Drug-resistant Plasmodium Falciparum Research Articles

Investigating the impact of solvation on p-Phenylenediamine - 2-Amino pyrimidine - Formaldehyde Terpolymer (P2APF) ligand's reactivity and drug suitability for malaria treatment: Insights from experimental and quantum calculations

This study aims at investigating the drug potential of p-Phenylenediamine – 2-Amino Pyrimidine – Formaldehyde terpolymer (P2APF) ligand against drug resistant Plasmodium falciparum. Plasmodium falciparum, an embodiment of cunning among malaria parasites, thrives in regions plagued by rampant mosquito transmission, subpar vector control, and limited access to effective treatments. To this end, this study explored the structural properties of the p-Phenylenediamine – 2-Amino Pyrimidine – Formaldehyde terpolymer (P2APF) ligand, (comprising p-Phenylenediamine, 2-Aminopyrimidine, and Formaldehyde) as a drug potent ligand against the common proteins coding for malaria infection. Various spectroscopic techniques such as FT-IR, UV–visible, and NMR (1H & 13C) spectroscopy were employed both experimentally and theoretically. Additionally, the ligand's surface morphology was investigated using scanning electron microscopy. To comprehend the influence of solvation, theoretical investigations utilizing density functional theory (DFT) at the B3LYPD(BJ)/6-311G++G (2d,2p) level were conducted in DMSO, water, and methanol solvents. The results from the frontier molecular orbital (FMO) analysis provided valuable insights into the electronic properties and solvation effects of P2APF. Specifically, the calculated energy gap of 3.671 eV for P2APF_methanol and 3.672 eV for P2APF_DMSO suggested these solvents as promising environments for reactivity and interactions. Molecular docking studies unveiled the most notable binding affinity between P2APF and the 5UMB chaperone protein receptor, exhibiting a binding affinity of -7.3 kcal/mol on serine and aspartic acid residues, with an average bond distance of 2.25 Å. This robust protein-ligand interaction signifies the promising potential of the P2APF ligand in targeting this specific receptor, thereby combating drug-resistant strains of Plasmodium falciparum for the present and the future.

Hybrid Peptide-Alkoxyamine Drugs: A Strategy for the Development of a New Family of Antiplasmodial Drugs.

The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite's food vacuoles, our approach is summarized as "to dig its grave with its fork". However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.

Unveiling potential repurposed drug candidates for Plasmodium falciparum through in silico evaluation: A synergy of structure-based approaches, structure prediction, and molecular dynamics simulations

The rise of drug resistance in Plasmodium falciparum, rendering current treatments ineffective, has hindered efforts to eliminate malaria. To address this issue, the study employed a combination of Systems Biology approach and a structure-based pharmacophore method to identify a target against P. falciparum. Through text mining, 448 genes were extracted, and it was discovered that plasmepsins, found in the Plasmodium genus, play a crucial role in the parasite's survival. The metabolic pathways of these proteins were determined using the PlasmoDB genomic database and recreated using CellDesigner 4.4.2. To identify a potent target, Plasmepsin V (PF13_0133) was selected and examined for protein-protein interactions (PPIs) using the STRING Database. Topological analysis and global-based methods identified PF13_0133 as having the highest centrality. Moreover, the static protein knockout PPIs demonstrated the essentiality of PF13_0133 in the modeled network. Due to the unavailability of the protein’s crystal structure, it was modeled and subjected to a molecular dynamics simulation study. The structure-based pharmacophore modeling utilized the modeled PF13_0133 (PfPMV), generating 10 pharmacophore hypotheses with a library of active and inactive compounds against PfPMV. Through virtual screening, two potential candidates, hesperidin and rutin, were identified as potential drugs which may be repurposed as potential anti-malarial agents.

Evaluation of the antimalarial activity of SAM13-2HCl with morpholine amide (SKM13 derivative) against antimalarial drug-resistant Plasmodium falciparum and Plasmodium berghei infected ICR mice.

Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.

Prevalence of polymorphisms in marker genes associated with antimalarial drug resistance in Plasmodium falciparum following 10 years of artemisinin-based combination therapy implementation in urban Kolkata.

Resistance to antimalarial drugs is one of the major challenges for malaria elimination. In India, artemisinin combination therapy (artesunate-sulfadoxin pyrimethamine) was introduced in place of chloroquine (CQ) for the treatment of uncomplicated falciparum malaria in 2010. Periodical monitoring of polymorphisms in antimalarial drug resistance marker genes will be useful for assessing drug pressure, mapping and monitoring of drug resistance status; and will be helpful for searching alternative treatments. This study was conducted to study the polymorphisms in antimalarial drug resistance marker genes among clinical Plasmodium falciparum isolates collected from Kolkata after 10 years of artemisinin-based combination therapie (ACT) implementation. Blood samples were collected from P. falciparum mono-infected patients and polymorphisms in P. falciparum CQ resistance transporter (pfcrt), P. falciparum multidrug resistance (pfmdr-1), P. falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthetase (pfdhps), pfATPase6 and pfK-13 propeller genes were analysed by polymerase chain reaction and DNA sequencing. In pfcrt gene, C72S, and K76T mutation was recorded in 100% isolates and no mutations was detected in any of the targeted codons of pfmdr-1 gene. A double mutant pfcrt haplotype SVMNT and wildtype haplotype NYD in pfmdr-1 were prevalent in 100% of study isolates. Triple mutant pfdhfr-pfdhps haplotype ANRNI-SGKAA was recorded. No polymorphism in pfK13 gene was documented in any of the isolates. Observed wild codon N86 along with Y184 and D1246 of pfmdr-1 gene might be an indication of the reappearance of CQ sensitivity. The absence of quadruple and quintuple haplotypes in pfdhfr-pfdhps gene along with the wild haplotype of pfK13 is evidence of ACT effectivity. Hence, similar studies with large sample size are highly suggested for monitoring the drug resistance status of P. falciparum.

A spatio-temporal model of multi-marker antimalarial resistance.

The emergence and spread of drug-resistant Plasmodium falciparum parasites have hindered efforts to eliminate malaria. Monitoring the spread of drug resistance is vital, as drug resistance can lead to widespread treatment failure. We develop a Bayesian model to produce spatio-temporal maps that depict the spread of drug resistance, and apply our methods for the antimalarial sulfadoxine-pyrimethamine. We infer from genetic count data the prevalences over space and time of various malaria parasite haplotypes associated with drug resistance. Previous work has focused on inferring the prevalence of individual molecular markers. In reality, combinations of mutations at multiple markers confer varying degrees of drug resistance to the parasite, indicating that multiple markers should be modelled together. However, the reporting of genetic count data is often inconsistent as some studies report haplotype counts, whereas some studies report mutation counts of individual markers separately. In response, we introduce a latent multinomial Gaussian process model to handle partially reported spatio-temporal count data. As drug-resistant mutations are often used as a proxy for treatment efficacy, point estimates from our spatio-temporal maps can help inform antimalarial drug policies, whereas the uncertainties from our maps can help with optimizing sampling strategies for future monitoring of drug resistance.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India

BackgroundResistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine–pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum.MethodsDried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method.ResultsA total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance.ConclusionThe study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Rhododendron subarcticum Leaf Essential Oil is Effective against Drug-Resistant Plasmodium falciparum and may therefore be Useful for Malaria Prophylaxis

Rhododendron subarcticum Leaf Essential Oil is Effective against Drug-Resistant Plasmodium falciparum and may therefore be Useful for Malaria Prophylaxis

Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana

Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide important information for malaria control policies, including detection of antimalarial drug resistance. However, genome sequencing capacity in malaria-endemic countries is limited. We designed and implemented an end-to-end workflow to detect Plasmodium falciparum antimalarial resistance markers and diversity in the vaccine target circumsporozoite protein (csp) using nanopore sequencing in Ghana. We analysed 196 clinical samples and showed that our method is rapid, robust, accurate and straightforward to implement. Importantly, our method could be applied to dried blood spot samples, which are readily collected in endemic settings. We report that P. falciparum parasites in Ghana are mostly susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine resistance and no evidence of artemisinin resistance. Multiple single nucleotide polymorphisms were identified in csp, but their significance is uncertain. Our study demonstrates the feasibility of nanopore sequencing for malaria genomic surveillance in endemic countries.

Structure-Activity Relationship Analysis of Novel Compounds Against Drug-Resistant Plasmodium Falciparum Strains

The escalating issue of drug-resistant Plasmodium falciparum strains poses a significant challenge to malaria treatment. This research paper presents a comprehensive analysis of the Structure-Activity Relationship (SAR) of innovative compounds designed to combat drug-resistant malaria. Through systematic examination of the chemical structures and their biological activity, we aim to identify potential drug candidates that can effectively address this global health concern. Our study contributes to the ongoing efforts in antimalarial drug development, offering insights into the design of novel compounds that may prove instrumental in countering drug resistance among Plasmodium falciparum strains and improving the efficacy of malaria treatment strategies. Keywords: Plasmodium falciparum, Antimalarial Compounds, Drug Resistance, Chemical Structure Analysis, Novel Compounds

Whole-genome surveillance identifies markers of Plasmodium falciparum drug resistance and novel genomic regions under selection in Mozambique.

Malaria is a devastating disease caused by Plasmodium parasites. The evolution of parasite drug resistance continues to hamper progress toward malaria elimination, and despite extensive efforts to control malaria, it remains a leading cause of death in Mozambique and other countries in the region. The development of successful vaccines and identification of molecular markers to track drug efficacy are essential for managing the disease burden. We present an analysis of the parasite genome in Mozambique, a country with one of the highest malaria burdens globally and limited available genomic data, revealing current selection pressure. We contribute additional evidence to limited prior studies supporting the effectiveness of SWGA in producing reliable genomic data from complex clinical samples. Our results provide the identity of genomic loci that may be associated with current antimalarial drug use, including artemisinin and lumefantrine, and reveal selection pressure predicted to compromise the efficacy of current vaccine candidates.

Nationwide spatiotemporal drug resistance genetic profiling from over three decades in Indian Plasmodium falciparum and Plasmodium vivax isolates

BackgroundDrug resistance is a serious impediment to efficient control and elimination of malaria in endemic areas.MethodsThis study aimed at analysing the genetic profile of molecular drug resistance in Plasmodium falciparum and Plasmodium vivax parasites from India over a ~ 30-year period (1993–2019). Blood samples of P. falciparum and/or P. vivax-infected patients were collected from 14 regions across India. Plasmodial genome was extracted and used for PCR amplification and sequencing of drug resistance genes in P. falciparum (crt, dhps, dhfr, mdr1, k13) and P. vivax (crt-o, dhps, dhfr, mdr1, k12) field isolates.ResultsThe double mutant pfcrtSVMNT was highly predominant across the country over three decades, with restricted presence of triple mutant CVIET from Maharashtra in 2012. High rates of pfdhfr-pfdhps quadruple mutants were observed with marginal presence of “fully resistant” quintuple mutant ACIRNI-ISGEAA. Also, resistant pfdhfr and pfdhps haplotype has significantly increased in Delhi between 1994 and 2010. For pfmdr1, only 86Y and 184F mutations were present while no pfk13 mutations associated with artemisinin resistance were observed. Regarding P. vivax isolates, the pvcrt-o K10 “AAG” insertion was absent in all samples collected from Delhi in 2017. Pvdhps double mutant SGNAV was found only in Goa samples of year 2008 for the first time. The pvmdr1 908L, 958M and 1076L mutations were highly prevalent in Delhi and Haryana between 2015 and 2019 at complete fixation. One nonsynonymous novel pvk12 polymorphism was identified (K264R) in Goa.ConclusionsThese findings support continuous surveillance and characterization of P. falciparum and P. vivax populations as proxy for effectiveness of anti-malarial drugs in India, especially for independent emergence of artemisinin drug resistance as recently seen in Africa.

In vitro and in vivo antiplasmodial evaluation of sugar-modified nucleoside analogues

Drug-resistant Plasmodium falciparum (Pf) infections are a major burden on the population and the healthcare system. The establishment of Pf resistance to most existing antimalarial therapies has complicated the problem, and the emergence of resistance to artemisinin derivatives is even more concerning. It is increasingly difficult to cure malaria patients due to the limited availability of effective antimalarial drugs, resulting in an urgent need for more efficacious and affordable treatments to eradicate this disease. Herein, new nucleoside analogues including morpholino-nucleoside hybrids and thio-substituted nucleoside derivatives were prepared and evaluated for in vitro and in vivo antiparasitic activity that led a few hits especially nucleoside-thiopyranoside conjugates, which are highly effective against Pf3D7 and PfRKL-9 strains in submicromolar concentration. One adenosine derivative and four pyrimidine nucleoside analogues significantly reduced the parasite burden in mouse models infected with Plasmodium berghei ANKA. Importantly, no significant hemolysis and cytotoxicity towards human cell line (RAW) was observed for the hits, suggesting their safety profile. Preliminary research suggested that these thiosugar-nucleoside conjugates could be used to accelerate the antimalarial drug development pipeline and thus deserve further investigation.

In Silico Screening and Molecular Dynamics Simulation of Potential Anti-Malarial Agents from Zingiberaceae as Potential Plasmodium falciparum Lactate Dehydrogenase (PfLDH) Enzyme Inhibitors.

Malaria continues to be a major public health issue in a number of countries, particularly in tropical regions-the emergence of drug-resistant Plasmodium falciparum encourages new drug discovery research. The key to Plasmodium falciparum survival is energy production up to 100 times greater than other parasites, primarily via the PfLDH. This study targets PfLDH with natural bioactive compounds from the Zingiberaceae family through molecular docking and molecular dynamic studies. Sulcanal, quercetin, shogosulfonic acid C, galanal A and naringenin are the Top 5 compounds with a lower binding energy value than chloroquine, which was used as a control in this study. By binding to NADH and substrate binding site residues, the majority of them are expected to inhibit pyruvate conversion to lactate and NAD+ regeneration. When compared to sulcanal and control drugs, the molecular dynamics (MD) simulation study indicated that quercetin may be the most stable molecule when interacting with PfLDH.

Development of a technique for analyzing the lengths of restriction fragments to assess the mefloquine resistance of plasmodium falciparum based on the identification of single-nucleotide polymorphisms (A958146T, A961013G, G961625T) of the PFMDR1 gene

ABSTRACT. A method for identifying single nucleotide polymorphisms of the PfMDR1 gene (A958146T, A961013G, G961625T) based on the analysis of the lengths of restriction fragments using polymerase chain reaction technology with specific primers is presented. To identify the polymorphism A958146T (amino acid substitution N86Y), it was proposed to amplify a fragment of parasitic deoxyribonucleic acid with a length of 417 bp, including the 86th codon of the PfMDR1 gene, followed by treatment of the amplicon with ApoI endonuclease, the restriction site of which includes the sequence of the analyzed mutation. With the Plasmodium falciparum genotype unchanged, the isolated deoxyribonucleic acid site is divided into two fragments of size 239 and 179 bp. In the case of an altered genotype containing a single nucleotide missense mutation A T, the original fragment 417 bp will be preserved. When designing the system to detect the amino acid substitution N1042D caused by the A961013G mutation, a pair of specific primers were selected that limit the 404 bp-long section of parasitic deoxyribonucleic acid. For restriction analysis, the most optimal was the use of AseI endonuclease, which in the case of an unchanged Plasmodium falciparum genotype divides the initial amplicon into 4 fragments (132, 116, 99 and 25 bp), and in the presence of A G mutation into 3 (248, 99, 25 bp). It was found that a fragment of the PfMDR1 gene, including the G961625T mutation leading to the amino acid substitution D1246Y, contains 1 site corresponding to the restriction site of endonuclease BglII. Therefore, in the case of the wild genotype of Plasmodium falciparum, the initial fragment of deoxyribonucleic acid is cut into 2 short sections (300 and 269 bp). With the D1246 mutation, the replacement of the nucleotide G T leads to the disappearance of the restriction site, so only one source fragment (509 bp) will be recorded on the electrophoregram. Based on the analysis of the data obtained, criteria for evaluating the drug resistance of Plasmodium falciparum have been developed. The decrease in sensitivity to mefloquine and its derivatives of pathogens of tropical malaria can be evidenced by positive results obtained using the developed methods for detecting haplotypes A T (N86Y (band 417 bp)), A G (N1042D (bands 248, 99 and 25 bp)), G T (D1246Y (509 bp)). The developed methods can be used to analyze the spread of drug-resistant tropical malaria.

Marmesin isolated from Celtis durandii Engl. root bioactive fraction inhibits β-hematin formation and contributes to antiplasmodial activity

Ethnopharmacological relevanceMalaria is a leading cause of death in many developing countries, especially in sub-Saharan Africa. Nigeria is endowed with an abundance of medicinal plants, many of which are used to treat malaria. Celtis durandii Engl. is one such plant used as a traditional antimalarial remedy in southeast Nigeria. However, its antiplasmodial potential is poorly explored. Aim of the studyThe study aimed at identifying the antiplasmodial components of C. durandii root extract through antiplasmodial activity-guided fractionation. Materials and methodsDichloromethane/methanol mixture extract (1:1 v/v) of C. durandii root was prepared and partitioned against water to obtain the organic phase, which was further separated by column chromatography into nine (C1 – C9) fractions. The antiplasmodial activity was evaluated by in vitro screening of the different fractions against drug-sensitive and drug-resistant Plasmodium falciparum strains. Further purification of the active column fractions resulted in a potent anti-Plasmodial compound that was subsequently investigated for its effect on β-hematin formation. Additionally, the isolated compound was characterized and identified as marmesin using mass spectrometry and nuclear magnetic resonance spectroscopy. ResultsCeltis durandii root extract exhibited promising antiplasmodial activity {IC50 (μg/ml) 5.92, 6.04, and 6.92} against PfW2mef, PfINDO, and Pf3D7 respectively. Pooled fractions with good antiplasmodial activity {IC50 (μg/ml) Pf3D7: 3.99; PfINDO: 2.24} and selectivity for the parasites (SI: 21) yielded a compound that was fourteen-fold potent in antiplasmodial activity against Pf3D7(IC50: 0.28 μg/ml). It also inhibited β-hematin formation with an IC50 = 150 μM. Further studies using spectral data, literature, and chemical databases identified the purified compound as marmesin. ConclusionThis work has demonstrated that Celtis durandii root extract has good antiplasmodial activity against drug-sensitive and drug-resistant P. falciparum. The inhibition of β-hematin formation by marmesin accounts in part for this activity.

A review of malaria molecular markers for drug resistance in Plasmodium falciparum and Plasmodium vivax in China.

China has now achieved the elimination of malaria, but it still faces severe challenges in the post-elimination stage. China continues to be plagued by imported malaria cases, and preventing re-transmission of imported malaria is critical. The effectiveness of antimalarial drugs for malaria control largely depends on the study of drug resistance markers in vitro. Monitoring molecular markers of parasite-associated drug resistance can help predict and manage drug resistance. There is currently a lack of systematic reviews of molecular markers for indigenous and imported malaria in China. Therefore, this review summarizes the published articles related to molecular marker polymorphism of indigenous and imported malaria cases in China in the past two decades, to study the mutation frequency and distribution of crt, mdr1, dhps, dhfr and K13 gene resistance-related loci. This can provide a whole picture of molecular markers and the resistance mutations of imported cases in China, which has certain significance for drug resistance surveillance planning, safe and effective treatment, and preventing the recurrence of local transmission by imported malaria in China in the future.

Identification of Potential Drug Targets in Erythrocyte Invasion Pathway of Plasmodium falciparum.

The erythrocyte invasion phase plays a critical role in multiplication, sexual determination, and drug resistance in Plasmodium falciparum. In order to identify the critical genes and pathways in the erythrocyte invasion phase, the gene set (GSE129949) and the RNA-Seq count data for the W2mef strain were used for further analysis. An integrative bioinformatics study was performed to scrutinize genes as potential drug targets. 487 differentially expressed genes (DEGs) with an adjusted P value < 0.001 enriched 47 Gene Ontology (GO) terms that were over-represented based on hyper-geometric analysis P value < 0.01. Protein-Protein interaction network analysis was done using DEGs with higher confidence interactions (PPI score threshold = 0.7). MCODE and cytoHubba apps were utilized to define the hub proteins and rank them based on multiple topological analyses and MCODE scores. Furthermore, Gene Set Enrichment Analysis (GSEA) was carried out by using 322 gene sets from the MPMP database. The genes involved in multiple significant gene sets were determined by leading-edge analysis. Our study identified six genes encoding proteins that could be potential drug targets involved in the erythrocyte invasion phase related to merozoites motility, cell-cycle regulation, G-dependent protein kinase phosphorylation in schizonts, control of microtubule assembly, and sexual commitment. The druggability of those proteins was calculated based on the DCI (Drug Confidence Index) and predicted binding pockets' values. The protein that showed the best binding pocket value was subjected to deep learning-based virtual screening. The study identified the best small molecule inhibitors in terms of drug-binding score against the proteins for inhibitor identification.

In vitro activity of rhinacanthin analogues against drug resistant Plasmodium falciparum isolates from Northeast Thailand

BackgroundNew anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand.MethodsUsing a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates.ResultsThe 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9–791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6–63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83–0.97, P < 0.001). There were no P. falciparum cytochrome b mutations observed in the field isolates, indicating that P. falciparum isolates from this area remained ATQ-sensitive. Pfkelch13 mutations and the ring-stage survival assay confirmed that most isolates were resistant to ART.ConclusionsTwo rhinacanthin analogues showed parasiticidal activity against multi-drug resistant P. falciparum isolates, although less potent than ATQ. Rhinacanthin analogue 2 was more potent than analogue 1, and can be a lead compound for further optimization as an anti-malarial in areas with multidrug resistance.

Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum.

Resistance of the human malaria parasites, Plasmodium falciparum, to artemisinins is now fully established in Southeast Asia and is gradually emerging in Sub-Saharan Africa. Although nonsynonymous SNPs in the pfk13 Kelch-repeat propeller (KREP) domain are clearly associated with artemisinin resistance, their functional relevance requires cooperation with other genetic factors/alterations of the P. falciparum genome, collectively referred to as genetic background. Here we provide experimental evidence that P. falciparum cyclophilin 19B (PfCYP19B) may represent one putative factor in this genetic background, contributing to artemisinin resistance via its increased expression. We show that overexpression of PfCYP19B in vitro drives limited but significant resistance to not only artemisinin but also piperaquine, an important partner drug in artemisinin-based combination therapies. We showed that PfCYP19B acts as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P). Curiously, artemisinin and piperaquine affect eIF2α-P in an inverse direction that in both cases can be modulated by PfCYP19B towards resistance. Here we also provide evidence that the upregulation of PfCYP19B in the drug-resistant parasites appears to be maintained by a short tandem repeat (SRT) sequence polymorphism in the gene's promoter region. These results support a model that artemisinin (and other drugs) resistance mechanisms are complex genetic traits being contributed to by altered expression of multiple genes driven by genetic polymorphism at their promoter regions.