Inhibiting the NLRP3 Inflammasome

molecules

Review Inhibiting the NLRP3 Inﬂammasome

Lina Y. El-Sharkawy 1, David Brough 2 and Sally Freeman 1,*

1 Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK; [email protected] 2 Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, AV Hill Building, Oxford Road, Manchester M13 9PT, UK; [email protected] * Correspondence: [email protected]

Academic Editor: Massimo Bertinaria Received: 9 October 2020; Accepted: 23 November 2020; Published: 25 November 2020

Abstract: Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1β and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1β and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches.

Keywords: inﬂammation; NLRP3; inﬂammasome; cytokine; cryo-EM; drug discovery; medicinal chemistry

1. Introduction Since our 2015 review titled “Inhibiting the Inflammasome: A Chemical Perspective” [1] there have been substantial developments in inflammasome biology and chemistry, with inflammasome inhibitors being tested in preclinical models for the treatment of several inflammatory disorders and entering clinical trials [2–6]. This review focuses on the design and development of small molecule inflammasome inhibitors, with attention on the mechanism of direct NLRP3 (NACHT, LRR, and PYD Domains-Containing Protein 3) inhibitors. We also discuss the potential of the published cryo-EM structure of NLRP3 bound to NEK7 (NIMA Related Kinase 7) [7] to further develop direct small molecule inhibitors of the inflammasome.

2. Inflammasomes and Innate Immunity Inflammation is generally regarded as a protective mechanism in case of infection or injury by the removal of toxins by immune cells. Non-sterile inflammation occurs from either parasites, fungi, viruses or bacteria. Sterile inflammation results from injury, foreign bodies, or hypersensitivity. Under normal conditions, immune cells are found circulating in the blood. However, upon recognition of toxins by host cells, immune cells, and their components are recruited to remove the damaging agents and promote tissue repair. Receptors that recognize pathogen-associated molecular patterns

Molecules 2020, 25, 5533; doi:10.3390/molecules25235533 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 14

Molecules 2020, 25, 5533 2 of 14 damaging agents and promote tissue repair. Receptors that recognize pathogen-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) are located on innate immune (PAMPs)cells and are and termed damage pattern associated recognition molecular receptors patterns (PRRs). (DAMPs) NOD- areLike located receptors on innate(NLRs) immune are cytosolic cells andPRRs are detecting termed patternboth PAMP recognition and DAMP receptors signals. (PRRs). Some NOD-Like NLRs form receptors an inflammatory (NLRs) are complex cytosolic called PRRs detectingan inflammasome both PAMP [8]. This and review DAMP focuses signals. on Somethe inhibition NLRs form of the an inflammasome inflammatory formed complex by called the NLR an inflammasomeNLRP3, a key protein [8]. This involved review in focuses sterile oninflammation the inhibition. NLRP3 of the is inflammasome a drug target of formed significant by the interest NLR NLRP3,to the pharmaceutical a key protein involved industry, in with sterile potential inflammation. for the NLRP3treatment isa of drug several target inflammatory of significant diseases interest toincluding the pharmaceutical cancer [9], autoimmune industry, with diseases potential [10], metabolic for the treatment disorders of [11] several, cardiovascular inflammatory disease diseases [12], includingCNS diseases cancer [13] [9, ],and autoimmune infectious diseases diseases including [10], metabolic COVID disorders-19 [14]. [11], cardiovascular disease [12], CNS diseases [13], and infectious diseases including COVID-19 [14]. 3. The NLRP3 Inflammasome 3. The NLRP3 Inflammasome The activation of the canonical NLRP3 inflammasome pathway requires two stages. The first is the primingThe activation stage which of the involves canonical myeloid NLRP3 differentiation inflammasome primary pathway response requires 88 two(MYD88), stages. and The Nuclear first is theFactor priming kappa stage-light which-chain involves-enhancer myeloid of activated differentiation B cells primary (NF-κB) response signaling 88 (MYD88), responsible and Nuclear for the Factorexpression kappa-light-chain-enhancer of NLRP3 and pro-IL- of1β activated [15]. Under B cells normal (NF-κ B) conditions signaling the responsible leucine-rich for the repeat expression (LRR) ofmaintains NLRP3 andthe NLRP3 pro-IL-1 inβ [an15 ].auto Under-repressed normal state conditions [15]. However, the leucine-rich in response repeat to (LRR) a second maintains PAMP the or NLRP3DAMP stimulus in an auto-repressed NLRP3 becomes state active [15]. However,in the secon ind response stage. The to pyrin a second domain PAMP (PYD) or DAMPof active stimulus NLRP3 NLRP3now binds becomes to the active PYD indomain the second of ASC stage. (apoptosis The pyrin-associated domain(PYD) speck- oflike active protein NLRP3 containing now binds a CARD to the PYD(caspase domain recruitment of ASC (apoptosis-associated domain)) resulting speck-like in a helical protein assembly containing of a CARD ASC (caspase to form recruitment the ASC domain))speck/pyroptosome. resulting in Afterwards a helical assembly, CARD- ofCARD ASC tointeraction form the betweenASC speck ASC/pyroptosome. and procaspase Afterwards,-1 takes CARD-CARDplace. Procaspase interaction-1 yields between caspase- ASC1 via andits own procaspase-1 proteolytic takes cleavage place. which Procaspase-1 subsequently yields caspase-1catalyzes viathe itscleavage own proteolytic of pro-IL cleavage-1β and pro which-IL- subsequently18 to their active catalyzes forms the which cleavage cause of inflammation pro-IL-1β and ( pro-IL-18Figure 1) to[16] their. Caspase active-1 formsalso leads which to a cause type inflammationof programmed (Figure cell death1)[ 16 known]. Caspase-1 as pyroptosis also leads resulting to a from type ofgasdermin programmed D (GSDMD) cell death cleavage. known GSDMD as pyroptosis binds resultingto lipids fromin the gasdermin cell membrane D (GSDMD) resulting cleavage. in pore GSDMDformation, binds with to cellular lipids in swelling the cell membrane and rupture resulting releasing in pore inflammatory formation, cytokines with cellular IL-1β swelling and IL and-18 rupture(Figure 1) releasing [17]. inflammatory cytokines IL-1β and IL-18 (Figure1)[17].

Figure 1. 1. PrimingPriming and and secondary secondary signals signals for NLRP3 for NLRP3 activation activation and its androle itsin inflammation. role in inflammation. Primary Primarysignal includes signal activation includes activationof Nuclear ofFactor Nuclear kappa Factor-light- kappa-light-chain-enhancerchain-enhancer of activated B of cells activated (NF-κB) B cells (NF-κB) mediated by myeloid differentiation primary response 88 (MYD88) by binding of P mediated by myeloid differentiation primary response 88 (MYD88) by binding of P pathogen- pathogen-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) to TLR, to TLR, and its translocation to the nucleus to undergo transcription and translation of NLRP3 and its translocation to the nucleus to undergo transcription and translation of NLRP3 inflammasome inflammasome components as well as pro-inflammatory cytokines. Secondary signals which result in components as well as pro-inflammatory cytokines. Secondary signals which result in NLRP3 NLRP3 inflammasome activation include ROS from mitochondrial disruption, cathepsins, VRAC efflux inflammasome activation include ROS from mitochondrial disruption, cathepsins, VRAC efflux of of anions and osmolytes and Ca2+ influx. Oligomerization and activation of NLRP3 inflammasome anions and osmolytes and Ca2+ influx. Oligomerization and activation of NLRP3 inflammasome results in cleavage of pro-inflammatory cytokines proIL-1β and proIL-18 through the action of results in cleavage of pro-inflammatory cytokines proIL-1β and proIL-18 through the action of caspase-1 to give the mature forms inflammatory cytokines interleukin (IL)-1β and IL-18, which cause caspase-1 to give the mature forms inflammatory cytokines interleukin (IL)-1β and IL-18, which cause inflammation. This figure was created under license using BioRender.com. https://biorender.com/. inflammation. This figure was created under license using BioRender.com. https://biorender.com/. Molecules 2020, 25, x FOR PEER REVIEW 3 of 14

4. Structure of NLRP3 and Cryo-EM Structure of NLRP3 Bound to NEK7 NLRP3 is composed of the nucleotide binding oligomerization domain (NOD, also called the NACHT domain), a pyrin domain (PYD) at the amino terminal and a leucine-rich repeat (LRR) at the carboxy terminal. The NACHT domain (ATPase domain) of NLRP3 is composed of the nucleotide binding domain (NBD), helical domains HD1 and HD2 and a winged helix domain (WHD) in Molecules 2020, 25, 5533 3 of 14 between HD1 and HD2 (Figure 2a) [7]. The cryo-EM structure of human NLRP3 bound to an artificially engineered dimer of NIMA (never4. Structure in mitosis of NLRP3 gene a)- andrelated Cryo-EM kinase 7 Structure (NEK7) (EMD of NLRP3-0476, BoundPDB-6NPY, to NEK7 resolution 3.8 Å) has been reported (Figure 2b) [7]. The serine/threonine kinase NEK7 is important for NLRP3 activation [7] and is shownNLRP3 to bind is composed through its of C the-terminal nucleotide lobe to binding NLRP3. oligomerization ATP is required domainfor NLRP3 (NOD, inflammasome also called the activation:NACHT domain), ATP binds a pyrinto the domainnucleotide (PYD) binding at the domain amino (NBD) terminal of NLRP3 and a leucine-rich and is hydrolyzed repeat to (LRR) ADP at the bycarboxy the ATPase terminal. function The (Figure NACHT 2c) domain[18,19]. The (ATPase energy domain) released offrom NLRP3 either isbinding composed and/or of hydrolysis the nucleotide ofbinding ATP domain is proposed (NBD), to helical induce domains a conformationally HD1 and HD2 modified and a winged protein, helix important domain (WHD) for NLRP3 in between inflammasomeHD1 and HD2 oligomerization (Figure2a) [7]. and activation (Figure 2d) [7].

(a)

(b) (c)

(d)

FigureFigure 2. 2. (a()a Structural) Structural domains domains of ofNLRP3; NLRP3; (b) (cryob) cryo-EM-EM structure structure of NLRP3 of NLRP3 with withbound bound ADP (green) ADP (green) (PDB:(PDB: 6NPY) 6NPY) [7] [7 ]focusing focusing on on the the active active site site of NLRP3 of NLRP3 with with ADP ADP bound bound in the in NACHT the NACHT domain domain (ribbon (ribbon diagram);diagram); (c ()c )Nucleotide Nucleotide Binding Binding Domain Domain (NBD) (NBD) with with the theresidues residues surrounding surrounding ADP ADPlabelled labelled (F506, (F506, EE150,150, I232, I232, G227, G227, Y166, Y166, R165, R165, T167, T167, W414, W414, C417). C417). Yellow Yellow dashed dashed lines represent lines represent hydrogen hydrogen bonds; (d bonds;) Steps(d) Steps involved involved in NLRP3 in NLRP3 activation activation and oligomerization and oligomerization:: (1) NEK7 (1) binding NEK7, binding,(2) ATP bind (2) ATPing and binding hydrolysisand hydrolysis with withinduced induced conformational conformational change change between between the NBD the NBDand HD2, and HD2,(3) dimerization (3) dimerization of of NLRP3NLRP3-NEK7-NEK7 activated activated complex, complex, ( (4)4) oligomerization of of the NLRP3-NEK7NLRP3-NEK7 complex with ASC andand pyrin pyrinto give to thegive NLRP3 the NLRP3 inﬂammasome, inflammasome, (5) cleavage (5) cleavage of procaspase-1 of procaspase to-1 mature to mature caspase-1. caspase- NLRP31. NLRP3 protein proteinimages images drawn drawn using PyMOLusing PyMOL from PDB:from PDB: 6NPY. 6NPY.

The cryo-EM structure of human NLRP3 bound to an artificially engineered dimer of NIMA (never in mitosis gene a)-related kinase 7 (NEK7) (EMD-0476, PDB-6NPY, resolution 3.8 Å) has been reported (Figure2b) [ 7]. The serine/threonine kinase NEK7 is important for NLRP3 activation [7] and is shown to bind through its C-terminal lobe to NLRP3. ATP is required for NLRP3 inflammasome activation: ATP binds to the nucleotide binding domain (NBD) of NLRP3 and is hydrolyzed to ADP by the ATPase function (Figure2c) [ 18,19]. The energy released from either binding and/or hydrolysis of ATP is proposed to induce a conformationally modified protein, important for NLRP3 inflammasome oligomerization and activation (Figure2d) [7]. Molecules 2020, 25, 5533 4 of 14

It is of interest to note that two of the ﬁve conserved motifs found in NLRP1-14NACHT include Walker A and Walker B [19], which are protein sequences found in ATP-requiring enzymes, initially described by Walker and co-workers [20]. Walker A (P loop), characterized by the sequence G-xxxx-GK-[S/T] (x: Any amino acid), forms part of the ATP binding site responsible for ATPase activity, with the lysine residue interacting with the γ-phosphate through hydrogen-bonding [21,22].

5. Role of NEK7 in Inflammasome Activation NEK7 is a ser/thr kinase that was found to be important for NLRP3 activation (PDB: 6NPY) [7]. To further elucidate the requirement of NEK7 for NLRP3 activation, a knockout experiment was carried out for NEK7 in iBMDMs:mutants prevented nigericin-induced NLRP3 activation whereas wild type NEK7 retained ASC-speck formation, caspase-1 processing and IL-1β release [7,23]. NEK7 was found to bind through its C-terminal lobe to LRR through half of its lobe whilst the other half is associated with the HD2 and NBD regions [7]. NEK7 forms a dimeric complex with NLRP3, proposed to be bound together via a flexible linker [7] (Figure2b,d). NEK7, present in low cellular amounts, binds to NEK9 for cell division and is only bound to NLRP3 in the interphase upon exposure of NLRP3 to primary signals, thus NEK7 is a crucial modulator of inflammasome activation [7,24]. It is important to note that NEK7 alone cannot activate NLRP3: ATP needs to be bound to the NBD domain, which phosphorylates S195 of NEK7 allowing NLRP3 to adopt its active conformation for oligomerization. The active conformer occurs when the NACHT domain rotates away from the HBD1 domain as a rigid body, resulting in a diminished point of contact between NEK7 and the NLRP3 NBD domain (Figure2d) [ 7]. It should be noted however that NEK7 dependence is not always required: When cells engage a Transforming growth factor-β-Activated Kinase 1 (TAK1)-dependent post-translational priming pathway, NEK7 is no longer required for NLRP3 activation [25].

6. Mechanism of Action of NLRP3 Inhibitors: Covalent Modiﬁers of the NACHT Domain Bertinaria and coworkers led the development of α,β-unsaturated “electrophilic warheads” as inﬂammasome inhibitors. Hits included ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate (Figure3) (Table1), which prevented the pyroptosis of THP-1 cells [ 26]. Chemically this Michael acceptor reacted covalently with glutathione (as a model thiol) and was also shown to inhibit NLRP3 ATPase activityMolecules 20 [2620]., 25, x FOR PEER REVIEW 5 of 14

Figure 3. StructuresStructures of direct NLRP3 inhibitors that have the potential to be covalent modiﬁers.modifiers.

NLRP3 inflammasome inhibitors MNS (3,4-methylenedioxy-β-nitrostyrene) (Figure 3) [29], INF39 (ethyl 2-(2-chlorobenzyl)acrylate) (Figure 3) [30], INF58 (Figure 4) [31] and OLT1177 (dapansutrile) (Figure 3) [32] also bind to the ATP binding site. A cysteine residue in the ATP-binding site of the NACHT domain reacts covalently with MNS through a Michael addition. MNS prevents the oligomerization and activation of the NLRP3 inflammasome, inhibiting LPS-induced NLRP3 activation and IL-1β release from mouse BMDMs with an IC50 of 2 µM (Table 1) [29]. INF39 was reported as a non-toxic irreversible NLRP3 inflammasome inhibitor with an IC50 of 10 µM (Table 1) [30]. INF58 inhibited the ATPase activity of the NLRP3 inflammasome with an IC50 of 74 µM, the proposed mechanism being through the covalent reaction of C419 with the Michael acceptor in INF58 (Figure 4) (Table 1) [31]. OLT1177 (dapansutrile) was found to prevent the NLRP3 interaction with ASC through the inhibition of ATPase, resulting in inhibition of IL-1β and IL-18 release from J774 macrophages with an IC50 of 1 nM (Table 1) [32]. Oridonin (Figure 4), an electrophilic natural ent-kaurane diterpenoid derivative obtained from Rabdosia rubescens, showed anti-inflammatory activity with an IC50 of ~0.75 M. In contrast to the other Michael acceptors which inhibit ATPase activity, oridonin forms a covalent link with C279, blocking the interaction between NLRP3 and NEK7 and preventing a conformational change, thus inhibiting NLRP3 inflammasome assembly and activation (Table 1) [28].

Y385 W416 HS C419 C419 S H K232 N H T233 N NH2 Cl O NH2 S Cl O R237 S Y381 O O INF58 O O H520

HS C279 HO OH HO OH O O O O S C279 H HO H HO H H OH OH Oridonin Figure 4. Structures of NLRP3 inhibitors INF58 and oridonin. Covalent bond formation of these inhibitors with cysteine residues in the NACHT domain of NLRP3 [28,31]. Molecules 2020, 25, x FOR PEER REVIEW 5 of 14

Molecules 2020, 25, 5533 5 of 14

CY-09 (Figure3) (Table1), a small molecule identified through screening in an in-house bioactive compound library in mouse bone marrow derived macrophages (BMDMs) against NLRP3, has an IC50 of 6µM. Furthermore, CY-09 causes inhibition of IL-1β and caspase-1 activity in synovial fluid cells fromFigure patients 3. Structures with gout. of direct CY-09 NLRP3 has been inhibitors proposed that have to be the a directpotential covalent to be covalent modifier modifiers. of the NLRP3 inflammasome, preventing its oligomerization by binding to the ATP-binding site of the NACHT domainNLRP3 [27]. CY-09 inflammasome has an α, β inhibitors-unsaturated MNS carbonyl (3,4-methylenedioxy Michael acceptor-β- groupnitrostyrene) which binds(Figure covalently 3) [29], withINF39 a nucleophile(ethyl 2-(2- onchlorobenzyl)acrylate the Walker A site [)27 (],Figure although 3) [30] this, reactionINF58 ( mayFigure be reversible 4) [31] and (see OLT1177 SI [28]). NACHT Computational(dapansutrile) ( dockingFigure 3 and) [32] molecular also bind dynamicsto the ATP simulations binding site. on A NLRP cysteine residueprotein in modelsthe ATP with-binding apo 2+ andsite haloof the ADP- NACHT/ATP-Mg domainwere reacts used covalently to investigate with MNS how through CY-09 inhibits a Michael ATPase addition. and inhibits MNS prevents NLRP3 activationthe oligomerization [27]. and activation of the NLRP3 inflammasome, inhibiting LPS-induced NLRP3 β activationNLRP3 and inflammasome IL-1β release inhibitorsfrom mouse MNS BMDMs (3,4-methylenedioxy- with an IC50 of 2 -nitrostyrene)µM (Table 1) [29] (Figure. INF393)[ was29], INF39reported (ethyl as 2-(2-chlorobenzyl)acrylate)a non-toxic irreversible NLRP3 (Figure inflammasome3)[ 30], INF58 (Figureinhibitor4)[ with31] and an OLT1177IC50 of 10 (dapansutrile) µM (Table 1) (Figure[30]. INF583)[ 32 inhibited] also bind the to the ATPase ATP binding activity site. of Athe cysteine NLRP3 residue inflammasome in the ATP-binding with an IC site50 ofof the74 NACHTµM, the domainproposed reacts mechanism covalently being with through MNS throughthe covalent a Michael reaction addition. of C419 MNSwith the prevents Michael the acceptor oligomerization in INF58 and(Figure activation 4) (Table of 1 the) [31] NLRP3. OLT1177 inflammasome, (dapansutrile) inhibiting was found LPS-induced to prevent NLRP3 the NLRP3 activation interaction and IL-1 withβ releaseASC through from mouse the inhibition BMDMs of with ATPase, an IC 50resultiof 2 µngM in (Table inhibition1)[ 29 ].of INF39 IL-1β wasand reportedIL-18 release as a non-toxicfrom J774 µ irreversiblemacrophages NLRP3 with an inflammasome IC50 of 1 nM ( inhibitorTable 1) [32] with. an IC50 of 10 M (Table1)[ 30]. INF58 inhibited the ATPaseOridonin activity (Figure of the NLRP34), an electr inflammasomeophilic natural with ent an- ICkaurane50 of 74 diterpenoidµM, the proposed derivative mechanism obtained being from throughRabdosia therubescens covalent, show reactioned anti of-inflammatory C419 with the activity Michael with acceptor an IC in50 INF58of ~0.75 (Figure M. In4) contrast (Table1)[ to 31the]. OLT1177other Michael (dapansutrile) acceptors was which found inhibit to prevent ATPase the activity, NLRP3 interactionoridonin forms with ASCa covalent through link the with inhibition C279, ofblocking ATPase, the resulting interaction in inhibition between ofNLRP3 IL-1β andand NEK7 IL-18 releaseand preventing from J774 a macrophagesconformational with change an IC, 50thusof 1inhibiting nM (Table NLRP31)[32]. inflammasome assembly and activation (Table 1) [28].

Y385 W416 HS C419 C419 S H K232 N H T233 N NH2 Cl O NH2 S Cl O R237 S Y381 O O INF58 O O H520

HS C279 HO OH HO OH O O O O S C279 H HO H HO H H OH OH Oridonin FigureFigure 4. 4. StructuresStructures of of NLRP3 NLRP3 inhibitors inhibitors INF58 INF58 and and oridonin. oridonin. Covalent Covalent bond bond formation formation of of these these inhibitorsinhibitors withwith cysteinecysteine residuesresidues inin thethe NACHTNACHT domaindomain of of NLRP3 NLRP3 [ 28[28,31,31].].

Oridonin (Figure4), an electrophilic natural ent-kaurane diterpenoid derivative obtained from Rabdosia rubescens, showed anti-inﬂammatory activity with an IC50 of ~0.75 µM. In contrast to the other Michael acceptors which inhibit ATPase activity, oridonin forms a covalent link with C279, blocking the interaction between NLRP3 and NEK7 and preventing a conformational change, thus inhibiting NLRP3 inﬂammasome assembly and activation (Table1)[28]. Molecules 2020, 25, 5533 6 of 14

Table 1. Direct inhibitors of the NLRP3 ATPase site and their activities.

Inhibitor % Inhibition at 10 µM /IC50 Mechanism of Action Ethyl 2-((2-chlorophenyl) 75.1 2.6% pyroptosis Inhibits NLRP3 ATPase [26] (hydroxy)methyl)acrylate ± CY-09 IC50 = 6 µM Walker A inhibitor [27] MNS IC50 = 2 µM Inhibits NLRP3 ATPase [29] OLT1177 (dapansutrile) IC50 = 1 nM ATPase inhibitor blocks NLRP3-ASC interaction [32] INF39 IC50 = 10 µM Irreversible NLRP3 inﬂammasome inhibitor [30] INF58 IC50 = 74 µM ATPase inhibitor [31] Oridonin IC50 = 0.75 µM ATPase inhibitor blocks NLRP3-NEK7 interaction [28] MCC950/CRID3 IC50 = 7.5 nM Walker B inhibitor [33–35]

7. Mechanism of Action of NLRP3 Inhibitors: MCC950 Binds to Walker B Walker B, an amino acid chain denoted by hLhhh[D/E] (h: Any hydrophobic residue), is present downstream of the Walker A motif. ATP hydrolysis requires a glutamate residue, which together with aspartate are involved in the formation of co-ordinate bonds with magnesium ions. Aspartate, the first acidic residue in the NLRP3 Walker B site, was found to be within 5Å of ATP-Mg2+ [19]. MCC950 (also known as CP-456773 and CRID3) (Figure5a) is a potent and well-studied inflammasome lead compound, which inhibits NLRP3-dependent ASC oligomerization, with an IC50 of 7.5 nM in BMDMs (Table1)[ 33]. MCC950 did not compete with ATP upon exposure to wild-type NLRP3∆LRR, showing that it was unlikely to bind at the Walker A site. The following mechanism of action studies provided evidence that MCC950 binds with high affinity to the Walker B site, and was proposed to form a complex with ATP. Vande Walle and coworkers [34] designed a cell-penetrant photo-affinity label (PAL) probe attached to an MCC950 analogue, PAL-CRID3, which inhibited NLRP3 [34]. Upon UV exposure of PAL-CRID3, the photo-reactive benzophenone undergoes covalent binding to the active site. The alkyne group of PAL-CRID3 reacts with a 5-carboxytetramethylrhodamine (TAMRA) fluorescent reporter using click chemistry, which after cell lysis allows for in-gel fluorescence detection and separation of the covalent MCC950-protein adduct by SDS-PAGE gel electrophoresis, showing direct binding of PAL-CRID3 to NLRP3 (Figure5b) [ 34]. Furthermore, Coll and coworkers also designed a photoaffinity probe of MCC950: PAP was covalently cross-linked to its target when activated by UV light, which was then reacted with biotin-PEG3-azide via Cu(1)-catalysed click chemistry and purified using streptavidin magnetic beads (Figure5c), again supporting binding of MCC950 close to the Walker B site [35]. Tapia-Abellán and coworkers [36] used Bioluminescence Resonance Energy Transfer (BRET) to study the molecular conformation of NLRP3 and showed that MCC950 results in closure of the open active conformation of NLRP3 to an inactive state [36]. Coll and coworkers [35] investigated the protein-ligand interaction formed between MCC950 and the NACHT domain using a drug affinity responsive target stability (DARTS) assay. The principle is based on the protein being resistant to degradation by proteases once it is stabilized by interaction with the inhibitor. MCC950 protected NLRP3 from proteolytic degradation supporting its direct interaction with NLRP3 [35]. In summary, these mechanism of action studies show that MCC950 is proposed to bind non-covalently with high affinity close to the Walker B site on NLRP3. This prevents a structural conformational change of the ATP-binding site keeping the NLRP3 inflammasome in an inactive state, preventing hydrolysis of ATP needed for NLRP3 assembly [34–36]. A small clinical study with MCC950 was carried out by Pfizer for rheumatoid arthritis, however increasing concentrations of MCC950 gave rise to an increased risk of liver toxicity, causing the development of MCC950 to cease after phase 1b testing [37]. Attention was then focused by some companies and academia to discover less toxic analogues of MCC950, which are discussed in the next section. Molecules 2020, 25, x FOR PEER REVIEW 6 of 14

Moleculescovalently2020, 25cross, 5533-linked to its target when activated by UV light, which was then reacted with biotin7 of- 14 PEG3-azide via Cu(1)-catalysed click chemistry and purified using streptavidin magnetic beads (Figure 5c), again supporting binding of MCC950 close to the Walker B site [35].

Figure 5. (a–c) Structure of inflammasome inhibitor MCC950 and its photoaffinity probes PAL-CRID3 and PAP Figure 5. (a–c) Structure of inflammasome inhibitor MCC950 and its photoaffinity probes PAL-CRID3 designed to probe the mechanism of action of MCC950; (b) In the presence of UV radiation, the benzophenone and PAP designed to probe the mechanism of action of MCC950; (b) In the presence of UV radiation, group in PAL-CRID3 cross-linked using radical chemistry to the NLRP3 protein. The alkyne group of the PAL- the benzophenone group in PAL-CRID3 cross-linked using radical chemistry to the NLRP3 protein. CRID3 protein-complex was reacted with TAMRA-azide using click chemistry and the fluorescent protein was isolatedThe alkyne by gel electrophoresis group of the PAL-CRID3; (c) In the presence protein-complex of UV radiation, was the reacted benzophenone with TAMRA-azide group in PAP using cross click-linked usingchemistry radical chemistry and the fluorescent to the NLRP3 protein protein. was The isolated alkyne by group gel electrophoresis;of the PAP protein (c) complex In the presence was reacted of UV with biotinradiation,-PEG3-azide, the benzophenone and the protein group isolated in PAP using cross-linked a streptavidin using column. radical Experiments chemistry to with the NLRP3both photoaffinity protein. probesThe supported alkyne group MCC950 of the binding PAP protein to the Walker complex B region was reacted of NLRP3 with [34,35 biotin-PEG3-azide,]. and the protein isolated using a streptavidin column. Experiments with both photoaffinity probes supported MCC950 binding to the Walker B region of NLRP3 [34,35].

8. Clinical Trials and Patents of NLRP3 Inflammasome Inhibitors in the Pharmaceutical Industry Pipeline Several companies, including Inflazome [4], IFM Therapeutics [3], NodThera [5], Novartis [2], Olatec therapeutics [6] and ZyVersa Therapeutics [38] have small-molecule NLRP3 inflammasome inhibitors that are currently in their drug discovery pipelines in either preclinical or clinical trials. The structures of the compounds have not been disclosed, with the exception of OLT1177 (dapansutrile) from Olatec therapeutics which is in phase 2 trials for oral use in systemic inflammation and treatment of Covid-19 [6,39,40]. Partnering with Novartis, IFM Therapeutics have IFM2427, a systemic-peripheral NLRP3 antagonist in phase 1 clinical trials for the treatment of chronic inflammatory diseases including gout, atherosclerosis and non-alcoholic steatohepatitis [2]. In addition, IFM therapeutics have a gut-directed and a CNS-penetrant NLRP3 antagonist in pre-clinical trials [3]. Nodthera has been granted patents on NLRP3 inhibitors covering carbamoyl derivatives [41]. NodThera has reported NT-0167, which has shown promising NLRP3 inhibition reducing IL-1β production in preclinical studies and is currently under investigation in healthy volunteers in phase I clinical trials [42]. Potent membrane permeable urea ester prodrugs, inspired by MCC950, have been reported as NLRP3 inhibitors, the most active being 2-[(phenylcarbamoyl)amino]acetates containing 2-pyrimidine (1) or 2-pyrazine (2) groups (Figure6), with IC 50s of 36 nM and 30 nM for the inhibition of IL-1β in PBMC cells (Table2). These two compounds were shown to be equipotent to MCC950, Molecules 2020, 25, x FOR PEER REVIEW 8 of 14

however they showed enhanced activity (1, 20-fold and 2, 8-fold) when tested in the whole blood assay when compared with MCC950 [43,44]. Inflazome, a biotech company whose vision is to develop potent, best-in-class, small molecule NLRP3 modulators for the treatment of a broad range of inflammatory diseases, has been granted US and Europe patents for its potent and selective lead NLRP3 inflammasome inhibitors: Somalix (peripheral) is in phase 2 clinical trials and inzomelid (CNS penetrant) has completed phase 1 [4,45,46]. O’Neill and coworkers hold a patent on sulfonylureas, including MCC950 (Figure 5) and MCC7840 (Figure 6) which inhibited NLRP3 with IC50 values of <100 nM [47]. The University of Queensland and Cadila have disclosed NLRP3 inhibitors, with the novel substituted sulfoximine Moleculescompound2020, 25, ( 55333) being the most active with a low nM IC50 value in THP1 cells (Figure 6) (Table8 of2)14 [48]. It has recently been reported that this series of N-cyano sulfoximine urea derivatives were highly potent and selective towards NLRP3 inhibition, showing good pharmacokinetic profiles with oral howeverbioavailability. they showed The enhanced most active activity compounds (1, 20-fold 4–7 and gave2, 8-fold)IC50 values when of tested 5, 7, 12 in theand whole 23 nM, blood respectively, assay whenin the compared IL-1β inhibition with MCC950 assay[ in43 ,mice44]. (Figure 6) (Table 2) [49].

FigureFigure 6. Structures 6. Structures of NLRP3 of NLRP3 inhibitors: inhibitors: MCC950 MCC950 analogues. analogues.

Inflazome, a biotechTable company 2. Activities whose of MCC950 vision analogues is to develop in the potent, NLRP3 best-in-class,inflammasome. small molecule NLRP3 modulators for the treatment of a broad range of inflammatory diseases, has been granted US and Inhibitor IC50 Europe patents for its potent and selective lead NLRP3 inflammasome inhibitors: Somalix (peripheral) MCC7840 <100 nM [47] is in phase 2 clinical trials and inzomelid (CNS penetrant) has completed phase 1 [4,45,46]. 1 36 nM [43] Table 2. Activities of MCC9502 analogues 30 in nM the NLRP3[43] inflammasome. 3 1.26 nM [48] Inhibitor4 5 nM IC50 [49] MCC7840 5 <1007 nM nM [49] [47] 16 3612 nMnM [ 43[49]] 27 3023 nMnM [ 43[49]] 3 1.26 nM [48] 48 <1 5 nM µM [49 [50]] 59 <1 7 nM µM [49 [50]] 610 12<1 nMµM [ 49[50]] 7 23 nM [49] µ 9. Computational Approaches to Design8 New NLRP3<1 M[ Inh50ibitors] 9 <1 µM[50] 10 <1 µM[50]

O’Neill and coworkers hold a patent on sulfonylureas, including MCC950 (Figure5) and MCC7840 (Figure6) which inhibited NLRP3 with IC 50 values of <100 nM [47]. The University of Queensland and Cadila have disclosed NLRP3 inhibitors, with the novel substituted sulfoximine compound (3) being the most active with a low nM IC50 value in THP1 cells (Figure6) (Table2)[ 48]. It has recently been reported that this series of N-cyano sulfoximine urea derivatives were highly potent and selective towards NLRP3 inhibition, showing good pharmacokinetic proﬁles with oral bioavailability. The most active compounds 4–7 gave IC50 values of 5, 7, 12 and 23 nM, respectively, in the IL-1β inhibition assay in mice (Figure6) (Table2)[49]. Molecules 2020, 25, 5533 9 of 14

MoleculesMolecules9. Computational 20202020,, 2255,, xx FORFOR Approaches PEERPEER REVIEWREVIEWto Design New NLRP3 Inhibitors 99 ofof 1414

9.1.9.1.9.1. MolecularMolecular Molecular DynamicsDynamics Dynamics AAA homologyhomology homology modelmodel model ofof of NLRP3NLRP3 NLRP3 waswas was preparedprepared prepared fromfrom from thethe the crystalcrystal crystal structurestructure structure ofof of NLRC4NLRC4 NLRC4 andand and thethe the FASTAFASTA FASTA sequencesequencesequence ofof of humanhuman human NLRP3NLRP3 NLRP3 ((www.uniprot.org (www.uniprot.orgwww.uniprot.org)).).. PerriconePerricone Perricone andand and coworkerscoworkers coworkers usedused used thisthis this homologyhomology homology modelmodel model ininin aa a MolecularMolecular Molecular DynamicsDynamics Dynamics (MD)(MD) (MD) studystudy study withwith with MCC950MCC950 MCC950 ((Figure (FigureFigure 55)) and and itsits analoguesanalogues [51] [[51]51.].. DetectionDetection Detection ofof of allall all potentialpotentialpotential druggabledruggable druggable bindingbinding binding sitessites sites closeclose toto thethe WalkerWalker B B region region werewere detecteddetected bybyby dockingdockingdocking MCC950 MCC950MCC950 as asas a aaprobe, probe,probe, using usingusing grid-based gridgrid--basedbased andandand geometry-basedgeometrygeometry--basedbased algorithmalgorithm approaches. approaches. DockingDocking Docking andand and MDMD MD simulationssimulations simulations gavegavegave aa a goodgood good RMSDRMSD RMSD ofof of 1.101.10 1.10 ÅÅ Å withwith with DFTDFT DFT ligandligand ligand optimizationoptimization optimization outcomesoutcomes outcomes comparedcompared compared withwith with (Epik)(Epik) (Epik) LigPrepLigPrep LigPrep tool.tool.tool. TheThe The GlideGlide Glide dockingdocking docking tooltool was was used used forfor MCC950MCC950MCC950 to toto give givegive extra extraextra precision precisionprecision with withwith and andand without withoutwithout ATP ATPATP in in thein thetheNLRP3. NLRP3. NLRP3. A favorable A A favorablefavorable docking dockingdocking score ofscore score8.8 of of Kcal −−8.88.8/mol Kcal/mol Kcal/mol was obtained was was obtaine obtainewhen ATPdd when when was bound, ATP ATP was was compared bound, bound, to − comparedcompared6.0 Kcal / tomolto −−6.06.0 for Kcal/molKcal/mol the docking forfor the posethe dockingdocking without posepose ATP. withoutwithout Analogues ATP.ATP. of AnaloguesAnalogues MCC950, compounds ofof MCC950,MCC950,8-10 compoundscompoundswere shown 88-- 10− were shown to have IC50 of < 1 µM in THP-1 cells [50] (Figure 7) (Table 2) were docked in the same 10to were have shown IC50 of to< 1haveµM IC in50 THP-1 of < 1 cellsµM in [50 THP] (Figure-1 cells7) (Table[50] (Figure2) were 7 docked) (Table in2) thewere same docked site asin MCC950,the same sitesiteshowing asas MCC950,MCC950, interactions showingshowing with interactionsinteractions amino acids withwith P281 aminoamino and S271acidsacids [ 51P281P281]. andand S271S271 [51][51]..

FigureFigureFigure 7.7. 7. AnaloguesAnaloguesAnalogues ofof of MCC9MCC9 MCC950:50:50: PPatented Patentedatented compoundscompounds compounds 88–8–10–1010 [[5050[50]].. ].

9.2.9.2.9.2. VirtualVirtual Virtual ScreeScree Screeningningning (VS)(VS) (VS) onon on ERER ER---ββ β linkedlinkedLinked toto to NLRP3NLRP3 NLRP3 InflamInflam Inflammasomemasomemasome ItItIt has has has been been been reported reported reported that that that NLRP3 NLRP3 NLRP3 inhibition inhibition inhibition is is is linked linked linked to to to estrogen estrogen estrogen receptor receptor receptor beta beta beta (ER (ER (ER---β)β)β ) upregulationupregulationupregulation [52][52] [52..]. AA A virtualvirtual virtual screening screening screening study study study was was was carried carried carried out out out on on on the the the ER ER ER---βββ crystal crystalcrystal structure structure structure (PDB: (PDB: (PDB: 5TOA)5TOA)5TOA) [53][53] [53 ] activeactive active sitesite site withwith with screeningscreening screening ofof of 20,00020,000 20,000 compoundscompounds compounds (ChemBridge(ChemBridge (ChemBridge library).library). library). LeadLead Lead compoundscompounds compounds benzo[cd]indolbenzo[cd]indolbenzo[cd]indol-2-one--22--oneone (IIIM (IIIM (IIIM-1266)--1266)1266) and and and benzylidene benzylidene benzylidene-thiazolidine-2,4-dione--thiazolidinethiazolidine--22,4,4--dionedione (IIIM (IIIM (IIIM-1268,--1268,1268, IIIM IIIM IIIM-1269,--1269,1269, IIIMIIIMIIIM-1270)--1270)1270) ((Figure (FigureFigure 88)) werewere alsoalsoalso docked dockeddocked into intointo the thethe ADP-binding ADPADP--bindingbinding site sitesite (Walker (Walker(Walker A A/A// Walker WalkerWalker B) B)B) of ofof the thethe cryo-EM cryocryo-- EMEMstructure structurestructure of of NLRP3of NLRP3NLRP3 (PDB: (PDB:(PDB: 6NPY), 6NPY),6NPY), giving givinggiving comparable comparablecomparable docking dockingdocking scores scoresscores toto MCC950 MCC950 [54] [[54]54]... IIIMIIIM IIIM-1266,--1266,1266, IIIMIIIMIIIM-1268,--1268,1268, IIIM IIIM-1269,IIIM--12691269,, andandand IIIM-1270 IIIM IIIM--12701270 showed showed showed inhibition inhibition inhibition of IL-1 of of β IL ILrelease--11ββ releaserelease in mouse in in mousemacrophage mouse macrophage macrophage (J774A.1) cells of 77.4%, 72.9%, 68.9%, and 74.7% at 10 µM, respectively. The IC values for IIIM-1268 and (J774A.1)(J774A.1) cellscells ofof 77.4%,77.4%, 72.9%,72.9%, 68.9%68.9%,, andand 74.7%74.7% atat 1010 µM,µM, respectively.respectively. TheThe50 ICIC5050 values values forfor IIIMIIIM--12681268 andandIIIM-1270 IIIMIIIM--12701270 were werewere 2.3 µ2.32.3M µMµM and andand 3.5 µ3.53.5M[ µMµM54], [54][54] although,, althoughalthough further furtherfurther biology biologybiology is desirable isis desirabledesirable to both ttoo bothboth confirm confirmconfirm their theirtheirdirect directdirect inhibition inhibitioninhibition of the ofof NLRP3 thethe NLRP3NLRP3 inflammasome inflamminflammaasomesome and validateandand validatevalidate its link itsits withlinklink withwith ER-β ERER. --β.β.

FigureFigureFigure 8. 8. 8. NLRP3NLRP3NLRP3 iinhibitorsnhibitors inhibitors identified identified identiﬁed from from from the the the virtual virtual virtual screening screening screening of of of a a a compound compound compound library library library with with with the the the estrogenestrogenestrogen receptorreceptor receptor betabeta beta crystalcrystal crystal structurestructure structure [54][54] [54.]..

9.3.9.3. ComputationalComputational StrategStrategiesies forfor thethe DevelDevelopmentopment ofof NLRP3NLRP3 InhiInhibitorsbitors byby DirectDirect BindingBinding toto NLRP3NLRP3 PyrinPyrin DomainDomain Molecules 2020, 25, 5533 10 of 14

9.3. Computational Strategies for the Development of NLRP3 Inhibitors by Direct Binding to NLRP3 Molecules 2020, 25, x FOR PEER REVIEW 10 of 14 Pyrin Domain TheThe crystal crystal structure structure of of NLRP3 NLRP3 pyrinpyrin domain (PDB: (PDB: 3QF2) 3QF2) [55] [55 ]has has widened widened the the scope scope for for the the discoverydiscovery of of NLRP3 NLRP3 inhibitors. inhibitors. TheThe pyrinpyrin domain at at the the amino amino terminus terminus of of NLRP3 NLRP3 binds binds to toASC ASC via via PYD-PYDPYD-PYD interactions. interactions. A A study study reported reported virtual virtual screening screening on on the the NLRP3 NLRP3 PYD PYD domain domain by by utilizing utilizing the ZINCthe databaseZINC database to screen to 6screen million 6 million compounds compounds using Schrodinger using Schrodinger A, LigPrep A, LigPrep properties properties along with along Glide  modulewith Glide in the module Schrodinger in the molecularSchrodinger simulations molecular package. simulationsβ-Carotene package. (provitamin-Carotene (provitamin A) (Figure9 )A) was (Figure 9) was identified from the virtual screen. Using surface plasmon resonance (SPR), an identified from the virtual screen. Using surface plasmon resonance (SPR), an experimental technique experimental technique used to detect molecular interactions, showed that -carotene bound directly used to detect molecular interactions, showed that β-carotene bound directly to the recombinant to the recombinant human pyrin domain of NLRP3 (KD = 3.41 × 10−6), blocking interaction of ASC human pyrin domain of NLRP3 (K = 3.41 10 6), blocking interaction of ASC with NLRP3 resulting with NLRP3 resulting in inhibitionD of inflammasome× − activation. Mutation studies and the BMDM in inhibition of inflammasome activation. Mutation studies and the BMDM gouty arthritis mouse gouty arthritis mouse model activated with MSU were used to further confirm the anti-inflammatory β modeleffect activated of -carotene with [56] MSU. were used to further confirm the anti-inflammatory effect of -carotene [56].

FigureFigure 9. 9.Structures Structuresof of NLRP3NLRP3 inhibitors β--carotenecarotene (left (left) )and and tranilast tranilast (right (right). ).

It isIt interesting is interesting to note to note that that tranilast tranilast (Figure (Figure9), a drug9), a that drug has that been has used been clinically used clinically for the treatment for the treatment of a number of inflammatory disorders, inhibits inflammasome activation with an IC50 of of a number of inflammatory disorders, inhibits inflammasome activation with an IC50 of 10–15 µM[57]. Tranilast10–15 µM has [57] been. Tranilast reported has tobeen have reported a related to have mechanism a related ofmechanism action as ofβ action-Carotene, as -Carotene binding, to thebinding NACHT to domain, the NACHT inhibiting domain, NLRP3-ASC inhibiting oligomerization NLRP3-ASC oligomerization and inflammasome and inflamm activationasome [57 ]. activation [57]. Inhibition of this protein-protein interaction is likely to be exploited in future drug Inhibition of this protein-protein interaction is likely to be exploited in future drug design of inhibitors design of inhibitors of the inflammasome. of the inflammasome. 10. Conclusions 10. Conclusions The NLRP3 inflammasome has been the focus of drug discovery research due to its involvement The NLRP3 inflammasome has been the focus of drug discovery research due to its involvement in several inflammatory diseases. Inhibitors either have an indirect effect or a direct effect on NLRP3 in several inflammatory diseases. Inhibitors either have an indirect effect or a direct effect on NLRP3 and in this review there is a focus on those with a direct effect. MCC950, a potent inflammasome and in this review there is a focus on those with a direct effect. MCC950, a potent inflammasome inhibitor with an IC50 of 7.5 nM, acts directly at or near the Walker B site, whereas CY09 is thought to inhibitoract directly with through an IC50 covalentof 7.5 nM, modification acts directly of atthe or Walker near the A binding Walker Bsite. site, The whereas cryo-EM CY09 structure is thought of tohuman act directly NLRP3 through bound covalent to NEK7 modification has provided of insights the Walker on the A mechanism binding site. of Thesignali cryo-EMng and structureassembly of humanof the NLRP3NLRP3 boundinflammasome, to NEK7 with has providedmolecular insightsdocking onin the the ADP mechanism-binding ofsite, signaling molecular and dynamic assembly ofsimulations, the NLRP3 inflammasome, pharmacophore- withbased molecular screening docking and virtual in the screening ADP-binding supporting site, molecularthe discovery dynamic of simulations,potent NLRP3 pharmacophore-based inhibitors with appropriate screening PK and properties virtual. screening supporting the discovery of potent NLRP3A inhibitors number of with pharmaceutical appropriate companies PK properties. have drug discovery programs to identify novel NLRP3 inflammasomeA number of inhibitors pharmaceutical, with several companies compounds have drug currently discovery undergoing programs clinical to identify trials. The novel outcome NLRP3 inflammasomeof these trials inhibitors,will of course with have several a major compounds impact on currently the future undergoing direction of clinical NLRP3 trials. inflammasome The outcome ofresearch these trials in the will pharmaceutical of course have industry a major. For impact example, on theinhibition future of direction the inflammasome of NLRP3 comes inflammasome with a researchrisk of ininfection, the pharmaceutical therefore formulations industry. designed For example, for localized inhibition or targeted of the inflammasome use may be benefi comescial. withIn a riskaddition, of infection, recent studies therefore have formulations shown that designed post-translational for localized modific ora targetedtion of the use inflammasome may be beneficial. is Inessential addition, [58,59 recent], for studies example have phosphorylation shown that post-translational and ubiquitination modification, which identifies of the potential inflammasome targets is essentialfor drug [58 discovery.,59], for example phosphorylation and ubiquitination, which identifies potential targets for drugAlthough discovery. this review focuses only on direct inhibition of the NLRP3 inflammasome, there is significant interest in the inhibition of upstream processes, which again may lessen the risk of Although this review focuses only on direct inhibition of the NLRP3 inflammasome, there is infection. For example, our research group has recently shown that chloride channels are important significant interest in the inhibition of upstream processes, which again may lessen the risk of infection. regulators of the inflammasome, with novel urea-based compounds showing promising indirect For example, our research group has recently shown that chloride channels are important regulators of inhibition of the NLRP3 inflammasome [60,61]. What is certain is that it will be a highly competitive the inflammasome, with novel urea-based compounds showing promising indirect inhibition of the race to discover the “First-in-Class” small molecule clinical NLRP3 inhibitor, which may have the NLRP3potential inflammasome to treat a range [60 of,61 therapeutic]. What is certainareas where is that inflammat it will beion a highlyis a significant competitive component. race to discover Molecules 2020, 25, 5533 11 of 14 the “First-in-Class” small molecule clinical NLRP3 inhibitor, which may have the potential to treat a range of therapeutic areas where inflammation is a significant component.

Author Contributions: Writing—manuscript preparation and Figures, L.Y.E.-S.; writing—review and editing, and supervision, S.F. and D.B. All authors have read and agreed to this version of the manuscript. Funding: L.S. was funded by Yasser El-Sharkawy and a University of Manchester Presidential Scholars Award. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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