Ex vivo and In vitro antiplasmodial activities of approved drugs predicted to have antimalarial activities using chemogenomics and drug repositioning approach.

High malaria mortality coupled with increased emergence of resistant multi-drug resistant strains of Plasmodium parasite, warrants the development of new and effective antimalarial drugs. However, drug design and discovery are costly and time-consuming with many active antimalarial compounds failing to get approved due to safety reasons. To address these challenges, the current study aimed at testing the antiplasmodial activities of approved drugs that were predicted using a target-similarity approach. This approach is based on the fact that if an approved drug used to treat another disease targets a protein similar to Plasmodium falciparum protein, then the drug will have a comparable effect on P. falciparum. In a previous study, in vitro antiplasmodial activities of 10 approved drugs was reported of the total 28 approved drugs. In this study, six out of 18 drugs that were previously not tested, namely epirubicin, irinotecan, venlafaxine, palbociclib, pelitinib, and PD153035 were tested for antiplasmodial activity. The drug susceptibility in vitro assays against five P. falciparum reference strains (D6, 3D7, W2, DD2, and F32 ART) and ex vivo assays against fresh clinical isolates were done using the malaria SYBR Green I assay. Standard antimalarial drugs were included as controls. Epirubicin and irinotecan showed excellent antiplasmodial ex vivo activity against field isolates with mean IC50 values of 0.044 ± 0.033 µM and 0.085 ± 0.055 µM, respectively. Similar activity was observed against W2 strain where epirubicin had an IC50 value of 0.004 ± 0.0009 µM, palbociclib 0.056 ± 0.006 µM, and pelinitib 0.057 ± 0.013 µM. For the DD2 strain, epirubicin, irinotecan and PD 153035 displayed potent antiplasmodial activity (IC50 < 1 µM). Epirubicin and irinotecan showed potent antiplasmodial activities (IC50 < 1 µM) against DD2, D6, 3D7, and F32 ART strains and field isolates. This shows the potential use of these drugs as antimalarials. All the tested drugs showed antiplasmodial activities with IC50 values below 20 µM, which suggests that our target similarity-based strategy is successful at predicting antiplasmodial activity of compounds thereby circumventing challenges in antimalarial drug discovery.

The Multistage Antimalarial Compound Calxinin Perturbates P. falciparum Ca2+ Homeostasis by Targeting a Unique Ion Channel.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, 'Calxinin'. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

Vitamin D Deficiency and Its Association with Iron Deficiency in African Children.

Vitamin D regulates the master iron hormone hepcidin, and iron in turn alters vitamin D metabolism. Although vitamin D and iron deficiency are highly prevalent globally, little is known about their interactions in Africa. To evaluate associations between vitamin D and iron status we measured markers of iron status, inflammation, malaria parasitemia, and 25-hydroxyvitamin D (25(OH)D) concentrations in 4509 children aged 0.3 months to 8 years living in Kenya, Uganda, Burkina Faso, The Gambia, and South Africa. Prevalence of iron deficiency was 35.1%, and prevalence of vitamin D deficiency was 0.6% and 7.8% as defined by 25(OH)D concentrations of <30 nmol/L and <50 nmol/L, respectively. Children with 25(OH)D concentrations of <50 nmol/L had a 98% increased risk of iron deficiency (OR 1.98 [95% CI 1.52, 2.58]) compared to those with 25(OH)D concentrations >75 nmol/L. 25(OH)D concentrations variably influenced individual markers of iron status. Inflammation interacted with 25(OH)D concentrations to predict ferritin levels. The link between vitamin D and iron status should be considered in strategies to manage these nutrient deficiencies in African children.

Hepcidin regulation in Kenyan children with severe malaria and non-typhoidal Salmonella bacteremia.

Malaria and invasive non-typhoidal Salmonella (NTS) are life-threatening infections that often co-exist in African children. The iron-regulatory hormone hepcidin is highly upregulated during malaria and controls the availability of iron, a critical nutrient for bacterial growth. We investigated the relationship between Plasmodium falciparum malaria and NTS bacteremia in all pediatric admissions aged <5 years between August 1998 and October 2019 (n=75,034). We then assayed hepcidin and measures of iron status in five groups: (1) children with concomitant severe malarial anemia (SMA) and NTS (SMA+NTS, n=16); and in matched children with (2) SMA (n=33); (3) NTS (n=33); (4) cerebral malaria (CM, n=34); and (5) community-based children. SMA and severe anemia without malaria were associated with a 2-fold or more increased risk of NTS bacteremia, while other malaria phenotypes were not associated with increased NTS risk. Children with SMA had lower hepcidin/ferritin ratios (0.10; interquartile range [IQR]: 0.03-0.19) than those with CM (0.24; IQR: 0.14-0.69; P=0.006) or asymptomatic malaria (0.19; IQR: 0.09-0.46; P=0.01) indicating suppressed hepcidin levels. Children with SMA+NTS had lower hepcidin levels (9.3 ng/mL; IQR: 4.7-49.8) and hepcidin/ferritin ratios (0.03; IQR: 0.01-0.22) than those with NTS alone (105.8 ng/mL; IQR: 17.3-233.3; P=0.02 and 0.31; IQR: 0.06-0.66; P=0.007, respectively). Since hepcidin degrades ferroportin on the Salmonella-containing vacuole, we hypothesize that reduced hepcidin in children with SMA might contribute to NTS growth by modulating iron availability for bacterial growth. Further studies are needed to understand how the hepcidin-ferroportin axis might mediate susceptibility to NTS in severely anemic children.

Prevalence and predictors of vitamin D deficiency in young African children.

BACKGROUND: Children living in sub-Saharan Africa have a high burden of rickets and infectious diseases, conditions that are linked to vitamin D deficiency. However, data on the vitamin D status of young African children and its environmental and genetic predictors are limited. We aimed to examine the prevalence and predictors of vitamin D deficiency in young African children. METHODS: We measured 25-hydroxyvitamin D (25(OH)D) and typed the single nucleotide polymorphisms, rs4588 and rs7041, in the GC gene encoding the vitamin D binding protein (DBP) in 4509 children aged 0-8 years living in Kenya, Uganda, Burkina Faso, The Gambia and South Africa. We evaluated associations between vitamin D status and country, age, sex, season, anthropometric indices, inflammation, malaria and DBP haplotypes in regression analyses. RESULTS: Median age was 23.9 months (interquartile range [IQR] 12.3, 35.9). Prevalence of vitamin D deficiency using 25(OH)D cut-offs of < 30 nmol/L and < 50 nmol/L was 0.6% (95% CI 0.4, 0.9) and 7.8% (95% CI 7.0, 8.5), respectively. Overall median 25(OH)D level was 77.6 nmol/L (IQR 63.6, 94.2). 25(OH)D levels were lower in South Africa, in older children, during winter or the long rains, and in those with afebrile malaria, and higher in children with inflammation. 25(OH)D levels did not vary by stunting, wasting or underweight in adjusted regression models. The distribution of Gc variants was Gc1f 83.3%, Gc1s 8.5% and Gc2 8.2% overall and varied by country. Individuals carrying the Gc2 variant had lower median 25(OH)D levels (72.4 nmol/L (IQR 59.4, 86.5) than those carrying the Gc1f (77.3 nmol/L (IQR 63.5, 92.8)) or Gc1s (78.9 nmol/L (IQR 63.8, 95.5)) variants. CONCLUSIONS: Approximately 0.6% and 7.8% of young African children were vitamin D deficient as defined by 25(OH)D levels < 30 nmol/L and < 50 nmol/L, respectively. Latitude, age, season, and prevalence of inflammation and malaria should be considered in strategies to assess and manage vitamin D deficiency in young children living in Africa.

Iron Deficiency Is Associated With Reduced Levels of Plasmodium falciparum-specific Antibodies in African Children.

BACKGROUND: Iron deficiency (ID) and malaria are common causes of ill-health and disability among children living in sub-Saharan Africa. Although iron is critical for the acquisition of humoral immunity, little is known about the effects of ID on antibody responses to Plasmodium falciparum malaria. METHODS: The study included 1794 Kenyan and Ugandan children aged 0-7 years. We measured biomarkers of iron and inflammation, and antibodies to P. falciparum antigens including apical merozoite antigen 1 (anti-AMA-1) and merozoite surface antigen 1 (anti-MSP-1) in cross-sectional and longitudinal studies. RESULTS: The overall prevalence of ID was 31%. ID was associated with lower anti-AMA-1 and anti-MSP-1 antibody levels in pooled analyses adjusted for age, sex, study site, inflammation, and P. falciparum parasitemia (adjusted mean difference on a log-transformed scale (ß) -0.46; 95 confidence interval [CI], -.66, -.25 P < .0001; ß -0.33; 95 CI, -.50, -.16 P < .0001, respectively). Additional covariates for malaria exposure index, previous malaria episodes, and time since last malaria episode were available for individual cohorts. Meta-analysis was used to allow for these adjustments giving ß -0.34; -0.52, -0.16 for anti-AMA-1 antibodies and ß -0.26; -0.41, -0.11 for anti-MSP-1 antibodies. Low transferrin saturation was similarly associated with reduced anti-AMA-1 antibody levels. Lower AMA-1 and MSP-1-specific antibody levels persisted over time in iron-deficient children. CONCLUSIONS: Reduced levels of P. falciparum-specific antibodies in iron-deficient children might reflect impaired acquisition of immunity to malaria and/or reduced malaria exposure. Strategies to prevent and treat ID may influence antibody responses to malaria for children living in sub-Saharan Africa.

Effects of vitamin D deficiency on neurobehavioural outcomes in children: a systematic review.

Introduction: Vitamin D plays an important role in brain development in experimental studies; however, the effect of vitamin D deficiency on child development remains inadequately characterized. We aimed to estimate the effects of vitamin D deficiency on neurobehavioural outcomes in children up to 18 years of age. Methods: We searched PubMed, EMBASE, PsycINFO, Scopus, Cochrane Library, Web of Science and Open Grey for published studies up to 10th January 2020. We included all studies that assessed the effects of maternal or child vitamin D status or vitamin D supplementation on neurobehavioural outcomes in children. Study findings were synthesized qualitatively as the high level of heterogeneity in study populations and methodologies precluded a quantitative meta-analysis. Results: Our search identified 5,633 studies, of which 31 studies with 31,375 participants from 18 countries were included in the systematic review. Of the studies identified, one was a randomised controlled trial (RCT) of vitamin D supplementation in children, while 30 were observational. The RCT (n=55) reported a beneficial effect of supplementation with lower doses compared to higher doses of vitamin D on motor development. Twelve mother-child studies (n=17,136) and five studies in children (n=1,091) reported an association between low maternal or child 25-hydroxyvitamin D levels and impaired neurobehavioural outcomes in children, while 15 mother-child studies (n=20,778) and eight studies in children (n=7,496) reported no association. Conclusions: Although animal studies point to an effect of vitamin D deficiency on brain development, there are few studies on the effects of vitamin D deficiency on neurobehavioural outcomes in children and their findings are inconsistent. There is a need for well-conducted, adequately powered studies to further determine these effects in children. Registration: PROSPERO ID CRD42018087619; registered on 15 February 2018.

Prevalence of vitamin D deficiency in Africa: a systematic review and meta-analysis.

BACKGROUND: Vitamin D deficiency is associated with non-communicable and infectious diseases, but the vitamin D status of African populations is not well characterised. We aimed to estimate the prevalence of vitamin D deficiency in children and adults living in Africa. METHODS: For this systematic review and meta-analysis, we searched PubMed, Web of Science, Embase, African Journals Online, and African Index Medicus for studies on vitamin D prevalence, published from database inception to Aug 6, 2019, without language restrictions. We included all studies with measured serum 25-hydroxyvitamin D (25[OH]D) concentrations from healthy participants residing in Africa. We excluded case reports and case series, studies that measured 25(OH)D only after a clinical intervention, and studies with only a meeting abstract or unpublished material available. We used a standardised data extraction form to collect information from eligible studies; if the required information was not available in the published report, we requested raw data from the authors. We did a random-effects meta-analysis to obtain the pooled prevalence of vitamin D deficiency in African populations, with use of established cutoffs and mean 25(OH)D concentrations. We stratified meta-analyses by participant age group, geographical region, and residence in rural or urban areas. The study is registered with PROSPERO, number CRD42018112030. FINDINGS: Our search identified 1692 studies, of which 129 studies with 21 474 participants from 23 African countries were included in the systematic review and 119 studies were included in the meta-analyses. The pooled prevalence of low vitamin D status was 18·46% (95% CI 10·66-27·78) with a cutoff of serum 25(OH)D concentration less than 30 nmol/L; 34·22% (26·22-43·68) for a cutoff of less than 50 nmol/L; and 59·54% (51·32-67·50) for a cutoff of less than 75 nmol/L. The overall mean 25(OH)D concentration was 67·78 nmol/L (95% CI 64·50-71·06). There was no evidence of publication bias, although heterogeneity was high (I2 ranged from 98·26% to 99·82%). Mean serum 25(OH)D concentrations were lower in populations living in northern African countries or South Africa compared with sub-Saharan Africa, in urban areas compared with rural areas, in women compared with men, and in newborn babies compared with their mothers. INTERPRETATION: The prevalence of vitamin D deficiency is high in African populations. Public health strategies in Africa should include efforts to prevent, detect, and treat vitamin D deficiency, especially in newborn babies, women, and urban populations. FUNDING: Wellcome Trust and the DELTAS Africa Initiative.

Antimalarials: Review of Plasmepsins as Drug Targets and HIV Protease Inhibitors Interactions.

Malaria is a major global health concern with the majority of cases reported in regions of South-East Asia, Eastern Mediterranean, Western Pacific, the Americas, and Sub-Saharan Africa. The World Health Organization (WHO) estimated 216 million worldwide reported cases of malaria in 2016. It is an infection of the red blood cells by parasites of the genus Plasmodium with most severe and common forms caused by Plasmodium falciparum (P. falciparum or Pf) and Plasmodium vivax (P. vivax or Pv). Emerging parasite resistance to available antimalarial drugs poses great challenges to treatment. Currently, the first line of defense includes artemisinin combination therapies (ACTs), increasingly becoming less effective and challenging to combat new occurrences of drug-resistant parasites. This necessitates the urgent need for novel antimalarials that target new molecular pathways with a different mechanism of action from the traditional antimalarials. Several new inhibitors and potential drug targets of the parasites have been reported over the years. This review focuses on the malarial aspartic proteases known as plasmepsins (Plms) as novel drug targets and antimalarials targeting Plms. It further discusses inhibitors of hemoglobin-degrading plasmepsins Plm I, Plm II, Plm IV and Histo-aspartic proteases (HAP), as well as HIV protease inhibitors of plasmepsins.

Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets.

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 µM to 50 µM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens.