Physicochemical, biological and β-haematin inhibiting activity of pyrido-dibemequines, pyrido[1,2-α]benzimidazoles and their derivatives

There is an urgent need for new antimalarials following the emergence of Plasmodium falciparum strains with reduced sensitivity to the currently used artemisinin combination therapies. Classical aminoquinoline-based drugs inhibit the formation of haemozoin (HZ) thereby causing parasite death from the cellular accumulation of toxic 'free' haem. Coincidentally, this immutable pathway also exists in Schistosoma mansoni, and presents a vulnerable target for drug design in these haematophagus organisms. Therefore, it would be of interest to explore novel scaffolds that can inhibit HZ formation as well as exploit the merits of established drugs via structural modifications that would harness their pharmacological and pharmacokinetic advantages while circumventing their therapeutic shortcomings. This project investigated the physicochemical, biological and mechanistic profiles of pyrido-dibemequine (pDBQ) and pyrido[1,2-α]benzimidazole (PBI) derivatives whose structural motifs were informed by previously synthesised prototype molecules. Specifically, the aqueous solubility, membrane permeability, lipophilicity, metabolic stability and potential for cardiotoxicity of seven pDBQs, their metabolites and ten PBIs were tested through computational and experimental methods. In addition, their antiplasmodial and antischistosomal activities were determined and correlated with their respective physicochemical properties. As regards mechanistic evaluation, their ability to inhibit formation of abiotic HZ, β-haematin (βH), was assessed and intracellular inhibition of HZ formation probed. The pDBQs constitute reversed chloroquines with a 4-aminoquinoline nucleus hybridised to a dibenzylmethylamine side group that serves as a chemosensitising moiety. The pDBQ derivatives showed moderate to high solubility (52 - 197 μM) and permeability (LogPₐₚₚ: -4.6 - -3.6) at pH 6.5. Their lipophilicity, indexed by cLogP, ranged between 3.7 and 5.6 while the mean LogD at both cytosolic (7.4) and vacuolar (5.0) pH was 3.15 and 0.93, respectively. The compounds also showed low-nanomolar range antiplasmodial activity against both chloroquine (CQ)-sensitive (CQS) and resistant (CQR) strains (IC₅₀ range CQS: 14.4 - 126.6 nM, CQRᴰᵈ²: 44.5 - 162 nM and CQR⁷ᴳ⁸: 69.6 - 307.1 nM), with no discernible cross-resistance with CQ and the antiplasmodial activity directly correlated with lipophilicity. Mechanistically, all the pDBQs inhibited βH formation (IC₅₀: 13 - 25 μM) and haem-pyridine fractionation profiles revealed they produced a CQ-like dose-dependent increase in toxic 'free' haem with corresponding decrease in HZ levels. Predicted human-Ether-a-Go-Go-Related Gene (hERG) channel inhibition pIC₅₀ ranged between 6.2 and 6.6, and correlated strongly with the cLogP and molecular weight. The derivatives were also highly susceptibility to microsomal metabolism, with N-dealkylation identified as the main biotransformation route. The pDBQ metabolites exhibited solubility and membrane permeability profiles similar to the parent compounds at pH 6.5, albeit with reduced lipophilicity (cLogP range: 2.3 - 3.5). Their βH inhibition activity (IC₅₀: 15 - 24 μM) was also comparable to the parent compounds as were the haem-pyridine fractionation profiles. However, they showed greater antiplasmodial activity, with 4/7 derivatives exhibiting IC₅₀ < 80 nM against PƒDd2 (CQR strain). The metabolites had reduced hERG channel inhibition potential (pIC₅₀: 5.0 - 5.7) and significantly improved metabolic stability upon incubation with mouse and human liver microsomes. The PBIs comprise molecules with structural likeness to CQ, including a planar heterocyclic moiety and a basic amine side group. PBI analogues showed low to moderate solubility (<5 - 80 μM) and were moderately lipophilic (mean LogD7.4: 3.04). Although most of the derivatives were stable in liver microsomes, their predicted hERG channel inhibition potential was higher (pIC₅₀: 6.11 - 7.50), presumably due to their high molecular weights. All but one derivative had submicromolar activity against CQS and CQR strains, with analogues bearing halo-substituents on the left of the PBI core showing the best antiplasmodial activity (mean IC₅₀: CQS = 26.7 nM and CQR = 30.0 nM), highest selectivity (188 - 341) as well as complete cures in P. berghei-infected mice. The PBIs also inhibited βH formation (IC₅₀: 6.8 - 120 μM) but did not all display intracellular inhibition of HZ formation. All derivatives were active against juvenile (mean IC₅₀: 1.97 μM) and adult (mean IC₅₀: 4.38 μM) schistosomes, with the 3, 4-dichloro-substituted analogue exhibiting 48% reduction of worm burden in vivo. In summary, the pDBQs evaluated in this project constitute potent antiplasmodial inhibitors of HZ formation but whose activity is compromised by metabolic and hERG liability while their metabolites seem to possess improved biological and physicochemical features. The observed activity of the PBIs against P. falciparum and S. mansoni complements the already-established broad antimicrobial potency of this chemotype

Pragmatic strategies in the use of Kiswahili demonstratives

This paper focuses on two spatial Kiswahili demontratives. In Kiswahili, demonstratives have been traditionally treated as morphosyntactic elements that modulate various elements and realize emphatic function. Demonstratives have also been studied as elements that express and facilitate cohesive relations and elements that realize deictic functions in discourse. In this paper we look at Kiswahili demonstratives as used in the standard Kiswahili language in Nairobi city. We argue that besides the traditionally recognized functions, demonstratives in standard Kiswahili are also used to pass subtle discourse messages which can only be explained by taking into account the pragmatic strategies employed in the use of demonstratives in specific discourse settings

Synthesis and biological characterisation of ester and amide derivatives of fusidic acid as antiplasmodial agents

A series of novel fusidic acid (FA) derivatives was synthesized by replacing the carboxylic acid group with various ester and amide groups and evaluated in vitro for their antiplasmodial activity against the chloroquine-sensitive NF54 and multidrug-resistant K1 strains of the malarial parasite Plasmodium falciparum. Most of these derivatives showed a 4-49 and 5-17-fold increase in activity against NF54 and KI strains, respectively, as compared to FA and had a good selectivity index. These derivatives are stable over the incubation period and do not appear to be prodrugs of fusidic acid

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisininbased combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nucleasebased gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bisaminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates

Understanding factors influencing home pregnancy test use among women in western Kenya: A qualitative analysis

BackgroundThere are limited data on home pregnancy test use among women in low-and-middle-income countries. A prior survey found that only 20% of women in western Kenya used a home pregnancy test to confirm their pregnancies before going to antenatal care. This qualitative study aims to understand why women do not use home pregnancy tests in early pregnancy.MethodsFrom April 2021 to July 2021, we interviewed women from four antenatal care clinics in Homa Bay and Siaya counties. We recruited women previously enrolled in the PrEP Implementation for Mothers in Antenatal care (PrIMA) study, a cluster-randomized trial that evaluated the best approaches to implementing PrEP in maternal and child health clinics in Western Kenya (NCT03070600). Interviews were conducted via phone, audio recorded, translated, and transcribed verbatim. We coded and analyzed the transcripts to capture factors influencing women's capability, opportunity, and motivation to use home pregnancy tests.ResultsWe conducted 48 semistructured interviews with women aged 21–42 years. Twenty-seven women did not use a home pregnancy test in their most recent pregnancy. Seventeen of these women reported not using a home pregnancy test before. Lack of knowledge, mistrust in the accuracy of tests, preferring to rely on signs and symptoms of pregnancy or get a test from the health facility, cost, and accessibility were key barriers to home pregnancy test use.ConclusionImproving the uptake of home pregnancy testing during early pregnancy will require efforts to enhance community knowledge of test use and associated benefits and reduce cost burdens by making tests more affordable and accessible

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5–8 fold elevation in the mutation rate, with an increase of 13–28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an “irresistible” compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this “mutator” parasite can be leveraged to drive P. falciparum resistome discovery

Advances in Malaria Pharmacology and the online Guide to MALARIA PHARMACOLOGY: IUPHAR Review X

Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology

Defining the clinical and cognitive phenotype of child savants with autism spectrum disorder

Objective: Whilst savant syndrome is most commonly observed in individuals with Autism Spectrum Disorder (ASD), it has historically been associated with intellectual impairment, and little is known about the clinical and cognitive characteristics of intellectually able individuals with ASD and savant skills. Methods: Participants with ASD and validated savant skills were compared with age and intelligence matched non-savants with ASD using a range of diagnostic and standardised tests. Results: Although the analysis of the clinical data revealed few differences between the groups, striking differences emerged during cognitive testing. Children with savant skills exhibited highly superior working memory and their scores on tests of analytic skills were also superior to those of non-savants. Conclusion: We propose that obsessionality, focused attention, superior working memory and analytic skills facilitate veridical mapping and pattern perception abilities characteristic in savant syndrome