Distribution and temporal dynamics of P. falciparum chloroquine resistance transporter mutations associated with piperaquine resistance in Northern Cambodia

Background Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009-2017. Methods The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative PCR was used to estimate pfpm2 and pfmdr1 copy number. Results Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being out-competed by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single copy pfpm2. Conclusions The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

In Silico Docking, ADMET and QSAR Study of few Antimalarial Phytoconstituents as Inhibitors of Plasmepsin II of P. falciparum Against Malaria

Background: Malaria is endemic in various parts of India particularly in the North- Eastern states with Plasmodium falciparum-the most prevalent human malaria parasite. Plantderived compounds have always received tremendous importance in the area of drug discovery and development and scientific study of traditional medicinal plants are of great importance to mankind. Objective: The present work deals with the computational study of some antimalarial compounds obtained from a few medicinal plants used by the tribal inhabitants of the North-Eastern region of India for treating malaria. Methods: In silico methodologies were performed to study the ligand-receptor interactions. Target was identified based on the pharmacophore mapping approach. A total of 18 plant-derived compounds were investigated in order to estimate the binding energies of the compounds with their drug target through molecular docking using Autodock 4.2. ADMET filtering for determining the pharmacokinetic properties of the compounds was done using Mobyle@RPBS server. Subsequent Quantitative-Structure Activity Relationship analysis for bioactivity prediction (IC50) of the compounds was done using Easy QSAR 1.0. Results: The docking result identified Salannin to be the most potent Plasmepsin II inhibitor while the QSAR analysis identified Lupeol to have the least IC50 value. Most of the compounds have passed the ADME/Tox filtration. Conclusion: Salannin and Lupeol were found to be the most potent antimalarial compounds that can act as successful inhibitors against Plasmepsin II of P. falciparum. The compounds Salannin and Lupeol are found in Azadirachta indica and Swertia chirata plants respectively, abundantly available in the North-Eastern region of India and used by many inhabiting tribes for the treatment of malaria and its symptoms.

Modulation of triple artemisinin-based combination therapy pharmacodynamics by Plasmodium falciparum genotype

The first-line treatments for uncomplicated Plasmodium falciparum malaria are artemisinin-based combination therapies (ACTs), consisting of an artemisinin derivative combined with a longer acting partner drug. However, the spread of P. falciparum with decreased susceptibility to artemisinin and partner drugs presents a significant challenge to malaria control efforts. To stem the spread of drug resistant parasites, novel chemotherapeutic strategies are being evaluated, including the implementation of triple artemisinin-based combination therapies (TACTs). Currently, there is limited knowledge on the pharmacodynamics and pharmacogenetic interactions of proposed TACT drug combinations. To evaluate these interactions, we established an in vitro high-throughput process for measuring the drug dose-response to three distinct antimalarial drugs present in a TACT. Sixteen different TACT combinations were screened against fifteen parasite lines from Cambodia, with a focus on parasites with differential susceptibilities to piperaquine and artemisinins. Analysis revealed drug-drug interactions unique to specific genetic backgrounds, including antagonism between piperaquine and pyronaridine associated with gene amplification of plasmepsin II/III, two aspartic proteases that localize to the parasite digestive vacuole. From this initial study, we identified parasite genotypes with decreased susceptibility to specific TACTs, as well as potential TACTs that display antagonism in a genotype-dependent manner. Our assay and analysis platform can be further leveraged to inform drug implementation decisions and evaluate next-generation TACTs.One Sentence SummaryIn vitro process to evaluate triple-drug combinations for prioritizing antimalarial combinations for in vivo evaluation.

Flap dynamics in pepsin-like aspartic proteases: a computational perspective using Plasmepsin-II and BACE-1 as model systems

Flexibility of β hairpin structure known as flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequence similarity. Tyrosine is a conserved residue present in the flap region of pepsin-like aspartic proteases. In apo protease, tyrosine remains in a dynamic equilibrium between normal and flipped states due to rotation of χ1 angle (distributions of the χ1 angle centred around radian or radian are denoted as normal, whereas distribution centred around ±π radian is denoted as flipped). In this study, we have used apo Plm-II and BACE-1 as model systems. Independent MD simulations starting with Plm-II and BACE-1 remained stuck either in normal or flipped state. Metadynamics simulations using torsion angles (χ1 and χ2 of Tyr) as CVs sampled transition between normal and flipped states. Qualitatively, flipped and normal states predicted to be equally populated. These states were stabilised by H-bond interactions to tryptophan (normal) and catalytic aspartate (flipped) respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine; reduced flap flexibility and resulted in a flap collapse. This is in accordance with previous experimental studies which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Using apo plasmepsin-II and BACE-1 as model systems, we have hypothesised that the rotation of tyrosine side-chain is the key movement which governs the flap dynamics in all pepsin-like aspartic proteases.

Baseline Ex Vivo and Molecular Responses of Plasmodium falciparum Isolates to Piperaquine before Implementation of Dihydroartemisinin-Piperaquine in Senegal

ABSTRACT Dihydroartemisinin-piperaquine, which was registered in 2017 in Senegal, is not currently used as the first-line treatment against uncomplicated malaria. A total of 6.6% to 17.1% of P. falciparum isolates collected in Dakar in 2013 to 2015 showed ex vivo-reduced susceptibility to piperaquine. Neither the exonuclease E415G mutation nor the copy number variation of the plasmepsin II gene (Pfpm2), associated with piperaquine resistance in Cambodia, was detected in Senegalese parasites.