Cryopyrin Associated Periodic Syndromes

Published Date: 02 Nov 2017

Abstract

Inflammation is part of the body’s mechanism for tissue repair and infection elimination. However, if the inflammatory response lasts beyond its protective phase, it can lead to conditions such as rheumatoid arthritis (RA), Alzheimer’s disease (AD), diabetes and stroke. The auto-inflammatory conditions, CAPS and FMF are thought to be caused by mutations in the NALP3 inflammasome which result in IL-1β over production. Current treatments for CAPS and FMF target the action of IL-1β; it would be more effective to inhibit maturation and release of IL-1β, hence the importance of synthesising inhibitors of the inflammasome.

A boron-containing lead compound (2-aminoethyl diphenylborinic acid, 2-APB) that inhibits IL-1β release was recently discovered (Dr Brough). In this study, six carbon analogues of BC-23 (a derivative of 2-APB) that were generated from the Zinc database (Panichakorn Jaiyong and Dr Bryce) are discussed. Synthesis of one of the analogues (4,4-diphenylbutenolide) was attempted using reaction sequences found through Scifinder.

During the study it was found that 4,4-Diphenylbutenolide had already been synthesised, therefore synthesis was stopped as it would have gone against research ethics to duplicate another study. The focus of the study switched to analysing samples of 4,4-Diphenylbutenolide, along with five other carbon analogues of BC-23, to test their purity and confirm the structures.

The structures of five of the samples were confirmed using 1H NMR and mass spectrometry. Analysis of the sixth sample (sample 23879) showed that the predicted structure was incorrect; the analytical section of this report mainly focuses on sample 23879.

Biological assays on the six samples showed that they had no activity against IL-1β release (Dr Bryce, Jaiyong, Dr Brough and Dr Freeman). These results show that boron is important for activity against inflammasome-mediated IL-1β release and should therefore be present in new analogues being designed against the inflammasome.

Introduction

Since the discovery of the inflammasome in 2002,2 there has been a substantial amount of research done to try and understand these molecular platforms. Most of the interest is focused on designing inhibitors of the inflammasome to treat auto-inflammatory conditions, which are thought to be caused by over-activation of the inflammasome.

Inflammation

Inflammation is often a protective response that helps the body to repair tissue and eliminate infections.3 But, if the inflammatory response lasts longer than needed it can lead to inflammatory conditions such as rheumatoid arthritis (RA), Alzheimer’s disease (AD), diabetes and stroke. Other inflammatory conditions such as CAPS (cryopyrin-associated periodic syndromes) and FMF (familial Mediterranean fever) are termed auto-inflammatory because they are not part of the body’s response to injurious stimuli; they result from the activation of the innate immune system without microbial or antibody activation. Auto-inflammatory conditions are thought to be driven by mutations in some of the genes involved in inflammatory responses and are largely driven by the pro-inflammatory cytokine interleukin-1beta (IL-1β). 4 Production of IL-1β has been shown to be increased in CAPS and FMF and this is thought to be due to mutations in some domains of a protein complex known as the inflammasome (below).5 The maturation and release of IL-1β is controlled by the inflammasome, therefore, if mutations lead to over activation of the inflammasome there will be over-secretion of IL-1β.

Current treatments for CAPS and FMF are focused on inhibiting IL-1β (e.g. Anakinra and canakinumab). Anakinra is a recombinant form of the endogenous IL-1 receptor antagonist (IL-1RA), which inhibits the action of IL-1β through inhibition of the IL-1 receptor (The inflammasome) whereas canakinumab is a recombinant human monoclonal antibody. Both drugs work by preventing IL-1β from binding to the IL-1 receptor, but, it would be more effective to have treatments that inhibit the production of IL-1β in the first place.

Cryopyrin-Associated Periodic Syndromes

CAPS is a collection of three diseases grouped together on the basis that they are all caused by autosomal-dominant mutations in a pattern recognition receptor known as NALP3 (NACHT, leucine-rich region and PYD domains containing protein 3). Autosomal dominant means that the person can inherit the disease even if the gene is only passed down by one parent. The three manifestations of CAPS are FCAS (familial cold autoinflammatory syndrome), MWS (Muckle-Wells syndrome) and NOMID (neonatal-onset multi-system inflammatory disease). NOMID is also known as CINCA (chronic infantile neurologic cutaneous articular syndrome).

Figure 1.1.1. WT (wild type) NLRP3 (NALP3) is involved in inflammasome formation only when there is an activating signal present, such as a PAMP/DAMP. Mutations in the NACHT (NOD) domain lead to a gain of function that allows the inflammasome to be activated even in the absence of an activating signal. (Figure from Ozkurede et al. 5)

FCAS is the least severe of the three forms of CAPS, with symptoms varying from a recurring rash to headaches and pain in the joints and muscles. These symptoms are very non-specific and can often be mistaken for flu or the common cold.

The severity of MWS is intermediate between FCAS and NOMID. Patients with MWS often experience symptoms similar to FCAS but these may be more severe and last longer. In addition, the patients may experience hearing loss and arthritis.

NOMID is the most severe form of CAPS.

Familial Mediterranean Fever

The Inflammasome

The inflammasome is a protein complex that is involved in activating caspase-1, which subsequently leads to maturation and release of IL-1β.2 The inflammasomes that are known to activate caspase-1 are composed of a pattern-recognition receptor of the nucleotide-binding domain leucine-rich repeat (NLR) family such as NALP1 (NACHT, leucine-rich region and PYD domains containing protein 1), NALP3 and NLRC4 (nucleotide-binding domain and leucine-rich repeat containing CARD 4), or the DNA-sensing AIM2 (absent in melanoma 2) and RIG-1 (retinoic-acid-inducible protein-1) receptors.3

Members of the NLR family are composed of three main domains:4

An N-terminal effector domain which will either be a PYD (pyrin domain), a CARD (caspase recruitment domain) or a BIR (baculovirus inhibitor of apoptosis protein repeat) domain.

A NACHT (NAIP, CIITA, HET-E and TP1) domain which is a nucleotide-binding domain involved in the formation of the oligomeric structure of the inflammasome.

A C-terminal leucine-rich repeat (LRR) domain which is thought to confer specificity and is involved in regulating inflammasome activation.

Two mechanisms of inflammasome activation have been proposed:4

Apoptosome formation where the NLR protein exists in its inactive form with the LRR domain bound to the PYD-NACHT domains. This prevents the NACHT domain from interacting with nucleotides. LRR can bind to PAMPs (pathogen-associated molecular patterns) or DAMPs (danger-associated molecular patterns) and when this happens LRR loses its binding with the NACHT and PYD domains. This allows for the exchange of ADP (adenosine diphosphate) for ATP (adenosine triphosphate), leading to a conformational change in the NLR that makes it capable of forming oligomers.

The NLR is kept inactive by a host guard complex that protects the NLR from proteasomal degradation. Activation of the host guard complex leads to the release of the PYD domain which is now free to interact with the PYD of ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and be involved in assembly of the inflammasome. ADP is exchanged for ATP leading to a conformational change that can form oligomers.

apoptosis-associated speck-like protein containing a caspase recruitment domain

The NALP3 inflammasome is the best characterised, and NALP3 is the receptor implicated in auto-inflammatory responses.3 For these reasons, this report will focus on the NALP3 inflammasome, details of which can be found under results and discussion.

Inhibition of the inflammasome

It may sometimes be essential to suppress activation of the inflammasome because inflammatory responses that last beyond dealing with foreign or endogenous insults can lead to excessive tissue damage. Mammalian cPOP1 (cellular pyrin domain (PYD) – only protein (POP) 1) and cPOP2 are thought to disrupt inflammasome activation by binding to ASC and blocking its interaction with NLRs.6 A number of CARD- and PYD-containing proteins are thought to suppress the activity of the inflammasome by blocking inflammasome component recruitment. PYD-containing proteins include pyrin, POP1 and POP2. CARD-containing proteins such as human Inca, iceberg and caspase-12 may suppress inflammasome activation by preventing the recruitment of caspase-1.7

The anti-apoptotic proteins bcl-2 and bcl-xl may suppress NALP1-dependent caspase-1 activation and IL-1B secretion by inhibiting ATP binding to the NALP1 NACHT domain, a necessary step for NACHT domain oligomerisation.7

Currently used drugs are based on neutralising cytokines that are already in circulation, but directly targeting the inflammasome could prevent cytokine generation and provide a novel approach to treating auto-inflammatory conditions.4

Interleukin-1 Beta

IL-1β is a pro-inflammatory cytokine produced primarily by macrophages. IL-1β mediates diverse biological processes such as the inflammatory response, fever, response to heat, chronic inflammatory response to antigenic stimuli and cell-cell signalling. The activity of IL-1β is tightly controlled via its expression, maturation and secretion. Pro-inflammatory stimuli are responsible for inducing the expression of pro-IL-1β, but IL-1β maturation and release are controlled by the inflammasome.7

Cleavage of IL-1β by caspase-1 is highly specific, such that other caspases, such as apoptotic caspases, are not likely to activate IL-1β directly. When IL-1β is produced for too long it causes tissue damage, bone resorption, collagen deposition and neovascularisation.

Ubiquitination

Ubiquitin (Ub) is a 76-amino acid protein that covalently binds to lysine residues of substrate proteins. Ubiquitination involves the attachment of ubiquitin to target proteins by a process regulated by three enzymes, E1, E2 and E3. E1 is an ubiquitin-activating enzyme and mediates the formation of an E1-ubiquitin thiol ester bond. E2 is an ubiquitin-conjugating enzyme which mediates the transfer of ubiquitin from E1 to the target protein and interacts with E3, an ubiquitin-protein ligase that finally conjugates Gly 76 on ubiquitin to a lysine of the target protein to form an isopeptide bond.8,9 Ubiquitin polymers can be formed between K48 residues on ubiquitin or through K63 residues. K48 polyubiquitination targets proteins for proteasomal degradation whereas K48 monoubiquitination regulates subcellular localisation and the recruitment of ubiquitin -binding proteins. K63 polyubiquitination is required for activating downstream molecules such as kinases, or protein recruitment.10

Figure 1.2.1. Amino acid sequence of ubiquitin

Figure 1.2.1. Isopeptide bond formed between ubiquitin and a target protein.

Deubiquitination

DUBs are proteases that process inactive ubiquitin precursors, proof-read ubiquitin -protein conjugates, remove ubiquitin from cellular adducts and keep the 26S proteasome free of unanchored ubiquitin chains that can compete with ubiquitinated substrates for ubiquitin binding sites. DUBs cleave ubiquitin from linked molecules after Gly 76 of ubiquitin.9

DUBs deconjugate ubiquitin substrates and save them from proteasomal degradation while helping to recycle ubiquitin at the same time. DUBs can be divided into two classes of proteases, cysteine proteases and metallo-proteases. Most DUBs are cysteine proteases and during catalysis the cysteine nucleophilically attacks the carbonyl of the isopeptide bond which is between the target protein and ubiquitin. The intermediate formed contains an oxyanion and is stabilised in the oxyanion hole provided by a glutamine, glutamate or asparagine residue and the main catalytic chain of the catalytic cysteine. As a result of this reaction the target protein is released and the DUB forms a covalent intermediate with ubiquitin. The enzyme is released when the intermediate reacts with water.10

Metallo-proteases use a Zn2+ bound polarised water molecule to generate a non-covalent intermediate with the substrate. The zinc atom is stabilised by an aspartate and two histidine residues. The DUB is released for the intermediate when a proton is transferred from a water molecule to the intermediate.10

Five classes of DUBs have been identified:

Ubiquitin C-terminal hydrolases (UCHs)

Ubiquitin-specific proteases (USPs)

Machado-Josephin disease protein domain proteases (MJDs)

Ovarian tumour proteases (OTU)

JAMM motif (zinc metallo) proteases (JAMM/MPN+ proteases)

UCHs, USPs, MJDs and OTUs are cysteine proteases whereas JAMM motif proteases are zinc-dependent metallo-proteases.9

JAMM motif proteases are made up of a subunit of the proteasome, Rpn11/POH1. The Rpn11 sequence, referred to as the MPN+ or the JAMM motif, bears a distinct motif that is a subtype of the MPN motif (Figure 1.2.1. Amino acid sequences of the conserved motifs surrounding catalytically active amino acid residues in some known JAMM motif proteases (*).9).9

Figure 1.2.1. Amino acid sequences of the conserved motifs surrounding catalytically active amino acid residues in some known JAMM motif proteases (*).9

AMSH (Associated Molecule with the SH3 domain of STAM) has the MPN+/JAMM motif and has been found to have DUB activity. The MPN+/JAMM motif includes two conserved His residues and an Asp residue both of which co-ordinate a zinc ion important for proteolytic activity. The His and Asp residues are essential for the function of the Rpn11 subunit when integrated into the proteasome. Glu48 is also conserved and is thought to act as an acid-base catalyst.9 The JAMM protease POH1 (Rpn11 in yeast), UCH-L5 and USP14 have been found in complex with the 19S proteasome-regulatory component. Deletion of the gene that encodes POH1 results in defective proteasomal degradation. This observation has led to the suggestion that POH1 generates the main DUB activity at the proteasome.10

Aims and Objectives

Evaluate the literature to find out about the target for inhibitors of the inflammasome such as the novel boron-containing compounds BC23, BC7 and BC18.

Because DUBs are involved in the activation of the inflammasome, it has been hypothesised that these compounds may inhibit DUBs and thereby prevent the activation of the inflammasome. The aim is to find out which DUB is involved in activating the inflammasome.

Synthesise an analogue of BC23 as a novel inhibitor of the inflammasome. A search of the literature revealed that sesquiterpene lactones such as parthenolide and other structurally-related compounds act as inhibitors of the inflammasome. Based on these results and the results obtained from screening for carbon analogues of BC-23 (Jaiyong and Bryce), a compound containing a gamma-lactone will be synthesised.

Use proton (1H) NMR spectroscopy and mass spectrometry to confirm the identity of six compounds obtained from the NCI.

Analytical Methods

Chromatography

Chromatography is a technique for separating a mixture into its constituents and testing the purity of the compounds. In all types of chromatography there is a stationary phase (a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase travels through the stationary phase by capillary action and carries the components of the mixture with it. The different components move through the stationary phase at different rates and can therefore be separated.

TLC and column chromatography were used for determining reaction progress and, in the case of column chromatography, also for separating the product from the rest of the components in the reaction mixture.

Thin Layer Chromatography

TLC is commonly used in synthetic chemistry to identify compounds, determine their purity and follow the progress of a reaction. In TLC the analyte(s) moves up a layer of stationary phase (usually silica gel), under the influence of a mobile phase (often a mixture of organic solvents). The distance moved by the analyte depends on its relative affinity for the stationary versus the mobile phase. Silica is polar therefore the distance moved by the analyte will depend on its polarity. The more polar the compound the more it partitions into the silica gel and thus the shorter the distance it travels up the TLC plate in a given time.

The distance travelled by the compound from the baseline (where the compound is spotted) divided by the distance travelled by the solvent (solvent front) from the baseline is referred to as the retention factor (Rf) value of the compound. The Rf value can be used to establish the identity of the spots on a TLC plate; the more polar the analyte the lower its Rf value.

The intensity of a spot is related to the concentration of the analyte producing it.

Procedure used for TLC - The baseline was made 1 cm from the bottom of the TLC plate and spotted with three samples (product, mixture of product and starting material, starting material). The solvent was allowed to dry before the TLC plate was placed in a developing beaker using the solvent systems mentioned above. The beaker was covered and the mobile phase was allowed to travel up the silica gel plate by capillary action. After about 10 minutes the TLC plate was removed and the solvent front was marked prior to drying the plate. The dried plate was dipped in potassium permanganate and then re-dried to reveal the spots. The Rf values for the starting material and the product were calculated as described in the results and discussion section.

Flash Column chromatography

General procedure for flash chromatography:

To a glass column that has either a glass frit or a plug of cotton wool directly above the stopcock, put a layer of clean sand above the plug of glass wool to obtain a flat surface.

With the column in a fume cardboard, pour in the silica gel using a funnel.

Pour elution solvent onto the silica gel and use pressurised gas to force the solvent through the silica until the entire silica plug becomes homogenous in appearance. Do not let the top of the column to run dry as this will force air back onto the top of the silica and homogeneity will be lost.

Allow the solvent which remains above the silica to drain down until it is flush with the surface of the silica.

Dissolve your sample into the minimum volume of the elution solvent.

Carefully apply the sample to the top of the column and allow it to soak into the silica.

Rinse the sides of the column with as little elution solvent as possible (a few millilitres) and then add a small amount of sand to protect the top surface of the silica when you add more solvent.

Add elution solvent to the column and apply pressure to force it through the column.

Collect the eluted solvent into separate fractions (each fraction is about one tenth of the column volume) until you use up all the elution solvent.

Use TLC to find out which fractions contain your compound of interest, combine these in a flask and concentrate the sample on a rotary evaporator.

Nuclear Magnetic Resonance Spectroscopy

In this study 1H NMR spectroscopy was used to confirm the structure of samples. The common solvents used in NMR are used in a deuterated form to avoid introducing extra signals. The solvents are incompletely deuterated (for example CDCl3 is only 99.9% deuterated) leading to easily identifiable signals which can serve as a reference. In this study CDCl3 was used and a weak signal at δ7.25 can be seen in most of the proton NMR spectra.

In addition to carrying out 1H NMR spectroscopy on the samples, ChemDraw Ultra was used to predict the 1H NMR spectra based on the suggested structures for the samples. The two spectra were compared and used to help decide whether the suggested structures were correct or not.

Due to the low energy of radio frequency radiation, the difference in energy states for NMR is small and means that NMR is less sensitive compared to other spectroscopy methods. For this reason mass spectrometry was also used to help confirm the structures. Infrared spectroscopy was also used for one compound whose structure was difficult to determine from 1H NMR spectroscopy or mass spectrometry.

The general principles of NMR spectroscopy are briefly discussed below.

NMR works because some atomic nuclei have a nuclear spin (I) which makes them behave like bar magnets. Nuclear which exhibit this magnetic moment include 1H, 11B, 13C, 14N, 15N, 17O, 19F and 31P. The protons in a nucleus have a positive charge and because the nucleus of an atom is always spinning, it generates a magnetic field along the spinning axis. When a proton with a magnetic moment is placed in a magnetic field it can align itself either with the field or against the field. Protons are more likely to be aligned with the field as this requires less energy. Radiowaves can be used to excite these protons so that they switch from being aligned with the field to being aligned against the applied magnetic field. When the protons relax back to their ground state they release a pulse of radiowaves which can be detected and analysed to predict the structure of the molecule.

When interpreting an NMR spectrum it is useful to consider the following:

Chemical shift.

The position of the peaks on the spectrum indicates the electromagnetic environment of the protons which produced the peaks. This is expressed as the chemical shift, δ (ppm).

Figure 4.3.2. 1H NMR chemical shift scale (reproduced from Pharmaceutical analysis text book)

Electron withdrawing groups lead to a reduced electron density around the proton. These protons are then said to be deshielded and appear at higher chemical shift values.

Protons that are involved in hydrogen bonding (such as OH or NH protons) are often observed over a large range of chemical shift values; the more hydrogen bonding there is the more deshielded the proton. These protons are said to be exchangeable and can often be difficult to predict; their presence in a compound can be confirmed by adding D2O to the sample and shaking to substitute the 1H atoms for 2H atoms.

Integration and proton equivalence.

The integration is the area under the peak of each signal and is proportional to the number of protons which caused the peak. The number of peaks is related to how many sets of chemically or magnetically equivalent protons are present in the compound.

Peak shape and spin – spin coupling

The shape of each signal is usually dependent on how exchangeable the proton which produced it is. For example OH groups produce short, wide peaks whilst CH3 groups produce tall thin peaks.

NMR peaks are often in groups because the magnetic field experienced by one set of protons is slightly affected by another set of adjacent protons in the same molecule; this effect is known as coupling. If a proton has one or more neighbours then the shape of its signal will be split into two or more lines following the n + 1 rule, where n is the number of neighbouring protons. The intensity of each peak can be predicted from Pascal’s triangle, depending on how many neighbours each set of protons has. However, if the coupling constants of the neighbouring protons are not equivalent then the pattern will become more complex.

Figure 4.3.2. Pascal's triangle showing the intensity of 1H NMR signals (adapted from wikipedia and Pharmaceutical analysis text book)

The amount that these protons affect each other depends on their spin state.

Figure 4.3.2. Possible spin states for a proton.

This effect is known as spin-spin coupling and protons have two possible spin states. Each state has a different effect on the magnetic environment of nearby protons. In a sample half of the protons will be in one state and half in the other, resulting in two peaks of equal height being produced. However, a group of equivalent protons can have a large number of combinations of spin states. In the example shown below four different peaks can be produced for the proton in black.

This is because there are four different values that the total energy of the CH3 protons can have:

All three protons in a low energy state.

The red proton in a higher energy state.

The red and blue protons in higher energy states.

All three protons in higher energy states.

Coupling constant – The coupling constant, J, is the distance between the peaks produced by the same coupling interaction and measures the interaction between the pair of coupled protons. The coupling constant in vicinal coupling (Ha–C–C–Hb) is usually in the range of 0 - 20 Hz. If two sets of peaks have the same coupling constant then it can be assumed that they are related by spin-spin coupling.

Mass spectrometry

In mass spectrometry the molecules of the sample are ionised and then identified according to their mass. The spectrometer only detects charged components because only they are deflected by the electromagnetic or electrostatic fields used. A mass spectrum is therefore a record of each ion reaching the detector against its mass-to-charge (m/z) value.

Electrospray ionisation (ESI) is one method used for sample ionisation and is the method that was used in this study. In ESI the analyte is usually dissolved in a mixture of an organic solvent (e.g. acetonitrile or methanol) and water with a pH modifier (e.g. formic acid or acetic acid for positive ion mode). The pH modifier ensures that ionisation (protonation or deprotonation) takes place in solution; therefore the only molecular species often detected is [M+H]+ in positive ion mode and [M+H]- in negative ion mode, and both these species undergo very little fragmentation. One disadvantage of ESI is that it is very sensitive to contaminants in the solvents, particularly alkali metals, and we often see ions which correspond to [M+Na]+ or [M+NH4]+.

Infrared spectrophotometry

The infrared (IR) spectra of organic compounds are associated with transitions between vibrational energy levels following the absorption of electromagnetic radiation. The electromagnetic radiation is passed through a sample and if the sample contains bonds that have a dipole, they will absorb the radiation. The energy from the radiation leads to the stretching or bending of these bonds. Different types of bonds absorb different wavelengths of infrared radiation and this enables the identification of the functional groups present in a sample.

The wavelengths used in IR spectrometry are between 2500 nm and 20 000. IR spectra are often expressed in terms of frequency (as wave numbers, cm-1) between 400 cm-1 and 4000 cm-1; the rational for the units of cm-1 for frequency can be given by the equation below:

where, ν = frequency (Hz)

c = speed of light (ms-1),

λ = wavelength (cm)

The speed of light is constant in vacuum, therefore.

Literature-based analysis of the NALP3 inflammasome

A review of the literature was done to find out which inflammasome is involved in inflammation and to try and find possible pathways to target in order to inhibit activation of the inflammasome. The searches carried out on PubMed and Medline point to the NALP3 inflammasome being the most important in auto-inflammatory conditions. The results obtained are discussed in the next sections.

The NALP3 inflammasome

NALP3 is an important pattern recognition receptor (PRR) involved in mediating inflammasome activation in response to pathogen-associated molecular patterns (PAMPs) and danger-associated molecular pattern (DAMPs).11 Inappropriate activation of NALP3 contributes to inflammatory syndromes such as CAPS and FMF and is associated with substitution mutations in the NALP3 gene, LPS-induced septic shock, gout, atherosclerosis, diabetes and Alzheimer’s disease.11 The structure of NALP3 is composed of a PAMP- or DAMP-sensing C-terminal leucine-rich repeat (LRR), a central nucleotide binding (NACHT) domain and an N-terminal effector pyrin domain (PYD)3

The high resolution structure of the NALP3 PYD showed that it includes six helices, H1 to H6 with two monomers in the asymmetric unit, called chain A and chain B, Figure 5.3.1. Ribbon diagram of the NALP3 PYD. Dashed box in chain B shows the structure of the C-terminus on chain B.. A number of residues found on the surface of the NALP3 PYD domain are also found on other PYD domains that interact with the PYD of ASC, (). These residues include Leu-17, Leu-22, Pro-33, Pro-34, His-51, Val-52, Ile-59, Gly-63, Ile-78 and Tyr-84 which are conserved as large hydrophobic residues on the surface of the NALP3 PYD. In addition to hydrophobic residues, Glu-15, Asp-21, Lys-23, Lys-24, lys-48, Asp-53, Glu-64, Glu-65, Arg-81 and Lys-89 are conserved among different PYDs.12

Figure 5.3.1. Ribbon diagram of the NALP3 PYD. Dashed box in chain B shows the structure of the C-terminus on chain B.

Figure 5.3.1. Structural-based sequence alignment of the NALP3 PYD with other ASC-binding PYDs. Yellow = residues at the hydrophobic core; green = conserved exposed hydrophobic residues; cyan = charged residues; red = residues involved in the interaction between chain A and chain B.12

The NALP3 protein is often present in its inactive form and undergoes a conformational change after activation which allows interaction of its PYD domain with the PYD domain of ASC. NALP3 can be activated by exposure to whole pathogens, PAMPs/DAMPs and environmental irritants.

Figure 5.3.1. Activation on NALP3 by PAMPs/DAMPs

ASC is an adaptor molecule containing a PYD and CARD domain. The CARD domain of ASC binds to the CARD domain of pro-caspase-1, thereby facilitating activation of pro-caspase-1 by the inflammasome.3 After caspase-1 activation, IL-1β is rapidly secreted. Activation of NALP3 occurs downstream of stimuli such as extracellular ATP, amyloid-β, hyperglycaemia, alum, bacterial toxins including nigericin and crystals such as silica.11 It is thought that NALP3 activators do not directly bind to the NALP3 protein but are involved in pathways that generate reactive oxygen species (ROS), destabilise lysosomal membranes or activate lysosomal proteases such as cathepsin B.3

Suggested mechanism of activation for the NALP3 inflammasome:4,7,13

A priming signal from a pattern recognition receptor or a cytokine receptor increases the amount of NALP3. This signal is regulated by mitochondrial ROS.

Figure 5.3.1. Different pathways for activating the NALP3 inflammasome

A second signal from the P2X7 receptor or pore-forming toxins is required to trigger formation of the inflammasome via three possible mechanisms as illustrated in Error: Reference source not found:

The NALP3 agonist, ATP, triggers P 2X7-dependent pore formation by the pannexin-1 hemi-channel. This allows extracellular NALP3 agonists to enter the cytosol and directly interact with NALP3.

The physical properties of engulfed NALP3 agonists such as MSU, silica, asbestos, amyloid-B and alum lead to lysosomal rupture. The NALP3 inflammasome senses lysosomal contents in the cytoplasm, for example via cathepsin-B-dependent processing of a direct NALP3 ligand.

DAMPs and PAMPs trigger the generation of ROS at mitochondrial membranes thereby triggering NALP3 inflammasome complex formation.

The second signal can also stimulate NALP3 deubiquitination by a mitochondrial ROS-independent mechanism.13

At high NALP3 expression levels, priming with TLR4 agonists is not required and treatment with ATP alone can activate NALP3.

However, at basal expression levels priming is required. This suggests that at basal levels NALP3 might be highly ubiquitinated at different domains by different polyubiquitin chains (e.g. K48 and K63). TLR4 signalling might activate a DUB enzyme that targets a specific polyubiquitin chain and/or a specific domain in NALP3, whereas ATP signalling might activate a second DUB enzyme that deubiquitinates a different domain therefore signals from multiple NALP3 agonists will be required.13

Potassium efflux is probably a necessary step upstream of NALP3 activation because it has been shown that macrophages cultured in a potassium-rich medium show decreased capacity for NALP3-dependent caspase-1 activation in response to a range of agonists.7

Some identified inhibitors of the NALP3 inflammasome

Parthenolide is an anti-inflammatory drug that is found in the medicinal plant feverfew. The anti-inflammatory activity of parthenolide is thought to be due to its ability to alkylate cysteine residues in NF-kB. The methylene moiety of parthenolide as part of a gamma-lactone group can serve as a site for covalent modification via a Michael addition reaction. The anti-inflammatory activity of parthenolide is lost when the methylene moiety is reduced. The epoxide moiety in parthenolide is also a potential site for nucleophilic attack by an amino acid side chain.14

Parthenolide inhibits the inflammasome by direct inhibition of caspase-1 and NALP3.15 Parthenolide inhibits the NALP3 inflammasome by inhibiting its ATPase activity that is required for activation. Parthenolide’s ability to inhibit the activity of multiple inflammasomes at low micromolar concentrations suggests that it may act on a target that is common to all inflammasome signalling pathways. Caspase-1 is the only component that is common to all inflammasomes. Parthenolide directly alkylates the p20 subunit of caspase-1. 16 The activity of parthenolide has been found to be related to the nucleophilic reaction of the methylene-gamma-lactone ring with glutathione or cysteine thiol groups of other target molecules.15

Bay 11-7082 and several vinyl sulfone compounds are selective inhibitors of NALP3. Phenyl vinyl (PV)-sulfone derivatives are also irreversible inhibitors of cysteine proteases such as cathepsins, raising the possibility that these compounds might also target a critical cysteine protease upstream of NALP3.17 BAY 11-7082 inhibits the ATPase activity of NALP3.16

Glibenclamide is a sulfonylurea anti-diabetic agent that has been shown to inhibit the NALP3 inflammasome to control the release of IL-1B.3 As an anti-diabetic it acts by inhibiting potassium channels in pancreatic β-cells. Glibenclamide also inhibits IL-1β production in response to multiple NALP3 stimuli but not NLRC4 or NALP1 activation. However, it has been shown that glibenclamide does not inhibit temperature-induced IL-1β release from monocytes of FCAS patients, suggesting that glibenclamide does not directly inhibit NALP3 but upstream signalling.16

The role of DUBs in regulating the NALP3 inflammasome

The activation of the NALP3 inflammasome is thought to be regulated by a deubiquitination mechanism.11,13 The deubiquitinating enzyme BRCC3 is a critical regulator of NALP3 activity by promoting its deubiquitination and characterising NALP3 as a substrate for the cytosolic BRCC3-containg BRISC complex. Murine BRCC3 and its human homolog BRCC36 are JAMM domain-containing ZN2+ metallo-proteases. BRCC3 is known to participate in DNA damage response in the BRCA1-A complex recruited by the chromatin at double-strand break sites, as well as in cytosolic BRISC complex. G5, a small molecule inhibitor of DUBs, inhibits the deubiquitination and subsequent activation of NALP3, suggesting that NALP3 activity is regulated by its ubiquitination. G5 inhibitory activity is selective for NALP3-dependent inflammasome stimuli, as G5 had no effect on caspase-1 cleavage and mature IL-1βsecretion triggered by S. Typhimurium or cytosolic poly(dA-dT) DNA, which are dependent on the NLRC4 and AIM2 inflammasomes, respectively, and independent of NALP3. Inhibition of deubiquitination by G5 triggers the polyubiquitination of both the NACHT and LRR domains of NALP3 with mixed K63 and K48 chains, which critically inhibits NALP3 inflammasome activation. Notably, G5 dramatically increased BRCC3 binding to high molecular weight ubiquitinated LRR, suggesting that BRCC3 specifically interacts with the ubiquitinated LRR domain.11

LPS-induced NALP3 deubiquitination is inhibited by mitochondrial ROS scavengers (NAC and Mito-TEMPO), whereas ATP-induced NALP3 deubiquitination is not inhibited by these agents. This suggests that the DUB enzyme involved in LPS-induced priming of NALP3 is different from that involved in ATP-induced activation of NALP3. These DUB enzymes might target different types of ubiquitin chains in NALP3 and/or different domains of NALP3.13

Inhibition of DUBs by ESI, b-AP15 or WP1130 blocked IL-1β processing and release induced by the NALP3 inflammasome. Data suggests that these effects were upstream of inflammasome formation as DUB inhibitors blocked ASC speck formation but had no direct effect on caspase-1 activity.8

DUB inhibitors could be used to target activation of the inflammasome and therefore prevent excessive inflammatory response.