Pathogenic variants in the NLRP3 LRR domain at position 861 are responsible for a boost-dependent atypical CAPS phenotype.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is associated with NLRP3 pathogenic variants, mostly located in the NACHT (neuronal apoptosis inhibitor protein, MHC class 2 transcription activator, incompatibility locus protein from Podospora anserina, telomerase-associated protein) domain. Cold-induced urticarial rash is among the main clinical features. However, this study identified a series of 14 patients with pathogenic variants of the Y861 residue (p.Tyr861) of the LRR domain of NLRP3 and minimal prevalence of cold-induced urticarial rash. OBJECTIVES: This study aimed to address a possible genotype/phenotype correlation for patients with CAPS and to investigate at the cellular levels the impact of the Y861C substitution (p.Tyr861Cys) on NLRP3 activation. METHODS: Clinical features of 14 patients with CAPS and heterozygous substitution at position 861 in the LRR domain of NLRP3 were compared to clinical features of 48 patients with CAPS and pathogenic variants outside the LRR domain of NLRP3. IL-1ß secretion by PBMCs and purified monocytes from patients and healthy donors was evaluated following LPS and monosodium urate crystal stimulation. RESULTS: Patients with substitution at position 861 of NLRP3 demonstrated a higher prevalence of sensorineural hearing loss while being less prone to skin urticarial. In contrast to patients with classical CAPS, cells from patients with a pathogenic variant at position 861 required an activation signal to secrete IL-1ß but produced more IL-1ß during the early and late phase of secretion than cells from healthy donors. CONCLUSIONS: Pathogenic variants of Y861 of NLRP3 drive a boost-dependent oversecretion of IL-1ß associated with an atypical CAPS phenotype.

Successful treatment of JAK1-associated inflammatory disease.

BACKGROUND: Gain-of-function variants of JAK1 drive a rare immune dysregulation syndrome associated with atopic dermatitis, allergy, and eosinophilia. OBJECTIVES: This study sought to describe the clinical and immunological characteristics associated with a new gain-of-function variant of JAK1 and report the therapeutic efficacy of Janus kinase (JAK) inhibition. METHODS: The investigators identified a family affected by JAK1-associated autoinflammatory disease and performed clinical assessment and immunological monitoring on 9 patients. JAK1 signaling was studied by flow and mass cytometry in patients' cells at basal state or after immune stimulation. A molecular disease signature in the blood was studied at the transcriptomic level. Patients were treated with 1 of 2 JAK inhibitors: either baricitinib or upadacitinib. Clinical, cellular, and molecular response were evaluated over a 2-year period. RESULTS: Affected individuals displayed a syndromic disease with prominent allergy including atopic dermatitis, ichthyosis, arthralgia, chronic diarrhea, disseminated calcifying fibrous tumors, and elevated whole blood histamine levels. A variant of JAK1 localized in the pseudokinase domain was identified in all 9 affected, tested patients. Hyper-phosphorylation of STAT3 was found in 5 of 6 patients tested. Treatment of patients' cells with baricitinib controlled most of the atypical hyper-phosphorylation of STAT3. Administration of baricitinib to patients led to rapid improvement of the disease in all adults and was associated with reduction of systemic inflammation. CONCLUSIONS: Patients with this new JAK1 gain-of-function pathogenic variant displayed very high levels of blood histamine and showed a variable combination of atopy with articular and gastrointestinal manifestations as well as calcifying fibrous tumors. The disease, which appears to be linked to STAT3 hyperactivation, was well controlled under treatment by JAK inhibitors in adult patients.

Mechanism-based and computational modeling of hydrogen sulfide biogenesis inhibition: interfacial inhibition.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in various signaling functions in health and diseases. The tetrameric cystathionine γ-lyase (CSE) contributes to H2S biogenesis and several investigations provide evidence on the pharmacological modulation of CSE as a potential target for the treatment of a multitude of conditions. D-penicillamine (D-pen) has recently been reported to selectively impede CSE-catalyzed H2S production but the molecular bases for such inhibitory effect have not been investigated. In this study, we report that D-pen follows a mixed-inhibition mechanism to inhibit both cystathionine (CST) cleavage and H2S biogenesis by human CSE. To decipher the molecular mechanisms underlying such a mixed inhibition, we performed docking and molecular dynamics (MD) simulations. Interestingly, MD analysis of CST binding reveals a likely active site configuration prior to gem-diamine intermediate formation, particularly H-bond formation between the amino group of the substrate and the O3' of PLP. Similar analyses realized with both CST and D-pen identified three potent interfacial ligand-binding sites for D-pen and offered a rational for D-pen effect. Thus, inhibitor binding not only induces the creation of an entirely new interacting network at the vicinity of the interface between enzyme subunits, but it also exerts long range effects by propagating to the active site. Overall, our study paves the way for the design of new allosteric interfacial inhibitory compounds that will specifically modulate H2S biogenesis by cystathionine γ-lyase.

Synthesis of Bisubstrate Analogues for RNA Methylation Studies using two Transition-Metal-Catalyzed Reactions.

RNA methyltransferases (RNA MTases) are a family of enzymes that catalyze the methylation of RNA using the cofactor S-adenosyl-L-methionine. While RNA MTases are promising drug targets, new molecules are needed to fully understand their roles in disease and to develop effective drugs that can modulate their activity. Since RNA MTases are suitable for bisubstrate binding, we report an original strategy for the synthesis of a new family of m6A MTases bisubstrate analogues. Six compounds containing a S-adenosyl-L-methionine (SAM) analogue unit covalently tethered by a triazole ring to the N-6 position of an adenosine were synthesized. A procedure using two transition-metal-catalyzed reactions was used to introduce the α-amino acid motif mimicking the methionine chain of the cofactor SAM. First, a copper(I)-catalyzed alkyne-azide iodo-cycloaddition (iCuAAC) reaction afforded the 5-iodo-1,4-disubstituted-1,2,3-triazole which was functionalized by palladium-catalyzed cross-coupling to connect the α-amino acid substituent. Docking studies of our molecules in the active site of the m6A ribosomal MTase RlmJ show that the use of triazole as a linker provides additional interactions and the presence of the α-amino acid chain stabilizes the bisubstrate. The synthetic method developed here enhances the structural diversity of bisubstrate analogues to explore the active site of RNA modification enzymes and to develop new inhibitors.

Persulfidation of DJ-1: Mechanism and Consequences.

DJ-1 (also called PARK7) is a ubiquitously expressed protein involved in the etiology of Parkinson disease and cancers. At least one of its three cysteine residues is functionally essential, and its oxidation state determines the specific function of the enzyme. DJ-1 was recently reported to be persulfidated in mammalian cell lines, but the implications of this post-translational modification have not yet been analyzed. Here, we report that recombinant DJ-1 is reversibly persulfidated at cysteine 106 by reaction with various sulfane donors and subsequently inhibited. Strikingly, this reaction is orders of magnitude faster than C106 oxidation by H2O2, and persulfidated DJ-1 behaves differently than sulfinylated DJ-1. Both these PTMs most likely play a dedicated role in DJ-1 signaling or protective pathways.

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae.

The universality of peptidoglycan in bacteria underlies the broad spectrum of many successful antibiotics. However, in our times of widespread resistance, the diversity of peptidoglycan modifications offers a variety of new antibacterials targets. In some Gram-positive species such as Streptococcus pneumoniae, Staphylococcus aureus, or Mycobacterium tuberculosis, the second residue of the peptidoglycan precursor, D-glutamate, is amidated into iso-D-glutamine by the essential amidotransferase MurT/GatD complex. Here, we present the structure of this complex at 3.0 Å resolution. MurT has central and C-terminal domains similar to Mur ligases with a cysteine-rich insertion, which probably binds zinc, contributing to the interface with GatD. The mechanism of amidation by MurT is likely similar to the condensation catalyzed by Mur ligases. GatD is a glutaminase providing ammonia that is likely channeled to the MurT active site through a cavity network. The structure and assay presented here constitute a knowledge base for future drug development studies.

Synthesis and in vitro antitumor activity of ring C and D-substituted phenanthrolin-7-one derivatives, analogues of the marine pyridoacridine alkaloids ascididemin and meridine.

A series of cycle C and D-substituted phenanthrolin-7-ones, analogues of the marine pyridoacridines meridine and ascididemin have been synthesized on the basis of Diels-Alder reactions involving quinoline-5,8-dione and 2- (or un)-substituted-N,N-dimethylhydrazones. All the compounds were evaluated for in vitro cytotoxic activity against 12 distinct human cancer cell lines. They all exhibit cytotoxic activity with IC(50) values at least of micromolar order.

Synthesis and in vitro antitumor activity of an isomer of the marine pyridoacridine alkaloid ascididemin and related compounds.

The isomer (9H-quino[4,3,2-de][1,7]phenanthroline-9-one) (2) of the marine alkaloid ascididemin (9H-quino[4,3,2-de][1,10]phenanthroline-9-one) (1) has been synthesized in six steps from 1,4-dimethoxyacridine (10) with an overall yield of 12%. Different related compounds were prepared and tested in vitro at six different concentrations on 12 different human cancer cell lines of various histopathological types (glioblastomas and breast, colon, lung, prostate and bladder cancers). Almost all the compounds present cytotoxic activity of micromolar order.

Synthesis and in vitro antitumor activity of phenanthrolin-7-one derivatives, analogues of the marine pyridoacridine alkaloids ascididemin and meridine: structure-activity relationship.

A series of substituted pyrido[4,3,2-de][1,7] or [1,10]-phenanthrolin-7-ones, analogues of the marine pyridoacridines meridine and ascididemin, have been synthesized on the basis of Diels-Alder reactions involving different quinoline-5,8-diones and N,N-aldehyde-dimethylhydrazones. All the compounds were evaluated for in vitro cytotoxic activity against 12 distinct human cancer cell lines. They all exhibit cytotoxic activity with IC(50) values at least of micromolar order.

Synthesis and in vitro antitumor activity of novel ring D analogues of the marine pyridoacridine ascididemin: structure-activity relationship.

Marine compounds with pyridoacridine skeletons are known to exhibit interesting antitumor activities. Ascididemin has already been reported as displaying significant antitumor activities in vitro and has also been found to have a relatively high global toxicity in vivo. We synthesized a series of 16 analogues (among which 11 compounds were different from previously described ones) with the aim of developing new anticancer agents with significantly improved efficacy/tolerability ratios. These compounds were obtained either by total synthesis from 5,8-quinolinedione and substituted 2-aminoacetophenones or by the direct substitution of ascididemin. The different compounds and ascididemin used as the control compound were tested at six different concentrations on 12 different human cancer cell lines of various histopathological types (glioblastomas and breast, colon, lung, prostate, and bladder cancers). The IC(50) value (i.e., the drug concentration inhibiting the mean growth value of the 12 cell lines by 50%) of these compounds ranged over five log concentrations, i.e., between 10 000 and 0.1 nM. For several new chemical entities, the antitumor activity (determined in vitro) and tolerability (determined in vivo) were superior to those of the parent alkaloids, i.e., ascididemin and 2-bromoleptoclinidone.