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Inhibiting the Inflammasome: A Chemical Perspective Alex George Baldwin, David Brough, and Sally Freeman J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 Sep 2015 Downloaded from http://pubs.acs.org on October 5, 2015

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Journal of Medicinal Chemistry

Inhibiting the Inflammasome: A Chemical Perspective Alex. G. Baldwin†, David Brough‡, Sally Freeman*† AUTHOR ADDRESS †

Manchester Pharmacy School, Faculty of Medical and Human Sciences, The University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK.

‡

Faculty of Life Sciences, The University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK.

KEYWORDS Inflammasome; inflammation; interleukin-1β; NLRP3 inhibitor, cysteine modification. ABSTRACT Inflammasomes are high molecular weight complexes that sense and react to injury and infection. Their activation induces caspase-1 activation and release of interleukin-1β, a proinflammatory cytokine involved in both acute and chronic inflammatory responses. There is increasing evidence that inflammasomes, particularly the NLRP3 inflammasome, act as guardians against non-infectious material. Inappropriate activation of the NLRP3 inflammasome contributes to the progression of many non-communicable diseases such as gout, type II diabetes

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and Alzheimer’s disease. Inhibiting the inflammasome may significantly reduce damaging inflammation and is therefore regarded as a therapeutic target. Currently approved inhibitors of interleukin-1β are rilonacept, canakinumab and anakinra. However, these proteins do not possess ideal pharmacokinetic properties and are unlikely to easily cross the blood-brain barrier. Since inflammation can contribute to neurological disorders, this review focusses on the development of small molecule inhibitors of the NLRP3 inflammasome.

Introduction

Inflammation is an essential host response to infection and injury. At the heart of inflammation research is an attempt to understand the regulation of the pro-inflammatory cytokine interleukin1β (IL-1β), which is central to host responses to infection,1 but also causes tissue injury when activated inappropriately.2 IL-1β is produced by many cells, most commonly those of hematopoietic lineage, as an inactive pro-IL-1β precursor. This precursor is expressed in response to pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) that bind to pattern recognition receptors (PRRs) on macrophages to upregulate pro-inflammatory gene expression.3,4 PAMPs are motifs carried by pathogens, such as bacterial endotoxin (or LPS) of Gram-negative bacteria, and DAMPs are commonly endogenous molecules released by necrosis. Inflammation that occurs in response to tissue injury in the absence of pathogen is mediated by DAMPs and is considered sterile, and contributes to tissue damage and disease.5 IL-1β is one of the primary inflammatory cytokines associated with sterile inflammatory responses.5 Pro-IL-1β is inactive and remains intracellular until a further PAMP or DAMP stimulation activates cytosolic PRRs, often of the NLR family, to form large multiprotein complexes called inflammasomes.5 These complexes consist of the PRR, pro-caspase-1,


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and an adaptor protein called ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), that interact via homology binding domains.5 A number of inflammasome forming PRRs have been identified including NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, IFI16, and RIG-I.5 Of these inflammasomes identified to-date, the best characterised, and most strongly associated with sterile inflammation, is formed by NLRP3.6 When NLRP3 senses PAMPs or DAMPs it recruits ASC, which in turn recruits caspase-1 causing its activation. Caspase-1 then processes pro-IL-1β (and pro-IL-18) to their mature forms that are rapidly secreted from the cell.5 In cellular models the NLRP3 inflammasome is proposed to follow a two stage activation process.7 The first stage is referred to as priming and requires the NF-kB-dependent upregulation of NLRP3 and IL-1β gene expression (Figure 1).7 This priming step is typically achieved by the activation of PRRs such as TLR4 by PAMPs like bacterial endotoxin (or LPS). IL-1β is expressed as an inactive precursor (pro-IL-1β). In the second activation stage, the primed cell now encounters a second stimulus (typically a DAMP in sterile inflammation) which causes activation of the NLRP3 inflammasome and promotes release of active IL-1β (Figure 2). During sterile inflammation there are a number of DAMPs that can activate the NLRP3 inflammasome. These include extracellular ATP acting via the P2X7 receptor,8,9 crystals of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD),10 aggregated amyloid beta (Aβ),11 sphingosine12 and hypotonic stress.13 These diverse DAMPs are not thought to interact with NLRP3 directly, but cause its activation via one or more of a number of mechanisms such as K+ efflux,14 lysosomal membrane destabilization,15 reactive oxygen species (ROS) production16 and ubiquitin/deubiquitination post-translational modifications (Figure 2).17



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Inappropriate activation of NLRP3 is associated with a number of non-communicable diseases including gout,10 atherosclerosis18 and type II diabetes.19 Aβ protein, which is associated with Alzheimer’s disease (AD), activates NLRP3 through phagocytosis and subsequent lysosomal destabilisation in microglial cells.11 These results support a recent study reporting NLRP3dependent inflammation contributing to disease progression in the APP/PS1 mouse model of AD.20 Heneka and colleagues showed that memory deficits in APP/PS1 mice were entirely dependent upon NLRP3 and APP/PS1 mice lacking NLRP3 increased the clearance of Aβ in microglial cells isolated from cerebral lysates.20 From these results, the authors suggest that blocking NLRP3 inflammasome activity could impede the progression of AD. Gain-of-function mutations in NLRP3 leads to severe autoinflammatory disorders such as familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal onset multisystem inflammatory disease (NOMID); these orphan diseases are classified as cyropyrinassociated periodic syndromes (CAPS) and can be treated with anti-IL-1β therapy.21 Given the importance of IL-1β in disease, a number of therapeutic biological IL-1β inhibitors were developed and are currently clinically available. These include the dimeric fusion protein rilonacept (Arcalyst®) and human immunoglobulin Gκ monoclonal antibody canakinumab (Ilaris®) which neutralise IL-1β directly. There is also a naturally occurring IL-1 receptor antagonist (IL-1Ra) or recombinant anakinra (Kineret®) that blocks IL-1 signalling.22,23 However, there are some problems associated with their use. For example, rilonacept was recently rejected for FDA approval recently for the treatment of gout flares due to safety concerns,24 and anakinra possesses poor pharmacokinetic properties with a short half-life,25 with acute gout patients requiring high dosages (100 mg daily).26 The major adverse reaction of rilonacept and anakinra is injection-site reactions.27,28 They are also high molecular weight


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proteins that show poor penetration of the blood-brain barrier (BBB): 0.2-0.3% anakinra crossed healthy BBB into the cerebrospinal fluid (CSF) in non-human primates and 1.2-2.0% anakinra was detected in the CSF of subarachnoid haemorrhage patients in which the BBB is likely to be compromised.25,29 Therefore, this limits their use for inflammatory diseases of the central nervous system, such as stroke or AD. A preferable approach may be the development of small molecule inhibitors of IL-1β release which may penetrate the brain more readily, have improved pharmacokinetic properties and be more cost effective.30 Small molecule inhibitors of the NLRP3 inflammasome will also be more valuable therapeutics as they can prevent inflammasome-dependent maturation of IL-18 (from pro-IL-18) and a form of ensuing cell death executed by caspase-1, termed pyroptosis (Figure 2).31 However, no approved small molecule inhibitor of IL-1β release currently exists and there is an unmet need for the development of such a drug. This review considers the NLRP3 inflammasome as a therapeutic target and primarily discusses synthetic small molecule inhibitors that have been developed to-date. It should be noted that a number of natural products also inhibit NLRP3 inflammasome activation and although are outside the scope of this review, are reviewed elsewhere.32 Glyburide Compound 1 (glyburide)33, also known as glibenclamide, is one of the front-line hypoglycaemic drugs used to treat type II diabetes mellitus (Figure 3).33 Compound 1 inhibits insulin release by blocking islet chloride channels34 and ATP-sensitive potassium (KATP) channels in pancreatic β cells.35 However, Gabel and colleagues later showed that 1 also inhibits IL-1β release in LPS-activated human monocytes with an IC50 of 12 µM36 but it is noteworthy that this concentration is likely to be much higher than the mean individual maximum serum concentration (Cmax) that can be achieved clinically. Initially, compound 1 was proposed to



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inhibit ATP-binding cassette (ABC) transporters based on comparison with the known ABC inhibitor 2 (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, DIDS)37 (Figure 3). It has been demonstrated that K+ efflux acts as a trigger for NLRP3 inflammasome activation when [K+]int falls below 90 mM,38 and therefore it was speculated that 1 disrupts potassium cation efflux from the cytosol. Indeed, compound 1 was suggested to inhibit IL-1β release during bronchial hyporesponsiveness through KATP channels.39 However, a later study showed that KATP channels are dispensable for inhibitory activity as IL-1β release was maintained even when KATP channel subunits Kir6.1, Kir6.2 and SUR2 were knocked out.40 The inhibitory effect of 1 on IL-1β release is NLRP3 specific as IL-1β secretion is not significantly affected in the presence of the NLRC4 activator Salmonella typhimurium or the AIM2 inflammasome activator Poly (dA:dT).41 Compound 1 does not directly suppress caspase-1 activity in vitro37,40 or in vivo42 unless NLRP3, NF-κB or P2X7 are knocked out.43,44 These results suggest that 1 may prevent ASC oligomerisation during NLRP3 inflammasome assembly or affect NLRP3 inflammasome activation upstream of its assembly. Preliminary SAR studies indicate that the cyclohexylurea moiety is dispensable for inhibitory activity but both the benzamido and sulfonyl groups of 1 contribute to inhibition of LPS+ATPstimulated caspase-1 activation and IL-1β secretion.40 Compound 1 has also been shown to block NLRP3 inflammasome activation and IL-1β production stimulated by the DAMP islet amyloid polypeptide (IAPP) associated with type II diabetes.45 Compound 1 also reduced mortality in 1160 patients suffering with infection of the Gram-negative bacterium Burkholderia pseudomallei, the causative agent of melioidosis,46 and completely blocked IL-1β release four hours following intraperitoneal injection of cyclophosphamide (CP) in a model of CP-induced cystitis.47 Obesity is one risk factor associated


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with severe acute pancreatitis, a host-driven disease caused by chronic pancreatic inflammation, and obese ob/ob mice treated with 1 have significantly decreased pancreatic damage and disease severity caused by the pro-inflammatory cytokines IL-1β and IL-6 in an NLRP3 inflammasomedependent manner.48 However, compound 1 is known to have some side effects. These are predominantly cardiovascular risks33 and a recent study on the clinical outcomes of diabetic patients on sulfonylurea drugs showed that 1 increases mortality rates over the more specific pancreatic β-cell sulfonylurea drugs gliclazide and glimepiride.49 In addition, the dosage required to significantly reduce IL-1β release would be in excess of the recommended dosage for type II diabetes treatment. A recently developed analogue of 1 that lacks the cyclohexylurea group responsible for hypoglycemic activity, compound 3 (16673-34-0)50, had no effect on glucose plasma levels with repeated injections of 500 mg/kg for 7 days and is largely protective in mouse models of reperfused and non-reperfused acute myocardial infarction (Figure 3).50,51 Like 1, compound 3 has good pharmacokinetic properties (t1/2 = 6-10 hours). It also has a comparable mode of action by suppressing formation of the NLRP3 (but not NLRC4 or AIM2) inflammasome in J774A.1 murine macrophage cells and primary adult rat cardiomyocytes in response to diverse NLRP3 stimuli.50,51 Importantly, 3 was still effective in bone marrow-derived macrophages (BMDMs) from a mutant mouse that elicits spontaneous NLRP3 inflammasome activation.51 These investigations are interesting for two reasons. Firstly, 3 is posited to bind ASC directly as: i) compound 3 blocks NLRP3 aggregation downstream of NLRP3 inflammasome activation, ii) inhibition of caspase-1 activation by 3 is unlikely since other inflammasome-forming protein complexes were unaffected, and iii) stained ASC aggregation (as a read-out of NLRP3 inflammasome speck formation) was significantly reduced in the presence of 3 as determined



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using immunocytochemistry. Secondly, the mutant mouse used for constitutive NLRP3 inflammasome activation by Marchetti and colleagues51 corresponds to the active NLRP3 mutation found in humans with MWS.21 The authors are currently investigating the efficacy of 3 in patients with cardiac injury or heart failure, but given that treatment with 3 was still effective in preventing IL-1β release in a mouse mutant model of MWS, it would be interesting to scope the therapeutic potential of 3 in other research areas involving the NLRP3 inflammasome. CRIDs and MCC950 One class of IL-1β release inhibitors derived from compound 1 are the cytokine release inhibitory drugs (CRIDs). These diarylsufonylurea analogues lack the cyclohexylurea moiety necessary for insulin release and showed improved inhibitory activity against LPS plus ATP-, cytotoxic T lymphocyte (CTL)-, and hypotonic stress-induced IL-1β maturation in human monocytes in vitro.36 Initially, reversible inhibitors 4 (CP-412,245) and 5 (CP-424,174)36 showed good potencies (IC50 of 260 and 210 nM, respectively) against IL-1β processing (Figure 4). The irreversible diarylsulfonylurea inhibitors 6 (CRID1) and 7 (CRID2)36 were later developed by Gabel and co-workers (IC50 of 350 and 250 nM, respectively). IL-6 and TNF-α was unaffected by CRID treatment and the CRIDs do not target caspase-1 activity directly. Along with the observation that pro-IL-1β levels are not affected36 suggests that the CRIDs do not target NLRP3 at the genetic level. Compounds 7 and 8 have been reported to modify cysteine residues on putative targets as a result of nucleophilic attack on the epoxide functional groups.52 However, more details on the mechanism of action of reversible inhibitors 4 and 5 is necessary as they do not contain epoxide functional groups and may not act through the same mode of action as 6/7. Continued work by O’Neill and colleagues demonstrated that the related sulfonylurea, compound 8 (MCC950)53, dose-dependently blocked LPS plus ATP-, nigericin-, MSU- and


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silica-induced IL-1β release in BMDMs, human monocyte-derived macrophages (HMDMs) and peripheral blood mononuclear cells (PBMCs) (Figure 4). Compound 8 inhibits IL-1β release with high potency (IC50 of 7.5 nM in BMDMs) and does not block TNF-α. The observed IL-1β inhibitory effect of 8 is NLRP3 specific and inhibits NLRP3-induced ASC speck formation in a manner independent of the NLRP3 priming, K+ efflux, Ca2+ influx or preventing direct NLRP3NLRP3/NLRP3-ASC interactions as determined by co-immunoprecipitation experiments. Compound 8 completely prevented the death of mice with MWS-associated NLRP3 mutations when compared to control mice. Even when the drug was withdrawn, MWS mice still survived for a further 10 days.53 Compound 8 was also active in ex vivo samples of MWS patients by suppressing LPS-induced caspase-1 activation and IL-1β secretion in PBMCs at a concentration of ~50 nM. In addition, 8 reduced the severity and occurrence of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis.53 Compound 8 has excellent pharmacokinetic properties with high stability in human or mouse liver microsomes. Compound 8 has a half-life of approximately 3 h and a bioavailability of 68% in C57BL/6 mice upon intravenous (3 mg/kg) and oral (20 mg/kg) administration,53 making the CRIDs and in particular 8 promising candidates for further development for the treatment of NLRP3-dependent pathologies. Auranofin Compound 9 (auranofin)54 is an Au-containing acetylated carbohydrate complex used for the treatment of rheumatoid arthritis (RA) (Figure 5). Monocytes and macrophages in the synovial fluid in RA patients secrete large quantities of IL-1β that leads to the pathophysiological changes associated with RA.55,56 Compound 9 suppresses pro-IL-1β mRNA expression in response to LPS, zymosan and Fusobacterium nucleatum/Peptostreptococcus anaerobius bacterial infection,



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and this reduces pro-IL-1β production in peritoneal cells and RAW 264.7 macrophages.55,57 Compound 9 also significantly suppresses LPS-induced NLRP3 and IL-1-related gene expression in J774 cells and primary mouse peritoneal macrophages,58 in addition to the expression of other pro-inflammatory cytokines, including IL-6 and TNF-α.55 These results are in line with the known mechanism of action of 9 by targeting inhibitor of kappa B (IκB) kinase (IKK), whose enzyme activity is critical for NF-κB activation. Compound 9 has been consistently shown to inhibit IKK phosphorylation of IκB protein bound to NF-κB and thus inhibits its ubiquitinylation and proteasomal degradation, preventing nuclear translocation of free NF-κB and induction of pro-IL-1β and NLRP3.56,59,60 Compound 9 also inhibits two other major transcription factors important for cell signalling in response to pathogens, AP-161,62 and IRF3.63 Given that NF-ᴋB, AP-1 and IRF3 are all regulated at the TLR4-MyD88 axis, it is posited that 9 prevents activation of the TLR4-MyD88 pathway. Indeed, 9 has also been demonstrated to prevent the homodimerisation of TLR4 required for NF-κB and IRF3 activation.63 Isakov and coworkers recently uncovered that thioredoxin reductase, a redox enzyme responsible in controlling macrophage activation, was another functionally relevant target of 9 in blocking LPS-induced pro-IL-1β and NLRP3 gene expression.58 Taken together, these studies suggest that 9 inhibits IL-1β secretion by blocking NF-ᴋB activation and subsequent NLRP3/pro-IL-1β transcription. Importantly, 9 also dose-dependently inhibits anthrax lethal toxin (LT)-induced NLRP1 and nigericin-stimulated NLRP3 inflammasome activation in a manner independent of LPS-induced NF-κB priming.64 How 9 blocks NLRP3 inflammasome formation without blocking the NF-ᴋB pathway has not been fully elucidated. Potential modes of action include inhibition of cathepsin


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B activity65 and enhancing protein S-nitrosylation of many proteins including Hsp90,66 both mechanisms of which are understood to repress NLRP3 inflammasome assembly.7,15 A brief mention of the role for the Au atom in compound 9 is warranted. Incorporation of Au in drugs is uncommon: only small molecule derivatives of 9 that contain Au suppress IL-1β secretion67 and the related Au compound myochrysine significantly inhibits caspase-1 activation and IL-1β maturation at micromolar concentrations.68 Compound 9 is suggested to act as a cysteine modifier at the Au centre and has been demonstrated to modify the important catalytic Cys-179 residue present in the IKKβ subunit.59,69 Importantly, derivatives of 9 react with cysteine at biologically relevant concentrations70 and a low energy cysteine complex has been confirmed by mass spectrometry.71 These derivatives contain various N6-benzyladenine groups (10)70 and show greater activity in suppressing IL-1β secretion than 9 (Figure 5). It is noteworthy that 10 complexes showed markedly greater anti-inflammatory effects compared to trichloridoAu(III)-N6-benzyladenine complexes (11)72 (Figure 5), indicating that the oxidation state of Au is important for its anti-inflammatory actions. Unfortunately, compound 9 possesses a number of poor pharmacological properties, including slow onset of action due to slow drug accumulation with blood concentration of the Au complex varying significantly from patient to patient.73 In particular, 9 binds readily to Cys-34 of human serum albumin in vitro 74 and in vivo 75 which strongly influences its bioavailability and transport in the blood. Additionally, 9 does have some side-effects: diarrhoea is the most common symptom associated with long-term use with 74% of patients affected.76 Parthenolide & Bay 11-7082 The herbal sesquiterpene lactone 12 (parthenolide)77 inhibits ATP-, nigericin-, MSU-induced NLRP3 inflammasome activation as well as directly targeting caspase-1 at a concentration of 10



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µM (Scheme 1). Compound 12 has been recently shown to inhibit IL-1β release from THP-1 cells with an IC50 of 2.59 µM.78 Compound 13 (Bay 11-7082)77 inhibits ATP-, nigericin- and MSU-induced NLRP3 inflammasomes at a concentration of 12 µM and does not inhibit caspase1 activity directly (Scheme 2). Compounds 12 and 13 prevent ASC pyroptosome formation and NLRP3 inflammasome activity by directly alkylating critical cysteine residues within the ATPase region of NLRP3 in NG5 cells and mouse primary BMDMs in a dose-dependent manner.77 Cysteine modification appears to be the major mode of action for these inflammasome inhibitors as addition of thiol-reactive agents including free glutathione and N-acetyl-L-cysteine (NAC) ameliorated the inhibitory activity of 12 and 13. SAR analysis of 13 (Figure 6) reveals that the olefin group and oxidized benzylic sulfone are essential for activity, whilst steric bulk on the benzyl substituent or at the C2 position are dispensable for inhibition of caspase-1 activation. Loss or exchange of the nitrile group for a weaker electrophile reduces caspase-1 activation. These observations support cysteine modification via Michael addition at the C3 position and highly electron withdrawing groups at the C1 position are responsible for activating the vinyl sulfone towards alkylation. It was later confirmed that 13 modifies cysteine residues via Michael addition with formation of the appropriate cysteine conjugate and concomitant release of 4methylbenzoic acid (Scheme 2).79 Similarly, 12 non-specifically modifies cysteine residues of caspase-1 p2077 through a Michael-type reaction with the α,β-unsaturated lactone, but the lack of α-hydrogen acidity leads to the addition product only (Scheme 1).79,80 Whilst the authors suggest that the inhibitory activity of 12 and 13 against the NLRP3 inflammasome was independent of NF-κB,77 there are reports that both compounds could inhibit NF-ᴋB-induced NLRP3 and pro-IL-1β gene expression in addition to directly targeting the NLRP3 inflammasome. Compound 12 has been shown to inhibit IKK81 and NF-κB directly82


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whilst 13 can inhibit IKK,83 the ubiquitin system (E2/3 enzymes)79 and a number of protein tyrosine phosphatases84 which are important for NF-κB activation. Compounds 12 and 13 interact with all these targets by cysteine modification and it is interesting to note that 12 modifies the exact same cysteine residue (Cys-179) present in the active site of the IKKβ subunit as 9.69,85 In the context of novel pharmacological therapeutics, 12 has been shown to be neuroprotective in a rat stroke model where decreased NF-κB and caspase-1 expression were observed in addition to decreased BBB permeability.86 However, the poor bioavailability and solubility of 12 means that it does not have suitable pharmacological properties for clinical trials and watersoluble analogues are being evaluated.87-89 Furthermore, the pharmacophore responsible for sesquiterpene lactone anti-inflammatory activity correlates with their cytotoxicity, suggesting that side effects such as cytotoxicity will be difficult to separate from their beneficial effects.90 In contrast, vinyl sulfones, such as 13, permeate cell membranes relatively easily77 and were tolerated well in mice and dogs in a preclinical trial as anti-parasitic agents.91 β-Hydroxybutyrate It has been recently demonstrated that the ketone body 14 (β-hydroxybutyrate, BHB)92 inhibits caspase-1 activation and IL-1β release in the presence of a large number of NLRP3 agonists, including ATP, nigericin, MSU, silica particles, sphingosine and lipotoxic fatty acids palmitate and ceramide (Figure 7). Youm and colleagues believe that a high concentration of circulating levels of 14 during periods of starvation or strenuous exercise may solve the link between nutritional state and altered immune cell action, with 14 acting as an immune effector. The authors suggest that 14 dampens the innate immune system, in part, by inhibiting NLRP3 inflammasome activation. Compound 14 specifically inhibits NLRP3 inflammasome formation,



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in a manner independent of its well established mechanisms of action, at physiologically relevant concentrations (5-10 mM) that are observed during periods of fasting or high-intensity exercise.92 Compound 14 is unlikely to be specific for NLRP3 at such high concentrations; indeed, one target of 14 is histone deacetylases (HDACs) and it was demonstrated that 14 induced H3 acetylation in BMDMs at similar concentrations.92 Further experiments revealed that the suppressive effect of 14 on ATP-induced NLRP3 inflammasome activation could not be abrogated by rotenone-induced ROS accumulation intracellularly but 14 efficiently prevented K+ efflux in response to ATP, MSU and ceramide in BMDMs. Compound 14 dose-dependently blocked constitutive NLRP3 inflammasome activation in mouse models of MWS and FCAS. Importantly, supplementation of FCAS mice with 14 in the form of 1,3-butanediol ketone diesters prior to a knock-in mutation of the NLRP3 gene (induced by tamoxifen-induced Cre mice containing mutant NLRP3L351P) protected mice from the aetiologies associated with FCAS such as neutrophilia.92 It should be noted that the related ketone bodies acetoacetate, butyrate and acetate, did not block NLRP3 activation and that the stereochemistry of 14 was not important for its suppressive effects on caspase-1 activation.92 Thus this suggests that the β-hydroxy group is essential for inhibitory activity against IL-1β release. Fc11a-2 More specific, synthetic inhibitors of inflammasome assembly have recently been developed such as the benzoimidazole 15 (Fc11a-2)93 (Figure 7). Compound 15 targets the NLRP3 inflammasome by interfering with the proximity-induced autocleavage of procaspase-1 which leads to less activated caspase-1 release from the NLRP3-ASC complex in a manner independent of NF-ᴋB activation.93 Compound 15 was shown be highly effective in suppressing IL-1β/18 production in LPS+ATP-stimulated THP-1 cells at 3 µM and dextran sulfate sodium (DSS)-


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induced experimental colitis in mice at 10-30 mg/kg dosage via intragastric adminstration.93 These results are promising and further studies are warranted to determine the target(s) of 15 and to evaluate efficacy in other NLRP3 inflammasome disease models. 3,4-Methylenedioxy-β-nitrostyrene Compound 16 (3,4-methylenedioxy-β-nitrostyrene, MNS)94 has recently been demonstrated to bind to the NACHT and LRR domains and inhibits NLRP3 ATPase activity required for ATPdependent oligomerisation of the NLRP3 inflammasome (Scheme 3). With an IC50 of 2 µM, compound 16 has a modest potency but high specificity against NLRP3 inflammasome activation compared to NLRC4 and AIM2 inflammasomes. In contrast to previous suggestions, 16 does not target spleen tyrosine kinase (Syk), a key kinase that regulates the NLRP3 inflammasome (further discussed later).95,96 In addition, excess L-cysteine or 13 prevented isolation of NLRP3 protein in the presence of biotinylated 16.94 These observations agree with the reported mode of action of 16 against tyrosine kinases96,97 and confirms that the mechanism of action of 13 and 16 is shared. Similarly to 13, a preliminary SAR study revealed that any modification to the nitrovinyl side chain ameliorates NLRP3 inhibitory activity (Figure 8). Ring opening of 16 to the trans-3-methoxy-4-hydroxy-β-nitrostyrene analogue retains NLRP3 inhibitory activity but removal of the 3,4-methylenedioxy group gives a derivative with reduced potency compared to 16. Taken together, compound 16 is proposed to irreversibly modify active site cysteine residues via Michael addition at the nitrovinyl group (Scheme 3). Caspase-1 inhibitors An approach to inhibit IL-1β secretion is to block the master protease responsible for maturation of the cytokine, caspase-1. Selected examples of caspase-1 inhibitors that prevent the cleavage of pro-IL-1β into IL-1β (and pro-IL-18 into IL-18) include compounds 17 (disulfiram)



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and 18 (ritonavir) (Figure 9).98 Two further caspase-1 inhibitors, 19 (VX-740)99 and analogue 20 (VX-765),100 are prodrugs which are metabolised by plasma esterases to the corresponding aldoacids 21 (VRT-18858) and 22 (VRT-043198), respectively (Scheme 4). Compound 19 showed activity in two murine models of osteoarthritis at 12.5 mg/kg administered orally99 and 20 significantly reduced the levels of IL-1β/18 in a mouse model of dermatitis caused by delayedtype hypersensitivity at 25 mg/kg administered orally.100 Compound 20 also significantly suppressed both acute seizures and chronic epilepsy in mice101 and was registered in clinical trials for psoriasis and epilepsy. Compound 20 appeared safe in a mouse model of RA as no toxicity was observed even when 100 mg/kg was administered twice daily for 28 days.100 Although compounds 19 and 20 entered clinical trials, they were not developed further due to potential hepatic toxicity.102 Other indirect inhibitors of the inflammasome Whilst there are compounds that may directly target the inflammasome, there are many more inhibitors that indirectly target the inflammasome by blocking the expression of NLRP3, procaspase-1 and pro-IL-1β (the ‘priming’ stage, Figure 1) or prevent deubiquitination, ion flux, ROS generation or lysosomal destabilisation (the ‘activation’ step, Figure 2). Some of these inhibitors are briefly described. TAK-242, bromoxone and 5Z-7-oxozeaenol The small molecules 23 (TAK-242) (Figure 10), 24 (bromoxone) (Figure 11) and 25 (5Z-7oxozeaenol)103 (Scheme 5) are effective NLRP3 inflammasome inhibitors that suppress LPS+ATP-induced IL-1β release upstream of IKK. Compound 23 binds to the intracellular domain of human TLR4 and Cys-747 is proposed to be irreversibly modified on TLR4 via Michael addition.104,105 Compound 23 inhibits NF-κB activation and subsequently the expression


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and processing of pro-inflammatory cytokines including IL-1, IL-6, IL-8 and TNF-α, in addition to TLR4 itself in vitro.104,106,107 Specifically, 23 blocks IL-1β release with an IC50 of 5.7 nM in LPS-stimulated peritoneal macrophages108 and has a fast onset of action in vitro104 and in vivo.109 The therapeutic benefit of 23 has been demonstrated in models of endotoxic shock and sepsis in mice and pigs by significantly reduced circulating serum levels of IL-1β.105,109-111 Compound 23 is also capable of crossing the BBB and can suppress neuroinflammation in ischemia/reperfusion brain injury in mice.112 Compound 24 is an irreversible broad-spectrum inflammasome inhibitor (NLRP1, NLRP3, AIM2) and suppresses caspase-1 activation in a manner independent of caspase-1 activity, ASC, ROS accumulation and transcription of NF-κB-encoded genes NLRP3 and TNF-α, with an IC50 of 170 nM in PMA-differentiated THP-1 cells.103 The target of 24 is currently unknown and whilst the known TAK1 inhibitor 25 specifically inhibited NLRP3 inflammasome activation in response to LPS, nigericin and alum, 24 did not affect TAK1/TAB1/TAB2 complex formation using a co-immunoprecipitation assay nor inhibit TAK1/TAB1-induced IᴋB phosphorylation via IKK.103 However, Gong and colleagues did demonstrate that 24 is an inhibitor of pro-IL-1β expression and abolished NF-κB nuclear translocation without directly affecting NF-κB or by suppressing IKKβ kinase activity, confirming the target of 24 is upstream of IKK. A SAR study of 24 isomers revealed that the hydroxyl group is required to prevent IL-1β secretion; a protected acetate derivative (26)103 showed no IL-1β inhibitory activity (Figure 11). However, hydroxyl and epoxide analogues of 24 (27-29)103 showed that the exact stereochemistry was not important for inhibitory activity (Figure 11). Meanwhile, 25 completely inhibits TAK1 enzyme activity at a concentration of 3 µM and its mechanism of action involves covalent, irreversible attachment of Cys-174 of TAK1 at the α,β-unsaturated ketone in an irreversible manner (Scheme 5).113



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7-Desacetoxy-6,7-dehydrogedunin Compound 30 (7-desacetoxy-6,7-dehydrogedunin, 7-DG)114 is a small molecule inhibitor of the NLRP1 and NLRP3 inflammasomes and affects protein-protein interactions between protein kinase R (PKR) and ASC, preventing activation of caspase-1 and production of IL-1β (Figure 12). PKR autophosphorylates target proteins in response to PAMPs/DAMPs and whilst one study demonstrates that a PKR kinase-dependent event was needed for inflammasome activation,115 another proposes that PKR kinase-independent events are sufficient for inflammasome activation.114 Compound 30 also interferes with PKR-IKK protein-protein interactions that prevents PKR-dependent IKK phosphorylation and subsequent proteasomal degradation, leaving NF-κB inactivated.114,116 Compound 30 protects anthrax LT-induced J774 macrophage cell death with an IC50 of 5 µM.114 Compound 30 has not yet been tried in clinical trials and is currently being used to study autoimmune and other inflammasome-component diseases involving release of IL-1β. DUB inhibitors NLRP3 is regulated at both transcriptional and post-translational levels in order to avoid spontaneous activation. The regulation of NLRP3 at the post-translational level is poorly characterised and an emerging hypothesis is the non-transcriptional regulation of inflammasome activation via deubiquitinylation, guided by deubiquitinating enzymes (DUBs). Juliana and coworkers initially discovered that, under normal resting conditions, NLRP3 is ubiquitinated and inactive.117 Activation of the NLRP3 inflammasome (but not NLRC4 or AIM2) is induced by deubiquitination in the presence of either LPS plus ATP or ATP alone.117 LPS treatment alone (i.e. engaging TLR signalling) causes deubiquitination of NLRP3 but is not sufficient for its activation. There are conflicting studies regarding the role of autophagy in NLRP3


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degradation118,119 but NLRP3 appears to be modified by mixed K48 and K63 ubiquitin chains on both the LRR and NACHT domains.17 BRCC3 was identified as the major DUB responsible for the specific deubiquitination of NLRP3 and its downregulation leads to polyubiquitination of Flag-NLRP3 in NG5 cell lysates and significant inhibition of ATP-induced IL-1β release.17 Importantly, a number of DUB inhibitors can block caspase-1 activation and subsequent IL-1β maturation such as 31 (PR-619), 32 (WP1130),117 33 (G5),17 34 (eeyarestatin I) and 35 (bAP15)118 (Figure 13). Therefore the potential use of DUB inhibitors to act as therapeutic agents to combat diseases involving NLRP3 inflammasome overactivation is an interesting area for research. P2X7R antagonists Blocking activation of the P2X7 purinoceptor (P2X7R) prevents NLRP3 inflammasomemediated IL-1β maturation and release by blocking ATP-induced K+ efflux in vitro120 and in vivo.121-123 P2X7R activation via extracellular ATP causes pannexin 1 recruitment to the plasma membrane and its interaction with P2X7R leads to K+ efflux from the cell8,9,124 and is sufficient for NLRP3 inflammasome activation.14 There has been significant interest in the design of P2X7R antagonists. A number of conformationally constrained analogues of 36 (KN62)125, one of the first-generation P2X7R antagonists discovered (Figure 14), have been synthesised which are highly potent and selective for P2X7R (IC50 for hP2X7R < 100 nM). Further P2X7R antagonists have been disclosed and have entered clinical testing including 37 (AZD9056)126, 38 (CE-224,535)127 and 39 (GSK1482160)128 (Figure 14). However, these antagonists were not efficacious in RA patients and were not considered an improvement over current therapies. The reasons are not fully understood but it should be noted that there is large genetic variation in the P2X7R gene with more than 260 single nucleotide polymorphisms (SNPs) known.129 In fact,



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P2X7R SNPs were recently shown to account for chronic pain variability in human cohorts of mastectomy and osteoarthritis.130 Additionally, the susceptibility of the Chinese population to the autoimmune disease, systemic lupus erthrymatosus (SLE), a condition that can cause excessive IL-1β secretion in response to host circulating dsDNA, was variable depending on P2X7R SNPs.131 Therefore P2X7R appears not to be a suitable therapeutic target, at least in the case of RA. Probenecid Compound 40 (probenecid)132 is a uricosuric drug (a drug that increases uric acid excretion in the urine) primarily used for the treatment of gout and conditions involving hyperuricemia (Figure 14). Urate-lowering therapy is required for patients with serum urate levels greater than the MSU saturation point and MSU crystal formation in joint synovial fluid is the main aetiology associated with arthritic gout.132 The role of MSU crystals in NLRP3 inflammasome activation is well established.10 Although compound 40 is widely believed to increase the rate of renal excretion of uric acid by inhibiting a renal anion tubular transporter,132,133 it was discovered that 40 also inhibits pannexin 1 channels.134,135 Silverman and colleagues first demonstrated that 40 binds pannexin 1 in multiple binding sites with an IC50 of ~ 150 µM and dose-dependently attenuated K+ efflux-induced ATP release in oocytes expressing pannexin 1.134,136 However, compound 40 is likely to be non-selective at this concentration as a number of its pharmacological targets, namely kidney organic anion transporters (OATs), are inhibited at IC50 values below 100 µM. Additionally, the mean plasma Cmax levels that can be achieved from the administration of 500 mg Probenecid Tablets used in the clinic is 35.3 µg/mL (123.7 µM).137 Thus at this dosage, compound 40 may not be sufficient to inhibit pannexin-1 therapeutically. Silverman et al. later showed that 40 inhibited K+-induced caspase-1 activation in neurons and


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astrocytes in which the importance of pannexin 1 was confirmed using cells where pannexin 1 was knocked down.136 Specifically, inhibition of NLRP1, NLRP2 and NLRP3 inflammasomes has been documented in the presence of 40.136,138-140 However, a recent study provides evidence against 40 targeting pannexin 1 and in fact, suggested to compete with ATP at the agonist binding site of P2X7R.141 A refinement of the mode of action of 40 is therefore required, particularly given the dispensable role of pannexin 1 in NLRP3-, NLRC4- or AIM2-dependent caspase-1 activation and IL-1β secretion using knock-out mice deficient in pannexin 1.142 Ion channel blockers Many reported inhibitors of IL-1β maturation appear to affect the electrochemical gradient across the plasma membrane. These include ATP-binding cassette (ABC) chloride transporter blockers 2 (Figure 3), 41 (tenidap), 42 (UK5099) and 43 (ethacrynic acid)143 (Figure 15). Ca2+ channel blockers 44 (nimodipine) and related compound 45 (nitrendipine)144 have also been demonstrated to inhibit Aβ-induced IL-1β release in vitro in microglial cells and in vivo (Figure 15). Whether ion channel blockers suppress inflammasome activation by stopping movement of ionic species across membranes or via unknown direct interactions with inflammasome components is not known. Compan and colleagues recently demonstrated that the movement of K+, Cl- and Ca2+ ions play an important role in regulatory cell volume-induced NLRP3 inflammasome activation during cell swelling. Blocking their translocation with the chloride channel blocker 46 (5-nitro-2-(3-phenylpropylamino)benzoic acid, NPPB) or with the selective intracellular Ca2+ chelator 47 (BAPTA-AM)13 abolished IL-1β release (Figure 15). These observations suggest that ion channel blockers or chemicals that prevent large cytosolic changes in K+, Cl- or Ca2+ concentrations are potential therapeutics against the NLRP3 inflammasome. ROS inhibitors



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In 2013, Latz and co-workers identified that intracellular ROS generation, produced by NADPH oxidase (NOX), causes NLRP3 inflammasome formation and caspase-1 activation in response to particulate matter.7 However, the role of ROS in inflammasome activation is now disputed. Phagocytes from patients suffering from chronic granulomatous disease (CGD), whose aetiology is well characterised as mutations in NOX subunits, have normal, if not increased, caspase-1 activity.145-147 In line with this, the NOX inhibitor 48 (diphenylene iodonium, DPI) and ROS inhibitor 49 (NAC)145,148 reduced intracellular ROS levels that inhibited IL-1β secretion in SK-N-MC cells and phagocytes isolated from both healthy volunteers and CGD patients (Figure 16). These results suggest that: i) increased intracellular ROS is strongly associated with NLRP3 inflammasome activation, and ii) compound 48 reduces ROS levels independently of its well characterised target, NOX. Indeed, compound 48 is beginning to be recognised as an inhibitor of NLRP3 priming by blocking the expression of pro-caspase-1, pro-IL-1β148 and NLRP360,149 at a concentration between 1-10 µM depending on cell type. Therefore the use of ROS inhibitors to demonstrate the interplay between ROS and activation of the inflammasome should be used with caution. Liao and colleagues further demonstrate that 48 inhibits IᴋB phosphorylation upstream of IKK/NF-ᴋB.149 It is debatable if 48 also suppresses NLRP3 activation in addition to NLRP3 expression.60,145,149,150 Surprisingly, one study suggests that 48 induces de novo pro-IL1β/NLRP3 translation, generates intracellular ROS production and exacerbates NLRP3-induced IL-1β secretion by disrupting mitochondrial membrane potential.151 It is clear that the mode of action of 48 and its ability to affect NLRP3 inflammasome priming and activation needs clarification but in general, studies appear to support a beneficial role of 48 in decreasing IL-1β secretion. Blocking signalling via lysosomal destabilisation


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It has been reported that the lysosomal cysteine protease cathepsin B is partially responsible for

crystalline

particle-induced

NLRP3

inflammasome

activation

via

lysosomal

destabilization.11,15,18,152 In this regard, it is likely that cathepsin B release into the cytosol presents an endogenous danger signal that directly or indirectly targets NLRP3 inflammasome activation.153 However, some studies suggest that NLRP3 inflammasome activation by cathepsin B release is independent of lysosomal destabilization.154 Nevertheless, the cathepsin B inhibitor 50 (Ca-074Me)15 significantly suppresses NLRP3 activation

154-157

(Figure 16) in addition to

NLRP1 inflammasome activation158 and may represent another potential therapeutic strategy to prevent excessive IL-1β release. Syk inhibitors The tyrosine kinase Syk is an essential modulator of NLRP3 inflammasome activation in both the priming step that leads to CARD9-dependent NF-κB-induced pro-IL-1β synthesis during fungal challenge and the activation step required for IL-1β processing in response to fungal or parasite pathogens.159,160 Mechanistically, Syk has been suggested to activate the NLRP3 inflammasome by triggering ROS production159 and phosphorylating ASC that is critical for promoting ASC oligomerisation and procaspase-1 recruitment.161 Importantly, Syk deletion or use of the Syk small molecule inhibitor 51 (R406)159 at 1 µM concentration inhibits both Candida albicans-stimulated pro-IL-1β synthesis and LPS+C.albincans-induced IL-1β/18 release in murine bone marrow-derived dendritic cells (BMDCs). Oral administration of 51 or 52 (R788)162, the phosphate pro-drug of 51 (Scheme 6), significantly prevented the progression and manifestations associated with the collagen-induced arthritis model of RA and prevented IL1β/18 expression in the synovial fluid at a dose of 30 mg/kg in rats. An initial phase II clinical trial demonstrated excellent efficacy of 52 in active RA patients receiving methotrexate



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compared to placebo receiving methotrexate.163 However, Astrazeneca discontinued the use of 52 for the treatment of RA in 2013 due to mixed efficacy in further phase II/III trials and observed adverse effects such as hypertension and gastrointestinal side effects. Despite this, further Syk inhibitors are currently being developed such as 53 (GS-9973)164 which is highly potent and selective for Syk (IC50 = 7.7 nM) and possesses favorable pharmacokinetic properties (Figure 17). Compound 53 is currently in clinical trials for autoimmune indications including RA. IRAK1/4 inhibitors As discussed previously, two signals (priming and activation) are required for NLRP3 inflammasome activation. However, two independent studies have recently uncovered a TLRMyd88-dependent pathway that causes early phase, acute NLRP3 inflammasome activation independent of priming.165,166 Interleukin-1 receptor-associated kinase 1/4 (IRAK1/4) were identified as key modulators of this rapid non-transcriptional activation of NLRP3 and IRAK1/4 knock-out BMDMs show significantly disrupted Listeria monocytogenes-induced or LPS+ATPstimulated early phase NLRP3 activation.165,166 The kinase activity of IRAK4 is required for the activation of IRAK1 as a IRAK4 kinase dead mutant cannot induce the rapid NLRP3 inflammasome activation pathway.166

Interestingly, immunoprecipitation experiments

investigating IRAK1 binding partners in addition to IRAK1 knock-out studies reveal that IRAK1 specifically associates with NLRP3 via direct binding to ASC and prevents both the relocalisation and oligomerisation of ASC. Additionally, IRAK1 knock-out BMDMs blocked the release of high mobility group box 1 (HMGB1) that is indicative of preventing pyroptosis.165 IL18 release is also prevented in IRAK1 knock-out BMDMs in vitro in response to LPS+ATP stimulus that is independent of pro-IL-18 expression and in vivo when L. monocytogenes is


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injected into the peritoneal cavity in mice.166 Importantly, the IRAK1/4 inhibitor 54 (IRAK1/4Inh)166 at 5 µM concentration abolished NLRP3 speck formation, co-localization of IRAK1 with NLRP3 and caspase-1 cleavage in unprimed cells (Figure 17). Despite few specific inhibitors of IRAK1 that currently exist, a number of potent and selective small molecule inhibitors for IRAK4 have been developed.167 It would be interesting to evaluate some of the new generation IRAK4 inhibitors as small molecule therapeutics against early phase, acute NLRP3 inflammasome activation. Conclusions Since the discovery of the inflammasome in 2002,168 at least 20 small molecules have been identified as inflammasome inhibitors (Table 1). Importantly, they could be protective in a number of sterile inflammatory diseases including, but not limited to, acute myocardial infarction, AD, colitis, cystitis, gout, RA and type II diabetes. Activation of the NLRP3 inflammasome is a strong indicator of sterile inflammation in these diseases and is therefore an important therapeutic target. Small molecule inhibitors of NLRP3 are likely to be protective in other non-communicable diseases involving sterile inflammation. Additionally, NLRP3 inflammasome inhibitors will block IL-1β, IL-18 and caspase-1-mediated pyroptosis in response to sterile DAMPs and are therefore considered more effective anti-inflammatory drugs than biologic agents in pathologies involving IL-18 and pyroptosis.31,169 It is increasingly evident that many of these inhibitors not only target the inflammasome directly but can also act as NF-κB inhibitors. The NF-κB pathway presents another major inflammatory cascade whose activation induces NLRP3 and pro-IL-1β gene expression and is required to prime the cell before encountering a DAMP stimulus.60,170 Thus some inflammasome inhibitors have a dual mode of action, targeting both the expression of NLRP3 protein governed



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by NF-κB and NLRP3 inflammasome formation directly. On one hand, inhibitors that act on two pathways rather than one maybe more effective therapeutics, requiring lower drug doses (and thus reducing risk of toxicity) as it presents an opportunity to simultaneously downregulate immature pro-IL-1β and NLRP3 expression.171 On the other hand, NF-κB regulates a host of other essential cellular processes beyond expression of NLRP3, pro-IL-1β and pro-caspase-1; therefore selectivity may become an issue. When investigating inflammasome biology, it is essential to selectively probe the NLRP3 inflammasome in order to fully appreciate its role, as well as the importance of other known and putative interacting proteins that regulate NLRP3, during sterile inflammation. Therefore the use of such small molecules in molecular biology to study the inflammasome such as NLRP3 should be used with caution. The predominant mode of action of current small molecule inflammasome inhibitors is reversible or irreversible cysteine modification. Two possibilities for cysteine modification exist: thiol(ate) nucleophilic substitution at a central electrophilic atom, such as the Au atom in compound 9 or a Michael reaction with an activated alkene such as compound 16. There is an interest in Michael acceptors as target covalent modifiers for the inflammasomes and a recent study demonstrated that this type of inflammasome inhibitor can efficiently block caspase-1driven pyroptosis.172 However, current inflammasome inhibitors target catalytically important cysteine residues in a non-specific manner, leading to off-target activities and raises issues concerning cytotoxicity. We believe that the future medicinal chemistry of useful drugs lies with a good structural understanding of NLRP3 and the generation of more ‘drug-like’ target covalent modifiers using a structure-based drug design approach. Such NLRP3 inhibitors that covalently target unconserved, suitably-positioned and accessible nucleophiles are likely to give high potency, selectivity and extended duration of action. The crystal structure of human NLRP3


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PYD has been solved and reveals that the unexpected Cys-8 and Cys-108 residues were highly conserved across species and the formation of a disulfide bond between these two cysteine residues could be important in regulating NLRP3 activation via ROS and redox potential.173 Studies such as these entertain the possibility that key, unconserved residues and pockets could be selectively targeted to prevent NLRP3 inflammasome activation. This leads to the related question: will small molecule inflammasome inhibitors that are cysteine modifiers be affected by the redox microenvironment? Indeed, cysteine modifying agents could also react with molecules associated with regulating redox such as glutathione, and the inhibitory activity of compound 17 was significantly reduced when the concentration of free cellular glutathione was increased.174 Regulation of the local redox microenvironment is essential for the secretion of proinflammatory cytokines including IL-1β.175 Therefore it is important to consider the effect of inflammasome inhibitors on the redox state of the cell, as well as their ability to suppress IL-1β processing and its release. IL-1β is an important mediator of macrophage and neutrophil recruitment to sites of infection, and its release is essential for neutrophil-mediated bacterial clearance in vivo.176-178 However, there is currently no evidence to indicate that the blockade of NLRP3 with small molecules is an improvement to biologic agents in terms of reducing the risk of infection. Indeed, it is documented that anakinra can increase the susceptibility of patients to serious life-threatening infections that require intravenous antibiotics and hospitalisation.179-181 Although inactivation of NLRP3 is unlikely to lead to complete blockade of IL-1β, administration of an NLRP3 inhibitor could still result in an increased risk of infection and should be carefully monitored during clinical trials. Ultimately, a consideration of the benefits and drawbacks of treatment with an



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NLRP3 inflammasome inhibitor is essential and the cost-benefit of treatment will be patientdependent. Whilst researchers have been synthesising molecules that target IL-1β release for some time,143 the identification of the inflammasome168 has focused the search around molecules that selectively target NLRP3. Whilst the field is still relatively young, this review highlights a number of chemical entities that block NLRP3 activation. This offers great promise for the future development of NLRP3 inhibitors where these molecules may act as scaffolds for further chemical refinement and optimization. Thus the design of inhibitors such as those reviewed herein could constitute an important class of anti-inflammatory drugs for the treatment of many non-communicable, sterile diseases involving excessive IL-1β release.


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AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed: Sally Freeman, Manchester Pharmacy School, Faculty of Medical and Human Sciences, The University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK Tel: 44-161-275-2366; Email: [email protected] Notes The authors declare no competing financial interest. Biographies Alex G. Baldwin received his B.Sc in Biological and Medicinal Chemistry from the University of Exeter, U.K., in 2013. Alex is the recipient of two scholarships and nine awards, including the Society of Biology Top Student Award and President’s Doctoral Scholar Award. Alex is currently studying for his Ph.D. in the Manchester Pharmacy School at the University of Manchester. Alex’s current work focuses on the design, synthesis and evaluation of novel inhibitors of the NLRP3 inflammasome. David Brough received his B.Sc in Biosciences and Chemistry (1999) from the Robert Gordon University, Aberdeen, and Ph.D in Biological Sciences (2002) from the University of Manchester. Following postdoctoral positions at the Universities of Cambridge, and then Manchester, David was awarded a Fellowship from the Wellcome Trust (2008) to establish research independence. Since 2013 David is a Lecturer at the University of Manchester.



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Sally Freeman received her B.Sc in Chemistry (1982) and Ph.D. (1985) from the Department of Chemistry at the University of Leicester, U.K., prior to post-doctoral studies with Professor Jeremy Knowles at Harvard University. Sally was a lecturer in the Aston Pharmacy School, Aston University, from 1987, moving to the Manchester Pharmacy School at the University of Manchester in 1995, where she is currently a Reader in Medicinal Chemistry. ACKNOWLEDGMENT The authors would like to thank the Manchester Pharmacy School and University of Manchester Presidential Doctoral Scholar award for financial support to A.G.B.


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ABBREVIATIONS IL-1(β), interleukin-1(β); PAMP, pathogen-associated molecular pattern; DAMP, damageassociated molecular pattern; PRR, pattern recognition receptor; LPS, lipopolysaccharide; ASC, apoptosis-associates speck-like protein containing a caspase recruitment domain; NLRP1-12, NOD-like receptor family, pyrin domain-containing protein 1-12; NLRC4, NOD-like receptor family, caspase recruitment domain-containing protein 4; AIM2, absent in melanoma 2; IFI16, interferon gamma-inducible protein 16; RIG-1, retinoic acid-inducible gene 1; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TLR, toll-like receptor; P2X7R, P2X7 receptor; MSU, monosodium urate; ROS, reactive oxygen species; AD, Alzheimer’s disease; APP/PS1, amyloid precursor protein/presenilin 1 mutant; IL-1Ra, IL-1 receptor antagonist; BBB, blood-brain barrier; CSF, cerebrospinal fluid; MyD88, myeloid differentiation primary response gene 88; Ub, ubiquitination; TAK1, transforming growth factor beta-activated kinase 1; TAB1-3, TAK1-binding proteins (TAB) 1-3; IκB, inhibitor of kappa B; IKK, IκB kinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NOD-like receptor family, pyrin domain-containing protein 3; LRR, leucine-rich repeat; NACHT, nucleotide-binding and oligomerisation; PYD, pyrin domain; CARD, caspase activation and recruitment domain; ASC, apoptosis-associates speck-like protein containing a CARD; DUBs, deubiquitinating enzymes; BHB, β-hydroxybutryrate; MNS, 3,4-methylenedioxy-β-nitrostyrene; 7-DG, 7-desacetoxy-6,7dehydrogedunin; KATP channel, ATP-sensitive potassium channel; SUR2, sulfonylurea receptor; IAPP, islet amyloid polypeptide; CP, cyclophosphamide; CAPS, cyropyrin-associated periodic syndrome; MWS, Muckle-Wells syndrome; FCAS, familial cold autoinflammatory syndrome; NOMID, neonatal onset multisystem inflammatory disease; CRIDs, cytokine release inhibitory drugs; CTL, cytotoxic T lymphocyte; BMDM, bone marrow-derived macrophage, HMDM, human monocyte-derived macrophage; PBMC, peripheral blood mononuclear cell; EAE,



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experimental autoimmune encephalomyelitis; RA, rheumatoid arthritis; TNF-α; tumor necrosis factor-alpha; AP-1, activator protein 1; IRF3, interferon regulatory factor 3; Cys, cysteine; DTT, dithiothreitol; LT, lethal toxin; NAC, N-acetyl-L-cysteine; DPI, diphenylene iodium; HDAC, histone deacetylase; MNS, 3,4-methylenedioxy-β-nitrostyrene; Syk, spleen tyrosine kinase; DSS, dextran sulfate sodium; PKR, protein kinase R; SNP, single nucleotide polymorphism; ABC transporter, ATP-binding cassette transporter; NOX, NADPH oxidase; CGD, chronic granulomatous disease; Cmax, maximum serum concentration.


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170. Cogswell, J. P., Godlevski, M. M., Wisely, G. B., Clay, W. C., Leesnitzer, L. M., Ways, J. P. & Gray, J. G. NF-κB regulates IL-1β transcription through a consensus NF-κB binding site and a nonconsensus CRE-like site. J. Immunol. 1994, 153, 712-723. 171. Small, B. G.; McColl, B. W.; Allmendinger, R.; Pahle, J.; Lopez-Castejon, G.; Rothwell, N. J.; Knowles, J.; Mendes, P.; Brough, D.; Kell, D. B. Efficient discovery of anti-inflammatory small-molecule combinations using evolutionary computing. Nat. Chem. Biol. 2011, 7, 902-908. 172. Cocco, M.; Garella, D.; Di Stilo, A.; Borretto, E.; Stevanato, L.; Giorgis, M.; Marini, E.; Fantozzi, R.; Miglio, G.; Bertinaria, M. Electrophilic warhead-based design of compounds preventing NLRP3 inflammasome-dependent pyroptosis. J. Med. Chem. 2014, 57, 10366-10382. 173. Bae, J. Y.; Park, H. H. Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly. J. Biol. Chem. 2011, 286, 39528-39536. 174. Nobel, C. S. I., Kimland, M., Nicholson, D. W., Orrenius, S. & Slater, A. F. G. Disulfiram is a potent inhibitor of proteases of the caspase family. Chem. Res. Toxicol. 1997, 10, 1319-1324. 175. Haddad, J. J. A redox microenvironment is essential for MAPK-dependent secretion of pro-inflammatory cytokines: modulation by glutathione (GSH/GSSG) biosynthesis and equilibrium in the alveolar epithelium. Cell Immunol. 2011, 270, 53-61. 176. Rider, P.; Carmi, Y.; Guttman, O.; Braiman, A.; Cohen, I.; Voronov, E.; White, M. R.; Dinarello, C. A.; Apte, R. N. IL-1α and IL-1β recruit different myeloid cells and promote different stages of sterile inflammation. J. Immunol. 2011, 187, 4835-4843.



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177. Miller, L. S.; O'Connell, R. M.; Gutierrez, M. A.; Pietras, E. M.; Shahangian, A.; Gross, C. E.; Thirumala, A.; Cheung, A. L.; Cheng, G.; Modlin, R. L. MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus. Immunity 2006, 24, 79-91. 178. Miller, L. S.; Pietras, E. M.; Uricchio, L. H.; Hirano, K.; Rao, S.; Lin, H.; O'Connell, R. M.; Iwakura, Y.; Cheung, A. L.; Cheng, G.; Modlin, R. L. Inflammasome-mediated production of IL-1β is required for neutrophil recruitment against Staphylococcus aureus in vivo. J. Immunol. 2007, 179, 6933-6942. 179. Galloway, J. B.; Hyrich, K. L.; Mercer, L. K.; Dixon, W. G.; Watson, K. D.; Lunt, M.; BSRBR Control Centre Consortium; Symmons, D. P.; on behalf of the British Society for Rheumatology Biologics Register. The risk of serious infections in patients receiving anakinra for rheumatoid arthritis: results from the British Society for Rheumatology Biologics Register. Rheumatology 2011, 50, 1341-1342. 180. Brassard, P.; Kezouh, A.; Suissa, S. Antirheumatic drugs and the risk of tuberculosis. Clin. Infect. Dis. 2006, 43, 717-722. 181. Salliot, C.; Dougados, M.; Gossec, L. Risk of serious infections during rituximab, abatacept and anakinra treatments for rheumatoid arthritis: meta-analyses of randomised placebo-controlled trials. Ann. Rheum. Dis. 2009, 68, 25-32. 182. Monteleone, M.; Stow, J. L.; Schroder, K. Mechanisms of unconventional secretion of IL-1 family cytokines. Cytokine 2015, 74, 213-218.


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TABLE OF CONTENTS GRAPHIC



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FIGURES

Figure

1.

NLRP3

priming

through

the

TLR4-MyD88

signaling

pathway.

Macrophages/microglia are primed by PAMP/DAMP stimulation of a toll-like receptor (TLR) to express

pro-IL-1β

and

NLRP3

in


an

inactive

state.

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Figure 2. NLRP3-dependent IL-1β release during sterile inflammation. 1. A DAMP signal induces recruitment of ASC and procaspase-1. 2. Recruitment causes oligomerisation and NLRP3 inflammasome formation. 3. Autoactivation induces procaspase-1 cleavage and caspase1 release. 4. Activated caspase-1 cleaves pro-IL-1β into mature IL-1β. 5. IL-1β is secreted in a caspase-1-dependent manner extracellularly through an as yet agreed mechanism.182



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Figure 3. Structures of NLRP3 inhibitors 1/3 and anion exchange inhibitor 2.

Figure 4. Structures of IL-1β processing inhibitors 4-7 and NLRP3 inhibitor 8.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cl Me

Me O

O Me

O O

Me O

P

O O

Au S

Cl Au Cl N

P Au

Me

N

N Me

N

N

O O

N H

Me R5 9

N

R5

R3 R4

N H

Cl

10

N N

R3 R4 11

Figure 5. Structure of NLRP1/NLRP3 inhibitor 9 and N6-benzyladenine analogues 10-11.

Figure 6. SAR summary of 13.

Figure 7. Structures of NLRP3 inhibitors 14-15.



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Figure 9. Structures of caspase-1 inhibitors 17-18.

Figure 10. Structure of TLR4 antagonist 23.


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Figure 12. Structure of IKK inhibitor 30.

Figure 13. Structures of DUB inhibitors 31-35.



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Page 68 of 74

Figure 14. Structures of P2X7R antagonists 36-39 and pannexin-1 inhibitor 40.

S O

Me

O

Cl OH

Cl

Me

Cl

O

O

Me

HO NH 2

O

O

O O

41

OMe

Me

N O

Me

O

CN

N

OH

H N

NO2 43

42

44 Me

H N

Me MeO

OEt O

O

Me

O

O

O O 2N

OH

Me

O O

O

O

O O

O

Me O

N O O

N H

O O

N

Me O

O

O

NO2 45

O

46

47

Figure 15. Structures of ABC anion transporter inhibitors 41-43, Ca2+ channel blockers 44-45, Cl- channel blocker 46 and intracellular Ca2+ chelator prodrug 47.


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Figure 16. Structures of ROS inhibitors 48-49 and cathepsin B inhibitor 50.

Figure 17. Structures of selective Syk inhibitor 53 and IRAK1/4 inhibitor 54.



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SCHEMES

Scheme 1. Mechanism of action of NLRP1/NLRP3 inhibitor 12 via Michael addition. 80

Scheme 2. Mechanism of action of NLRP3 inhibitor 13 via Michael addition.79

Scheme 3. Proposed mechanism of action of NLRP3 inhibitor 16 via Michael addition.


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Scheme 4. Metabolism of caspase-1 prodrugs (A) 19 into 20 and (B) 21 into 22.99,100

Scheme 5. Mechanism of action of TAK1 inhibitor 25 via Michael addition.113



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Scheme 6. Metabolism of Syk prodrug 52 into 51.183


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TABLES Compound

IC50 (µM, IL1β release)

1

Target(s)

Mechanism of action

Reference

12 ± 5

NLRP3 (indirectly)

Prevents ASC oligomerisation, independent of KATP channels

36,40

3

-

NLRP3 (indirectly), ASC?

Prevents ASC oligomerisation

50,51

4

0.21 ± 0.06

NLRP3 (indirectly)

n/a, reversible inhibitor

36

5

0.26 ± 0.05

NLRP3 (indirectly)

n/a, reversible inhibitor

36

6

0.35

GSTO 1-1

Cysteine modification, blocks NLRP3 activation

36,52

7

0.25

GSTO 1-1


36,52

8

0.0075

NLRP3

Prevents ASC oligomerisation

53

-

NLRP1b, NLRP3, TLR4, IKK, TrxR,

Cysteine modification, blocks NLRP3 priming

58,63,64,69

2.59

NLRP1, NLRP3, caspase-1, IKK, NF-κB

Cysteine modification, inhibits NLRP3 ATPase activity

77,78,81,82

13

-

NLRP3, IKK, E2/3 enzymes, PTPs

Cysteine modification, inhibits NLRP3 ATPase activity

77,79,83,84

14

-

NLRP3 (indirectly)

Prevents K+ efflux, independent of its known targets

92

15

-

NLRP3 (indirectly)

Prevents autocleavage of procaspase1

93

16

~2

NLRP3

Cysteine modification? Inhibits NLRP3 ATPase activity

94

19

0.42

Caspase-1

Blocks pro-IL-1β cleavage

99

20

0.67 ± 0.55

Caspase-1

Blocks pro-IL-1β cleavage

100

23

0.0057

TLR4


103-105

24

~0.17

Upstream of IKK

Cysteine modification? Blocks NLRP3 priming

103

9

12



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Page 74 of 74

25

-

TAK1


103,113

30

-

PKR

Cysteine modification?, prevents PKR-IKK and PKR-ASC interactions

114

31-35

-

DUBs


17,117,118

36-39

-

P2X7R

Prevents K+ efflux, blocks NLRP3 activation

125-128

40

-

Panx1?

Blocks NLRP1-3 activation

134-136,138-140

2, 41-47

-

Various ion channels, Ca2+

Prevents cytosolic fluxes, blocks NLRP3 activation

13,143,144

48-49

-

NOX, ROS

Blocks NLRP3 priming

60,145,148

50

-

Cathepsin B

Blocks lysosomal destabilisationinduced NLRP3 inflammasome activation?

15,154-156

51, 53

-

Syk

Blocks ROS synthesis, prevents ASC oligomerisation

159,161,164

54

-

IRAK1/4

Blocks NLRP3 priming, prevents ASC oligomerisation

165,166

Table 1. Current small molecule inhibitors of the NLRP3 inflammasome.


Inhibiting the Inflammasome: A Chemical Perspective - Journal of

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