Plasmodium Proteins Research Articles

Microtubule inner proteins of Plasmodium are essential for transmission of malaria parasites

Microtubule inner proteins (MIPs) are microtubule-associated proteins that bind to tubulin from the luminal side. MIPs can be found in axonemes to stabilize flagellar beat or within cytoplasmic microtubules. Plasmodium spp. are the causative agents of malaria that feature different parasite forms across a complex life cycle with both unique and divergent microtubule-based arrays. Here, we investigate four MIPs in a rodent malaria parasite for their role in transmission to and from the mosquito. We show by single and double gene deletions that SPM1 and TrxL1, MIPs associated with subpellicular microtubules, are dispensable for transmission from the vertebrate host to the mosquito and back. In contrast, FAP20 and FAP52, MIPs associated with the axonemes of gametes, are essential for transmission to mosquitoes but only if both genes are deleted. In the absence of both FAP20 and FAP52, the B-tubule of the axoneme partly detaches from the A-tubule, resulting in the deficiency of axonemal beating and hence gamete formation and egress. Our data suggest that a high level of redundancy ensures microtubule stability in the transmissive stages of Plasmodium, which is important for parasite transmission.

Expression and purification of E140 protein antigen fragments of Plasmodium vivax and Plasmodium berghei for serological assays.

Malaria, a life-threatening disease caused by Plasmodium parasites, continues to pose a significant global health threat, with nearly 250 million infections and over 600 000 deaths reported annually by the WHO. Fighting malaria is particularly challenging partly due to the complex life cycle of the parasite. However, technological breakthroughs such as the development of the nucleoside-modified mRNA lipid nanoparticle (mRNA-LNP) vaccine platform, along with the discovery of novel conserved Plasmodium antigens such as the E140 protein, present new opportunities in malaria prevention. Importantly, production of recombinant proteins for malaria vaccine evaluation by serological assays often represents an additional hurdle because many Plasmodium proteins are complex and often contain transmembrane domains that make production and purification particularly difficult. This research protocol provides a step-by-step guide for the production and purification of P. berghei and P. vivax E140 protein fragments that can be used to test humoral immune responses against this novel malaria vaccine target. We demonstrate that the purified proteins can be successfully used in enzyme-linked immunosorbent assay (ELISA) to evaluate antigen-specific binding antibody responses in sera obtained from E140 mRNA-LNP-vaccinated mice. Therefore, these proteins can contribute to the development and evaluation of E140-based malaria vaccines.

Targeting T-Cell Activation for Malaria Immunotherapy: Scoping Review.

Malaria remains a critical global health issue due to high mortality rates, drug resistance, and low treatment efficacy. The genetic variability of Plasmodium proteins complicates the development of long-lasting immunity, as it impedes the human immune system's ability to sustain effective responses. T cells play a crucial role in combating malaria, but the parasite's complex life cycle-spanning liver and blood stages-presents significant challenges in effectively activating and targeting these cells. Immunotherapy, which enhances the immune response and promotes durable T cell activity, offers a promising avenue for more effective and lasting malaria treatments. This review systematically analyzed 63 studies published in the last decade, focusing on the role of T cells in malaria. Among the studies, 87.2% targeted T cells as immunotherapy candidates, with CD4+ and CD8+ T cells each accounting for 47.6% of the studies. γδ T cells were the focus in 7.9% of cases, while 12.7% explored non-T cell contributions to enhancing T cell-mediated responses. The findings underscore the potential of T cells, particularly CD8+ T cells, in liver-stage defense and advocate for the exploration of advanced vaccine platforms and novel therapies, such as mRNA-based vectors and monoclonal antibodies.

Structural characterization of the ACDC domain from ApiAP2 proteins, a potential molecular target against apicomplexan parasites.

The apicomplexan AP2 (ApiAP2) proteins are the best characterized family of DNA-binding proteins in Plasmodium spp. malaria parasites. Apart from the AP2 DNA-binding domain, there is little sequence similarity between ApiAP2 proteins. However, a conserved AP2-coincident domain mostly at the C-terminus (ACDC domain) is observed in a subset of the ApiAP2 proteins. The structure and function of this domain remain unknown. We report two crystal structures of ACDC domains derived from distinct Plasmodium ApiAP2 proteins, revealing a conserved, unique, noncanonical, four-helix bundle architecture. We used these structures to perform in silico docking calculations against a library of known antimalarial compounds and identified potential small-molecule ligands that bind in a highly conserved hydrophobic pocket that is present in all apicomplexan ACDC domains. These ligands provide a new molecular basis for the future design of ACDC inhibitors.

APEX-based proximity labeling in Plasmodium identifies a membrane protein with dual functions during mosquito infection.

Transmission of the malaria parasite Plasmodium to mosquitoes necessitates gamete egress from red blood cells to allow zygote formation and ookinete motility to enable penetration of the midgut epithelium. Both processes are dependent on the secretion of proteins from distinct sets of specialized vesicles. Inhibiting some of these proteins has shown potential for blocking parasite transmission to the mosquito. To identify new transmission blocking vaccine candidates, we aimed to define the microneme content from ookinetes of the rodent model organism Plasmodium berghei using APEX2-mediated rapid proximity-dependent biotinylation. Besides known proteins of ookinete micronemes, this identified over 50 novel candidates and sharpened the list of a previous survey based on subcellular fractionation. Functional analysis of a first candidate uncovered a dual role for this membrane protein in male gametogenesis and ookinete midgut traversal. Mutation of a putative trafficking motif in the C-terminus affected ookinete to oocyst transition but not gamete formation. This suggests the existence of distinct functional and transport requirements for Plasmodium proteins in different parasite stages.

Phosphorylation in the Plasmodium falciparum Proteome: A Meta-Analysis of Publicly Available Data Sets.

Malaria is a deadly disease caused by Apicomplexan parasites of the Plasmodium genus. Several species of the Plasmodium genus are known to be infectious to humans, of which P. falciparum is the most virulent. Post-translational modifications (PTMs) of proteins coordinate cell signaling and hence regulate many biological processes in P. falciparum homeostasis and host infection, of which the most highly studied is phosphorylation. Phosphosites on proteins can be identified by tandem mass spectrometry (MS) performed on enriched samples (phosphoproteomics), followed by downstream computational analyses. We have performed a large-scale meta-analysis of 11 publicly available phosphoproteomics data sets to build a comprehensive atlas of phosphosites in the P. falciparum proteome, using robust pipelines aimed at strict control of false identifications. We identified a total of 26,609 phosphorylated sites on P. falciparum proteins, split across three categories of data reliability (gold/silver/bronze). We identified significant sequence motifs, likely indicative of different groups of kinases responsible for different groups of phosphosites. Conservation analysis identified clusters of phosphoproteins that are highly conserved and others that are evolving faster within the Plasmodium genus, and implicated in different pathways. We were also able to identify over 180,000 phosphosites within Plasmodium species beyond falciparum, based on orthologue mapping. We also explored the structural context of phosphosites, identifying a strong enrichment for phosphosites on fast-evolving (low conservation) intrinsically disordered regions (IDRs) of proteins. In other species, IDRs have been shown to have an important role in modulating protein-protein interactions, particularly in signaling, and thus warranting further study for their roles in host-pathogen interactions. All data have been made available via UniProtKB, PRIDE, and PeptideAtlas, with visualization interfaces for exploring phosphosites in the context of other data on Plasmodium proteins.

Comparative genomics of two protozoans Dictyostelium discoideum and Plasmodium falciparum reveals conserved as well as distinct regulatory pathways crucial for exploring novel therapeutic targets for Malaria

Plasmodium falciparum, which causes life-threatening cerebral malaria has rapidly gained resistance against most frontline anti-malarial drugs, thereby generating an urgent need to develop novel therapeutic approaches. Conducting in-depth investigations on Plasmodium in its native form is challenging, thereby necessitating the requirement of an efficient model system. In line, mounting evidence suggests that Dictyostelium discoideum retains both conformational and functional properties of Plasmodium proteins, however, the true potential of Dictyostelium as a host system is not fully explored. In the present study, we have exploited comparative genomics as a tool to extract, compare, and curate the extensive data available on the organism-specific databases to evaluate if D. discoideum can be established as a prime model system for functional characterization of P. falciparum genes. Through comprehensive in silico analysis, we report that despite the presence of adaptation-specific genes, the two display noteworthy conservation in the housekeeping genes, signaling pathway components, transcription regulators, and post-translational modulators. Furthermore, through orthologue analysis, the known, potential, and novel drug target genes of P. falciparum were found to be significantly conserved in D. discoideum. Our findings advocate that D. discoideum can be employed to express and functionally characterize difficult-to-express P. falciparum genes.

A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be capable of killing parasites in their liver and blood stage forms, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis, as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits; both of which are parasite-specific quinoline-4-carboxamides, and analogs of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for the specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing the identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.

Epitope prediction of malarial MSP-1 protein of plasmodium sp. using in silico method

Due to its pivotal function in parasite adherence to the RBC membrane during erythrocyte invasion, Msp1 represents a potential vaccine candidate. Three or four fragments of MSP1 are produced during the proteolytic maturation process, and these fragments stay together as a complex on the cell surface. Msp1 fragments have been investigated as potential vaccine candidate, and malaria antibody immunoassays have made use of the fragmented polypeptide, which exhibits a considerable degree of intra-species conservation. Because of its polymorphism, Msp1 is also useful in strain differentiation and carries a crucial genetic marker. To comprehend genetic variation among species and find epitopes for the development of a disease vaccine, Msp1 needs to be investigated. The preliminary investigation on the evolutionary history using the Maximum Likelihood method and the General Time Reversible model (GTR) models were used. The phylogenetic relationship of the MSP1 gene was inferred and it represents the level of similarity between the MSP1 gene of Plasmodium species from various locations in South-East Asia. The top 30 MHC class II epitopic regions were predicted using Bepipred server with epitope mean score above 0.05.

Extracellular Proteomic Profiling from the Erythrocytes Infected with Plasmodium Falciparum 3D7 Holds Promise for the Detection of Biomarkers.

Plasmodium falciparum (P. falciparum), which causes the most severe form of malaria, if left untreated, has 24h window in which it can cause severe illness and even death. The aim of this study was to create the most comprehensive and informative secretory-proteome possible by combining high-accuracy and high-sensitivity protein identification technology. In this study, we used Plasmodium falciparum 3D7 (Pf3D7) as the model parasite to develop a label-free quantification proteomic strategy with the main goal of identifying Pf3D7 proteins that are supposed to be secreted outside the infected erythrocytes in the spent media culture during the in-vitro study. The spent culture media supernatant was subjected to differential and ultra-centrifugation steps followed by total protein extraction, estimation, and in-solution digestion using trypsin, digested peptides were analyzed using Nano-LC coupled with ESI for MS/MS. MS/MS spectra were processed using Maxquant software (v2.1.4.0.). Non-infected erythrocytes incubated spent cultured media supernatant were considered as control. Out of discovered 38 proteins, proteins belonging to P. falciparum spp. were EGF-like protein (C0H544), Endoplasmic reticulum chaperone GRP170 (C0H5H0), Small GTP-binding protein sar1 (Q8I1S0), Erythrocyte membrane protein 1, PfEMP1 (Q8I639), aldehyde reductase (Q8ID61), Conserved Plasmodium proteins (Q8IEH3, Q8ILD1), Antigen 332, DBL-like protein (Q8IHN4), Fe-S cluster assembly protein (Q8II78), identified and chosen for further in-depth investigation. This study highlights the value of secretory Plasmodium proteins play crucial roles in various aspects of the disease progression and host-pathogen interactions which can serve as diagnostic markers for malaria infection.

In vitro, in vivo and in silico antiplasmodial profiling of the aqueous extract of Hibiscus asper HOOK F. Leaf (Malvaceae)

Ethnopharmacological relevancePlasmodium resistance to antimalarial drugs raises the urgent need to seek for alternative treatments. Aqueous extract of Hibiscus asper leaves is currently used in malaria management but remains less documented.Aim of the study: The study aims to evaluate antimalarial effects of the aqueous extract of Hibiscus asper. UHPLC/MS, was used to identify some likely compounds present in the plant that were thereafter docked to some malaria parasite proteins. Study designIn vitro anti-plasmodium and antioxidant, UHPLC/Ms analysis, in vivo antimalarial of the plant extract, and in silico molecular docking prediction of some identified compounds were performed to investigate the pharmacological effects of H. asper. Material and methodsThe in vitro antiplasmodial activity of the extract was carried out on Plasmodium falciparum strains using SYBR-green dye; then, the curative antimalarial activity was conducted on Plasmodium berghei NK65-infected male Wistar rats. The UHPLC/MS analysis was used to identify plant compounds, followed by interactions (docking affinity) between some compounds and parasitic enzymes such as P. falciparum purine nucleoside phosphorylase (2BSX) and 6-phosphogluconate dehydrogenase (6FQY) to explore potential mechanisms of action at the molecular level. ResultsNo hemolysis effect of the extract was observed at concentrations up to 100 mg/mL. In vitro test of the aqueous leaves extract of H. asper showed inhibitory activity against P. falciparum Dd2 and 3D7 strains with IC50 values of 19.75 and 21.97 μg/mL, respectively. The curative antimalarial test of the H. asper extract in infected rats exhibited significant inhibition of the parasite growth (p < 0.001) with inhibition percentage of 95.11%, 97.68% and 95.59% at all the doses (50, 100 and 200 mg/kg) respectively. The extract corrected major physiological alterations such as liver and kidney impairments, oxidative stress and architectural disorganization in liver, spleen and kidneys tissues. The UHPLC/MS analysis identified 7 compounds, namely chlorogenic acid, azulene, quercetin, rhodine, 1-ethyl-2,4-dimethyl benzene and phthalan. Out of seven compounds identified in the extract quercetin and phthalan showed higher in silico inhibitory activity against P. falciparum purine nucleoside phosphorylase and Plasmodium falciparum 6-phosphosgluconate dehydrogenase parasite enzymes. ConclusionThese findings indicate that H. asper could be a promising complementary medicine to manage malaria. Meanwhile, the affinity of annoted compounds with these enzymes should be further confirmed.

Post-Translational Modifications of Proteins of Malaria Parasites during the Life Cycle.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host.

A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be multistage actives, capable of killing parasites in the liver and blood, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits, both of which are parasite-specific quinoline-4-carboxamides, and analogues of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.

Evaluation of naturally acquired immune responses against novel pre-erythrocytic Plasmodium vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin

BackgroundPlasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity.MethodsTwelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used.ResultsIn those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58–99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9–35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses.ConclusionsThese results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.

Repurposing DrugBank compounds as potential Plasmodium falciparum class 1a aminoacyl tRNA synthetase multi-stage pan-inhibitors with a specific focus on mitomycin

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC50 < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Novel quinolinepiperazinyl-aryltetrazoles targeting the blood stage of Plasmodium falciparum.

The emergence of drug resistance against the frontline antimalarials is a major challenge in the treatment of malaria. In view of emerging reports on drug-resistant strains of Plasmodium against artemisinin combination therapy, a dire need is felt for the discovery of novel compounds acting against novel targets in the parasite. In this study, we identified a novel series of quinolinepiperazinyl-aryltetrazoles (QPTs) targeting the blood stage of Plasmodium. In vitro anti-plasmodial activity screening revealed that most of the compounds showed IC50 < 10 μM against chloroquine-resistant PfINDO strain, with the most promising lead compounds 66 and 75 showing IC50 values of 2.25 and 1.79 μM, respectively. Further, compounds 64-66, 68, 75-77 and 84 were found to be selective (selectivity index >50) in their action against Pf over a mammalian cell line, with compounds 66 and 75 offering the highest selectivity indexes of 178 and 223, respectively. Explorations into the action of lead compounds 66 and 75 revealed their selective cidal activity towards trophozoites and schizonts. In a ring-stage survival assay, 75 showed cidal activity against the early rings of artemisinin-resistant PfCam3.1R539T. Further, 66 and 75 in combination with artemisinin and pyrimethamine showed additive to weak synergistic interactions. Of these two in vitro lead molecules, only 66 restricted rise in the percentage of parasitemia to about 10% in P. berghei-infected mice with a median survival time of 28 days as compared to the untreated control, which showed the percentage of parasitemia >30%, and a median survival of 20 days. Promising antimalarial activity, high selectivity, and additive interaction with artemisinin and pyrimethamine indicate the potential of these compounds to be further optimized chemically as future drug candidates against malaria.

Plasmodium, the Apicomplexa Outlier When It Comes to Protein Synthesis.

Plasmodium is an obligate intracellular parasite that has numerous interactions with different hosts during its elaborate life cycle. This is also the case for the other parasites belonging to the same phylum Apicomplexa. In this study, we bioinformatically identified the components of the multi-synthetase complexes (MSCs) of several Apicomplexa parasites and modelled their assembly using AlphaFold2. It appears that none of these MSCs resemble the two MSCs that we have identified and characterized in Plasmodium. Indeed, tRip, the central protein involved in the association of the two Plasmodium MSCs is different from its homologues, suggesting also that the tRip-dependent import of exogenous tRNAs is not conserved in other apicomplexan parasites. Based on this observation, we searched for obvious differences that could explain the singularity of Plasmodium protein synthesis by comparing tRNA genes and amino acid usage in the different genomes. We noted a contradiction between the large number of asparagine residues used in Plasmodium proteomes and the single gene encoding the tRNA that inserts them into proteins. This observation remains true for all the Plasmodia strains studied, even those that do not contain long asparagine homorepeats.

Flavonoids from aerial parts of Cissampelos pareira L. as antimalarial agents: Computational validation of ethnopharmacological relevance

The diverse chemical scaffolds of the Cissampelos pareira make it pharmacologically distinct because of its pleiotropic therapeutical applications. Phytochemically, C. pareira is primarily known for its isoquinoline alkaloids, but flavonoids and their glycosides have also been reported from the aerial part. Among the several traditional uses of C. pareira, the juice of leaves that are enriched with flavonoids has been used to cure malarial fever. In silico molecular docking of phytomolecules from C. pareira revealed that flavonoids exhibited good binding affinity with the selected antimalarial targets. Among the screened molecules, quercetin, quercetin-3-O-sophoroside, and kaempferol-3-O-β-D-glucuronopyranoside showed the most prominent binding with the selected Plasmodium proteins. However, quercetin-3-O-sophoroside (Q3S) showed a broad-spectrum binding affinity with falcipain-2 (-9.53 Kcal/mol), plasmepsin-2 (-12.08 Kcal/mol), Pf1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) (-14.35 Kcal/mol), Pf lactose dehydrogenase (PfLDH) (-9.57 Kcal/mol), and Pf dihydrofolate reductase (PfDHFR) (-11.62 Kcal/mol). The pharmacokinetics evaluation showed that identified hits follow several ADME-Tox properties. MD Simulation suggests that Q3S forms a highly stable complex with PfLDH. The RMSF value range of docked complexes of Q3S with Plasmepsin-2, PfDHFR, PfDXR, and Falcipain-2 is 0.1 to 0.5, 0.8, 0.7, and 0.6 nm, respectively. These findings suggest that C. pareira and its flavonoids hold potential as broad-spectrum antimalarial agents, which could pave the way for further investigation and development of novel therapeutics to combat this deadly disease.

Identification of Novel, Potent, and Selective Compounds against Malaria Using Glideosomal-Associated Protein 50 as a Drug Target.

Phylum apicomplexan consists of parasites, such as Plasmodium and Toxoplasma. These obligate intracellular parasites enter host cells via an energy-dependent process using specialized machinery, called the glideosome. In the present study, we used Plasmodium falciparum GAP50, a glideosome-associated protein, as a target to screen 951 different compounds from diverse chemical libraries. Using different screening methods, eight compounds (Hayatinine, Curine, MMV689758 (Bedaquiline), MMV1634402 (Brilacidin), and MMV688271, MMV782353, MMV642550, and USINB4-124-8) were identified, which showed promising binding affinity (KD < 75 μM), along with submicromolar range antiparasitic efficacy and selectivity index > 100 fold for malaria parasite. These eight compounds were effective against Chloroquine-resistant PfINDO and Artemisinin-resistant PfCam3.1R359T strains. Studies on the effect of these compounds at asexual blood stages showed that these eight compounds act differently at different developmental stages, indicating the binding of these compounds to other Plasmodium proteins, in addition to PfGAP50. We further studied the effects of compounds (Bedaquiline and USINB4-124-8) in an in vivoPlasmodium berghei mouse model of malaria. Importantly, the oral delivery of Bedaquiline (50 mg/kg b. wt.) showed substantial suppression of parasitemia, and three out of seven mice were cured of the infection. Thus, our study provides new scaffolds for the development of antimalarials that can act at multiple Plasmodium lifecycle stages.