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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. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1b and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered an attractive therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of chloride channels in the regulation of NLRP3 in murine macrophages. Specifically, we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to chloride channel inhibitors, suggesting there are additional and specific chloride sensing and regulating mechanisms controlling NLRP3. [ABSTRACT FROM AUTHOR]

Inhibitors of the enzyme NQO2 (NRH: quinone oxidoreductase 2) are of potential use in cancer chemotherapy and malaria. We have previously reported that non-symmetrical furan amidines are potent inhibitors of NQO2 and here novel analogues are evaluated. The furan ring has been changed to other heterocycles (imidazole, N -methylimidazole, oxazole, thiophene) and the amidine group has been replaced with imidate, reversed amidine, N -arylamide and amidoxime to probe NQO2 activity, improve solubility and decrease basicity of the lead furan amidine. All compounds were fully characterised spectroscopically and the structure of the unexpected product N -hydroxy-4-(5-methyl-4-phenylfuran-2-yl)benzamidine was established by X-ray crystallography. The analogues were evaluated for inhibition of NQO2, which showed lower activity than the lead furan amidine. The observed structure-activity relationship for the furan-amidine series with NQO2 was rationalized by preliminary molecular docking and binding mode analysis. In addition, the oxazole-amidine analogue inhibited the growth of Plasmodium falciparum with an IC 50 value of 0.3 μM. [ABSTRACT FROM AUTHOR]

NRH:quinone oxidoreductase 2 enzyme (NQO2) is a potential therapeutic target in cancer and neurodegenerative diseases, with roles in either chemoprevention or chemotherapy. Here we report the design, synthesis and evaluation of non-symmetrical furan-amidines and their analogues as novel selective NQO2 inhibitors with reduced adverse off-target effects, such as binding to DNA. A pathway for the synthesis of the non-symmetrical furan-amidines was established from the corresponding 1,4-diketones. The synthesized non-symmetrical furan-amidines and their analogues showed potent NQO2 inhibition activity with nano-molar IC 50 values. The most active compounds were non-symmetrical furan-amidines with meta - and para -nitro substitution on the aromatic ring, with IC 50 values of 15 nM. In contrast to the symmetric furan-amidines, which showed potent intercalation in the minor grooves of DNA, the synthesized non-symmetrical furan-amidines showed no affinity towards DNA, as demonstrated by DNA melting temperature experiments. In addition, Plasmodium parasites, which possess their own quinone oxidoreductase PfNDH2, were inhibited by the non-symmetrical furan-amidines, the most active possessing a para -fluoro substituent (IC 50 9.6 nM). The high NQO2 inhibition activity and nanomolar antimalarial effect of some of these analogues suggest the lead compounds are worthy of further development and optimization as potential drugs for novel anti-cancer and antimalarial strategies. [ABSTRACT FROM AUTHOR]

Abstract: The diethyl ester of [18F]ML-10 is a small molecule apoptotic PET probe for cancer studies. Here we report a novel multi-step synthesis of the diethyl ester of ML-10 in excellent yields via fluorination using Xtal-Fluor-E. In addition, a one-pot radiosynthesis of the diethyl ester of [18F]ML-10 from nucleophilic [18F]fluoride was completed in 23% radiochemical yield (decay corrected). The radiochemical purity of the product was ⩾99%. The diethyl ester of [18F]ML-10 was used in vivo to detect apoptosis in the testes of mice. In parallel studies, the dansyl-ML-10 diethyl ester was prepared and used to detect apoptotic cells in an in vitro cell based assay. [Copyright &y& Elsevier]

Abstract: Sterically constrained probes 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8) and 2,4-O-bispyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene (9) exhibit specific dimer fluorescent characteristics (λ max 555 nm and 511 nm, respectively), attributed to the 2,4-diaxial arrangement of the dansyl or pyrene groups. Reduction of the azo-conformational locking group in (8) and (9) yielded 1,3-bisdansyl-4,6-diaminocyclohexane (16) and 1,3-bispyrenoyl-4,6-diaminocyclohexane (17) in the tetra-equatorial chair conformation, thus minimising interaction of the bisdansyl or bispyrenoyl groups. This induces a change in fluorescence from a cooperative green emission dimer band to a blue-shifted, monomer type fluorescence, with λ max 448 nm and 396 nm for the reduced forms (16) and (17), respectively. The azo-bond conformational lock can either be reduced under biomimetic conditions (using sodium dithionite) or with bacteria (Clostridium perfringens or Escherichia coli) utilising azo-reductase enzymes. These fluorescent probes have the potential to specifically detect azo-reductase expressing bacteria. [Copyright &y& Elsevier]

Abstract: Ester prodrugs have the potential to eliminate the gastrotoxicity associated with the carboxylic acid group of indomethacin. 4,6-Bis-O-2′-[1′-(4″-chlorobenzoyl)-5′-methoxy-2′-methyl-1′H-indol-3′-acetyl]-myo-inositol-1,3,5-orthoacetate (2) was synthesised and evaluated as a COX-2 inhibitor. It adopts a conformationally restricted chair with two indomethacin groups in the sterically hindered 1,3-diaxial positions. Acid-induced cleavage of the orthoacetate lock of the prodrug leads to a ring flip of the myo-inositol ring with the two indomethacin groups now in 1,3-diequatorial positions. This increases the susceptibility of hydrolysis of the ester groups to release indomethacin under acidic conditions. The long half-life (152min) of decomposition of (2) at ∼pH 1–2 suggests that it may bypass the stomach with minimal hydrolysis upon oral administration. Indomethacin ester (2) was completely stable at pH 4.0–8.5 over 24h at 37°C and showed comparable activity to indomethacin in a COX-2 assay (pH 8.0). [Copyright &y& Elsevier]

Abstract: Among small cellular molecules, certain inositol phosphates (InsPs) excel at complexing Fe3+ in a safe manner in vitro. In particular Ins(1,2,3)P 3 is probably an important chelator of iron cations not strongly bound to proteins, which avoids iron redox cycling and the associated production of free radicals. In this work we report the first thermodynamic data of the Fe3+/Fe2+ redox potential in the presence of Ins(1,2,3)P 3 which demonstrate the capability of this ligand to inhibit iron(III) reduction in the intracellular media. The Fe3+–Ins(1,2,3)P 3 speciation was also studied by 31P NMR measurements, focusing on the structure of the complexes. The work is completed by theoretical calculations which lead us to predict the geometries of the species at different pH values and their relative stabilities. The influence of the ring conformation in the Fe3+ complexed forms is also discussed. [Copyright &y& Elsevier]

Abstract: Animal cells contain a pool of inositol phosphates whose biological function is still under current investigation. Ins(1,2,3)P 3 is probably an important safe chelator of iron cations not strongly bound to proteins. In order to clarify its biological functions, Ins(1,2,3)P 3 chemistry under physiological conditions must be completely elucidated. The protonation and complexation behaviour of Ins(1,2,3)P 3 has been recently studied under these conditions by potentiometry. Under simulated physiological conditions it forms the protonated species H2L4− and H3L3−. The presence of high concentrations of potassium in intracellular compartments causes the formation of two predominant Ins(1,2,3)P 3 complexes: [K(HL)]4− and [K(H2L)]3−, in the absence of iron. In this work we expand part of this macroscopic knowledge to the inframolecular level, by 31P NMR measurements and focusing on the protonation and complexation of this biologically relevant molecule to potassium. We complete this study with theoretical calculations which lead us to predict the geometries of every form of the ligand and their relative stabilities. The influence of the ring conformation in protonated and complexed forms is also discussed. [ABSTRACT FROM AUTHOR]

Abstract: myo-Inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], a component in mammalian cells, possesses the correct chemical properties of an intracellular iron transit ligand. Here we have examined the conformation of the Ins(1,2,3)P3–Fe3+ complex. The synthesis and antioxidant properties of 4,6-carbonate-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-carbonate Ins(1,2,3,5)P4], which is locked in the unstable penta-axial chair conformation and 1,2,3-trisphosphoglycerol, a flexible acyclic analogue of Ins(1,2,3)P3, are reported. 4,6-Carbonate Ins(1,2,3,5)P4 caused complete inhibition of iron-catalysed hydroxyl radical (HO ) formation at 100 μM, thereby resembling Ins(1,2,3)P3 and supporting a penta-axial chair binding conformation. In contrast, 1,2,3-trisphosphoglycerol was shown to have incomplete antioxidant properties. In support of experimental observations, we have applied high-level density functional calculations to the binding of Ins(1,2,3)P3 to iron. This study provides evidence that Fe3+ binds tightly to the less stable penta-axial conformation of Ins(1,2,3)P3 using terminal and bridging phosphate oxygens, thought to also contain a tightly bound water molecule or hydroxyl ligand in the complex. [ABSTRACT FROM AUTHOR]

Abstract: The autoinducer (4S)-4,5-dihydroxypentane-2,3-dione ((S)-DPD, AI-2) facilitates chemical communication, termed ‘quorum sensing’, amongst a wide range of bacteria, The synthesis of (S)-DPD is challenging in part due to its instability. Herein we report a novel synthesis of (S)-DPD via (2S)-2,3-O-isopropylidene glyceraldehyde, through Wittig, dihydroxylation and oxidation reactions, with a complimentary asymmetric synthesis to a key precursor. Its enantiomer (R)-DPD, was prepared from d-mannitol via (2R)-2,3-O-isopropylideneglyceraldehyde. The synthesized enantiomers of DPD have AI-2 bioluminescence-inducing properties in the Vibrio harveyi BB170 strain. [Copyright &y& Elsevier]

Restricted rotation in indol-3-yl-N-alkyl- and indol-3-yl-N,N-dialkyl-glyoxalylamides can in principle give the syn-periplanar and anti-periplanar rotamers. In asymmetrically disubstituted glyoxalylamides, steric effects lead to the occurrence of both rotamers, as observed by NMR spectroscopy. The predominant peak corresponds with the anti rotamer, in which the bulkier alkyl group is orientated trans to the amide carbonyl group. In monoalkylated glyoxalylamides, only one set of peaks is observed, consistent with the presence of only one rotamer. Crystal structures of 5-methoxyindole-3-yl-N-tert-butylglyoxalylamide, indole-3-yl-N-tert-butylglyoxalylamide, and indole-3-yl-N-isopropylglyoxalylamide reported here reveal a syn conformation held by an intramolecular N-H...O hydrogen bond. [ABSTRACT FROM AUTHOR]

Mammalian cells contain a pool of iron that is not strongly bound to proteins, which can be detected with fluorescent chelating probes. The cellular ligands of this biologically important “chelatable”, “labile” or “transit” iron are not known. Proposed ligands are problematic, because they are saturated by magnesium under cellular conditions and/or because they are not “safe”, i.e. they allow iron to catalyse hydroxyl radical formation. Among small cellular molecules, certain inositol phosphates (Ins Ps) excel at complexing Fe3+ in such a “safe” manner in vitro. However, we previously calculated that the most abundant Ins P, inositol hexakisphosphate, cannot interact with Fe3+ in the presence of cellular concentrations of Mg2+. In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3) P 3], a cellular constituent of unknown function and the simplest Ins P to display high-affinity, “safe”, iron complexation. We report thermodynamic constants for the interaction of Ins(1,2,3) P 3 with Na+, K+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+. Our calculations indicate that Ins(1,2,3) P 3 can be expected to complex all available Fe3+ in a quantitative, 1:1 reaction, both in cytosol/nucleus and in acidic compartments, in which an important labile iron subpool is thought to exist. In addition, we calculate that the fluorescent iron probe calcein would strip Fe3+ from Ins(1,2,3) P 3 under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3) P 3. Therefore Ins(1,2,3) P 3 is the first viable proposal for a transit iron ligand. [ABSTRACT FROM AUTHOR]

Abstract: The aromatic rings in 4-O-dabsyl-6-O-dansyl-myo-inositol-1,3,5-orthoformate (6) participate in electron transfer causing static quenching as detected by the absence of fluorescence. Upon addition of acid, the orthoformate lock is cleaved, with subsequent conformational change of the myo-inositol ring from penta-axial to the more stable penta-equatorial chair, which causes some increase in fluorescence due to spatial separation of fluorophore and a quencher and reduction in static quenching. In the case of 4,6-O-bisdansyl-myo-inositol-1,3,5-orthoformate (3), the acid-induced removal of the orthoformate lock leads to substantial change of fluorescence following spatial separation of two dansyl groups. [Copyright &y& Elsevier]

Abstract: Thymidine phosphorylase (TP) is over-expressed in various tumour types and plays an important role in tumour angiogenesis, growth, invasion and metastasis. The enzymatic activity of TP is required for the angiogenic effect of TP, therefore, inhibitors of TP are of significant interest in cancer chemotherapy. A series of xanthine oxidase (XO) activated prodrugs of known inhibitors of TP have been designed and synthesized with the ultimate intent of improving tumour selectivity and pharmacokinetic characteristics. These prodrugs were not inhibitors of TP, but were selectively oxidized by XO at C-2 and/or C-4 of the uracil ring moiety to generate the desired TP inhibitor. Molecular modelling of both the TP inhibitors and XO-activated prodrugs rationalized their binding in the active site of the human TP crystal structure. [Copyright &y& Elsevier]

Abstract: Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein–protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring. [Copyright &y& Elsevier]

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil. [Copyright &y& Elsevier]

(S)-4,5-Dihydroxypentane-2,3-dione [(S)-DPD, (1)] is a precursor for AI-2, a quorum sensing signalling molecule for inter- and intra-species bacterial communication. The synthesis of its fluoro-analogue, 4-fluoro-5-hydroxypentane-2,3-dione (2) is reported. An intermediate in this route also enables a new, shorter synthesis of the native (S)-DPD. 4-Fluoro-DPD (2) completely inhibited bioluminescence and bacterial growth of Vibrio harveyi BB170 strain at 12.5 μM and 100 μM, respectively. [ABSTRACT FROM AUTHOR]

(NRH):quinone oxidoreductase 2 (NQO2) is associated with various processes involved in cancer initiation and progression probably via the production of ROS during quinone metabolism. Thus, there is a need to develop inhibitors of NQO2 that are active in vitro and in vivo. As part of a strategy to achieve this we have used the 4-aminoquinoline backbone as a starting point and synthesized 21 novel analogues. The syntheses utilised p -anisidine with Meldrum's acid and trimethyl orthoacetate or trimethyl orthobenzoate to give the 4-hydrazin-quinoline scaffold, which was derivatised with aldehydes or acid chlorides to give hydrazone or hydrazide analogues, respectively. The hydrazones were the most potent inhibitors of NQO2 in cell free systems, some with low nano-molar IC 50 values. Structure-activity analysis highlighted the importance of a small substituent at the 2-position of the 4-aminoquinoline ring, to reduce steric hindrance and improve engagement of the scaffold within the NQO2 active site. Cytotoxicity and NQO2–inhibitory activity in vitro was evaluated using ovarian cancer SKOV-3 and TOV-112 cells (expressing high and low levels of NQO2, respectively). Generally, the hydrazones were more toxic than hydrazide analogues and further, toxicity is unrelated to cellular NQO2 activity. Pharmacological inhibition of NQO2 in cells was measured using the toxicity of CB1954 as a surrogate end-point. Both the hydrazone and hydrazide derivatives are functionally active as inhibitors of NQO2 in the cells, but at different inhibitory potency levels. In particular, 4-((2-(6-methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol has the greatest potency of any compound yet evaluated (53 nM), which is 50-fold lower than its toxicity IC 50. This compound and some of its analogues could serve as useful pharmacological probes to determine the functional role of NQO2 in cancer development and response to therapy. Image 1 • Hydrazone analogues of 4-aminoquinolines inhibit NQO2, with nM IC 50 values. • Hydrazone analogues had low cytotoxicity to ovarian cancer SKOV-3 & TOV-112 cells. • Useful probes to interrogate the role of NQO2 in cancer/other biological conditions. [ABSTRACT FROM AUTHOR]

Summary NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca 2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca 2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca 2+ . The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome is a key target for drug discovery due to its implication in a range of inflammation-related diseases. In this work, we identify new inhibitors of the NLRP3 inflammasome via a hierarchical virtual screening strategy using molecular similarity, docking and MD simulation. The most potent inhibitors identified from a subsequent biological assay (IC 50 of 1 - 4 μM) feature a sulfonamide group, a motif known to favour NLRP3 inhibition, in conjunction with an indole, benzofuran or tricyclic 6,7-dihydro-5H-indeno[5,6-b]furan ring, yielding novel scaffolds. These structures provide a basis for the design of more potent, selective NLRP3 inhibitors., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inhibitors of NQO2 (NRH: quinone oxidoreductase) have potential application in several areas of medicine and pharmacology, including cancer, neurodegeneration (PD and AD), stroke, and diabetes. Here, resveratrol, a known inhibitor of NQO2, was used as the lead by replacing the double bond in resveratrol with a benzothiazole scaffold. Fifty-five benzothiazoles were designed as NQO2 inhibitors and synthesized, comprising five benzothiazole series with 3,5-dimethoxy, 2,4-dimethoxy, 2,5-dimethoxy, 3,4-dimethoxy, and 3,4,5-trimethoxy substituents, the key synthetic step being a Jacobson cyclisation with the appropriate thiobenzamide. All compounds were evaluated in an NQO2 enzyme inhibition assay, with four compounds having IC 50 values of <100 nM. The most active (IC 50 25 nM) was 6-hydroxy-2-(3',5'-dihydroxyphenyl)benzo[d]thiazole ( 15 ), a good mimetic of resveratrol. Three of the 3',4',5'-trimethoxybenzothiazole analogues, with 6-methoxy ( 40 , IC 50 51 nM), 6-amino ( 48 , IC 50 79 nM), and 6-acetamide ( 49 , IC 50 31 nM) substituents, were also potent inhibitors of NQO2. Computational modelling indicated the most active compounds exhibited good shape complementarity and polar interactions with the NQO2 active site. Through the inhibition of NQO2, benzothiazole-based compounds may have the potential to enhance the efficiency of cancer therapies or minimise oxidative damage in neuroinflammation.

Excessive or aberrant NLRP3 inflammasome activation has been implicated in the progression and initiation of many inflammatory conditions; however, currently no NLRP3 inflammasome inhibitors have been approved for therapeutic use in the clinic. Here we have identified that the natural product brazilin effectively inhibits both priming and activation of the NLRP3 inflammasome in cultured murine macrophages, a human iPSC microglial cell line and in a mouse model of acute peritoneal inflammation. Through computational modeling, we predict that brazilin can adopt a favorable binding pose within a site of the NLRP3 protein which is essential for its conformational activation. Our results not only encourage further evaluation of brazilin as a therapeutic agent for NLRP3-related inflammatory diseases, but also introduce this small-molecule as a promising scaffold structure for the development of derivative NLRP3 inhibitor compounds., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

The NLRP3 inflammasome is a component of the inflammatory response to infection and injury, orchestrating the maturation and release of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and triggering pyroptotic cell death. Appropriate levels of NLRP3 activation are needed to avoid excessive tissue damage while ensuring host protection. Here we report a role for symmetrical diarylsquaramides as selective K + efflux-dependent NLRP3 inflammasome enhancers. Treatment of macrophages with squaramides potentiated IL-1β secretion and ASC speck formation in response to K + efflux-dependent NLRP3 inflammasome activators without affecting priming, endosome cargo trafficking, or activation of other inflammasomes. The squaramides lowered intracellular K + concentration which enabled cells to respond to a below-threshold dose of the inflammasome activator nigericin. Taken together these data further highlight the role of ion flux in inflammasome activation and squaramides as an interesting platform for therapeutic development in conditions where enhanced NLRP3 activity could be beneficial., (© 2023. The Author(s).)

The emergence of resistance to first-line antimalarial drugs calls for the development of new therapies for drug-resistant malaria. The efficacy of quinoline-based antimalarial drugs has prompted the development of novel quinolines. A panel of 4-aminoquinoline hydrazone analogues were tested on the multidrug-resistant K1 strain of Plasmodium falciparum : IC 50 values after a 48 h cycle ranged from 0.60 to 49 µM, while the 72 h cycle ranged from 0.026 to 0.219 μM. Time-course assays were carried out to define the activity of the lead compounds, which inhibited over 50% growth in 24 h and 90% growth in 72 h. Cytotoxicity assays with HepG2 cells showed IC 50 values of 0.87-11.1 μM, whereas in MDBK cells, IC 50 values ranged from 1.66 to 11.7 μM. High selectivity indices were observed for the lead compounds screened at 72 h on P. falciparum . Analyses of stage specificity revealed that the ring stages of the parasite life cycle were most affected. Based on antimalarial efficacy and in vitro safety profiles, lead compound 4-(2-benzylidenehydrazinyl)-6-methoxy-2-methylquinoline 2 was progressed to drug combination studies for the detection of synergism, with a combinatory index of 0.599 at IC 90 for the combination with artemether, indicating a synergistic antimalarial activity. Compound 2 was screened on different strains of P. falciparum (3D7, Dd2), which maintained similar activity to K1, suggesting no cross-resistance between multidrug resistance and sensitive parasite strains. In vivo analysis with 2 showed the suppression of parasitaemia with P. yoelii NL (non-lethal)-treated mice (20 mg/kg and 5 mg/kg).

Many transmembrane proteins are modulated by intracellular or extracellular pH. Investigation of pH dependence generally proceeds by mutagenesis of a wide set of amino acids, guided by properties such as amino-acid conservation and structure. Prediction of pKas can streamline this process, allowing rapid and effective identification of amino acids of interest with respect to pH dependence. Commencing with the calcium-activated chloride channel bestrophin 1, the carboxylate ligand structure around calcium sites relaxes in the absence of calcium, consistent with a measured lack of pH dependence. By contrast, less relaxation in the absence of calcium in TMEM16A, and maintenance of elevated carboxylate sidechain pKas, is suggested to give rise to pH-dependent chloride channel activity. This hypothesis, modulation of calcium/proton coupling and pH-dependent activity through the extent of structural relaxation, is shown to apply to the well-characterised cytosolic proteins calmodulin (pH-independent) and calbindin D9k (pH-dependent). Further application of destabilised, ionisable charge sites, or electrostatic frustration, is made to other human chloride channels (that are not calcium-activated), ClC-2, GABA A , and GlyR. Experimentally determined sites of pH modulation are readily identified. Structure-based tools for pKa prediction are freely available, allowing users to focus on mutagenesis studies, construct hypothetical proton pathways, and derive hypotheses such as the model for control of pH-dependent calcium activation through structural flexibility. Predicting altered pH dependence for mutations in ion channel disorders can support experimentation and, ultimately, clinical intervention.

Inflammation driven by DNA sensors is now understood to be important to disease pathogenesis. Here, we describe new inhibitors of DNA sensing, primarily of the inflammasome forming sensor AIM2. Biochemistry and molecular modeling has revealed 4-sulfonic calixarenes as potent inhibitors of AIM2 that likely work by binding competitively to the DNA-binding HIN domain. Although less potent, these AIM2 inhibitors also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against DNA-driven inflammatory responses. The 4-sulfonic calixarenes inhibited AIM2-dependent post-stroke T cell death, highlighting a proof of concept that the 4-sulfonic calixarenes could be effective at combating post-stroke immunosuppression. By extension, we propose a broad utility against DNA-driven inflammation in disease. Finally, we reveal that the drug suramin, by virtue of its structural similarities, is an inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an increasing clinical need., Competing Interests: The authors declare that they have no conflict of interest., (© 2023 The Authors.)

Bestrophin 1 (Best1) is a chloride channel that localises to the plasma membrane of retinal pigment epithelium (RPE) cells. Mutations in the BEST1 gene are associated with a group of untreatable inherited retinal dystrophies (IRDs) called bestrophinopathies, caused by protein instability and loss-of-function of the Best1 protein. 4PBA and 2-NOAA have been shown to rescue the function, expression, and localisation of Best1 mutants; however, it is of interest to find more potent analogues as the concentration of the drugs required is too high (2.5 mM) to be given therapeutically. A virtual docking model of the COPII Sec24a site, where 4PBA has been shown to bind, was generated and a library of 1416 FDA-approved compounds was screened at the site. The top binding compounds were tested in vitro in whole-cell patch-clamp experiments of HEK293T cells expressing mutant Best1. The application of 25 μM tadalafil resulted in full rescue of Cl - conductance, comparable to wild type Best1 levels, for p.M325T mutant Best1 but not for p.R141H or p.L234V mutants.

The NLRP3 inflammasome is a cytoplasmic complex that regulates the activation of inflammatory cytokines and, given its implication in a range of diseases, is an important therapeutic target. The cofactor ATP and the centrosomal kinase NEK7 are important for NLRP3 activation. Here we have constructed and simulated computational models of full-length monomeric NLRP3 to shed light on the importance of NEK7 and cofactor interactions for its conformation and dynamics in aqueous solution. We find that molecular dynamics simulation reproduces well the features of the recently published cryo-EM structure of the ADP-bound NLRP3-NEK7 complex; on the removal of NEK7, the NLRP3 molecule adopts a more compact closed form during simulations. Replacement of ADP by ATP promotes a rearrangement of hydrogen-bonding interactions, domain interfaces, and a degree of opening of the NLRP3 conformation. We also examine the dynamics of an acidic loop of the LRR domain of NLRP3, which samples in a region observed in the NEK7-bound cryo-EM structure but not in an oligomeric form of inactive NLRP3. During the molecular dynamics simulations of NLRP3, we find some plasticity in its topology that suggests access routes for ATP to the cofactor pocket not immediately evident from the existing NEK7-bound cryo-EM structure. These computed dynamical trajectories of NLRP3 provide insight into coordinates of deformation that may be key for cofactor binding and inflammasome activation., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The NLRP3 inflammasome is currently an exciting target for drug discovery due to its role in various inflammatory diseases; however, to date, no NLRP3 inhibitors have reached the clinic. Several studies have used natural products as hit compounds to facilitate the design of novel selective NLRP3 inhibitors. Here, we review selected natural products reported in the literature as NLRP3 inhibitors, with a particular focus on those targeting gout. To complement this survey, we also report a virtual screen of the ZINC20 natural product database, predicting favored chemical features that can aid in the design of novel small molecule NLRP3 inhibitors., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: QT prolongation and intracellular Ca 2+ loading with diastolic Ca 2+ release via ryanodine receptors (RyR2) are the predominant mechanisms underlying hypokalaemia-induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VK-II-86, a carvedilol analogue lacking antagonist activity at β-adrenoceptors, in hypokalaemia., Experimental Approach: Surface ECG and ventricular action potentials (APs) were recorded from whole-heart murine Langendorff preparations. Ventricular arrhythmia incidence was compared in hearts perfused with low [K + ], and those pretreated with dantrolene or VK-II-86. Whole-cell patch clamping was used in murine and canine ventricular cardiomyocytes to study effects of dantrolene and VK-II-86 on AP parameters in low [K + ] and effects of VK-II-86 on the inward rectifier current (I K1 ), late sodium current (I Na&#95;L ) and the L-type Ca 2+ current (I Ca ). Effects of VK-II-86 on I Kr were investigated in transfected HEK-293 cells. A fluorogenic probe quantified the effects of VK-II-86 on oxidative stress in hypokalaemia., Key Results: Dantrolene reduced the incidence of ventricular arrhythmias induced by low [K + ] in explanted murine hearts by 94%, whereas VK-II-86 prevented all arrhythmias. VK-II-86 prevented hypokalaemia-induced AP prolongation and depolarization but did not alter AP parameters in normokalaemia. Hypokalaemia was associated with decreased I K1 and I Kr , and increased I Na-L , and I Ca . VK-II-86 prevented all hypokalaemia-induced changes in ion channel activity and oxidative stress., Conclusions and Implications: VK-II-86 prevents hypokalaemia-induced arrhythmogenesis by normalizing calcium homeostasis and repolarization reserve. VK-II-86 may provide an effective treatment in hypokalaemia and other arrhythmias caused by delayed repolarization or Ca 2+ overload., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

A set of meta -substituted 3-arylisoquinolinones have been identified that show substantial cytotoxicity in breast, liver, lung and colon cancer cell lines; these are up to 700-fold more active than the corresponding para analogues. These compounds were initially proposed as inhibitors of N -ribosyl dihydronicotinamide (NRH): quinone oxidoreductase 2 (NQO2) but were found to be inactive against the enzyme. Instead, COMPARE analysis suggested that 6-fluoro-3-( meta -fluorophenyl)isoquinolin-1(2 H )-one ( 4 ) could mimic colchicine and interact with microtubules, a recognized target for cancer therapy. Subsequent docking, molecular dynamics simulations, and free energy analysis further suggested that compound 4 bound well into the colchicine-binding pocket of tubulin. Indeed, 4 suppressed tubulin polymerization, caused G 2 / M cell cycle arrest, and induced apoptosis. Also, 4 inhibited the formation of endothelial cell capillary-like tubes and further disrupted the structure of preestablished tubes; the effects were not observed with para analogue 5 . In accordance with this, the computed free energy of binding of 5 to tubulin was lower in magnitude than that for 4 and appeared to arise in part from the inability of the para substituent to occupy a tubulin subpocket, which is possible in the meta orientation. In conclusion, the antiproliferative potential of the novel 3-arylisoquinolinones is markedly influenced by a subtle change in the structure ( meta versus para ). The meta -substituted isoquinolinone 4 is a microtubule-destabilizing agent with potential tumor-selectivity and antiangiogenic and vascular disrupting features.

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum . This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC 50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC 50 of 68.6 µM, d -erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum . Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P . falciparum is the reason for their weak in vitro anti-malarial activities.

The pathogenesis of Alzheimer's disease (AD) is correlated with the misfolding and aggregation of amyloid- beta protein (Aβ). Here we report that the antibiotic benzylpenicillin (BP) can specifically bind to Aβ, modulate the process of aggregation and supress its cytotoxic effect, initially via a reversible binding interaction, followed by covalent bonding between specific functional groups (nucleophiles) within the Aβ peptide and the beta -lactam ring. Mass spectrometry and computational docking supported covalent modification of Aβ by BP. BP was found to inhibit aggregation of Aβ as revealed by the Thioflavin T (ThT) fluorescence assay and atomic force microscopy (AFM). In addition, BP treatment was found to have a cytoprotective activity against Aβ-induced cell cytotoxicity as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell toxicity assay. The specific interaction of BP with Aβ suggests the possibility of structure-based drug design, leading to the identification of new drug candidates against AD. Moreover, good pharmacokinetics of beta -lactam antibiotics and safety on long-time use make them valuable candidates for drug repurposing towards neurological disorders such as AD., Competing Interests: 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., (© 2021 The Authors.)

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, and contributes to inflammation exacerbating disease. Fenamate non-steroidal anti-inflammatory drugs (NSAIDs) were recently described as NLRP3 inflammasome inhibitors via chloride channel inhibition. Fenamate NSAIDs inhibit cyclooxygenase (COX) enzymes, limiting their potential as therapeutics for NLRP3-associated diseases due to established side effects. The aim here was to develop properties of the fenamates that inhibit NLRP3, and at the same time to reduce COX inhibition. We synthesised a library of analogues, with feedback from in silico COX docking potential, and IL-1β release inhibitory activity. Through iterative screening and rational chemical design, we established a collection of chloride channel inhibiting active lead molecules with potent activity at the canonical NLRP3 inflammasome and no activity at COX enzymes, but only in response to stimuli that activated NLRP3 by a K + efflux-dependent mechanism. This study identifies a model for the isolation and removal of unwanted off-target effects, with the enhancement of desired activity, and establishes a new chemical motif for the further development of NLRP3 inflammasome inhibitors., Competing Interests: The authors have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Aggregation of amyloid β 1-42 (Aβ 1-42 ) peptide within the brain is considered one of the main causes of the neuropathological changes associated with Alzheimer's disease. Resveratrol is a well-known antioxidant but has also been reported to bind to Aβ 1-42 peptide, thereby reducing aggregation. However, little is known of the precise mechanism by which resveratrol reduces Aβ 1-42 peptide aggregation. Using the thioflavin-T assay, the ability of resveratrol to reduce the extent of Aβ 1-42 peptide aggregation was investigated. The findings of the present study demonstrate that interaction of resveratrol with Aβ 1-42 peptide resulted in the cleavage of Aβ 1-42 peptide into smaller fragments, as detected by matrix assisted laser desorption ionization-time of flight mass spectrometry. Atomic force microscopy analyses revealed Aβ 1-42 peptide, under control conditions, aggregated into oligomers, protofibrils, and fibrils, whereas there was a distinct lack of these structures when Aβ 1-42 peptide was incubated with resveratrol. Following 10 days incubation of Aβ 1-42 peptide with resveratrol, particles with a mean z-height of 1.940 nm (range 0.675-3.275 nm) were observed, which are characteristic of shorter peptide species. In cell-based studies, resveratrol significantly reduced the cytotoxicity of Aβ 1-42 peptide toward SH-SY5Y human neuroblastoma cells, suggesting a protective effect of the polyphenol. We therefore propose a novel mechanism by which resveratrol disrupts Aβ 1-42 aggregation by mediating fragmentation of Aβ 1-42 into smaller peptides, which have no propensity to aggregate further., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy., Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp., Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1., Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

Neuroinflammation is becoming increasingly recognized as a critical factor in the pathology of both acute and chronic neurological conditions. Inflammasomes such as the one formed by NACHT, LRR, and PYD domains containing protein 3 (NLRP3) are key regulators of inflammation due to their ability to induce the processing and secretion of interleukin 1β (IL-1β). IL-1β has previously been identified as a potential therapeutic target in a variety of conditions due to its ability to promote neuronal damage under conditions of injury. Thus, inflammasome inhibition has the potential to curtail inflammatory signaling, which could prove beneficial in certain diseases. In this review, we discuss the evidence for inflammasome contributions to the pathology of neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease, epilepsy, and acute degeneration following brain trauma or stroke. In addition, we review the current landscape of drug development targeting the NLRP3 inflammasome.

Inflammation is an established contributor to disease and the NLRP3 inflammasome is emerging as a potential therapeutic target. A number of small molecule inhibitors of the NLRP3 pathway have been described. Here we analysed the most promising of these inhibitor classes side by side to assess relative potency and selectivity for their respective putative targets. Assessed using ASC inflammasome-speck formation, and release of IL-1β, in both human monocyte/macrophage THP1 cells and in primary mouse microglia, we compared the relative potency and selectivity of P2X7 inhibitors, inflammasome inhibitors (diarylsulfonylurea vs. the NBC series), and caspase-1 inhibitors. In doing so we are now able to provide a well characterised small molecule tool kit for interrogating and validating inflammasome-dependent responses with a range of nanomolar potency inhibitors against established points in the inflammasome pathway.

The NLRP3 inflammasome is an important regulator of the sterile inflammatory response, and its activation by host-derived sterile molecules leads to the intracellular activation of caspase-1, processing of the pro-inflammatory cytokines interleukin-1β (IL-1β)/IL-18, and pyroptotic cell death. Inappropriate activation of NLRP3 drives a chronic inflammatory response and is implicated in several non-communicable diseases, including gout, atherosclerosis, type II diabetes and Alzheimer's disease. In this study, we report the design, synthesis and biological evaluation of novel boron compounds (NBCs) as NLRP3 inflammasome inhibitors. Structure-activity relationships (SAR) show that 4-fluoro substituents on the phenyl rings retain NLRP3 inhibitory activity, whereas more steric and lipophilic substituents diminish activity. Loss of inhibitory activity is also observed if the CCl 3 group on the oxazaborine ring is replaced by a CF 3 group. These findings provide additional understanding of the NBC series and will aid in the development of these NLRP3 inhibitors as tool compounds or therapeutic candidates for sterile inflammatory diseases., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

N -Methyl carbamoylimidazole is a safe and practical alternative to methyl isocyanate for carbamoylation reactions. We have developed a new chemical route for its synthesis from methyl iodide and applied this to the synthesis of N -[ 11 C]methyl carbamoylimidazole as a new [ 11 C]synthon to radiolabel biomolecules for PET imaging research. N -[ 11 C]methyl carbamoylimidazole was prepared from [ 11 C]methyl iodide in 70-74% radiochemical yield (decay corrected) and can be used in situ for further reaction without purification. The reactivity of N -[ 11 C]methyl carbamoylimidazole was demonstrated in a series of [ 11 C]carbamoylation reactions.

Dehydroacetic acid is a common pyrone derivative used commercially as an antibacterial and antifungal agent. Based on the synthesis of dehydroacetic acid ( 1 ) from N -hydroxysuccinimdyl acetoacetate, a novel series of enamine-based derivatives were synthesised in order to improve the antibacterial activity of dehydroacetic acid. The antibacterial activities of the synthesised analogues were evaluated against Escherichia coli and Staphylococcus aureus . Derivative 4d (N-Ph) was identified as the most potent inhibitor of S. aureus growth. Overall, derivative 4b (N-Me) showed the best broad-spectrum activity with five-fold greater minimum inhibitory concentration and 11-fold greater minimum biocidal concentration against E. coli when compared to dehydroacetic acid, in addition to improved antibacterial activity against S. aureus ., Competing Interests: Compliance with ethical standardsThe authors declare that they have no competing interests.