diseases; therefore, harnessing the full potential of NOD inhibitors is a key topic in medicinal chemistry. Although they are among the best studied NOD-like receptors (NLRs), the therapeutic potential of pharmacological modulation of NOD1 and NOD2 is largely unexplored. This review is focused on the scientific progress in the field of NOD inhibitors over the past decade, including the recently reported selective inhibitors of NOD1 and NOD2. In addition, the potential approaches to inhibition of NOD signaling as well as the advantages and disadvantages linked with inhibition of NOD signaling are discussed. Finally, the potential directions for drug discovery are also discussed.

1. INTRODUCTION Humans and other multicellular organisms are constantly challenged by an enormous diversity of pathogens, including viruses, bacteria, and fungi. These are, for the most part, neutral or beneficial to the host but may cause infectious diseases. A complex network, the innate immune system, that is able to detect and respond to such threats has evolved in all multicellular organisms, while mammals have additionally developed adaptive immunity. The initial defense response is orchestrated by evolutionarily conserved sentinels, designated pattern recognition receptors (PRRs), that are highly expressed in innate immune cells like macrophages and dendritic cells but also in epithelial cells and fibroblasts. They are located on the cell surface or in the cytosol and detect conserved pathogenassociated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN), nucleic acids, and flagellin, that are usually present in pathogens but not in the host.1 On recognition of their respective ligands, PRRs trigger signaling cascades, mediated mostly by activation of the transcription factor nuclear factor κB (NF-κB) and mitogen-associated protein kinases (MAPKs), that lead to inflammatory responses. PRR subfamilies comprise Toll-like receptors (TLRs) and the C-type lectin receptors that are membrane-bound and usually located extracellularly but also present in the intracellular vesicular compartment (in the cases of TLR3, -7, -8, and -9), while nucleotide-binding oligomerization domain (NOD) like receptors (NLRs) and RIG-I-like receptors (RLRs) monitor only the intracellular milieu.1−3 In addition, the innate immune system detects endogenous products of tissue injury (DNA, ATP, uric acid), named danger-associated molecular patterns (DAMPs), that are released in the circulation following injury or cell rupture. The innate immune system in this way maintains an appropriate homeostasis between overstimulation and suppression of host immune responses. © 2014 American Chemical Society

NLRs are cytoplasmic proteins with a characteristic tripartite domain architecture comprising a C-terminal sensor domain, a centrally located nucleotide-binding domain (NACHT), and an N-terminal effector domain. The 23 identified mammalian NLRs have been divided into subgroups based on the structural motif of an N-terminal domain that consists of caspase-recruitment domains (CARDs), pyrin domains, or baculovirus inhibitory repeats.4 NLRs are further classified into two groups, depending on their function: inflammasome and nodosome NLRs.5 NOD1 and NOD2, which belong to the nodosome subgroup of NLRs, play the main role in host defense, since they are the only ones that recognize bacterial peptidoglycan components, which leads to activation of NF-κB and MAPKs, resulting in an inflammatory response.2,3 This response, however, is under tight supervision of numerous regulators that keep it under control. Mutations in the NOD1 and NOD2 genes and ligand-elicited overactivation have both been shown to cause dysregulation of NLR function, which is linked to a variety of human diseases, including inflammatory disorders and cancer. This review is focused on the largely unexplored therapeutic potential of pharmacological modulation of NOD1 and NOD2 signaling and on the medicinal chemistry of NOD inhibitors.

2. NOD1 AND NOD2 2.1. Structure. NOD1 and NOD2 have a tripartite domain architecture composed of (i) a C-terminal sensor domain consisting of leucine-rich repeats (LRRs) that supposedly mediate the recognition of ligands, (ii) a centrally located nucleotide-binding domain NACHT that mediates self-oligomerization and activation, and (iii) N-terminal effector domains, Received: November 28, 2013 Published: April 7, 2014 6897

dx.doi.org/10.1021/jm401841p | J. Med. Chem. 2014, 57, 6897−6918

Journal of Medicinal Chemistry

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consisting of one (NOD1) or two CARD domains (NOD2) that mediate protein−protein interactions and are therefore essential for the transduction of signal (Figure 1).4

Like all NLRs, NOD1 and NOD2 belong to the AAA+ (extended ATPase associated with various cellular activities) ATPase family of proteins, where they are further classified as signal transduction ATPases with numerous domains (STAND).6−8 The AAA+ proteins have in common a structurally conserved ATPase domain that assembles into oligomeric rings and converts the chemical energy of nucleotide binding/hydrolysis into conformational changes. The ATPase domain of most AAA+ proteins consists of several characteristic and conserved features, namely, the Walker A and Walker B boxes and the Sensor 1 and Sensor 2 motifs, which are involved in nucleotide binding and hydrolysis of the β−γ phosphate diester bond leading to specific conformational changes.4,8

Figure 1. NOD protein domain architecture.

Scheme 1. Major Signaling Pathways of NOD1 and NOD2

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Scheme 2. Noncanonical Signaling Pathways of NOD1 and NOD2: (A) Antiviral Signaling, (B) Xenophagy, (C) Apoptosis, and (D) Inflammasome Formation

The Walker A box is characterized by a short GK(T/S) sequence (where the last residue is either Thr or Ser) that interacts with the phosphate moiety through bonding with the Lys residue. The Walker B box is usually characterized by a longer sequence pattern hhhhD(D/E) (h represents a hydrophobic residue).4,9 While the proximal Asp is crucial for coordinating binding of the Mg2+ cation that is required for nucleotide hydrolysis, the second acidic residue, usually Glu, primes a water molecule for the hydrolysis of ATP.6 The “extended Walker B box”, common to almost all NLRs (including NOD1 and NOD2), is a modified version of the original sequence in which the second acidic residue is missing. Instead, it consists of a conserved DE motif located three residues downstream of the first Asp and was most likely developed to functionally compensate for the missing second Asp, thus retaining the ATP hydrolysis activity.4 The Sensor 1 motif, which is common to all AAA+ proteins and consists of a conserved Arg, interacts with the γ-phosphate of ATP and coordinates nucleotide hydrolysis and conformational changes between subunits. On the other hand, the Sensor 2 motif, which is characterized by a conserved Arg/Lys and is involved in

nucleotide binding and hydrolysis, is generally missing in STAND class. In fact, it is replaced by a conserved His in the winged helix domain.4 An additional auxiliary residue GxP consists of a conserved Pro residue that interacts with the adenine moiety of the bound ATP.4,9 2.2. Signaling Pathways. Bacterial PGN motifs are supposedly recognized by the LRRs of the C-terminal domain, as supported by recent biochemical evidence. However, there is no structural data to corroborate these findings.10−12 Conversely, Mo et al. have shown that MDP binds to the central NACHT domain.13 Both NOD1 and NOD2 have been shown to bind nucleotides (ATP, ADP), the central NACHT domain, which possesses ATPase activity, being crucial for activation and oligomerization.13−15 NOD1 and NOD2 are normally kept in an inactive state, either bound to ADP or free of nucleotide, autoinhibited through intramolecular shielding by LRRs folded over the NACHT domain.13 This state is also maintained by interaction with chaperone proteins including Hsp90 and SGT1.16−18 On recognition of their cognate elicitors they undergo nucleotide exchange (ATP for ADP), causing conformational 6899



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and JNK, while it has no, or only a modest, effect on the activation of NF-κB.37 2.2.2. Noncanonical Signaling Pathways. In addition to the canonical NF-κB and MAPK pathways, NOD1 and NOD2 activate other noncanonical innate immunity mechanisms. They have been shown to induce activation of interferon regulatory factors (IRF) IRF3 and IRF7, resulting in induction of type I interferon (IFN), IFNβ, via formation of a multiprotein complex consisting of RIP2 and TRAF3 (shown in Scheme 1).38−41 In addition, they are also involved in other signaling pathways of innate immunity. Viral ssRNA is yet another PAMP that has been reported to activate NOD2, causing its translocation toward the mitochondria. There it supposedly binds to, and subsequently activates, the protein MAVS (mitochondrial antiviral signaling) through its NACHT and LRR domains, resulting in IRF3 and IRF7 activation and the induction of type I IFN response (shown in Scheme 2).40 NOD1 and NOD2 also play prominent roles in facilitating autophagosome formation via interaction with ATG16L1 (autophagy related 16-like 1), thereby enhancing xenophagy, a pivotal process leading to the clearance of pathogens, thus reducing the microbial burden (shown in Scheme 2).42 On the one hand, NOD1 and NOD2 have been shown to recruit ATG16L1 to the membrane to target bacteria at point of entry independently of RIP2.42 On the other hand, NOD2-stimulated xenophagy has been shown to be dependent on RIP2 tyrosine kinase activity which activates p38 MAPK and ERK as well as relieves the repression of xenophagy by protein phosphatase 2A (PP2A).43 In addition, a xenophagy-independent role of ATG16L1 was recently uncovered; namely, it serves as a negative regulator of NOD-driven inflammatory processes.44 Following the fusion of autophagosome with lysosome, the bacteria are degraded, yielding antigens that are then presented to T cells by MHC II molecules (except in the case of epithelial cells which typically do not express MHC II).39,42−45 NOD1 and NOD2 have also been reported to interact with the apoptotic pathway indirectly via interaction of RIP2 with the IAP family of proteins (XIAP, cIAP1, and CIAP2).29,30 Moreover, NOD1 has been shown to activate caspase 8, thereby inducing caspase-8-mediated apoptosis which has been linked to its reported antitumor actions.46 Finally, NODs have been reported to interact with certain other members of the NLR family, namely, NLRP1 (NACHT, LRR, and pyrin domain-containing protein 1) and NLRP3, thus assembling into inflammasome superstructures.47−49 Inflammasomes are signaling platforms capable of detecting a broad range of molecular signatures, including pathogenic microorganisms and sterile stressors, that activate proinflammatory cytokines IL1β and IL-18.50 Following recognition of their cognate ligand (DAMPs, asbestos, urea or cholesterol crystals), the inflammasome NLRs interact with adaptor protein ASC (apoptosisassociated specklike protein containing CARD) and procaspase 1, forming large multimeric protein complexes. This in turn leads to activation of caspase 1, which proteolytically activates the proinflammatory cytokines IL-1β and IL-18 (shown in Scheme 2).50 2.3. Regulation of the NOD Signaling Pathway. The NOD1 and NOD2 signaling cascades have to be tightly controlled because of potentially harmful effects arising from overactivation or suppression of the inflammatory response. Tipping the scale in any direction would disturb this delicate balance and could lead either to serious infectious diseases or to chronic inflammatory and autoimmune diseases. Even though

changes that lead to exposure of the NACHT domains. The binding of ATP, with its subsequent hydrolysis, drives the process of self-oligomerization that results in the activated state of NOD1/NOD2 with reduced affinity to Hsp90.15,18 Of note, the ATP hydrolysis step is also essential for deactivation of the NOD2 signaling platform, nodosome disassembly, whereas NOD1 may use a mechanism other than ATP hydrolysis for deactivation.15 In fact, the NOD1 CARD domain was recently reported to form dimers, which could be involved in a different path to formation of the signaling complex.19 These reactions are followed by migration to the plasma membrane, since it has recently been observed that the activation status of NOD1 and NOD2 in intestinal epithelial cells is also reflected by their subcellular localization. This pattern is consistent in various epithelial cell lines in which elicitor-mediated activation of NOD1 and NOD2 causes their localization to actin-rich regions at the plasma membrane (the sites of bacterial entry), which is essential for activation of NF-κB.15,20,21 These findings have been corroborated by the recent discovery that receptor-interacting protein 2 (RIP2), the downstream component of the NOD signaling platform, nodosome, is also recruited to the plasma membrane and that its activity is dependent on its subcellular localization.22 However, although plasma membrane localization is important in certain settings and may contribute to maximize efficiency, it is not essential, since signaling can also occur from other regions.23 2.2.1. Canonical NF-κB Pathway and Stress Kinases (Depicted in Scheme 1). At the molecular level, the signaling pathways downstream of NOD1 and NOD2 both converge on the signaling kinase RIP2. This serine−threonine/tyrosine kinase, with dual kinase specificity, is recruited and activated through homophilic CARD−CARD interactions, thus creating a signaling platform for subsequent downstream signal transduction.24−27 Tumor necrosis factor receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase, is then recruited to RIP2 and subsequently autoubiquitinated. The RIP2 kinase domain has also been demonstrated to bind other E3 ubiquitin ligases such as cellular inhibitors of apoptosis (cIAPs), cIAP 1 and cIAP2, which are required for K63 ubiquitination of RIP2, and X-linked IAP (XIAP). This mediates its ubiquitination, thus enabling the recruitment of linear ubiquitin assembly complex (LUBAC). LUBAC subsequently attaches ubiquitin chains to, and thus activating, the regulatory protein NF-κB essential modulator (NEMO).28−31 Poly-ubiquitination of RIP2 attracts the adapter proteins TAB1/2/3 (TAK-binding proteins 1, 2, and 3) and their associated kinase, transforming growth factor (TGF) β activated kinase 1 (TAK1).24,32,33 IκB kinase (IKK) complex is then recruited and activated, thereby assembling a multiprotein conglomerate that triggers phosphorylation of IκB, the inhibitory protein of NF-κB, causing translocation of NF-κB to the nucleus where it initiates activation of the target genes and the onset of an inflammatory response.34 NOD1 and NOD2, moreover, also activate the three main families of mitogen-activated protein kinases (MAPKs) (p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), resulting in the induction of activator protein 1 (AP-1) activity. The AP-1 transcription factor controls several processes, including cell proliferation and differentiation and apoptosis.33,35,36 Furthermore, in myeloid cells (dendritic cells, macrophages), NOD2 has been shown to interact with CARD9 adapter protein, which is required for connecting NOD2 PGN recognition function to downstream activation of MAPKs, p38, 6900



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Table 1. Regulators of NOD-Signaling Networka protein

pathway

effect

mechanism of action

ref

Rac1GTPase and β-PIX

NOD2

−

erbin CD147 vimentin FRMPD2

NOD2 NOD2 NOD2 NOD1 and NOD2

− − + +

Regulators of Cellular Localization Catalyzes association of NOD2 with the actin cytoskeleton, facilitating erbin−NOD2 interaction; β-PIX reinforces the Rac1GTPase effect on NOD2 Colocalizes with NOD2 at the membrane, preventing overwhelming activation Competes directly with RIP2 for binding to NOD2 Unclear mode of action; interacts with NOD2 at the plasma membrane Associates with the erbin−NOD2 complex; provides a spatial control mechanism for NOD2-mediated responses

CARD8 JNKBP1 NOD2-S and NOD2-C NLRC4

NOD2 NOD2 NOD2 NOD1 and NOD2 NOD2 NOD1 and NOD2 NOD1

− − − −

Regulators of Oligomerization Interacts with NOD2 NACHT domain, interfering with oligomerization; hinders membrane localization Forms a complex with NOD2, blocking NOD2 oligomerization Truncated forms of NOD2; interfere with NOD2 oligomerization Hetero-oligomerization with NOD1/2 NACHT domain, preventing downstream signaling

59 60 61, 62 65

+ +

Interaction with NOD2 NACHT domain and formation of a large complex that prevents proteasomal degradation Exerts a stabilizing function, promoting maturation

63 16, 64

+

17

−

Exerts a stabilizing function, promoting maturation Regulators of Ubiquitination Polyubiquitinates activated RIP2 and cIAP1, targeting them for deactivation/degradation

−

Interferes with XIAP−RIP2 interaction

68

−

Involved in deubiquitination of RIP2

69, 70

− − − +

K48 ubiquitinates NOD2, rendering it prone to proteasomal degradation Interacts with an active NOD2−RIP2 complex, inhibiting subsequent ubiquitination and phosphorylation Promotes K48 polyubiquitination of NOD1, rendering it prone to proteasomal degradation K63-ubiquitinates RIP2

71 72 73 24

+

K63-ubiquitinates RIP2; cIAP1 preferentially binds to RIP2; cIAP1 promotes XIAP degradation

29

+

Polyubiquitinates RIP2 (not K63!) and recruits LUBAC

30, 31

+

K63-ubiquitinates RIP2

74

−

Regulators of RIP2 Recruitment Interferes with polyubiquitination of RIP2 adapter and recruitment of RIP2 into large signaling complexes

44

−

Competes with RIP2 as a binding partner to NOD CARD domains

75

−

Combines with CARD domain and prevents formation of NOD2−RIP2

76

− − +

Binds to RIP2 in unstimulated cells and sequesters it from NOD2 Binds to various regions of NOD2, blunting the NOD2-induced NF-κB activation Mediates Tyr phosphorylation of RIP2 by Src kinase; recruited into bacterial invasion sites and interacts with NOD1

77 78 79, 80

− −

Regulators via Unknown Mechanisms Unclear mode of action Unclear mode of action

81 82

− − +

Unclear mode of action; keeps NOD2 in an inactive state Unclear mode of action Unclear mode of action

83 84 85

+

Unclear mode of action; interacts with NOD2

86

p62 Hsp90 SGT-1 ITCH SHIP-1 A20 TRIM27 TRAF4 PSMA7 TRAF2, -5, and -6 cIAP1 and -2 XIAP Pellino3

ATG16L1 ubiquitin AAMP MEKK4 RIG-I GEF-H1

PRDX4 CENTβ1 CAD PTPN22 Ankrd17 GRIM19

NOD2 (NOD1?) NOD1 and NOD2 NOD1 and NOD2 NOD2 NOD2 NOD1 NOD1 and NOD2 NOD1 and NOD2 NOD1 and NOD2 NOD1 and NOD2 NOD1 and NOD2 NOD1 and NOD2 NOD1 and NOD2 NOD2 NOD2 NOD1 and NOD2 NOD2 NOD1 and NOD2 NOD2 NOD2 NOD1 and NOD2 NOD2

52, 55 53, 54 56 57 58

66, 67

a

Abbreviations: A20, ubiquitin E3 ligase A20; AAMP, angio-associated migratory protein; Ankrd17, ankyrin repeat-containing protein; ATG16L1, autophagy related 16-like 1; β-PIX, p21-activated kinase interacting exchange factor; CAD, carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase; CARD8, caspase-recruitment domain 8; CENTβ1, centaurin β1; cIAP, cellular inhibitor of apoptosis; FRMPD2, FERM and PDZ domain-containing 2 protein; GEF-H1, guanine nucleotide exchange factor H1; GRIM-19, eukaryotic gene associated with retinoic-interferon-induced mortality 19; Hsp90, heat shock protein 90; ITCH, itchy E3 ubiquitin protein ligase; JNKBP1, c-Jun N-terminal kinase binding protein; MEKK4, mitogen-activated protein kinase kinase kinase 4; NLRC4, NOD-like receptor family CARD domain-containing protein 4; PSMA-7, proteasome subunit α type 7; PTPN22, protein tyrosine phosphatase non-receptor type 22; Rac1, Ras-related C3 botulinum toxin substrate 1; RIG-I, retinoic acid induced gene I; SGT-1, suppressor of the G2 allele of Skp1; SHIP-1, Src homology 2 domain containing inositol 5-phosphatase 1; TRAF, tumor necrosis factor receptor associated factor; TRIM27, tripartite motif containing protein 27; XIAP, X-linked inhibitor of apoptosis. 6901



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PGN of Gram-negative and some Gram-positive bacteria, is the minimal sequence necessary and sufficient for binding to NOD1.89−92 The presence of a preceding alanine in L-Ala-DGlu-meso-DAP (TriDAP) enhances this response.10 NOD2, in contrast, is a general sensor, since it recognizes and binds the MDP present in the PGN of both Gram-negative and Grampositive bacteria.11,93,94 The mechanism underlying the uptake of extracellular NOD1 and NOD2 ligands into the cytosol is still not completely clear, but several possible mechanisms have been proposed by which NOD ligands can be delivered: (i) Helicobacter pylori delivers NOD1 ligands into epithelial cells through type IV secretion systems.95 (ii) Intestinal epithelial cells and, to a minor extent, monocytes express peptide transporter PepT1, known to facilitate specifically the delivery of MDP to the cytosol,96−98 whereas PepT2 mediates the uptake of iE-DAP.99 (iii) Both MDP and M-Tri-DAP undergo clathrin-dependent endocytosis followed by ligand processing and subsequent transfer from the endosome into the cytosol. Transport takes place either out of early endosomes by SLC15A4 transporter, which enables NOD1 ligand transport, or out of mature endosomes, in which case acidification, provided by vacuolar ATPases, is required for NOD2 ligand transport.100,101 (iv) Bacterial outer membrane vesicles constitute another possible means of intracellular delivery of NOD1 ligands to epithelial cells.102,103 (v) The fact that a variety of methods of delivery (calcium shock, microinjection) and introduction of hydrophobic groups into NOD1 and NOD2 agonists strongly enhance the potency of the compounds suggests that these synthetic derivatives are capable of crossing the membrane by passive absorption.104 NOD1 and NOD2 induce NF-κB activation following recognition of bacterial components, resulting in proinflammatory cytokine and chemokine secretion, the hallmark of an inflammatory response. In addition, NODs are involved in the production and secretion of antimicrobial peptides, such as βand α-defensins, in the gastrointestinal tract. NOD1 and NOD2 are also capable of initiating another essential part of the antimicrobial response characterized by formation of reactive oxygen and nitrogen species via induction of nitric oxide synthase and NADPH oxidases.105,106 Their role in gastrointestinal tract innate immunity appears to be indispensable for intestinal homeostasis, since intestinal cells lacking TLRs are hyporesponsive to TLR ligands and rely mostly on NOD1 activity for orchestrating an appropriate immune response. Signaling through NOD1 also provides backup in cases where the cells are tolerant to MDP107 or NOD2 function is impaired.108 The NOD2 genotype has been shown to lack any influence on responses to specific NOD1 agonists, suggesting the independent action of the NOD1 and NOD2 pathways.108 Of note, the degradation of NODs is tightly controlled by the chaperone Hsp90, which dissociates from activated NODs. Moreover, NOD2 in MDP-stimulated macrophages was shown to undergo a proteasome-dependent degradation, leading to a tolerant state (to restimulation with MDP) to protect the host from septic shock.18 Several investigations of the function of NODs in host defense have demonstrated their essential role in controling bacterial infections caused by Salmonella typhimurium, Citrobacter rodentium, Listeria monocytogenes, Legionella pneumophila, Clostridium dif f icile, Helicobacter pylori, to name only a few.38,88,105 NOD1 has been reported to recognize and initiate a response against a variety of Gram-negative bacteria, while NOD2 senses both Gram-positive and Gram-negative bacteria.

the main events leading to NF-κB activation are established, the regulatory networks that control positive and negative regulation are still poorly understood. Several studies have shown that NOD1 and NOD2 interact with accessory proteins that, mostly negatively but also positively, regulate their signaling pathway, thus constituting an important part of the NOD-signaling network (listed in Table 1). The regulation of signaling occurs at different stages and is also dependent on the cell type. During the early stages of NOD signaling, regulation by the cytoskeleton takes place, since disruption of cytoskeleton was shown to potentiate the response of macrophages to NOD ligands.51 In addition, given that activation of NOD depends on the concentration of ligand at the membrane, the total activation level seems to be regulated at the level of cellular localization, especially in polarized intestinal epithelial cells.52 Moreover, the oligomerization process also represents an important step in the NOD-mediated signaling cascade and is controlled by several regulatory components, which interact with the central NACHT domain. RIP2 undergoes autophosphorylation and ubiquitination in a site-specific and time-specific manner to effect the activation and subsequent deactivation of NOD signaling. In fact, the ubiquitination of RIP2 is considered to be one of the most tightly regulated steps in NOD-signaling pathway, since RIP2 kinase domain has been shown to bind various E3 ubiquitin ligases, that facilitate the posttranslational modifications involved in protein activation. However, RIP2 interacts not only with NOD1 and NOD2 through CARD− CARD interactions but also with several other proteins; therefore, numerous CARD-containing molecules can be involved in the innate immune response. Interestingly, additional regulators of NOD signaling pathways have recently been revealed. Their mechanisms of action, however, have yet to be disclosed. Finally, a recent, comprehensive small interfering RNA (siRNA) screen has revealed several previously unknown positive and negative regulators of the NOD2 signaling pathway. Identification of the genes coding for the various classes of proteins will further increase our understanding of how this pathway is regulated.87 The negative regulation is of special importance, since NOD activation triggers many signaling pathways, including NF-κB, MAPK, and type I IFN pathways, which can result in an overwhelming inflammatory response. Mutations in the NOD2 NACHT domain, more precisely in the extended Walker B box, can cause an enhanced NF-κB activation, in contrast to the corresponding mutations in NOD1. This implies that NOD2 is more susceptible to autoactivation than NOD1 and hence needs to be more tightly controlled. The latter conclusion is corroborated by the facts that (i) many more negative regulators are reported for NOD2 than for NOD1, (ii) mutations found in NOD2 are more likely to be associated with disease, and (iii) NOD2 expression is more restricted than that of NOD1.15 2.4. Physiological Relevance of NOD1 and NOD2. The cell response to bacterial peptidoglycan is mediated largely by NOD1 and NOD2, two members of the cytosol localized receptor family, which recognize PGN breakdown products following exposure to the cytosol. NOD1 recognizes primarily Gram-negative bacteria and is expressed in epithelial, immune, and various other types of cells. The pattern of expression of NOD2 appears to be more restricted, since it is expressed mainly in professional immune cells (macrophages, dendritic cells, Paneth cells) but also in epithelial cells of the intestine and lung.88 The dipeptide D-Glu-meso-DAP (iE-DAP), present in the 6902



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NF-κB activation, while the mutation of the corresponding amino acid in NOD1 completely abolishes NF-κB activation. These findings imply that NOD2 is more susceptible to autoactivation than NOD1 and that the mutations found in NOD2 are more likely to be associated with disease. Certain NOD2 variants in the NACHT region, more precisely in the extended Walker B box, which is essential for ATP hydrolysis, have been identified in the genetic disorders Blau syndrome (BS) (three missense mutations: R334Q, R334W, L469F)122−124 and early onset sarcoidosis (EOS) (D382E, A612T).125 These diseases are characterized by granulomatous inflammation of multiple organs and have been shown to lead to gain of function, resulting in increased basal NOD2-mediated NF-κB activity and autoactivation (mostly due to prominent membrane recruitment).125 Similarly, NOD2 has also been associated with arthritis.126,127 Furthermore, a new autoinflammatory disease, designated NOD2-associated autoinflammatory disease (NAID), has recently been reported by Yao et al. In NAID, the gene mutations IVS8 + 158, R702W, and R703C are located between the NACHT and LRR regions, and the fact that one of the variants (R703C) shows increased NF-κB activation, a gainof-function mutation is indicated.128 In addition to its strong association with inflammatory diseases, NOD2 has also been linked to atopy and asthma.129−131 There is also increasing evidence for its detrimental role in certain autoimmune diseases, multiple sclerosis, and systemic lupus erythematosus.132 Inappropriate activation of NOD2 may also lead to uncontrolled proliferation of certain cell types, but the role of NOD2 mutations is still unclear.133−136 Increased incidence of colorectal cancer was observed in CD patients (mutations L1007fsinsC, R702W, and G908R),133−136 while on the other hand a protective role of NOD2 in cancer was reported.137 Genomewide association studies have also linked SNPs in NOD2 to prostate134 and endometrial135 cancer. Finally, SNPs in the NOD2 gene have been linked to increased susceptibility to infectious diseases, such as leprosy138 and tuberculosis.139,140 In spite of the similarity between NOD1 and NOD2, mutations of the NOD2 gene frequently lead to development of a disease, while correlations between NOD1 gene variants and diseases are not as common. A recent study of NOD proteins has revealed that these receptors have different modes of activation, given that a single mutation in the NACHT domain has contrasting effects on NOD1- and NOD2-mediated activation.15 NOD1 has a somewhat controversial role in the development of cancer and cancer predisposition. While certain SNPs have been shown to disturb NOD1 function and to predispose to certain types of cancer,133 a protective role of NOD1 in cancer was reported in sharp contrast.141 Of note, NOD1 has also been shown to exhibit a tumor-suppressor function.46 Moreover, an insertion−deletion polymorphism ND1 + 32656 within NOD1 has been linked to increased susceptibility to asthma and inflammatory bowel disease. This mutation caused the expression of isoforms lacking the LRRs, rendering these proteins constitutively active.142,143 Finally, NOD1 polymorphisms have also been linked to atopic diseases.144 Besides SNPs, ligand-elicited overactivation has been shown to cause dysregulation of NOD1, thus exhibiting a detrimental effect. For instance, PGN-dependent NOD1 activation has been associated with the progression of multiple sclerosis.145 Moreover, there is emerging evidence that administration of NOD1 agonists induces coronary arteritis, suggesting a role for NOD1 in cardiovascular inflammation.146−148 In addition, artificially induced NOD1 activation also contributes to the development

Recent reports have revealed that NOD1 and NOD2 are also involved in host defense against nonbacterial pathogens, such as protozoan parasites (Toxoplasma gondii, Trypanosoma cruzi) and viral infections (respiratory syncytial virus).88 Although NOD ligands are capable of triggering an inflammatory response characterized by the secretion of cytokines and chemokines, antimicrobial peptides, and reactive oxygen and nitrogen species, the response may not be strong enough. Several in vitro studies have demonstrated that NOD1 and NOD2 ligands, in synergy with TLR ligands, act on the production of TLR-mediated inflammatory cytokines, which could potentiate the cellular response against pathogens.107,109−114 This cross-talk between TLRs and NLRs is crucial for the enhancement of innate and adaptive immune responses. NOD1 and NOD2 ligands have been shown to induce Th1- and Th2-type immune responses, thus controlling the onset of an appropriate adaptive immune response.35,115 Interestingly, while NOD1 and NOD2 ligands drive Th2-type immunity, combined TLR and NOD stimulation enhances Th1type immunity.115,116 Finally, a synergistic interaction between a complement protein C5a and NOD2 has also been demonstrated in the regulation of chemokine expression, thereby promoting sepsis.117

3. NOD PROTEINS AND DISEASES 3.1. Role in Disease Etiology. Human NOD1 and NOD2 proteins are encoded by the CARD4 and CARD15 genes, respectively. Single nucleotide polymorphisms (SNPs) and mutations in the CARD4 and CARD15 genes cause dysregulation of NLR function, which has been linked to a multitude of human diseases, ranging from chronic inflammatory disorders and autoimmunity to cancer. The functions of NOD1 and NOD2 proteins can be classified either as gain of function or loss of function, depending on the activation state of NF-κB. Furthermore, the importance of the regulation of level of ubiquitination and phosphorylation must not be overlooked. Namely, the defective NOD signaling might arise not only because of mutant NODs but also because of faulty components of the NOD signaling pathway. For instance, ITCH deficiency was shown to result in insufficient clearance of activated RIP2, leading to enhanced NF-κB activation in spite of wild type NOD2.67,118 Crohn’s disease (CD) is a multifactorial inflammatory bowel disease characterized by transmural granulomatous inflammation of the gastrointestinal tract. It has been associated with three major variants of NOD2 polymorphisms in the LRR domain either by single amino acid changes, such as R702W and G908R, or by a frameshift insertion of L1007fsinsC which causes a reading frame shift and a complete loss of function.119−121 This latter mutation which results in truncated isoform also prevents NOD2 from localizing to plasma membrane because of deletion of the terminal LRRs.20 Several other less common variants have also been shown to be more frequent in CD patients; however, the majority of patients who develop CD actually have wild type NOD2, which indicates that in these cases the defect might lie in other components of NOD2 signaling pathway (e.g., lower expression of E3 ubiquitin ligases, mutated ATG16L1).67 The function of the extended Walker B box residues in NOD1 and NOD2 signaling is fundamentally different. On the one hand, the mutations of the first acidic residue of the extended Walker B box of both NOD1 and NOD2 lead to a complete loss of NF-κB activation.15 In sharp contrast, mutation of the second acidic residue in NOD2 results in a significantly increased basal 6903



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Figure 2. NOD inhibitors of natural and endogenous origin.

of ocular inflammation149 and fetal inflammation.150 Finally, NOD1 activation by NOD1 ligands has been shown to stimulate insulin resistance and adipose tissue inflammation in doses that elicit only minor changes in cytokine levels.151,152 3.2. Therapeutic Potential of NOD1 and NOD2 Inhibition. There are a number of potential targets for NOD1 and NOD2 inhibition within the innate immune system; however, they need to be exploited with care, since the system is a major coordinating node of host defense, inflammation, and apoptosis. Targeted and controlled modulation of the innate immune response have great potential for the treatment of chronic inflammatory and autoimmune diseases characterized by increased NF-κB activation. Dysregulation of NLRs is implicated in the pathology of various inflammatory disorders. Given that therapeutic intervention of NLR dysfunction is currently only possible by the administration of strong, broad-range antiinflammatory drugs, which often display severe side effects, specific modulators of NLR signaling would be beneficial. NOD1 and NOD2 are of particular interest, since they activate the NFκB pathway directly, and inhibition of NOD1 and NOD2 signaling could be advantageous in treating many acute and chronic diseases in which suppression of the proinflammatory response would be beneficial.153−155 NOD2 is mostly recognized for its involvement in the pathogenesis of CD, which is associated with a loss-of-function mutation resulting in loss of downstream NOD2 signaling. However, some NOD2-associated diseases are caused by mutations in the NOD2 gene that lead to gain-of-function constitutive activation (BS and EOS) or by hyperactivity of wild type NOD2. Given that hyperactivity of NOD2 is implicated in the pathophysiology of these disorders, inhibition of NOD2 and the components of its signaling pathway would appear to be a logical therapeutic strategy. Current therapy for NOD2associated diseases consists of potent anti-inflammatory medicines (corticosteroids) and biologicals (TNF-α inhibitors and IL-1 receptor antagonists). Although the use of TNF-α inhibitors is usually effective in treating CD, there is no correlation between the response and NOD2 variants. TNF-α inhibitors are effective in alleviating BS symptoms.128 Although SNPs in NOD2 gene increase susceptibility to leprosy and tuberculosis, this resulted from a loss-of-function mutation, so inhibition of NOD2 signaling does not appear to be a viable strategy. Similarly, the NOD2 mutation, which has been shown to predispose to cancer, also exhibited loss of function.136 PGN has been shown to play a role in the development of sepsis, a systemic inflammatory response. Although NOD1 and NOD2 agonists are only weak inducers of inflammatory

responses, this activity can be amplified significantly by the synergistic crosstalk between NOD receptors and other PRRs.112−114,117 In addition, signals of endogenous damage are also presumed to be capable of activating NOD1 and NOD2. Thus, therapeutic inhibition of NOD1 and NOD2 could be useful for treating (cardio)vascular complications arising from both infectious- and noninfectious-related multiorgan failure.146−148 Saturated fatty acids have been shown to act as agonists of NOD1 and NOD2 receptors.156 In the setting of a high-fat diet, human intestinal cells can be exposed to high concentrations of fatty acids, which may cause excessive inflammation. Intestinal mast cells are activated by orally administered PGN, which in turn causes diarrhea. This effect has been inhibited by blocking the NOD1 signaling, again implying potential therapeutic possibilities.157 Thus, NOD1 inhibition is thought to posit an interesting alternative in treating the excessive inflammation associated with cardiovascular, intestinal, and metabolic diseases.153 Finally, certain types of cancer have been associated with NOD1 mutations. However, given their role in sensing potentially carcinogenic infectious agents (Helicobacter pylori, Escherichia coli, Shigella f lexneri) and the antitumor effect of NOD1 activation, NOD1 inhibitors could only exacerbate the progress of the disease. Mechanistically, suppressing NOD1 or NOD2 activity should lead to impaired host defense, which raises the main issue of concern, that of safety. However, the fact that pharmacological agents only reduce, and rarely completely inhibit, the activity of their targets reinforces the suggestion that inhibition of NOD signaling could be employed safely. Targeting the upstream pathways responsible for NF-κB signaling would be expected to leave most innate immunity defense mechanisms intact, which could be beneficial for diseases in which the specific target is involved in the disease pathogenesis.38 It has been speculated that brief cycles of therapy with NOD1 or NOD2 inhibitors (administered concomitantly with antibiotics) could reduce inflammation and help to re-establish immune homeostasis.38 In considering therapeutic targeting of NOD receptors with inhibitors, it is important to take into account the extensive crosstalk between NOD receptors and other PRRs, as exemplified by synergistic interaction with TLRs.153 Finally, small molecule inhibitors would also provide useful pharmacological tools for elucidating the roles of these proteins in acute and chronic inflammatory diseases, as well as in normal hostdefense mechanisms. Harnessing the full potential of NOD inhibitors is thus a “hot topic” in medicinal chemistry. 6904



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Figure 3. SAR of indoline scaffold-based NOD inhibitors.

4. INHIBITORS OF NOD1 AND NOD2 SIGNALING 4.1. Natural Inhibitors of NOD1/2 Signaling. Numerous natural compounds possess anti-inflammatory properties. Curcumin (1, Figure 2), a polyphenol present in the plant Curcuma longa, has long been known as a potent antiinflammatory agent because of its inhibition of various signaling pathways (TNF-α, TLR4), which in turn leads to a general inhibition of NF-κB activation. It has recently been shown to suppress not only MDP-induced but also lauric acid induced NOD2 signaling, resulting in the suppression of NF-κB activation and IL-8 expression in a dose-dependent manner. Although no conclusive evidence for a specific effect of 1 on NOD2 has been provided, it has been speculated that it acts by interfering with NOD2 oligomerization. In addition, 1 has also been shown to inhibit NOD1-induced NF-κB activation.158 Furthermore, parthenolide (2), a representative sesquiterpene lactone, has also been shown to exert anti-inflammatory effects via down-regulation of MDP-induced and lauric acid induced NOD2 signaling, which leads to suppression of NF-κB activation and IL-8 expression.158 The latter effect has also been ascribed to the inhibition of ligand-dependent NOD2 oligomerization. Strikingly, 1 and 2 inhibited not only the activation of NOD2 signaling induced by a ligand of bacterial origin (MDP) but also its activation induced by an endogenous ligand, lauric acid.158 It is noteworthy that both compounds also inhibited NF-κB activation induced by NOD2 overexpression. The mechanism by which they inhibit NOD2 oligomerization is unknown; however, it has been proposed that their structures contain reactive centers, in the form of an α-methylene-γ-lactone group or an α,β-unsaturated carbonyl group, that enable reaction with biological nucleophiles such as the sulfhydryl groups present in the NACHT domains of NLRs and p65 subunit of NF-κB.159 2 has also been shown to interfere with the ATPase activity of NLRP3, thereby hindering NLRP3 inflammasome activation.160 Similar inhibitory effects on NF-κB activation were also observed for helenalin (3) and several other sesquiterpene lactones; however, with the exception of 3, their mechanism of its action was not elucidated.158,159,161,162 3 has been shown to alkylate and thus inhibit the p65 subunit of NF-κB. Importantly, the IC50 values reported for inhibition by NF-κB were not influenced by the cytotoxicity of the compounds. Of note, it has also been suggested that pseudopterosins such as pseudopterosin A (4),

diterpenoid glycosides of marine origin, target the NOD receptors, since their anti-inflammatory actions are not due to the inhibition of LPS-induced inflammatory responses.163,164 4.2. Endogenous Inhibitors of NOD1/2 Signaling. NOD proteins can also be modulated by certain endogenous molecules, namely, dietary fatty acids. While saturated fatty acids, such as lauric acid, induce the activation of NOD1/2 signaling, some unsaturated fatty acids, particularly the n − 3 polyunsaturated type, exert inhibitory effects on this signaling pathway.156 Among the polyunsaturated fatty acids, docosahexaenoic acid (DHA) (5) and eicosapentaenoic acid (EPA) (6) exert the most prominent NOD1/2 inhibitory effects, although this suppression of NF-κB activation can, to a greater extent, be ascribed to their action on the NOD2 signaling pathway. They have been shown to prevent the self-oligomerization process of NOD2, resulting in inhibition of downstream signaling, seen as suppression of NF-κB activation and IL-8 expression.156 However, it has to be taken into account that fatty acids, in addition to NOD signaling, can also inhibit several other signaling pathways. Further, the inhibitory effects of 5 and 6 on NOD activation could also be ascribed to their enzymatic metabolites. However, these results underline the important role of dietary fatty acids in inflammatory processes. 4.3. Inhibitors of NOD1/2 Signaling from the Molecular Libraries Network Initiative. In the search for small molecule NOD1 inhibitors, an exhaustive high-throughput screening (HTS) program was conducted under the auspices of the NIH-sponsored Molecular Libraries Probe Production Center Network. Several active scaffolds with IC50 values less than 10 μM were identified from a library of 290 000 compounds from the NIH Molecular Libraries Small Molecule Repository compound collection, using cell-based NF-κB driven luciferase reporter gene activity as a measure of NOD1 modulation. The main goal was to identify a compound with IC50 < 1 μM and selectivity against NOD2 and TNF-α. Secondary function assays were performed in order to confirm that such compounds inhibit IL-8, a biologically relevant downstream target of NOD1 pathway. The indoline scaffold, the tetrahydroisoquinoline scaffold, and the benzimidazole scaffold met the initial screening criteria of (i) inhibition of NOD1 with IC50 < 1 μM, (ii) inhibition of NOD1-induced IL-8 secretion with IC50 < 1 μM, and (iii) a target selectivity over NOD2 of 5× and over TNF-α of 10×.165,166 6905



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Figure 4. SAR of tetrahydroisoquinoline scaffold-based NOD inhibitors.

Figure 5. SAR of 2-aminobenzimidazole scaffold-based selective NOD1 inhibitors.

to complete loss of activity. The stereoelectronic influence of different electron-deficient and electron-rich functionalities was determined by varying the substituent R, and electron-donating groups were found to augment the activity of the compounds.165,166 The compounds present in both the indoline and tetrahydroquinoline scaffold series exhibited nonselective inhibitory activity, inhibiting not only NOD1- but also NOD2mediated and TNF-α-mediated NF-κB activation. SAR analysis of the benzimidazole series (representative compounds 13−15, Figure 5) revealed that the presence of an NH2 group at 2-position is essential, since its replacement (with SH, Me, or H) rendered the compounds inactive. Moreover, replacing the hydrogens at R1 and R2 with methyl groups completely abrogated the activity. In addition, variation of the type of spacer X (e.g., sulfonyl, carbonyl, methylene) demonstrated that the sulfonyl group provides the most active compounds of the series and is also essential for their selectivity. Finally, the stereoelectronic nature of the substituent at R3 considerably affected the bioactivity. Compound 14, with the 4-chloro substituent on the aromatic ring, was 6-fold more potent than the hit compound 13, while other substituents reduced the potency.165,166 Compound 13, also designated noditinib 1, was found to be noncytotoxic and to inhibit selectively NOD1-dependent NF-κB activation (IC50 < 1 μM). It exhibited >36-fold selectivity against NOD2-dependent and TNF-α-dependent NF-κB activation,

The indoline scaffold was rationally modified at three different sites R1, R2, and R3 to investigate the structure−activity relationship (SAR). Numerous analogues were prepared in order to determine the effect of the size of the aliphatic group at R2, the stereoelectronic influence of different electron-deficient and electron-rich functionalities at R3, and the effect of the indoline substitution at R1 (representative compounds 7−9 are shown in Figure 3). The bioactivity of these compounds is dependent on the nature of the substituent R1; replacement of the methyl group with hydrogen resulted in complete loss of activity. Small alkyl groups such as cyclopropyl, methyl, and ethyl are tolerated at the R2 position. Significantly, the effect of substituent R2 depends on the electronic nature of R3. In general, electron-withdrawing groups (NO2, COMe) at R3 increase the activity of compounds, while neutral or electron-donating groups result in decreased activity. Unfortunately, none of the compounds from this series exerted selectivity toward NOD1 and NOD2; therefore, this scaffold was abandoned.165,166 The tetrahydroisoquinoline scaffold incorporating series was modified at three sites (representative compounds 10−12 are shown in Figure 4). The tetrahydroisoquinoline moiety was found to be essential for bioactivity, since its replacement with other heterocycles (e.g., piperidine, indoline) resulted in complete loss of activity. Having the CONH group as the spacer separating the two phenyl rings was mandatory for bioactivity, since replacement with its alternative, the retroamide NHCO, led 6906



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Figure 6. SAR of purine scaffold-based selective NOD1 inhibitors.

Figure 7. SAR of benzimidazole diamides as selective NOD2 inhibitors.

conformational change in NOD1, increase its concentration in the membrane fraction, and at the same time, reduce the concentration of RIP2 at the membrane.167 During the same attempts to identify small molecule NOD1 inhibitors employing the HTS campaign, a compound of another chemical class, purine-2,6-diones (a xanthine), was identified as an active and NOD1-selective inhibitor (compound 16 (ML146)). Several compounds of this class were synthesized to gain more insight into SAR (representative compounds 16− 19 shown in Figure 6).168

thus meeting all the required criteria. Moreover, it showed excellent selectivity against more than 400 different kinases.165,166 The secondary assay confirmed the functional activity of 13 by inhibiting NOD1-dependent IL-8 secretion from cells. The 2aminobenzimidazole scaffold based series is thus the first class of NOD1-selective inhibitors to be identified.165 The mechanism of its action was investigated using 1H NMR spectroscopy with purified NOD1 protein and 13. The latter was found to interact directly with NOD1 protein, while ATP binding was not affected. Further experiments suggest that the compound may induce a 6907



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Figure 8. SAR of hydrophenalene−chromium complexes as NOD2 inhibitors.

The nature of the substituent R1 has a profound effect on the bioactivity of compounds that was influenced not only by the number of carbons and the branching pattern but also by the degree of unsaturation of substituent R1. The presence of a thioalkyl group (compounds 16−18) as the substituent R2 on the xanthine ring was essential for NOD1 versus NOD2 selectivity. Replacing it by NO2 (compound 19) enhanced the activity but resulted in complete loss of selectivity. Hydrogen, as a R3 substituent, provided the most active compounds of the series and its replacement by larger groups (e.g., methyl) resulted in complete loss of selectivity.168 The physicochemical properties of the identified probes of different class type are consistent with those of druglike molecules. The calculated log P values were 3.2 (13) and 3.5 (16). The polar surface areas ranged from 67 Å2 (16) to 86 Å2 (13), and both compounds contained fewer than five H-bond donors and acceptors. Their pharmacological properties were evaluated using in vitro ADMET (absorption, distribution, metabolism, excretion, and toxicity) assays. While 13 was moderately soluble at 5.9−7 μM, 16 was the least soluble (1.5−1.8 μM) across the whole pH range (5−7.4). Both probes exhibited good in vitro cell permeability using PAMPA (parallel artificial membrane permeability assay). Their metabolic stability was very low in the presence of murine microsomes (0.8% remained after 1 h of exposure) but less so in the presence of human hepatic microsomes. Finally, none of the compounds, at 50 μM concentration, were cytotoxic to immortalized human hepatocytes.169 4.4. Inhibitors of NOD2 Signaling Developed at GlaxoSmithKline. The HTS screening campaign to find NOD2-selective small molecule inhibitors has also been conducted within the molecular libraries network but without success. By use of a cell-based HTS approach, namely, MDPinduced IL-8 release in HEK293 cells, with approximately 1.9 million compounds, a benzimidazole diamide designated GSK669 (20) (IC50 = 3.3 μM) was identified as a selective inhibitor capable of diminishing MDP-induced IL-8 release without affecting the function of RIP2 kinase.170

Even more potent and selective NOD2 inhibitors were developed using a rational SAR approach (representative compounds 20−26 are shown in Figure 7). Varying the R1 substituent proved to be the most tolerant of alterations in terms of retaining bioactivity. The dihydroindane moiety in compound 20 was successfully replaced by many groups (e.g., 3-isopropylphenyl (compound 21) and naphthyl (compound 22)), resulting in compounds more potent than the lead. However, replacement of R2 proved to be a less appropriate modification, since it only allowed for minor alterations such as ortho-substitutions on the phenyl ring or its replacement by a naphthyl ring (compound 24). Introduction of a chlorine atom at position 5 or 6 (R3) of the benzimidazole ring (e.g., compound 23) enhanced the inhibitory activity, while other changes were not tolerated. The mono- and dimethylation of amides (R4, R5) brought about significant changes in activity. While the methyl group introduced at position R4 significantly enhanced the activity of the parent compound (GSK717 (25); IC50 = 0.4 μM), methylation of the amide functionality at R5 or dimethylation (R4 = R5 = Me) of amides resulted in loss of activity. The final set of compounds, combining the most promising fragments, showed no significant improvement, the most potent inhibitor being compound 26 (IC50 = 0.2 μM). The mode of action of the benzimidazole diamide class of NOD2-inhibitors was then subjected to in-depth investigation. These inhibitors were shown not to interfere with the cellular uptake of MDP. Moreover, they inhibited dose-dependently the release of cytokines from MDP-stimulated primary human monocytes. Their inhibitory activity was inversely related to MDP concentration, and further exploration revealed a competitive interaction between MDP and 25 by binding to the same site on NOD2 protein. Interestingly, neither ssRNAelicited nor NOD2-overexpression-elicited NOD2 activation was blocked by 20. This inability of 20 to block the response to ssRNA could mean that it acts on other proteins of the NOD2 signaling complex. Most importantly, the inhibitors proved to be specific and selective; they failed to inhibit the activation of TLR2-, TNFR1-, and NOD1-mediated pathways (>30 μM), which share components with NOD2 downstream of TAB/ 6908



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Scheme 3. Depiction of NOD1/NOD2 Primary HTS Inhibition Assay

inhibitory effect of this compound, the authors speculated that it acts at the level of NOD2 in a competitive manner. The nondruglike properties of organochromium compounds could, however, limit their use in vivo. Further, selectivity against NOD1 was not tested, so the compounds cannot be regarded as NOD2-selective inhibitors.164,171 4.6. Overview of NOD1/NOD2 Inhibition Assays. Several assays were developed in order to evaluate the compounds for their potential inhibitory activity. HEK293T cells express low levels of endogenous NOD1 and NOD2; however, they do not express TLR2, -4, and -8 and therefore do not respond to stimulation with other classical bacterial PAMPs, in particular to contaminations with LPS. The primary HTS assay employing NOD1-overexpressing HEK293T cells transfected with NF-κBdriven luciferase reporter gene was described, which measures luciferase activity upon exposure to compounds and the subsequent stimulation with TriDAP as a measure of modulation of NOD1 activity. An analogous HTS luciferase reporter assay employing NOD2-overexpressing HEK293T cells measuring modulation of NOD2-dependent activation upon stimulation with MDP was also developed. Both assays (depicted in Scheme 3) can also be used as the initial counterscreen when searching for either NOD1- or NOD2-specific inhibitors.165,166,168 Moreover, an adaptation of the classical luciferase reporter gene assay employing an NF-κB reporter plasmid and a constitutive β-galactosidase plasmid has recently been reported to measure NOD1- and NOD2-mediated NF-κB activation. The β-galactosidase reporter assay allows for variations in the

TAK1. In addition, these compounds did not inhibit the activity of more than 300 kinases, including RIP2 kinase.170 4.5. Pseudopterosin-Based Inhibitors of NOD2 Signaling. Arenechromium diterpene complexes, based on the structure of pseudopterosins, have been reported as specific inhibitors of NOD2-mediated NF-κB signaling.164 When screened for their influence on TLR2-, TLR4-, and TNFmediated NF-κB activation, they appear to be selective for NODmediated inflammatory pathways. They exhibit certain structural requirements for the specific inhibition of NOD2 signaling (representative compounds 27−29 are shown in Figure 8). The benzylic substituent at position C3 and the lipophilic side chain at position C7 are very important for both specificity and optimal activity on NOD2. It is noteworthy that the benzylamines are considerably more active than their ether congeners. Moreover, bulky side chains at position C7 are not tolerated, whereas compounds with a small 2-methyl-2-propenyl, allyl, or propargyl side chain exhibit pronounced inhibition of NODsignaling. On the other hand, the stereochemistry at the benzylic position was found not to be essential for NOD2 specificity but appeared to affect the activity. Compounds lacking the benzyl group blocked all pathways tested. Strikingly, the presence of the Cr(CO)3 moiety significantly affected the cytotoxicity, since the decomplexed ligand exhibited a pronounced cytotoxic effect.164,171 Compound 27 was the most potent and specific inhibitor of the series, with IC50 of 5−15 μM, depending on the cell line. Since increased concentrations of MDP diminished the 6909



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Scheme 4. Potential Target Sites for Inhibition of NOD Signaling

unexplored, raising several questions. Can other downstream targets also be exploited for clinical gain? Would global inhibition be preferred over inhibition of a specific downstream effector? Deeper insights into the underlying mechanisms of NOD activation and its regulation are essential for understanding NOD-related diseases and for the design of pharmacological agents targeting NOD signaling (potential target sites are shown in Scheme 4). 5.1. Targeting the Ligand Binding. NOD1 and NOD2 ligands bind to the LRR domains of these proteins,10−12 but no crystal structure of ligand-bound LRR is available, making rational structure-based design impossible, although homology models of NOD1 and NOD2 have been published.4 The recently discovered selective inhibitors of NOD2 signaling of the benzimidazole diamide and hydrophenalene structural classes compete with MDP for binding to NOD2, suggesting that they bind to the same site, while the first selective NOD1 inhibitor, 13, has been shown to bind to NOD1 and alter its conformation and subcellular localization. Competition experiments with TriDAP have not been performed, although the inability of ligand-bound NOD1 to migrate to the plasma membrane suggests that 13 binds to the LRR domain, which is responsible for the membrane interaction of NOD1. Given that most NOD-related diseases are not caused by excessive ligand concentration but usually by mutations in genes coding for NOD1 and NOD2 that lead to constitutive activation, targeting of ligand binding would appear to be pointless. In fact, the most rational target has to be located downstream of ligand binding. 5.2. Targeting the ATP-Binding Site. There are many successful examples in medicinal chemistry of how to target nucleotide-binding domains of proteins by small-molecule drugs, so it is reasonable to assume that the NOD proteins could

transfection efficiency and to monitor cell death (eventually induced by experimental settings).172 Both reporter assay methods use transfected HEK293T cells overexpressing NOD1 or NOD2, which yield a robust readout; thus, the methods are well suited as HTS primary assay. Of note, Grimes et al. developed a different surface plasmon resonance-based assay to screen for inhibitors/activators of the NOD2 signaling pathway using recombinant NOD2.11 In terms of functional characterization of the compounds, secondary assays were developed to measure their effect on the authentic downstream effect of NF-κB activation, namely, the production of IL-8 in TriDAP- or MDP-stimulated human breast cancer epithelial cell line MCF-7 overexpressing NOD1 or NOD2, respectively.165,168 In addition, colonic epithelial HCT116 cells express both NOD1 and NOD2 and respond to their activation with increased NF-κB activity and secretion of IL8 but do not respond to agonists of TLR2 and TLR4 receptors and therefore also represent a suitable model for the functional characterization of NOD1 as well as NOD2 ligands.156,158,170 Finally, assays for specificity of the inhibitors against activation of NF-κB through other non-NOD pathways in the NF-κB driven luciferase reporter cell line were also developed (e.g., TNF-α assay).165,166,168

5. RATIONAL TARGETING OF THE NOD SIGNALING PATHWAY The NLR signaling pathway is one of a multitude of pathways that converge on the IKK complex and activate the transcription factor NF-κB. NOD1 and NOD2 are among the best studied NLR family proteins, but the therapeutic potential of pharmacological modulation of NOD1 and NOD2 is largely 6910



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Figure 9. Inhibitors of NOD signaling that function via targets other than NOD receptors.

they have exhibited a significant in vitro efficiency in alleviating the exacerbated cytokine responses in NOD2 hyperactive states (primary macrophages that lack ITCH and macrophage cell lines overexpressing the BS R334Q mutation).175 Further downstream is another kinase, TAK1, involved in NOD signaling, which is an interesting target, since several TAK1 inhibitors (e.g., (5Z)-7-oxozeaenol (34)) have already been reported.33 However, this inhibition would be highly nonselective, since both NLR and TLR pathways converge on TAK1, which makes it an inappropriate target for NOD related diseases. The compounds that exhibit nonselective activity and inhibit not only the NOD1 but also NOD2- and TNFα-mediated NF-κB activation may hold great potential for use as general antiinflammatory agents, as dual or even multiple inhibitors in the low micromolar range. 5.5. Targeting the Regulatory Network of Signal Transduction. Activation and deactivation of NOD signaling are tightly controlled by posttranslational modifications, in particular by ubiquitination and phosphorylation of RIP2 in a site-specific and time-specific manner. Hence, NOD-associated hyperactive states could also be treated by targeting the ubiquitin-regulated signaling network.67,176 Numerous small molecule inhibitors of both cIAPs and XIAP, such as SM-122 (35), are currently in clinical trials as anticancer agents.177 Since they correct excessive NOD responses in vitro, they could also be examined for down-regulation of NOD signaling.67,176 In fact, cIAP1 inhibitor has been shown as efficacious in correcting the excessive cytokine responses in NOD2 hyperactive disease models.67 However, even if these inhibitors were repurposed for down-regulation of NOD2 signaling, they would represent a lessspecific alternative to NOD2 inhibitors.176 Numerous proteins are known as positive and negative regulators of NOD signaling, but none are selective pharmacologic targets for modulation of NOD function, since they play multiple roles in the cell machinery. Two strategies could be employed to inhibit NOD signaling: activation of negative regulators or suppression of positive regulators. In general, it is easier to design protein inhibitors, which makes the suppression of positive regulators a viable therapeutic strategy. However, the TRAF4 binding motif in the structure of NOD2, composed of two consecutive glutamate residues Glu279 and Glu280, could

similarly be targeted. Small-molecule inhibitors targeting the ATP binding pocket of the catalytic domain of kinases have potential to become drugs devoid of major side effects; however, one of the most important challenges is the optimization of selectivity.173 In order to become activated, both NOD1 and NOD2 have to bind ATP.13 Nucleotide binding and hydrolysis are both important in the activation and deactivation of signaling.15 The ATP binding site is thus an interesting target, since mutations predicted to disrupt ATP binding have also prevented activation of NOD signaling.13,15 However, one has to take into consideration that when mutated, ATP hydrolysis but not ATP binding is usually abolished.9 This suggests that small molecules targeting nucleotide binding or hydrolysis specifically could have both inhibiting and activating effects. The mechanism of action of 16, a representative of the second known structural class of NOD1 inhibitors, purine-2,6-diones, has not been described. Considering its similarity to nucleotides, it could be speculated that it binds to the nucleotide-binding domain of NOD1. 5.3. Targeting the NOD Self-Oligomerization. Several natural products (curcumin, parthenolide) have been reported to inhibit NOD oligomerization, supposedly by preventing the formation of the disulfide bonds joining NACHT domains.158 However, they also act via unrelated mechanisms and inhibit several other inflammatory pathways. As opposed to lauric acid, which induces NOD signaling, the polyunsaturated fatty acids DHA and EPA have been strikingly demonstrated to inhibit this process by interfering with NOD oligomerization.156 5.4. Targeting the RIP2 Recruitment/RIP2 Kinase Activity. The signaling pathways downstream of NOD1 and NOD2 oligomerization converge on the signaling kinase RIP2, which is an interesting modulator of synergistic and inhibitory interactions between the two receptors and is a viable target for pharmacological intervention with RIP2 selective inhibitors, such as SB203580 (31, Figure 9).33,153,174 A recent study has reported the unexpected finding of RIP2 tyrosine kinase activity. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib (32) and erlotinib (33), have been shown to inhibit both RIP2 tyrosine phosphorylation and MDPinduced cytokine release at nanomolar concentrations, indicating their possible use in treating inflammatory disorders.175 In fact, 6911



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What is more, there is structural data that indicate that NOD1 CARD domains form dimers.19 This CARD−CARD interaction surface might also be exploited in terms of achieving NOD1 inhibition. Apart from medicinal chemistry, much remains to be done. Further in vitro as well as in vivo disease model experiments are urgently needed to investigate and establish their clinical usefulness, since little is known for certain about the actual efficacy of NOD inhibitors in NOD-hyperactive states. In conclusion, there is a strong basis for suggesting that NOD1 and NOD2 inhibitors could have an important therapeutic role. Moreover, these inhibitors can be used as pharmacological probes in research to define their role in disease and in normal host-defense mechanisms. Harnessing the full potential of NOD inhibitors certainly constitutes a “hot topic” in medicinal chemistry.

also be targeted. TRAF4 acts as a negative regulator of NOD2; hence, a rationally designed TRAF4 agonistic peptidomimetic might provide an opportunity to selectively inhibit NOD2.72 Further insight into the regulation of NOD membrane trafficking may provide novel therapeutic strategies. Vimentin acts as a positive regulator of NOD2 signaling by supervising plasma membrane trafficking. Withaferin A (36), a steroidal lactone, can inhibit vimentin and thus also selectively inhibit NOD2 signaling.57 Vimentin thus represents a potential selective therapeutic target for treating NOD2-hyperactive states. Moreover, Hsp90 has been shown to positively regulate NOD1 and NOD2. It has been shown to maintain NOD2 stability, since its inhibition led to the proteasomal degradation of NOD2.18 However, it plays a major role in many other cellular processes and hence may not be suitable as a target, although numerous Hsp90 inhibitors such as geldanamycin (37) exist and have been designed as anticancer agents, which are currently in clinical trials. This target also lacks NOD1 versus NOD2 selectivity.16,178 Finally, proto-oncogene tyrosine-protein kinase (Src) has been shown to phosphorylate RIP2 in the presence of GEF-H1 which acts as an intersection between Src kinase function and NLR signaling. Several selective Src kinase inhibitors, such as saracatinib (38), are already known and could be used to treat NOD2-hyperactive states.79,179

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AUTHOR INFORMATION

Corresponding Author

*Phone: +386 1 4769 574. Fax: + 386 1 4258 031. E-mail: ziga. jakopin@ffa.uni-lj.si. Notes

The authors declare no competing financial interest. Biography Ž iga Jakopin received his degree in Pharmacy in 2005 and his Ph.D. in Pharmaceutical Sciences in 2010 from the University of Ljubljana (Faculty of Pharmacy, Ljubljana, Slovenia). At present he is an Assistant Professor of Medicinal Chemistry at the Faculty of Pharmacy, University of Ljubljana. His main research interests concern design and synthesis of peptidomimetics and immunomodulatory compounds. His research focus at present is on the development of new ligands (agonists, inhibitors) of NOD receptors.

6. FUTURE DIRECTIONS AND CONCLUSIONS A number of potential targets reside within the innate immune system, a major junction of host defense, inflammation, and apoptosis. Innate immunity is characterized by complex networks, rather than simple linear pathways, of interacting proteins that are involved in its regulation, so side effects of compounds influencing innate immunity pathways have to be anticipated. The target has to be selected to minimize the possibility of side effects, and to enhance the overall safety of this approach, the compounds were designed to specifically and selectively bind to that target. With this in mind, targeted modulation of the innate immune response holds great potential for the treatment of chronic inflammatory and autoimmune diseases, characterized by increased NF-κB activation. Dysregulation of NLRs is implicated in the pathology of various inflammatory disorders, and since therapeutic intervention of NLR dysfunction is currently only possible by the administration of strong broad-range anti-inflammatory drugs, which often display severe side effects, new, specific modulators of NLR signaling are essential. However, suppressing the NOD1 or NOD2 activity is likely to impair host defense, raising the question of safety. Targeting the upstream pathways responsible for NF-κB signaling is expected to leave most innate immunity defense mechanisms intact while still being beneficial for diseases in which the specific target is involved in the disease pathogenesis.38 It is also worth mentioning that several anticancer agents, including EGFR, Src, and Hsp90 inhibitors, which incidentally all belong to the class of ATP-competitive kinase inhibitors, have shown potential in correcting excessive NOD2 responses; therefore, they could be repurposed as inhibitors of NOD2 signaling. Novel and selective NOD inhibitors, however, are urgently needed. Two distinct chemical classes of selective NOD1 inhibitors, 2-aminobenzimidazole and purine-2,6-dione, have already been identified.165−168 However, only the benzimidazole diamides have so far been established as selective NOD2 inhibitors.170 More insight into the structures of NOD1 and NOD2 is needed to pave ways for the development of inhibitors.

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ACKNOWLEDGMENTS This work was supported financially by the Slovenian Research Agency (Grant P1-0208). The author thanks Dr. Roger Pain for critical reading of the manuscript. The author apologizes to the authors of important contributions, which have been left out because of space constraints.

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ABBREVIATIONS USED A20, ubiquitin E3 ligase A20; AAA+, extended ATPase associated with various cellular activities; AAMP, angioassociated migratory protein; ADMET, absorption, distribution, metabolism, excretion, and toxicity; Ankrd17, ankyrin repeatcontaining protein; AP-1, activator protein 1; ASC, apoptosisassociated specklike protein containing caspase-recruitment domain; ATG16L1, autophagy related 16-like 1; β-PIX, p21activated kinase interacting exchange factor; BS, Blau syndrome; CAD, carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase; CARD, caspase-recruitment domain; CD, Crohn’s disease; CENTβ1, centaurin β1; cIAP, cellular inhibitor of apoptosis; DAMP, danger-associated molecular pattern; DHA, docosahexaenoic acid; EGFR, epidermal growth factor receptor; EOS, early onset sarcoidosis; EPA, eicosapentaenoic acid; ERK, extracellular signal-regulated kinase; FRMPD2, FERM and PDZ domain-containing 2 protein; GEF-H1, guanine nucleotide exchange factor H1; GRIM-19, eukaryotic gene associated with retinoic-interferon-induced mortality 19; Hsp90, heat shock protein 90; HTS, high-throughput screening; iEDAP, dipeptide D-Glu-meso-DAP; IFN, interferon; IKK, IκB kinase; IL, interleukin; Ipaf, interleukin-1β-converting enzyme protease-activating factor; IRF, interferon regulatory factor; 6912



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D. L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotidebinding oligomerization domain 1. J. Biol. Chem. 2011, 286, 31003− 31013. (11) Grimes, C. L.; De Zoysa Ariyananda, L.; Melnyk, J. E.; O’Shea, E. K. The innate immune protein Nod2 binds directly to MDP, a bacterial cell wall fragment. J. Am. Chem. Soc. 2012, 134, 13535−13537. (12) Tanabe, T.; Chamaillard, M.; Ogura, Y.; Zhu, L.; Qiu, S.; Masumoto, J.; Ghosh, P.; Moran, A.; Predergast, M. M.; Tromp, G.; Williams, C. J.; Inohara, N.; Nuñez, G. Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. EMBO J. 2004, 23, 1587−1597. (13) Mo, J.; Boyle, J. P.; Howard, C. B.; Monie, T. P.; Davis, B. K.; Duncan, J. A. Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP. J. Biol. Chem. 2012, 287, 23057−23067. (14) Askari, N.; Correa, R. G.; Zhai, D.; Reed, J. C. Expression, purification, and characterization of recombinant NOD1 (NLRC1): a NLR family member. J. Biotechnol. 2012, 157, 75−81. (15) Zurek, B.; Proell, M.; Wagner, R. N.; Schwarzenbacher, R.; Kufer, T. A. Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation. Innate Immun. 2012, 18, 100−111. (16) Mayor, A.; Martinon, F.; De Smedt, T.; Pétrilli, V.; Tschopp, J. A crucial function for SGT1 and Hsp90 in inflammasome activity links mammalian and plant innate immune responses. Nat. Immunol. 2007, 8, 497−503. (17) da Silva Correia, J.; Miranda, Y.; Leonard, N.; Ulevitch, R. SGT is essential for Nod1 activation. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6764−6769. (18) Lee, K. H.; Biswas, A.; Liu, Y. J.; Kobayashi, K. S. Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components. J. Biol. Chem. 2012, 287, 39800−39811. (19) Srimathi, T.; Robbins, S. L.; Dubas, R. L.; Hasegawa, M.; Inohara, N.; Park, Y. C. Monomer/dimer transition of the caspase-recruitment domain of human Nod1. Biochemistry 2008, 47, 1319−1325. (20) Barnich, N.; Aguirre, J. E.; Reinecker, H. C.; Xavier, R.; Podolsky, D. K. Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition. J. Cell Biol. 2005, 170, 21−26. (21) Kufer, T. A.; Kremmer, E.; Adam, A. C.; Philpott, D. J.; Sansonetti, P. J. The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction. Cell. Microbiol. 2008, 10, 477−486. (22) Lécine, P.; Esmiol, S.; Métais, J. Y.; Nicoletti, C.; Nourry, C.; McDonald, C.; Nuñez, G.; Hugot, J. P.; Borg, J. P.; Ollendorff, V. The NOD2-RICK complex signals from the plasma membrane. J. Biol. Chem. 2007, 282, 15197−15207. (23) Philpott, D. J.; Sorbara, M. T.; Robertson, S. J.; Croitoru, K.; Girardin, S. E. NOD proteins: regulators of inflammation in health and disease. Nat. Rev. Immunol. 2014, 14, 9−23. (24) Hasegawa, M.; Fujimoto, Y.; Lucas, P. C.; Nakano, H.; Fukase, K.; Nuñez, G.; Inohara, N. A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-κB activation. EMBO J. 2008, 27, 373−383. (25) Inohara, N.; Koseki, T.; Lin, J.; del Peso, L.; Lucas, P. C.; Chen, F. F.; Ogura, Y.; Nuñez, G. An induced proximity model for NF-kappaB activation in the Nod1/RICK and RIP signaling pathways. J. Biol. Chem. 2000, 275, 27823−27831. (26) Abbott, D. W.; Wilkins, A.; Asara, J. M.; Cantley, L. C. The Crohn’s disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Curr. Biol. 2004, 14, 2217− 2227. (27) Fridh, V.; Rittinger, K. The tandem CARDs of NOD2: intramolecular interactions and recognition of RIP2. PLoS One 2012, 7, e34375. (28) Abbott, D. W.; Yang, Y.; Hutti, J. E.; Madhavarapu, S.; Kelliher, M. A.; Cantley, L. C. Coordinated regulation of Toll-like receptor and

ITCH, itchy E3 ubiquitin protein ligase; JNK, c-Jun N-terminal kinase; JNKBP1, c-Jun N-terminal kinase binding protein; LPS, lipopolysaccharide; LRR, leucine-rich repeat; LUBAC, linear ubiquitin assembly complex; MAPK, mitogen-associated protein kinase; MAVS, mitochondrial antiviral signaling; MDP, muramyl dipeptide; MHC II, major histocompatibility complex II; MEKK4, mitogen-activated protein kinase kinase kinase 4; MoA, mode of action; NACHT, nucleotide binding domain; NADPH, nicotinamide adenine dinucleotide phosphate; NAID, nucleotide-binding oligomerization domain 2 associated autoinflammatory disease; NF-κB, nuclear factor κB; NEMO, nuclear factor κB essential modulator; NIH, National Institutes of Health; NLR, nucleotide binding oligomerization domain like receptor; NLRC4, nucleotide binding oligomerization domain like receptor family caspase-recruitment-domain-containing protein 4; NLRP, NACHT-, LRR-, and PYD-domain-containing protein; NOD, nucleotide-binding oligomerization domain; Pak, p21-activated kinase; PAMP, pathogen-associated molecular pattern; PAMPA, parallel artificial membrane permeability assay; PGN, peptidoglycan; PP2A, protein phosphatase 2A; PRDX4, peroxiredoxin 4; PRR, pattern recognition receptor; PSMA-7, proteasome subunit α type 7; PTPN22, protein tyrosine phosphatase non-receptor type 22; Rac1, Ras-related C3 botulinum toxin substrate 1; RIG-I, retinoic acid induced gene I; RIP2, receptor-interacting protein 2; RLR, retinoic acid induced gene I like receptor; SAR, structure−activity relationship; SGT-1, suppressor of the G2 allele of Skp1; SHIP-1, Src homology 2 domain-containing inositol 5-phosphatase 1; siRNA, small interfering ribonucleic acid; SNP, single nucleotide polymorphism; SQSTM1, sequestosome 1; Src, proto-oncogene tyrosine-protein kinase; STAND, signal transduction ATPase with numerous domains; TAB, transforming growth factor binding protein; TAK1, transforming growth factor β activated kinase 1; TLR, Toll-like receptor; TNF-α, tumor necrosis factor α; TRAF, tumor necrosis factor receptor associated factor; TriDAP, L-Ala-D-Glu-meso-DAP; TRIM, tripartite motif-containing protein; XIAP, X-linked inhibitor of apoptosis

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