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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. This study shows how web-based calculation of pKas allows 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 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, GABAA, and GlyR. Experimentally-determined sites of pH modulation are readily identified. The structure-based tool is freely available, allowing users to focus 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.

The NLRP3 inflammasome is a cytoplasmic complex that regulates the activa- tion of inflammatory cytokines and, given its implication in a range of dis- eases, 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 struc- ture. These computed dynamical trajectories of NLRP3 provide insight into coordinates of deformation that may be key for cofactor binding and inflam- masome activation.

Inflammation driven by DNA sensors is now understood to be central to disease pathogenesis. Here we describe new inhibitors of pathogenic DNA sensing, primarily of the inflammasome forming sensor AIM2. Molecular modelling and biochemistry has revealed potent inhibitors of AIM2 that work by binding competitively to the DNA binding site. Though less potent, these AIM2 inhibitors, 4-sulfonic calixarenes, also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against pathogenic 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 combatting post-stroke immunosuppression. By extension, we propose a broad utility against DNA driven inflammation in disease. Finally, we reveal that the ancient drug suramin, by virtue of its structural similarities, is an excellent inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an ever increasing clinical need.

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_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.

Aggregation of amyloid β1–42 (Aβ1–42) 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 Aβ1–42 peptide, thereby reducing aggregation. However, little is known of the precise mechanism by which resveratrol reduces Aβ1–42 peptide aggregation. Using the thioflavin-T assay, the ability of resveratrol to reduce the extent of Aβ1–42 peptide aggregation was investigated. The findings of the present study demonstrate that interaction of resveratrol with Aβ1–42 peptide resulted in the cleavage of Aβ1–42 peptide into smaller fragments, as detected by matrix assisted laser desorption ionization-time of flight mass spectrometry. Atomic force microscopy analyses revealed Aβ1–42 peptide, under control conditions, aggregated into oligomers, protofibrils, and fibrils, whereas there was a distinct lack of these structures when Aβ1–42 peptide was incubated with resveratrol. Following 10 days incubation of Aβ1–42 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 Aβ1–42 peptide toward SH-SY5Y human neuroblastoma cells, suggesting a protective effect of the polyphenol. We therefore propose a novel mechanism by which resveratrol disrupts Aβ1–42 aggregation by mediating fragmentation of Aβ1–42 into smaller peptides, which have no propensity to aggregate further.

Manipulating neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, no anticonvulsant compounds have yet been reported that directly manipulate neuronal homeostasis. Here, we describe a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB), with a mode-of-action that includes increased expression of the homeostatic regulator Pumilio (Pum). In Drosophila and mouse we use a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy. The pyrazole analogue (RAB216) demonstrates best efficacy, protecting 50% of mice from PTZ-induced seizure. Knock-down of Pum, in Drosophila, blocks anticonvulsive effects, whilst analysis of validated Pum targets show significant reductions following exposure of mouse brain to 4-TBB. This study provides proof-of-principle that anticonvulsant effects can be achieved through regulation of neuronal homeostasis and identifies a chemical lead compound for future development.

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 IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 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 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., The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, and contributes to inflammation exacerbating disease.

Background and Purpose Hypokalaemia is the most common electrolyte disturbance with a significant mortality due to the occurrence of ventricular arrhythmia. There is currently no available treatment to prevent ventricular arrhythmia in patients susceptible to hypokalaemia, such as those with heart failure. Potassium supplementation alone is not sufficient to alter mortality. QT prolongation and intracellular Ca2+ loading with subsequent diastolic Ca2+ release via ryanodine receptors (RyR2) are considered the predominant arrhythmogenic mechanisms in hypokalaemia-induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VKII-86, a carvedilol analogue with no β-blocking activity, in hypokalaemia. Methods 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 pre-treated with dantrolene or VKII-86. Whole-cell patch clamping was used in murine and canine ventricular cardiomyocytes to study the effects of dantrolene and VK-II-86 on AP parameters in normal and low [K+] and the effects of VK-II-86 on the inward rectifier current (IK1), late sodium current (INa_L) and the L-type Ca2+ current (ICa). Effects of VKII-86 on IKr were investigated in HEK-293 cells transfected with KCNH2. Results Dantrolene significantly reduced the incidence of ventricular arrhythmias induced by low [K+] in explanted murine hearts by 94%, whereas VKII-86 prevented all arrhythmias, p Conclusions and Implications VKII-86 prevents hypokalaemia-induced arrhythmogenesis by normalising intracellular calcium homeostasis and repolarization reserve. The fact that VKII-86 does not have beta-blocking activity and does not alter AP parameters in normal [K+] is promising for future tolerability and electrophysiological drug safety testing, respectively. This unique pharmacological profile may provide an exciting treatment option in hypokalaemia and other arrhythmias caused by delayed repolarization or Ca2+ overload such as heart failure and the Long QT Syndromes. Conflict of Interest None

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1β 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., eLife digest Inflammation is a critical part of a healthy immune system, which protects us against harmful pathogens (such as bacteria or viruses) and works to restore damaged tissues. In the immune cells of our body, the inflammatory process can be activated through a group of inflammatory proteins that together are known as the NLRP3 inflammasome complex. While inflammation is a powerful mechanism that protects the human body, persistent or uncontrolled inflammation can cause serious, long-term damage. The inappropriate activation of the NLRP3 inflammasome has been implicated in several diseases, including Alzheimer’s disease, heart disease, and diabetes. The NLRP3 inflammasome can be activated by different stimuli, including changes in cell volume and exposure to either molecules produced by damaged cells or toxins from bacteria. However, the precise mechanism through which the NLRP3 becomes activated in response to these stimuli was not clear. The exit of chloride ions from immune cells is known to activate the NLRP3 inflammasome. Chloride ions exit the cell through proteins called anion channels, including volume-regulated anion channels (VRACs), which respond to changes in cell volume. Green et al. have found that, in immune cells from mice grown in the lab called macrophages, VRACs are the only chloride channels involved in activating the NLRP3 inflammasome when the cell’s volume changes. However, when the macrophages are exposed to molecules produced by damaged cells or toxins from bacteria, Green et al. discovered that other previously unidentified chloride channels are involved in activating the NLRP3 inflammasome. These results suggest that it might be possible to develop drugs to prevent the activation of the NLRP3 inflammasome that selectively target specific sets of chloride channels depending on which stimuli are causing the inflammation. Such a selective approach would minimise the side effects associated with drugs that generically suppress all NLRP3 activity by directly binding to NLRP3 itself. Ultimately, this may help guide the development of new, targeted anti-inflammatory drugs that can help treat the symptoms of a variety of diseases in humans.

Background Hypokalaemia-induced arrhythmia is a major cause of morbidity and mortality, affecting approximately 14% of outpatients and 20% of inpatients. Hypokalaemic inpatients have a mortality 10 times higher than their counterparts. This is due to ventricular arrhythmias initiated by early afterdepolarisations (EADs) and triggered activity. There is no antiarrhythmic drug approved for the prevention of hypokalaemia-induced ventricular arrhythmia. Recent evidence suggests that EADs can arise because of the synchronised release of Ca2+ from the sarcoplasmic reticulum, rather than the reactivation of L-type Ca2+ channels during action potential prolongation. Therefore, ryanodine receptor inhibition should prevent hypokalaemia-induced ventricular arrhythmia. Purpose To test the hypothesis that the ryanodine receptor inhibitors dantrolene and VKII-86 (a carvedilol analogue) are effective in preventing hypokalaemia-induced ventricular arrhythmia. Methods Whole heart Langendorff preparations isolated from C57BL/J6 mice were perfused with Krebs-Henseleit solution containing normal (4mM) or low (2mM) concentrations of [K+]. Surface ECG and subepicardial ventricular action potentials were simultaneously recorded. After establishing that the model reproduces EAD-mediated triggered arrhythmias in low extracellular [K+], hearts in the treatment groups were pre-treated with dantrolene or VKII-86 for 30 minutes prior to being exposed to low extracellular [K+]. The frequency of non-sustained and sustained ventricular arrhythmias occurring during a 30-minute period of exposure to low extracellular [K+] were compared between the treatment groups and controls treated with the solvent DMSO (0.1% v/v) (n=6 in each group). Results The mean number of non-sustained arrhythmias was 29.3±9.6 (Mean±SEM) in the control group, 3.2±1.2 in the dantrolene group and 0 in the VKII-86 group; p Conclusions These results provide the first demonstration of ryanodine receptor inhibition as an effective treatment for the prevention of ventricular arrhythmia in a murine model of hypokalaemia. This study is consistent with recent studies indicating that intracellular Ca2+ load is an important mechanism underlying the development of EAD-mediated triggered arrythmia. Further studies are needed to determine whether these drugs could be re-purposed as antiarrhythmics in the setting of hypokalemia. Further development of the carvedilol analogue VKII-86 should also be considered. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The British Heart Foundation, The Lankenau Institute for Medical Research.

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1β and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered a 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 the identity of which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of Cl- channels in the regulation of NLRP3 activation. 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 Cl- channel inhibitors, suggesting there are additional and specific Cl- sensing and regulating mechanisms controlling NLRP3.

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.

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-[11C]methyl carbamoylimidazole as a new [11C]synthon to radiolabel biomolecules for PET imaging research. N-[11C]methyl carbamoylimidazole was prepared from [11C]methyl iodide in 70–74% radiochemical yield (decay corrected) and can be used in situ for further reaction without purification. The reactivity of N-[11C]methyl carbamoylimidazole was demonstrated in a series of [11C]carbamoylation reactions. Electronic supplementary material The online version of this article (10.1007/s10967-018-5948-4) contains supplementary material, which is available to authorized users.

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 furanamidine series with NQO2 was rationalized by preliminary molecular docking and binding mode analysis. In addition, the oxazole-amidine analogue inhibited the growth of Plasmodium\ud falciparum with an IC50 value of 0.3 M.

Aggregation of amyloid β

(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 IC50 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 IC50. 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.

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 IC50 values. The most active compounds were non-symmetrical furan-amidines with meta- and para-nitro substitution on the aromatic ring, with IC50 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 (IC50 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.

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.

Inflammasomes are high molecular weight complexes that sense and react to injury and infection. Their activation induces caspase-1 activation and release of interleukin-1β, a pro-inflammatory cytokine involved in both acute and chronic inflammatory responses. There is increasing evidence that inflammasomes, particularly the NLRP3 inflammasome, act as guardians against noninfectious material. Inappropriate activation of the NLRP3 inflammasome contributes to the progression of many noncommunicable diseases such as gout, type II diabetes, and Alzheimer's disease. Inhibiting the inflammasome may significantly reduce damaging inflammation and is therefore regarded as a therapeutic target. Currently approved inhibitors of interleukin-1β are rilonacept, canakinumab, and anakinra. However, these proteins do not possess ideal pharmacokinetic properties and are unlikely to easily cross the blood-brain barrier. Because inflammation can contribute to neurological disorders, this review focuses on the development of small-molecule inhibitors of the NLRP3 inflammasome.

The pentapeptide, tert-Prenyl4th-NH2 (DMePhe-DTrp-Phe-DTrp(N-tert-prenyl)-Leu-NH2), has recently been reported by our group to exhibit properties of substance P (SP) antagonist G against small cell lung cancer (SCLC). In this study, we undertook a systematic structure activity investigation to optimise this lead compound to improve its in vitro anti-tumour activity and biocompatibility. A series of d-tryptophan (D-Trp) derivatives were synthesised, with a range of aliphatic N-alkyl chains (methyl to pentyl) on the indole nitrogen (Nind). These were incorporated into the pentapeptide sequence by substitution of the Nind-tert-prenylated D-Trp 4th residue with the Nind-alkylated D-Trp derivatives. These pentapeptides were significantly more potent than tert-Prenyl4th-NH2, with the Nind-butyl modification generating the most cytotoxic peptides. Compared to tert-Prenyl4th-NH2, a single butyl modification on the 4th D-Trp residue (Butyl4th-NH2) showed a ∼3 fold enhancement in cytotoxicity in either the chemo-naive H69 or the DMS79 (originating from a patient treated with chemotherapeutics and radiation therapy) SCLC cell lines. In addition, the di-butylated sequence on the 2nd and 4th D-Trp residues (Butyl2nd,4th-NH2) gave ∼4.5 times higher cytotoxicity against the H69 cell line and a ∼2 fold increase against the DMS79 cell line, compared to tert-Prenyl4th-NH2. The favoured position for butyl modification was the 4th D-Trp residue, as the Butyl2nd-NH2 peptide gave lower cytotoxicity on both cell lines. Butylated peptide sequences, when exposed to neat mouse plasma for 24 h at 37 °C, were found to resist degradation with >80% remaining intact compared to ∼58% for tert-Prenyl4th-NH2. The degradation pathway in plasma occurs via de-amidation of the C-terminus, confirmed by mass spectrometry and RP-HPLC analysis. The butyl modification also conferred resistance to metabolism when tested using S9 liver fraction from mouse. The optimum analogue responsive against the DMS79 cell line was the Butyl4th-NH2 pentapeptide, which revealed a concentration dependent increase in apoptosis: the level of late apoptotic cells rose from ∼36% at 2 μM to ∼96% at 6 μM, as determined by flow cytometry, compared to the unmodified peptide that showed no such effect. Concluding, the butyl substitutions offered the best perspective for high cytotoxicity, induction of apoptosis and metabolic compatibility thereby comprising an improved broad spectrum SP antagonist candidate for treatment of SCLC., Graphical abstract Image 1, Highlights • 8 novel pentapeptide analogues of SPG with N-indole alkylation were synthesised. • Butylation enhanced cytotoxicity against H69 and DMS79 cells; IC50 = 0.63–2.31 μM. • DTrp(N-butyl) at 4th position (16) was effective at inducing apoptosis; 96% at 6 μM. • Butylation enhanced peptides' stability in plasma and S9 liver fraction. • To date, 16 & 19 (dibutylated) are the most potent peptides against SCLC.

The diethyl ester of [(18)F]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 [(18)F]ML-10 from nucleophilic [(18)F]fluoride was completed in 23% radiochemical yield (decay corrected). The radiochemical purity of the product was ≥99%. The diethyl ester of [(18)F]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.

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.

5-(2-Aminopropyl)indole (5-IT) and 3-(2-aminopropyl)indole (α-methyltryptamine, AMT) are isomeric substances and their differentiation can be a challenge under routine analytical conditions, especially when reference material is unavailable. 5-IT represents a very recent addition to the battery of new psychoactive substances that are commercially available from online retailers. This report illustrates how subtle differences observed under mass spectral and UV conditions can help to facilitate the differentiation between the two isomers. Analyses included 1 H and 13C NMR, GC-EI/CI ion trap MS, applications of several U/HPLC-DAD and HPLC-MS methods. Investigations currently underway also highlight the confirmation that AMT was detected in a number of fatal intoxications. These findings also demonstrate that there is a potential risk of misidentification when dealing with both substances. Copyright © 2012 John Wiley & Sons, Ltd.

Among small cellular molecules, certain inositol phosphates (Ins P s) excel at complexing Fe 3+ 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 Fe 3+ /Fe 2+ 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 Fe 3+ –Ins(1,2,3) P 3 speciation was also studied by 31 P 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 Fe 3+ complexed forms is also discussed.

Animal cells contain a pool of inositol phosphates whose biological function is still under current investigation. Ins(1,2,3)P3 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)P3 chemistry under physiological conditions must be completely elucidated. The protonation and complexation behaviour of Ins(1,2,3)P3 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)P3 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.

A large number of cathinone derivatives have shown a wide range of bioactive properties, attracting great interest from communities associated with pharmaceutical research. Some of these derivatives have gained popularity as so-called recreational ‘legal highs’ due to their availability on the Internet and high street shops. A previous study described the qualitative analysis of 24 ‘legal high’ Energy-1 (NRG-1) and NRG-2 products obtained from 18 websites following the ban on mephedrone and derivatives in April 2010. The majority of these products contained a mixture of cathinones just carrying a new label. Here, three additional cathinone products have been detected; two from an NRG-1 sample and one from an NRG-3 sample. This report describes their identification. NRG-1 sample 1 consisted of a mixture of 4 cathinones namely 4-fluoromethcathinone (1), 1(3,4-methylenedioxyphenyl)-2-(methylamino)pentan-1-one (pentylone, 2), 3,4-methylenedioxy-α-pyrrolidinobutyrophenone (MDPBP, 3) and 3,4-methylenedioxypyrovalerone (MDPV, 4). The sample labelled as NRG-3 (mislabelled with the chemical structure of mephedrone) consisted of am ixture of 4-methyl-α-pyrrolidinopropiophenone (MPPP, 5) and (2), whereas the remaining NRG-1 sample 2 (also mislabelled with the chemical structure of mephedrone) consisted of a mixture of (2) and (3). Qualitative analyses were carried out by GC-(EI/CI)-MS, NMR spectroscopy and confirmation by preparation of standards. The preparation of brominated precursors carrying the 3,4-methylenedioxyphenyl nucleus revealed extensive α,α-dibromination: the mass spectral and NMR data of these intermediates are also presented and discussed. Copyright c � 2010 John Wiley & Sons, Ltd.

myo -Inositol 1,2,3-trisphosphate [Ins(1,2,3)P 3 ], 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)P 3 –Fe 3+ 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)P 4 ], which is locked in the unstable penta-axial chair conformation and 1,2,3-trisphosphoglycerol, a flexible acyclic analogue of Ins(1,2,3)P 3 , are reported. 4,6-Carbonate Ins(1,2,3,5)P 4 caused complete inhibition of iron-catalysed hydroxyl radical (HO ) formation at 100 μM, thereby resembling Ins(1,2,3)P 3 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)P 3 to iron. This study provides evidence that Fe 3+ binds tightly to the less stable penta-axial conformation of Ins(1,2,3)P 3 using terminal and bridging phosphate oxygens, thought to also contain a tightly bound water molecule or hydroxyl ligand in the complex.

Naphyrone (naphthylpyrovalerone, O-2482) has been recently advertised for purchase on a number of websites. This compound has been viewed as a so-called ‘legal high’ and was classified as a controlled drug under the UK Misuse of Drugs Act 1971 in mid-July 2010. So far, naphyrone is commonly equated with 1-naphthalen-2-yl-2-pyrrolidin-1-yl-pentan-1-one (β-naphyrone) but analytical characterization of two naphyrone samples revealed the existence of a novel isomer consistent with 1naphthalen-1-yl-2-pyrrolidin-1-yl-pentan-1-one (α-naphyrone). Analyses of both α -a ndβ-naphyrone were carried out using gas chromatography ion trap (EI/CI) mass spectrometry and 1D/2D nuclear magnetic resonance spectroscopy. This provides the first report of α-naphyrone in the scientific literature and the ability to differentiate it from the β-isomer should be of interest to forensic and clinical communities. Copyright c � 2010 John Wiley & Sons, Ltd.

University students with Learning Difficulties (LD) undergo lengthy, expensive assessment by an educational psychologist to provide a detailed cognitive profile on which to base accommodations to enable each individual to study without disadvantage. However, reports are often hard for students to understand and, without trained LD tutors, this information remains underutilized. We trialed an alternative instrument based on the York Adult Assessment developed in the UK, hoping to enable university staff in disabilities and academic skills units to identify students with dyslexia quickly, easily and at no cost to the student and to recommend a limited range of appropriate accommodations based on the result. The trial produced significant group effects, but unacceptable false negatives; we cannot recommend the instrument, therefore, and the need for a reliable alternative remains. This article considers the problems surrounding the present method of assessment and discusses the methodological problems of dev...

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-HO hydrogen bond.