Antifungal benzo[b]thiophene 1,1-dioxide IMPDH inhibitors exhibit pan-assay interference (PAINS) profiles

Fungi cause serious life-threatening infections in immunocompromised individuals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor

Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome

MicroRNA (miRNA)-mediated RNA interference regulates many immune processes, but how miRNA circuits orchestrate aberrant intestinal inflammation during inflammatory bowel disease (IBD) is poorly defined. Here, we report that miR-223 limits intestinal inflammation by constraining the nlrp3 inflammasome. miR-223 was increased in intestinal biopsies from patients with active IBD and in preclinical models of intestinal inflammation. miR-223-/y mice presented with exacerbated myeloid-driven experimental colitis with heightened clinical, histopathological, and cytokine readouts. Mechanistically, enhanced NLRP3 inflammasome expression with elevated IL-1β was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2+ inflammatory monocytes and pharmacologic blockade of IL-1β or NLRP3 abrogated this phenotype. Generation of a novel mouse line, with deletion of the miR-223 binding site in the NLRP3 3′ untranslated region, phenocopied the characteristics of miR-223-/y mice. Finally, nanoparticle-mediated overexpression of miR-223 attenuated experimental colitis, NLRP3 levels, and IL-1β release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation

Design, synthesis and evaluation of an NLRP3 inhibitor diazirine photoaffinity probe

The NLRP3 inhibitor MCC950/CRID3 ameliorates a remarkable number of inflammatory disorders in animal models. Herein we describe a trifluoromethyl phenyl diazirine (TPD) photoaffinity probe, called TPD-950-Br, to probe the molecular interactions of MCC950. We show that TPD-950-Br covalently captures proximal species upon photo-activation and inhibits IL-1β production in an NLRP3 inhibitor assay

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1 production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1 production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1 production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1 production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1 maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria

Non-canonical Caspase-1 signaling drives RIP2-dependent and TNF-α-mediated inflammation in vivo

Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1 mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo.Reinke et al. show that enzymatically inactive caspase-1-C284A mediates non-canonical caspase-1 signaling. This pathway is RIP2 dependent and mediated by TNF-α but independent from IL-1 cytokines

NLRP3 inflammasome inhibition by MCC950 in aged mice improves health via enhanced autophagy and PPARα activity

The NLRP3 inflammasome has emerged as an important regulator of metabolic disorders and age-associated diseases as reported in NLRP3 deficient mice. Here we asked whether in old mice C57BL6J the NLRP3 inflammasome inhibitor MCC950 is able to attenuate age- assocociated metabolic syndrome providing health benefits. We report that MCC950 attenuates metabolic and hepatic dysfunction in aged mice. MCC950 inhibited the Pi3K/AKT/mTOR pathway, and enhanced autophagy and activated peroxisome proliferator-activated receptor alpha (PPARα) in vivo and in vitro. The data suggest that MCC950 mediates the protective effects by mTOR inhibiton activating autophagy and PPARα. In conclusion, pharmacological inhibition of NLRP3 in aged mice has a significant impact on health. Thus, NLRP3 may be a therapeutic target of human age-associated metabolic syndrome

Multiple inflammasomes may regulate the interleukin-1-driven inflammation in protracted bacterial bronchitis

Protracted bacterial bronchitis (PBB) in young children is characterised by prolonged wet cough, prominent airway interleukin (IL)-1β expression and infection, often with nontypeable Haemophilus influenzae (NTHi). The mechanisms responsible for IL-1-driven inflammation in PBB are poorly understood. We hypothesised that the inflammation in PBB involves the NLRP3 and/or AIM2 inflammasome/IL-1β axis. Lung macrophages obtained from bronchoalveolar lavage (BAL), peripheral blood mononuclear cells (PBMCs), blood monocytes and monocyte-derived macrophages from patients with PBB and age-matched healthy controls were cultured in control medium or exposed to live NTHi. In healthy adult PBMCs, CD14+ monocytes contributed to 95% of total IL-1β-producing cells upon NTHi stimulation. Stimulation of PBB PBMCs with NTHi significantly increased IL-1β expression (p<0.001), but decreased NLRC4 expression (p<0.01). NTHi induced IL-1β secretion in PBMCs from both healthy controls and patients with recurrent PBB. This was inhibited by Z-YVAD-FMK (a caspase-1 selective inhibitor) and by MCC950 (a NLRP3 selective inhibitor). In PBB BAL macrophages inflammasome complexes were visualised as fluorescence specks of NLRP3 or AIM2 colocalised with cleaved caspase-1 and cleaved IL-1β. NTHi stimulation induced formation of specks of cleaved IL-1β, NLRP3 and AIM2 in PBMCs, blood monocytes and monocyte-derived macrophages. We conclude that both the NLRP3 and AIM2 inflammasomes probably drive the IL-1β-dominated inflammation in PBB

Multiple inflammasomes may regulate the interleukin-1-driven inflammation in protracted bacterial bronchitis

Protracted bacterial bronchitis (PBB) in young children is characterised by prolonged wet cough, prominent airway interleukin (IL)-1β expression and infection, often with nontypeable Haemophilus influenzae (NTHi). The mechanisms responsible for IL-1-driven inflammation in PBB are poorly understood. We hypothesised that the inflammation in PBB involves the NLRP3 and/or AIM2 inflammasome/IL-1β axis. Lung macrophages obtained from bronchoalveolar lavage (BAL), peripheral blood mononuclear cells (PBMCs), blood monocytes and monocyte-derived macrophages from patients with PBB and age-matched healthy controls were cultured in control medium or exposed to live NTHi. In healthy adult PBMCs, CD14+ monocytes contributed to 95% of total IL-1β-producing cells upon NTHi stimulation. Stimulation of PBB PBMCs with NTHi significantly increased IL-1β expression (p<0.001), but decreased NLRC4 expression (p<0.01). NTHi induced IL-1β secretion in PBMCs from both healthy controls and patients with recurrent PBB. This was inhibited by Z-YVAD-FMK (a caspase-1 selective inhibitor) and by MCC950 (a NLRP3 selective inhibitor). In PBB BAL macrophages inflammasome complexes were visualised as fluorescence specks of NLRP3 or AIM2 colocalised with cleaved caspase-1 and cleaved IL-1β. NTHi stimulation induced formation of specks of cleaved IL-1β, NLRP3 and AIM2 in PBMCs, blood monocytes and monocyte-derived macrophages. We conclude that both the NLRP3 and AIM2 inflammasomes probably drive the IL-1β-dominated inflammation in PBB