key: cord-0782279-k53o7qw7
authors: Ulgheri, Fausta; Spanu, Pietro; Deligia, Francesco; Loriga, Giovanni; Fuggetta, Maria Pia; de Haan, Iris; Chandgudge, Ajay; Groves, Matthew; Domling, Alexander
title: Design, synthesis and biological evaluation of 1,5-disubstituted α-amino tetrazole derivatives as non-covalent inflammasome-caspase-1 complex inhibitors with potential application against immune and inflammatory disorders
date: 2021-11-18
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2021.114002
sha: d9453a2ff76adcaec947801a28a211fa702c6474
doc_id: 782279
cord_uid: k53o7qw7

Compounds targeting the inflammasome-caspase-1 pathway could be of use for the treatment of inflammation and inflammatory diseases. Previous caspase-1 inhibitors were in great majority covalent inhibitors and failed in clinical trials. Using a mixed modelling, computational screening, synthesis and in vitro testing approach, we identified a novel class of non-covalent caspase-1 non cytotoxic inhibitors which are able to inhibit IL-1β release in activated macrophages in the low μM range, in line with the best activities observed for the known covalent inhibitors. Our compounds could form the basis of further optimization towards potent drugs for the treatment of inflammation and inflammatory disorders including also dysregulated inflammation in Covid 19.

Inflammasomes are multiprotein complexes emerged as key regulators of innate immune response and inflammation [1] . Their assembly in cytosol, in response to molecules derived from microorganism (pathogen-associated molecular patterns -PAMPs) or endogenous danger signals (damage-associated molecular patterns -DAMPs), induces the activation of the holoenzyme inflammasome-caspase 1 complex, that triggers the release of the mature form of inteleukin-1 (IL-1) and IL-18 and drives pyroptosis. The release in the cytosol of these potent proinflammatory mediators, culminate in beneficial immune responses and antimicrobial defense [2] .

However, a deregulated activation and secretion of inflammatory mediators induced by endogenous danger signals, is linked to the onset or progression of cardiovascular diseases, inflammatory pathologies, neurodegenerative, metabolic and autoimmune diseases and cancer [3] [4] [5] [6] [7] [8] [9] [10] . There is a growing evidence on the relation between innate immunity, excessive release of pro-inflammatory IL-1 and various immune and inflammatory disorders, including CNS diseases such as Alzheimer's (AD), Parkinson's (PD) and Huntington's (HD) diseases, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS) [11] [12] [13] [14] [15] . Activation of microglia and other cell types in the brain, in response to DAMPs in the form of misfolded proteins, mislocalized nucleic acids or aggregated peptides such as amyloid- (A for AD, -synuclein for PD, superoxide dismutase for ALS and huntingtin for HD, leading to uncontrolled release of pro-inflammatory mediators, is emerged as a key mechanism in the development and progression of major neurodegenerative disease [16] [17] [18] . Moreover, there is also a growing evidence that the pathway inflammasome NLRP3/Caspase-1 is overactivated by the SARS-Cov-2 and may be responsible for the high mortality observed in the Covid-19 patients due to the inflammatory internal organs collapse driven by the cytokine storm induced by the virus [19] [20] [21] [22] [23] . It is now quite clear that an effective J o u r n a l P r e -p r o o f pharmacological approach to Covid-19 must comprise an antiviral drug in combination with an inflammation modulator able to regulate the innate immunity system response preserving its regular activity but quenching its overactivation.

Caspase-1 activity is essential in immune and inflammatory response irrespective of molecular signals that induce the assembly of the different holoenzyme inflammasome-caspase-1 complexes.

In addition to pro-IL-1 and pro-IL18, also gasdermin D (GSDMD) is a recognized substrate for caspase-1 to generate an N-terminal cleavage product (GSDMD-NT) that induces plasma membrane pores and pyroptosis [24] . Recent studies have shed some light on the nature of the caspase-1 active species (p33/p10 linked to inflammasome to form the inflammasome-caspase-1 complex), on the location of caspase-1 activity, on the intrinsic time limiting mechanism of caspase-1 activity of the inflammasome-dependent inflammatory responses, as well as on the role of caspase-1 in inducing pyroptosis by mediating GSDMD cleavage [24, 25] . Compounds All these peptidomimetic molecules and the currently used peptidic caspase-1 tool compounds are often unselective and covalent as mode of action (MoA), peptidic in nature, and with very low blood-brain barrier (BBB) penetration, if at all. The lack of selectivity and the off-target interaction with other nucleophiles in vivo due to their covalent MoA, may have contributed to the effects observed in long term toxicity studies in dogs accompanying the human phase II study of Pralnacasan (VX-740) [39] . Novel improved compounds are urgently needed for testing the hypothesis if suitable caspase-1 inhibitors are able to slow down or prevent inflammatory diseases in humans also because there are no caspase-1 inhibitor drugs approved for clinical use on the market. Here we report the design and synthesis of a new class of potent and stable nonpeptidomimetic and non-covalent caspase-1 inhibitors designed to overcome the drawbacks of the previous compounds for therapeutic applications.

J o u r n a l P r e -p r o o f

We have designed a new class of non-covalent, non-peptidic, small molecule caspase-1 inhibitors by structure-based drug design. Our goal was to obtain a new class of stable and bioavailable inhibitors rationally designed to interact with the catalytic site of the enzyme in a competitive and non-covalent MoA in order to avoid off-target interactions with other nucleophiles leading to side and toxic effects previously described in clinical trials for covalent inhibitors. The multicomponent reaction (MCR) approach was used for the synthesis of our molecules, because it offers a significant advantage over conventional linear step synthesis permitting the one pot assembly of very complex structures and the easy synthesis of a collection of derivatives.

Our design of non-covalent caspase-1 inhibitors is based on a substrate mimicry approach (Fig. 2) .

The WEAD (trp-glu-ala-asp) sequence is generally recognized by caspase-1 and target proteins are always aspartyl-cleaved (P1=Asp) [40] . Thus, we aimed to design non cleavable aspartyl mimicking scaffolds which would allow for easy modification in further positions to address P2-P4 sites. Multicomponent reaction chemistry is known to address a large drug-like scaffold space and is compatible to multiple functional (often unprotected) groups [41] [42] [43] . For example, MCR has been recently used for the improved synthesis of the drugs atorvastatin, praziquantel and ivosidenib [44] [45] [46] . Multicomponent reaction chemistry allows for the simultaneous introduction of several side chains via different building blocks which potentially can mimic P2-P4 sites.

Multicomponent reactions we are experienced in are isocyanide-based MCRs [47] . Specifically, tetrazole yielding MCRs recently attracted considerable attention due to their scaffold diversity, ease of access and drug-like properties [48] . Thus, we decided to investigate the Ugi tetrazole J o u r n a l P r e -p r o o f variation (UT-4CR) and search the UT-4CR chemical space for potential caspase-1 inhibitors. The UT-4CR variation consists of the reaction of an isocyanide with an oxo component and a secondary or primary amine as variable building blocks to yield 1,5-disubstituted -amino tetrazole [49, 50] .

Our design includes a 4-amino-3-hydroxy butanoic acid mimicking the aspartyl P1 side chain. 7 We pursued a computational prescreening to decide which compounds to synthesize and test. The ChemAxon suite was used in order to create a virtual library of compounds based on multicomponent reaction (MCR) chemistry [51] . Babel and Moloc molecular design software were used to generate 3D conformations, to fix the unique molecular library to a fragment and to optimize the energy and the overlap of the library with the protein [52] . As the receptor for modelling we were using the 2HBQ PDB structure [53] . It is a crystal structure of wildtype human caspase-1 in complex with covalent 3-[2-(2-benzyloxycarbonylamino-3-methyl-butyrylamino)propionylamino]-4-oxo-pentanoic acid (z-VAD-FMK) inhibitor. A typical result from virtual screening the UT-4CR chemical space is shown in Fig. 3 . J o u r n a l P r e -p r o o f

In order to explore the UT-4CR chemical space for potential caspase-1 inhibitors the series of the selected 1,5-disubstituted -amino tetrazole 5 and 16 were synthesized via MCRs. The four components of the UT-4CR needed to meet all the requirements established by the computational study were methyl-4-amino-3-hydroxybutanoate as the amine component 1, an aliphatic or aromatic aldehyde 2, a 4-isocyanobutanamide derivative 3, and trimethylsilyl azide 4 (Scheme 1).

A collection of derivatives 5 and 16 has been then synthesised exploiting variations to the aldehyde and isocyanide components of the MCR. J o u r n a l P r e -p r o o f tetrahydroisoquinoline moiety that showed to induce the best enzymatic activity with respect to the other isocyanides 3b and 3c (see below). Compounds 16aa-16ad were obtained in good yields after basic hydrolysis and purification on a silica gel pad (Scheme 4). a IC50 is the concentration of the inhibitor where the enzyme activity is reduced by half (curve fits were performed when the activities at the highest concentration of compounds were less than 60%); ND not determined, and IA inactive.

Comparing the activity of the three series of compounds, the better potency was observed for 1,5disubstituted -amino tetrazole 5aa-5ah, showing that the tetrahydroisoquinoline moiety determines a better P3-P4 sites interactions with respect to benzyl or 6,7-dimethoxy-J o u r n a l P r e -p r o o f tetrahydroisoquinoline. Tuning of the hydrophobic P2 site interactions by aliphatic and aromatic R2 residues gave tetrazole derivatives with IC50 values in the M range. The best compound of the series was the tetrazole derivative 5ae (R2=neopentyl) with an IC50 of 15.1 M and 8.12% of residual enzymatic activity at the 100 M concentration. Regarding the (3R) or (3S) diastereomeric mixture of 1,5-disubstituted -amino tetrazole derivatives 5 we observed that the (3R) configured stereoisomers were slightly more active with respect to their (3S) stereoisomers, in agreement with the results of our computational studies, therefore the subsequent experiments on stereoisomers were performed by using only the (3R) diastereomeric mixture of the tetrazole derivatives. In any case, no significative differences in enzymatic activities were observed between racemic compounds and the same tetrazole derivatives with (3R) stereochemistry. a IC50 is the concentration of the inhibitor where the enzyme activity is reduced by half (curve fits were performed when the activities at the highest concentration of compounds were less than 60%); ND not determined, and IA inactive. 2-Thiophenyl ND IA a IC50 is the concentration of the inhibitor where the enzyme activity is reduced by half (curve fits were performed when the activities at the highest concentration of compounds were less than 60%); ND not determined, and IA inactive.

Finally, in order to investigate additional hydrophobic P2' site interactions, two N-alkyl substituents were introduced in the tetrazole derivatives containing tetrahydroisoquinoline and isobutyl or neopentyl residues. Benzyl and 4-fluoro-benzyl R3 substituents were introduced to give compounds 16aa-16ad (Scheme 4). A reduction of the IC50 value was observed for compounds 16aa-16ab (R3= isobutyl) with respect to their not N-alkylated tetrazole derivates but the same effect was not confirmed for compounds 16ac-16ad (R3= neopentyl) ( Table 4 ). No significative difference in enzymatic activity was observed between compound 16ac and the same tetrazole derivative with (3R) stereochemistry as we found in the previous cases. Taken together, these results on enzymatic activity of diastereomeric mixture support the decision to synthesize and test only the racemic mixture of compound 5 or compound 16 in the subsequent cell-based assays. a IC50 is the concentration of the inhibitor where the enzyme activity is reduced by half (curve fits were performed when the activities at the highest concentration of compounds were less than 60%); ND not determined, and IA inactive.

The 1,5-disubstituted -amino tetrazole 16aa and 5ae were selected for in vitro evaluation of the cytotoxicity and immunomodulatory effect because of their very good caspase-1 inhibition enzymatic activity. The cell line U-937 was used to reproduce in vitro a biological model of inflammation. U-937 is a human cell line expressing many of the monocytic like characteristics.

U-937 was differentiated into macrophages with PMA and stimulated with LPS to induce IL-1 production as described in literature [54, 55] .

To exclude a cytotoxic effect of 16aa and 5ae on human U937 cell line, cells were exposed to increasing concentrations of the synthesized compounds 16aa and 5ae ranging from 1nM to 100μM for 48h and then evaluated for cell viability and cell growth inhibition by MTT assay. The results illustrated in Fig. 4 show that at the higher concentration the viability for both compounds J o u r n a l P r e -p r o o f was reduced by almost 50% while at lowest concentrations the cytotoxic effect is low or is not significant.

In order to evaluate if the toxicity observed at the100 M concentration was dependent from the solvent used (DMSO) rather than from compound 16aa or 5ae, the experiments were repeated by adding DMSO alone at the same concentration used to dilute 16aa and 5ae. As showed in Fig. 4 , at the concentrations of 100 M or 10M, it can be assumed that the reduction of viability was clearly due to the presence of DMSO rather than to 16aa or 5ae. Our results showed that the secretion of IL-1 was significantly suppressed by 16aa and that the required concentration is related to the schedule of treatment. We could speculate that during the simultaneous treatment with LPS, 16aa acts early during the LPS priming. LPS is toll-like receptor (TLR4) ligand, the binding LPS/TLR4 is defined as the priming step, which provides the first signal for NLRP3 inflammasome activation that in turn active Caspase-1 that is involved in the maturation of interleukin IL-1β [56] . After 4 hours of pretreatment with LPS, inflammatory machinery is complete and this may require a greater concentration of 16aa to inhibit Caspase 1.

The treatment with graded concentrations of compound 5ae ranging from 10μM to 1 nM has been also performed simultaneously to LPS (1μg/ml) treatment for 24h. The results show that also J o u r n a l P r e -p r o o f compound 5ae is able to significantly inhibit the IL-1 production after LPS stimulation with IC50= 50.06 μM when used in the presence of LPS. 

Through the rational design and an MCR based synthetic approach of new non-covalent caspase-1 inhibitors we were able to obtain, a noncytotoxic 1,5-disubstituted -amino tetrazoles able to target the inflammasome caspase-1 pathway that could be of use for the treatment of inflammatory driven diseases. This new class of non-covalent inhibitors was able to inhibit IL-1 release in activated macrophages in the low M range that is in line with the best activities observed for the known covalent inhibitors that failed in clinical trials, although they showed a much higher 

SOCl2 (190 L, 2.52 equiv.) was added dropwise at 0 °C to a flask containing CH3OH (5 mL), then 4-amino-3-hydroxybutanoic acid (250 mg, 2.10 equiv.) was added. The reaction mixture was stirred at r.t. overnight, then the solvent was removed under vacuum. The residue was stirred with J o u r n a l P r e -p r o o f hexanes and concentrated under vacuum to give the clean product in a quantitative yield as a thick light-yellow oil. 1 

A solution of 4-aminobutanoic acid 6 (4 g, 38.75 mmol) in propyl formate HCOOC3H7 (40 mL) and formic acid (2 mL 

To a suspension of 4-formamidobutanoic acid 7 (1 equiv) in THF (0.3 M) at r.t., the corresponding amines 8, 9 or 10 (1 equiv.), HOBT (1 equiv.) and DCC (1.1 equiv.) were added. The reaction mixture was stirred at r.t. overnight, then filtered on a celite pad washing with a minimal amount of THF. The solvent was removed under vacuum and the crude product was purified by filtration on a silica gel pad under vacuum eluting first with AcOEt and then with CH2Cl2/CH3OH 9/1 to recover the desired formamides 11-13.

Thick light-yellow oil obtained in 97% yield. 1 77 (m, 2Ha,b) . 13 

Thick light-yellow oil obtained in 91% yield. 1 

To a solution of formamide 11-13 (1 equiv.) in dry CH2Cl2 (0.3 M), under nitrogen, Et3N (5 equiv.) and POCl3 (1.5 equiv.) were added at 0°C. The reaction mixture was stirred at r.t. for 1 h, then treated with a 20% aqueous solution of Na2CO3. The aqueous layer was extracted three times with CH2Cl2. The organic phase, dried on Na2SO4, was filtered and concentrated under vacuum. The crude product was purified by filtration on a silica gel pad under vacuum, eluting first with CH2Cl2 and then with CH2Cl2/AcOEt 9/1 to recover the desired products 3a-c.

J o u r n a l P r e -p r o o f Light yellow oil obtained in 87% yield. 1 

Light yellow oil obtained in 85% yield. 1 °C and NaBH4 (64 mg, 1.68 mmol) was added, and the mixture was stirred for 4.5h at r.t., then concentrated under vacuum to give a thick colorless oil that was dissolved in CH3OH (5 mL), cooled to 0 °C and treated dropwise with SOCl2 (42 mL, 5.75 mmol) under nitrogen. The reaction mixture was stirred at r.t. overnight then the solvent was removed under vacuum to give the crude product 15a or 15b as a solid that was used as such in the following MCRs.

White solid obtained in quantitative yield. m.p.= 138-140 °C. 1 

White solid obtained in quantitative yield. m.p.= 163-165 °C. 1 and TMSN3 (1 equiv.) were added. The reaction mixture was stirred at room temperature for 5 days and then filtered on a celite pad to give the crude product that was purified by filtration on a silica gel pad eluting with CH2Cl2/Et2O 9/1, then EtOAc/Acetone 9/1 to give a tick light yellow oil with yields ranging from 20% to 92%. The 4-MCR product was then solved in CH3OH (0.05 M) was treated with a solution of NaOH 1M (0.1 M in CH3OH). The solution was stirred at room temperature for 5h, then the solvent was removed under vacuum and the crude product was purified by filtration on a silica gel pad eluting with EtOAc/Acetone 9/1 and CH2Cl2/CH3OH 8/2 to give the desired product 5 or 16, with yields ranging from 50% to 96%.

The title compound was prepared from compound 3a and 2 (R1=methyl) following the general procedure of 5. Yield MCR 31%, yield hydrolysis 52%, light yellow oil, mixture of atropoisomers. 

The title compound was prepared from compound 3a and 2 (R1=isopropyl) following the general procedure of 5. Yield MCR 51%, yield hydrolysis 96%, light yellow oil, mixture of atropoisomers. 

The title compound was prepared starting from compounds 3a and 2 (R1= t butyl) following the 

The title compound was prepared from compound 3a and 2 (R1=isobutyl) following the general procedure of 5. Yield MCR 55%, yield hydrolysis 89%, colorless oil, mixture of atropoisomers. 

The title compound was prepared starting from compounds 3a and 2 (R1=neopentyl) following the 

The title compound was prepared starting from compounds 3a and 2 (R1=neopentyl) and the enantiopure compound 1(3R) following the general procedure of 5. 

J o u r n a l P r e -p r o o f 

The title compound was prepared starting from compounds 3a and 2 (R1=phenyl) following the 

The title compound was prepared starting from compounds 3a and 2 (R1=phenyl) and the enantiopure compound 1(3R) following the general procedure of 5. 

The title compound was prepared starting from compounds 3a and 2 (R1=phenyl) and the enantiopure compound 1(3S) following the general procedure of 5. 

The title compound was prepared starting from compounds 3a and 2 (R1=2-thiophenyl) following the general procedure of 5. Yield MCR 26%, hydrolysis 69%, thick light-yellow oil, mixture of atropoisomers. 1 J o u r n a l P r e -p r o o f

The title compound was prepared starting from compounds 3a and 2 (R1=2-thiophenyl) and the enantiopure compound 1(3R) following the general procedure of 5. Yield MCR 31%, hydrolysis 57%, thick light-yellow oil, mixture of atropoisomers. 1 

The title compound was prepared starting from compounds 3a and 2 (R1=2-thiophenyl) and the enantiopure compound 1(3S) following the general procedure of 5. 

J o u r n a l P r e -p r o o f

The title compound was prepared starting from compounds 3b and 2 (R1=methyl) following the general procedure of 5. Yield MCR 55%, hydrolysis 65%, light-yellow oil, mixture of atropoisomers. 1 

The title compound was prepared starting from compounds 3b and 2 (R1=isopropyl) following the 

The title compound was prepared starting from compounds 3b and 2 (R1=neopentyl) following the 

The title compound was prepared starting from compounds 3b and 2 (R1=phenyl) following the 

The title compound was prepared starting from compounds 3b and 2 (R1=phenyl) and the enantiopure compound 1(3R) following the general procedure of 5. Yield MCR 52%, hydrolysis 54%, light-yellow oil, mixture of atropoisomers. 1 

The title compound was prepared starting from compounds 3b and 2 (R1=2-tiophenyl) and the enantiopure compound 1(3R) following the general procedure of 5. Yield MCR 39%, hydrolysis 55%, light-yellow oil, mixture of atropoisomers. 1 

The title compound was prepared starting from compounds 3b and 2 (R1=2-tiophenyl) and the enantiopure compound 1(3S) following the general procedure of 5. 

The title compound was prepared starting from compounds 3c and 2 (R1= t butyl) following the 

The title compound was prepared starting from compounds 3c and 2 (R1=neopentyl) following the general procedure of 5. Yield MCR 60%, hydrolysis 60%, light-yellow oil, mixture of atropoisomers. 1 

The title compound was prepared starting from compounds 3c and 2 (R1=2-thiophenyl) following the general procedure of 5. J o u r n a l P r e -p r o o f SIGMA) for 24h. To induce IL-1 production the U937 differentiated cells were treated with 1μg/ml of LPS (SIGMA) for 4h [26] .

The effect of 16aa and 5ae were evaluated on cell viability of U937 cells. The cells were seeded in 98-well plates with 10000 cells per well and treated with different concentration of 16aa and 5ae ranging from 100M to 1nM for 48 hrs. U937 were also treated with RPM11640 containing DMSO at the same concentration used to dilute 16aa and 5ae at the beginning and in the following dilutions. After incubation with 16aa, 5ae or DMSO an MTT test has been performed to evaluate cells viability. Briefly, after 2 days of culture, 0.1 mg of MTT (in 20 l of PBS) was added to each well and cells were incubated at 37°C for 4 h. Cells were then lysed, and the absorbance was read at 595 nm using a microplate reader.

Frozen culture SN of cells treated with LPS and 16aa or 5ae were thawed and immediately tested for the presence of human IL-1 The test was carried out by ELISA quantitative sandwich enzyme immunoassay technique (ELISA kit Quantikine, Human Il-1/Il-1F2 immunoassay, R&D Systems, Minneapolis, USA) specific for natural and recombinant human IL-1.

* Corresponding Author.

** Corresponding Author.

1H)-yl)-4-oxobutyl)-1H-tetrazol-5-yl)-3-methylbutyl)amino)-3-hydroxybutanoate (16aa

The title compound was prepared starting from compounds 3a, 15 (R1= H) and 2 (R2=isobutyl) following the general procedure of 16. Yield MCR 22%, hydrolysis 69%, colorless oil, mixture of atropoisomers. 1 H NMR CD3OD δ 7.36-7.26 (m, 4Ha,b), 7.23-7.13 (m, 5Ha,b)

1H)-yl)-4-oxobutyl)-1H-tetrazol-5-yl)-3-methylbutyl)(4-fluorobenzyl)amino)-3-hydroxybutanoate (16ab

m, 1Ha), 3.99-3.87 (m, 1Hb), 3.80-3.66 (m, 4Ha-c), 2.92 (t, J=5.6 HZ, 2Ha), 2.86-2.78 (m, 2Hb,c), 2.71-2.60 (m, 1Ha-c), 2.54-2.44 (m, 3Ha-c), 2.32-1.83 (m, 5Ha-c), 1.56-1.44 (m, 1Ha-c), 0.94 (d, J=6.8 Hz, 3Ha-c

1H)-yl)-4-oxobutyl)-1H-tetrazol-5-yl)-3,3-dimethylbutyl)(benzyl)amino)-3-hydroxybutanoate (16ac

The title compound was prepared starting from compounds 3a, 15 (R1=H) and 2 (R2=neopentyl) following the general procedure of 16. Yield MCR 92%, hydrolysis 40%, colorless oil, mixture of atropoisomers. 1 H NMR CD3OD δ 7.33-7.10 (m, 9Ha,b)

0.80 (bs, 9Ha,b). 13 C NMR CD3OD δ 177.05, 172.33, 172.21, 157

(1H)-yl)-4-oxobutyl)-1H-tetrazol-5-yl)-3,3-dimethylbutyl)(benzyl)amino)-3-hydroxybutanoate

(m, 4Ha,b), 3.86-3.53 (m, 4Ha,b), 2.92 (t, J=6 Hz, 2Ha), 2.85 (t, J=6Hz, 2Hb), 2.72-1.70 (m, 10 Ha,b), 0.82 (bs, 9Ha,b). 13 C NMR CD3OD

1H)-yl)-4-oxobutyl)-1H-tetrazol-5-yl)-3,3-dimethylbutyl)(4-fluorobenzyl)amino)-3-hydroxybutanoate (16ad

(m, 5Ha,b), 2.26-1.79 (m, 5Ha,b), 0.81 (bs, 9Ha,b). 13 C NMR CD3OD δ 176

Caspase-1 inhibition assays (Provided by Reaction Biology CRO)

YVAD-AFC emits blue light (Em=400 nm)the fold increase in caspase-1 activity. Compounds exhibit no fluorescent background that could interfere with the assay. All synthesized compounds were tested 10-dose IC50 with 3-fold serial dilution starting at 100 M or 10 M against caspase-1

Cell line, Differentiation, LPS stimulation

The cells were cultured in a humidified incubator with 5% CO2 at 37 °C

differentiate the U937 monocytes into macrophages, the cells were seeded in CM in 24 well plates treated with 50 μg/ml phorbol-12-myristate-13-acetate (PMA

Inflammasomes: Mechanism of Assembly, Regulation and Signalling

Regulation of Inflammasome Activation

Inflammasomes: Mechanism of Action

NLRP3 Inflammasome in Cancer and Metabolic Diseases

Focus on the Role of NLRP3 Inflammasome in Diseases

Advances in Inflammasome Research: Recent Breakthroughs and Future Hurdles

Protective and Detrimental Roles of Inflammasomes in Disease

The Pathogenic Role of the Inflammasome in Neurodegenerative Diseases

Inflammasomes and Metabolic Disorders: Old Genes in Modern Diseases

The Inflammasomes and Autoinflammatory Syndromes

The Role of Microglia and the Nlrp3 Inflammasome in Alzheimer's Disease

Targeting the NLRP3 Inflammasome in Chronic Inflammatory Diseases: Current Perspectives

Pharmacological Inhibition of the NLRP3 Inflammasome as a Potential Target for Multiple Sclerosis Induced Central Neuropathic Pain

Therapeutics Targeting the Inflammasome after Central Nervous System Injury

NLRP3 Inflammasome in Neurological Diseases, from Functions to Therapies

NALP3 Inflammasome Activation in Protein Misfolding Diseases

Degradation of Misfolded Proteins in Neurodegenerative Diseases: Therapeutic Targets and Strategies

Innate Immunity in Alzheimer's Disease

SARS-CoV-2 Infection: NLRP3 Inflammasome as Plausible Target to Prevent Cardiopulmonary Complications?

Age-Induced NLRP3 Inflammasome Over-Activation Increases Lethality of SARS-CoV-2 Pneumonia in Elderly Patients

Novel Coronavirus-Induced NLRP3 Inflammasome Activation: A Potential Drug Target in the Treatment of COVID-19

SARS-CoV-2 Infection and Overactivation of NLRP3 Inflammasome as a Trigger of Cytokine "Storm" and Risk Factor for Damage of Hematopoietic Stem Cells

Inflammasome Regulation in Driving COVID-19 Severity in Humans and Immune Tolerance in Bats

Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death

Self-Cleavage Is an Intrinsic Mechanism to Terminate Inflammasome Activity

Caspases as Therapeutic Targets

Caspase-1 Inhibition Prevents Neuronal Death by Targeting the Canonical Inflammasome Pathway of Pyroptosis in a Murine Model of Cerebral Ischemia

Caspase-1 Has a Critical Role in Blood-Brain Barrier Injury and Its Inhibition Contributes to Multifaceted Repair

Pre-Symptomatic Caspase-1

Inhibitor Delays Cognitive Decline in a Mouse Model of Alzheimer Disease and Aging

Reducing C-Terminal Truncation Mitigates Synucleinopathy and Neurodegeneration in a Transgenic Model of Multiple System Atrophy

ICE Cleaved Alpha-Synuclein as a Biomarker

IL-Converting Enzyme/Caspase-1 Inhibitor VX-765 Blocks the Hypersensitive Response to an Inflammatory Stimulus in Monocytes from Familial Cold Autoinflammatory Syndrome Patients

Caspase-1 Inhibitor Regulates Humoral Responses in Experimental Autoimmune Myasthenia Gravis via IL-6-Dependent Inhibiton of STAT3

Inflammatory Caspases: Targets for Novel Therapies

Caspase-1 Inhibition Alleviates Cognitive Impairment and Neuropathology in an Alzheimer's Disease Mouse Model

Caspase Inhibitors: A Review of Recently Patented Compounds

Pharmacological Caspase Inhibitors: Research towards Therapeutic Perspectives

Strategies for Targeting the NLRP3 Inflammasome in the Clinical and Preclinical Space

Small Molecule Active Site Directed Tools for Studying Human Caspases

Design of Caspase Inhibitors as Potential Clinical Agents

Multicomponent Reaction-Derived Covalent Inhibitor Space

Chemistry and Biology of Multicomponent Reactions

Multicomponent Reactions (MCR) in Medicinal Chemistry: a Patent Review

Atorvastatin (Lipitor) by MCR

Efficient Multicomponent Reaction Synthesis of the Schistosomiasis Drug Praziquantel

Applications of Fluorine-Containing Amino Acids for Drug Design

Editorial: Isocyanide-Based Multicomponent Reactions

Tetrazoles via Multicomponent Reactions

With Unprotected Amino Acids to Tetrazolo Peptidomimetics

Dömling, α-Amino Acid-Isosteric α-Amino Tetrazoles

Reactor was used for enumeration and reaction modeling

A Common Allosteric Site and Mechanism in Caspases

Macrophage Differentiation Induced by PMA Is Mediated by Activation of RhoA/ROCK Signaling

Ameloblastin Upregulates Inflammatory Response Through Induction of IL-1β in Human Macrophages

Mechanism and Regulation of NLRP3 Inflammasome Activation

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

Supplementary data to this article can be found online at …..