Activity of alkoxyamide-based histone deacetylase inhibitors against Plasmodium falciparum malaria parasites.

There are more than 240 million cases of malaria and 600,000 associated deaths each year, most due to infection with Plasmodium falciparum parasites. While malaria treatment options exist, new drugs with novel modes of action are needed to address malaria parasite drug resistance. Protein lysine deacetylases (termed HDACs) are important epigenetic regulatory enzymes and prospective therapeutic targets for malaria. Here we report the antiplasmodial activity of a panel of 17 hydroxamate zinc binding group HDAC inhibitors with alkoxyamide linkers and different cap groups. The two most potent compounds (4a and 4b) were found to inhibit asexual P. falciparum growth with 50% inhibition concentrations (IC50's) of 0.07 µM and 0.09 µM, respectively, and demonstrated >200-fold more selectivity for P. falciparum parasites versus human neonatal foreskin fibroblasts (NFF). In situ hyperacetylation studies demonstrated that 4a, 4b and analogs caused P. falciparum histone H4 hyperacetylation, suggesting HDAC inhibition, with structure activity relationships providing information relevant to the design of new Plasmodium-specific aliphatic chain hydroxamate HDAC inhibitors.

Isolation and In Vitro and In Vivo Activity of Secondary Metabolites from Clerodendrum polycephalum Baker against Plasmodium Malaria Parasites.

Chemical investigation of the antimalarial medicinal plant Clerodendrum polycephalum led to the isolation of five new diterpenoids, including ajugarins VII-X (1-4) and teuvincenone K (5), along with four known compounds, namely, 12,16-epoxy-6,11,14,17-tetrahydroxy-17(15 → 16)-abeo-5,8,11,13,15-abietapentaen-7-one (6), methyl pheophorbide A (7), loliolide (8), and acacetin (9). The chemical structures of the new compounds were elucidated using NMR spectroscopy, mass spectrometry, circular dichroism, as well as density functional theory calculations. All compounds were evaluated for in vitro activity against Plasmodium falciparum 3D7 malaria parasites with methyl pheophorbide A (7) showing the strongest activity (IC50 4.49 µM). Subsequent in vivo testing in a Plasmodium berghei chemosuppression model showed that compound 7 significantly attenuated peripheral blood parasitemia, leading to 79% and 87% chemosuppression following oral doses at 10 and 20 mg/kg, respectively.

An exploratory analysis examining differences in physical activity and motor competence in children with and without asthma: brief report.

OBJECTIVE: The purpose of this pilot study was to examine potential differences in motor competence (MC) and physical activity (PA) between children with and without asthma. METHODS: Thirty-seven children and adolescents completed the Exercises for a Healthy Asthma Lifestyle and Enjoyment study (46% with asthma, 51% female, 11.1 ± 0.4 years, and 46% White). Motor competence was assessed using the Movement Assessment Battery for Children 2nd edition (MABC-2). PA was assessed using accelerometry. RESULTS: Children with asthma had significantly lower MC in the domain of aiming and catching (with asthma: 8.2 ± 0.4 vs. without asthma: 9.9 ± 0.5; p = 0.03) and fewer daily minutes spent in moderate-to-vigorous PA (MVPA) (with asthma: 18.0 ± 2.3 min vs. without asthma: 27.2 ± 3.6 min; p = 0.047). There were no significant group differences in manual dexterity, balance, total MABC-2 score, or total daily PA (all ps > 0.05). CONCLUSIONS: This study provides confirmatory evidence that children with asthma display lower MC and spend less time in MVPA compared to children without asthma. Because MC is a prerequisite for engaging in PA, future research should seek to determine if the differences observed in MC contribute to disparities in MVPA observed in this clinical population.

Two birds in the hand: Concurrent and switching cognitive flexibility in preschoolers.

Cognitive flexibility, the ability to think of something in more than one way, has been studied in preschoolers from two different approaches. Within executive function research, most studies operationalize cognitive flexibility using sequential tasks in which children must think of a specific stimulus first in one way and then in another way (switching cognitive flexibility). In contrast, Piagetian multiplicative classification tasks also require cognitive flexibility by asking children to consider multiple dimensions of a single stimulus at the same time. Piagetian tasks are typically inductive and require abstraction skills. Although research with Piagetian tasks suggests that children are unable to consider multiple dimensions simultaneously until they reach the concrete operational stage, there is some evidence that preschoolers can coordinate two dimensions simultaneously in deductive tasks. In this study, we examined a deductive version of a matrix completion task in which preschoolers (N = 102) were asked to consider two dimensions of a given stimulus simultaneously. We show that 4- and 5-year-olds can succeed on this task and compare children's performance on the matrix completion task with their performance on a widely used switching cognitive flexibility task, the Dimensional Change Card Sort. We discuss the implications of relating the two aspects of cognitive flexibility development.

Psychological Responses to Intermittent Activities in Children With and Without Asthma.

PURPOSE: The purpose of this study was to examine the psychological responses to intermittent activities of varying intensities and types among children with and without asthma. METHODS: A total of 37 children and adolescents (51% male, aged 8-16 y, 54% nonwhite, and 54% without asthma) participated in this study. Participants completed 5 exercises in the same order: self-paced walking, resistance activities, dance video, gamified obstacle course, and step test. In-task mood was assessed using the Feeling Scale, in-task perceived exertion was assessed via the ratings of perceived exertion scale, and postactivity enjoyment was assessed using the Physical Activity Enjoyment Scale. RESULTS: There was a significant main effect of exercise type on mood (P < .001), ratings of perceived exertion (P < .001), and enjoyment (P < .002). There was not a significant main effect of asthma status on mood, ratings of perceived exertion, or enjoyment (Ps > .05). Children with asthma reported significantly lower in-task mood during the step exercise (P < .037) and reported significantly lower postactivity enjoyment after the walk and obstacle course exercises (Ps < .03). CONCLUSIONS: Regardless of differences by asthma status for in-task mood during the obstacle course and for postactivity enjoyment during the walk and step exercises, both children with and without asthma reported high in-task mood and postactivity enjoyment during all 5 exercises.

Empathetic Concern in Emerging Adolescents: The Role of Theory of Mind and Gender Roles.

The current study examined the roles of gender, and gender-role orientation in young adolescents' empathetic concern. In addition, this study aimed to explore the contribution of Theory of Mind in participants' empathetic concern. Finally, this study examined whether gender and gender-role orientation were implicated in emerging adolescents' Theory of Mind understanding. One-hundred-fifty 11- to 12-year-olds (79 self-identified females) completed questionnaires measuring their empathetic concern, Theory of Mind, and their perceived gender-role orientation. Results showed that gender-role orientation, specifically, femininity and masculinity predicted empathetic concern above and beyond gender. In addition, the effects of cognitive and affective Theory of Mind are explored and discussed in relation to empathetic concern. Finally, neither gender nor gender-role orientation was found to contribute to participants' Theory of Mind understanding. These findings suggest that emerging adolescents' perceived gender roles, as well as their ability to consider another's beliefs, play a role in their expression of empathetic concern.

Parents accurately perceive problematic eating behaviors but overestimate physical activity levels in preschool children.

PURPOSE: The aim of this study is to examine the associations between parents' perceptions of their child's physical activity and eating behaviors to actual physical activity, body mass index percentage (BMI%), and body fat percentage (BF%). A secondary aim is to examine additional parental determinants to child's physical activity. METHODS: Participants were preschool children (N = 114, 59 females, Mage= 4.06) from three University-sponsored centers and parents (N = 114, 107 mothers). Parents self-reported physical activity, perceptions of child's physical activity, and completed "The Child Eating Behavior Questionnaire". Children physical activity was collected with accelerometers. RESULTS: Whereas 97% of the 68 parents with children meeting physical activity guidelines accurately identified their child as active, 93% of the 14 parents with children not meeting physical activity guidelines inaccurately identified their child as active (X2(1, N = 82) = 0.58, p = 0.446)). Regarding eating behaviors, child BMI% was moderately correlated with parent's perceptions of their child's Emotional Overeating (r(74) = 0.416, p < 0.001) and Food Responsiveness (r(74) = 0.543, p < 0.001). Parent's engagement in vigorous physical activity demonstrated a positive relationship to child's physical activity (r(78) = 0.297, p = 0.008). CONCLUSION: Parents of inactive children have inaccurate perceptions of their child's physical activity. The association between children's BMI% and eating behaviors indicates parents can accurately perceive problematic eating behaviors. Parents, who accurately perceive their child's behaviors, may be in a better position to identify deficiency and seek early intervention. Additionally, parent's physical activity may have implications to children's physical activity. LEVEL OF EVIDENCE: Level V: Descriptive cross-sectional study.

Pharmacodynamic Evaluation of Plasma and Epithelial Lining Fluid Exposures of Amikacin against Pseudomonas aeruginosa in a Dynamic In Vitro Hollow-Fiber Infection Model.

Given that aminoglycosides, such as amikacin, may be used for multidrug-resistant Pseudomonas aeruginosa infections, optimization of therapy is paramount for improved treatment outcomes. This study aims to investigate the pharmacodynamics of different simulated intravenous amikacin doses on susceptible P. aeruginosa to inform ventilator-associated pneumonia (VAP) and sepsis treatment choices. A hollow-fiber infection model with two P. aeruginosa isolates (MICs of 2 and 8 mg/liter) with an initial inoculum of ∼108 CFU/ml was used to test different amikacin dosing regimens. Three regimens (15, 25, and 50 mg/kg) were tested to simulate a blood exposure, while a 30 mg/kg regimen simulated the epithelial lining fluid (ELF) for potential respiratory tract infection. Data were described using a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model. Whole-genome sequencing was used to identify mutations associated with resistance emergence. While bacterial density was reduced by >6 logs within the first 12 h in simulated blood exposures following this initial bacterial kill, there was amplification of a resistant subpopulation with ribosomal mutations that were likely mediating amikacin resistance. No appreciable bacterial killing occurred with subsequent doses. There was less (<5 log) bacterial killing in the simulated ELF exposure for either isolate tested. Simulation studies suggested that a dose of 30 and 50 mg/kg may provide maximal bacterial killing for bloodstream and VAP infections, respectively. Our results suggest that amikacin efficacy may be improved with the use of high-dose therapy to rapidly eliminate susceptible bacteria. Subsequent doses may have reduced efficacy given the rapid amplification of less-susceptible bacterial subpopulations with amikacin monotherapy.

Proteomic analysis of Plasmodium falciparum histone deacetylase 1 complex proteins.

Plasmodium falciparum histone deacetylases (PfHDACs) are an important class of epigenetic regulators that alter protein lysine acetylation, contributing to regulation of gene expression and normal parasite growth and development. PfHDACs are therefore under investigation as drug targets for malaria. Despite this, our understanding of the biological roles of these enzymes is only just beginning to emerge. In higher eukaryotes, HDACs function as part of multi-protein complexes and act on both histone and non-histone substrates. Here, we present a proteomics analysis of PfHDAC1 immunoprecipitates, identifying 26 putative P. falciparum complex proteins in trophozoite-stage asexual intraerythrocytic parasites. The co-migration of two of these (P. falciparum heat shock proteins 70-1 and 90) with PfHDAC1 was validated using Blue Native PAGE combined with Western blot. These data provide a snapshot of possible PfHDAC1 interactions and a starting point for future studies focused on elucidating the broader function of PfHDACs in Plasmodium parasites.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mitoribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Measuring on the go: Response to Morra, Panesi, Traverso, and Usai.

Morra, Panesi, Traverso, and Usai's (Journal of Experimental Child Psychology, 2017, Vol. 167, pp. 246-258) effort to clarify theoretical models and nomenclature confusion surrounding young children's executive functions development is laudable and important. In this article, we address some of the points these authors raised regarding our previous article (Journal of Experimental Child Psychology, 2017, Vol. 159, pp. 199-218). Although we agree that the Multidimensional Card Selection Task makes working memory demands, it goes beyond working memory to measure concurrent cognitive flexibility in preschoolers. Using this task will allow researchers to fine-tune our models of cognitive flexibility and executive functions development.

Catalyst-Controlled Stereoselective Synthesis Secures the Structure of the Antimalarial Isocyanoterpene Pustulosaisonitrile-1.

Three new isocyanoditerpenes (5-7) have been characterized from Australian specimens of the nudibranch Phyllidiella pustulosa. The planar structure and (3R,6S,7R) absolute configuration of pustulosaisonitrile-1 were deduced by spectroscopic analyses at 900 MHz informed by molecular modeling, DFT calculations, and computational NMR chemical shift predictions and by comparison of experimental electronic circular dichroism (ECD) data with TDDFT-ECD calculations for the truncated model compound 8. A catalyst-controlled enantio- and diastereoselective total synthesis of the two most likely diastereomeric candidates for the structure of 5 solidified its (3R,6S,7R,10S,11R,14R) absolute configuration. Three individual enantioselective methods provided stereochemical control at key positions, permitting an unambiguous final structural assignment. Isocyanide 5 and synthetic diastereomers 5a and 5c showed activity against Plasmodium falciparum malaria parasites (IC50 ∼1 µM).

The Multidimensional Card Selection Task: A new way to measure concurrent cognitive flexibility in preschoolers.

Most executive function research examining preschoolers' cognitive flexibility, the ability to think about something in more than one way, has focused on preschoolers' facility for sequentially switching their attention from one dimension to another (e.g., sorting bivalent cards first by color and then by shape). We know very little about preschoolers' ability to coordinate more than one dimension simultaneously (concurrent cognitive flexibility). Here we report on a new task, the Multidimensional Card Selection Task, which was designed to measure children's ability to consider two dimensions, and then three dimensions, concurrently (e.g., shape and size, and then shape, size, and color). More than half of the preschoolers in our sample of 107 (50 3-year-olds and 57 4-year-olds) could coordinate three dimensions simultaneously and consistently across three test trials. Furthermore, performance on the Multidimensional Card Selection Task was related, but not identical, to performance on other cognitive tasks, including a widely used measure of switching cognitive flexibility (the Dimensional Change Card Sort). The Multidimensional Card Selection Task provides a new way to measure concurrent cognitive flexibility in preschoolers, and opens another avenue for exploring the emergence of early cognitive flexibility development.

Design and Synthesis of Novel Anti-Plasmodial Histone Deacetylase Inhibitors Containing an Alkoxyamide Connecting Unit.

Despite recent declines in mortality, malaria remains an important global health problem. New therapies are needed, including new drugs with novel modes of action compared to existing agents. Among new potential therapeutic targets for malaria, inhibition of parasitic histone deacetylases (HDACs) is a promising approach. Homology modeling of PfHDAC1, a known target of some anti-plasmodial HDAC inhibitors, revealed a unique threonine residue at the rim of the active site in close proximity to the location of the cap group of vorinostat-type HDAC inhibitors. Aiming to obtain HDAC inhibitors with potent and preferential anti-plasmodial activity, we synthesized a mini-library of alkoxyamide-based HDAC inhibitors containing hydrogen bond acceptors in the cap group. Using a 5-step synthetic route, 12 new inhibitors were synthesized and assayed against Plasmodium falciparum asexual blood stage parasites (clones 3D7 and Dd2) and human cells (HepG2). The most active compound 6h (Pf3D7 IC50 : 0.07 µM; PfDd2 IC50 : 0.07 µM) was 25-fold more toxic against the parasite versus human HepG2 cells. Selected compounds were shown to cause hyperacetylation of P. falciparum histone H4, indicating inhibition of one or more PfHDACs.

Adaptation of the [3H]Hypoxanthine Uptake Assay for In Vitro-Cultured Plasmodium knowlesi Malaria Parasites.

The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [(3)H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC50s) consistent with those obtained with P. falciparum This assay provides a platform to use P. knowlesi in vitro for drug discovery.

Synthesis and antimalarial evaluation of amide and urea derivatives based on the thiaplakortone A natural product scaffold.

A series of amide (8­32, 40­45) and urea (33, 34, 36­39) analogues based on the thiaplakortone A natural product scaffold were synthesised and screened for in vitro antimalarial activity against chloroquine-sensitive (3D7) and chloroquine- and mefloquine-resistant (Dd2) Plasmodium falciparum parasite lines. Several analogues displayed potent inhibition of P. falciparum growth (IC50 <500 nM) and good selectivity for P. falciparum versus human neonatal foreskin fibroblast cells (selectivity index >100). Two of these compounds, 8 and 33, exhibited good aqueous solubility and metabolic stability, and when administered subcutaneously to mice (32 mg kg(-1)), plasma concentrations remained above 0.2 µM for at least 8 h. Both 8 and 33 were well tolerated in mice after subcutaneous administration of 32 mg kg(-1) twice daily for 4 days. Using this regimen blood stage P. berghei was suppressed by 52% for 8 and 26% for 33, relative to the vehicle control.

Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum.

The η-carbonic anhydrases (CAs, EC 4.2.1.1) were recently discovered as the sixth genetic class of this metalloenzyme superfamily, and are so far known only in protozoa, including various Plasmodium species, the causative agents of malaria. We report here an inhibition study of the η-CA from Plasmodium falciparum (PfCA) against a panel of sulfonamides and one sulfamate compound, some of which are clinically used. The strongest inhibitors identified were ethoxzolamide and sulthiame, with KIs of 131-132 nM, followed by acetazolamide, methazolamide and hydrochlorothiazide (KIs of 153-198 nM). Brinzolamide, topiramate, zonisamide, indisulam, valdecoxib and celecoxib also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM. An interesting observation was that the more efficient PfCA inhibitors are representative of several scaffolds and chemical classes, including benzene sulfonamides, monocyclic/bicyclic heterocyclic sulfonamides and compounds with a more complex scaffold (i.e., the sugar sulfamate derivative, topiramate, and the coxibs, celecoxib and valdecoxib). A comprehensive inhibition study of small molecules for η-CAs is needed as a first step towards assessing PfCA as a druggable target. The present work identifies the first known η-CA inhibitors and provides a platform for the development of next generation novel PfCA inhibitors.

Antimalarial Isocyano and Isothiocyanato Sesquiterpenes with Tri- and Bicyclic Skeletons from the Nudibranch Phyllidia ocellata.

Five new isocyano/isothiocyanato sesquiterpenes (1-5) with tri- or bicyclic carbon skeletons have been characterized from Australian specimens of the nudibranch Phyllidia ocellata. Spectroscopic analyses at 900 MHz were informed by DFT calculations. The 1S, 5S, 8R configuration of 2-isocyanoclovene (1) was determined by X-ray crystallographic analysis of formamide 6. A biosynthetic pathway to clovanes 1 and 2 from epicaryolane precursors is proposed. Isocyanides 1, 2, and 4 showed activity against Plasmodium falciparum (IC50 0.26-0.30 µM), while isothiocyanate 3 and formamide 6 had IC50 values of >10 µM.