Falciparum Protein Research Articles

Proteome-Wide Identification of RNA-dependent proteins and an emerging role for RNAs in Plasmodium falciparum protein complexes

Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.

Computational Molecular Docking Studies for Antimalarial Drug Discovery Against Plasmodium falciparum

Malaria, a deadly disease caused by the Plasmodium falciparum parasite, poses a global health crisis with limited treatment options due to drug resistance. In the quest for new antimalarial drugs, computational molecular docking has emerged as a pivotal approach. This study delves into the application of computational docking techniques to identify potential drug candidates targeting critical proteins within the parasite. Leveraging genetic and structural data, we scrutinize key Plasmodium falciparum proteins involved in essential biological processes. The evaluation of various computational methodologies, including molecular dynamics simulations and scoring functions, aids in the identification of promising compounds. Additionally, we highlight recent advances in machine learning and artificial intelligence for more efficient virtual screening. The results of these studies provide a promising pool of candidate compounds, accelerating the development of novel antimalarial drugs to combat this persistent global health threat. Keywords: Computational Molecular Docking, Plasmodium falciparum, Molecular Dynamics, Protein- Ligand Interactions, Protein Structure.

Potentials of Terpenoids as Inhibitors of Multiple Plasmodium falciparum Protein Drug Targets.

The resistance of parasite to readily affordable antimalarial drugs, the high cost of currently potent drugs, and the resistance of vector mosquitoes to insecticides threaten the possibility of malaria eradication in malaria endemic areas. Due to the fact that quinine and artemisinin were isolated from plants sources, researchers have been encouraged to search for new antimalarials from medicinal plants. This is especially the case in Africa where a large percentage of the population depends on medicinal plant to treat malaria and other ailments. In this study, we evaluated previously characterized Plasmodium-cidal compounds obtained from the African flora to identify their likely biochemical targets, for an insight into their possible antimalarial chemotherapy. Molecular docking study was first conducted, after which remarkable compounds were submitted for molecular dynamic (MD) simulations studies. From a total of 38 Plasmodium-cidal compounds docked with confirmed Plasmodium falciparum protein drug targets [plasmepsin II (PMII), histo-aspartic protein (HAP) and falcipain-2 (FP)], two pentacyclic triterpene, cucurbitacin B and 3 beta-O-acetyl oleanolic acid showed high binding affinity relative to artesunate. This implies their capacity to inhibit the three selected P. falciparum target proteins, and consequently, antimalarial potential. From the MD simulations studies and binding free energy outcomes, results confirmed that the two compounds are stable in complex with the selected antimalarial targets; they also showed excellent binding affinities during the 100 ns simulation. These results showed that cucurbitacin B and 3 beta-O-acetyl oleanolic acid are potent antimalarials and should be considered for further studies.

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.

Antimalarial Activities of a Therapeutic Combination of Azadirachta indica, Mangifera indica and Morinda lucida Leaves: A Molecular View of its Activity on Plasmodium falciparum Proteins.

The search for new antimalarial drugs remains elusive prompting research into antimalarial combinations from medicinal plants due to their cheapness, efficacy and availability. Azadirachta indica (AI), Morinda lucida (ML) and Mangifera indica (MI) have all been reported as potent antimalarial plants. This study evaluated the efficacy of an antimalarial combination therapeutics prepared from leaves of AI, ML and MI using in vitro, in vivo and molecular methods. Refined extracts of the plants combination was made by partitioning the aqueous extract of plants combinations (AI + MI, AI + ML, MI + ML, AI + MI + ML) using methanol and ethyl acetate consecutively. The resulting ethyl acetate partitioned fraction was evaluated for its antimalarial activity. Molecular docking and molecular dynamics simulation were employed to determine the possible mechanism of action of the constituent of the most active combination against four important P. falciparum proteins. The result revealed that the refined extract from combinations AI + ML and MI + ML at 16mg/kg bodyweight have the highest chemo-suppressive effect of 90.7% and 91.0% respectively compared to chloroquine's 100% at 10mg/kg. Also, refined extract from MI + ML combination improved PCV levels significantly (p < 0.05) compared to controls. Molecular docking revealed oleanolic acid and ursolic acid as multiple inhibitors of plasmepsin II, hiso-aspartic protease, falcipain-2 and P. falciparum Eonyl acyl-carrier protein reductase with relative stability during 100ns of simulation. The study unveiled the potentials of ML and MI as good candidates for antimalarial combination therapy and further established their use together as revealed in folklore medicine.

PlasmoFAB: a benchmark to foster machine learning for Plasmodium falciparum protein antigen candidate prediction.

Machine learning methods can be used to support scientific discovery in healthcare-related research fields. However, these methods can only be reliably used if they can be trained on high-quality and curated datasets. Currently, no such dataset for the exploration of Plasmodium falciparum protein antigen candidates exists. The parasite P.falciparum causes the infectious disease malaria. Thus, identifying potential antigens is of utmost importance for the development of antimalarial drugs and vaccines. Since exploring antigen candidates experimentally is an expensive and time-consuming process, applying machine learning methods to support this process has the potential to accelerate the development of drugs and vaccines, which are needed for fighting and controlling malaria. We developed PlasmoFAB, a curated benchmark that can be used to train machine learning methods for the exploration of P.falciparum protein antigen candidates. We combined an extensive literature search with domain expertise to create high-quality labels for P.falciparum specific proteins that distinguish between antigen candidates and intracellular proteins. Additionally, we used our benchmark to compare different well-known prediction models and available protein localization prediction services on the task of identifying protein antigen candidates. We show that available general-purpose services are unable to provide sufficient performance on identifying protein antigen candidates and are outperformed by our models that were trained on this tailored data. PlasmoFAB is publicly available on Zenodo with DOI 10.5281/zenodo.7433087. Furthermore, all scripts that were used in the creation of PlasmoFAB and the training and evaluation of machine learning models are open source and publicly available on GitHub here: https://github.com/msmdev/PlasmoFAB.

Expression and copper binding studies of a Plasmodium falciparum protein with Cox19 copper binding motifs

Copper can exist in an oxidized and a reduced form, which enables the metal to play essential roles as a catalytic co-factor in redox reactions in many organisms. Copper confers redox activity to the terminal electron transport chain cytochrome c oxidase protein. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11 in association with Cox19. When copper is chelated in growth medium, Plasmodium falciparum parasite development in infected red blood cells is inhibited and excess copper is toxic for the parasite. The gene of a 26 kDa Plasmodium falciparum PfCox19 protein with two Cx9C Cox19 copper binding motifs, was cloned and expressed as a 66 kDa fusion protein with maltose binding protein and affinity purified (rMBP-PfCox19). rMBP-PfCox19 bound copper measured by: a bicinchoninic acid release assay; an in vivo bacterial host growth inhibition assay; ascorbate oxidation inhibition and differential scanning fluorimetry. The native protein was detected by antibodies raised against rMBP-PfCox19. PfCox19 binds copper and is predicted to associate with PfCox11 in the insertion of copper into the CuB site of Plasmodium cytochrome c oxidase. Characterisation of the proteins involved in Plasmodium spp. copper metabolism will help us understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

Proteome-wide comparison of tertiary protein structures reveals molecular mimicry in Plasmodium-human interactions

IntroductionMolecular mimicry is a strategy used by parasites to evade the host’s immune system and facilitate transmission to a new host. To date, high-throughput examples of molecular mimicry have been limited to comparing protein sequences. However, recent advances in the prediction of tertiary structural models, led by Deepmind’s AlphaFold, enable the comparison of thousands of proteins from parasites and their hosts at the structural level, allowing for the identification of more mimics. Here, we present the first proteome-level search for tertiary structure similarity between proteins from Plasmodium falciparum, a malaria-causing parasite, and humans.MethodsWe assembled a database of experimentally-characterized protein tertiary structures (from the Protein Data Bank) and AlphaFold-generated protein tertiary structures from P. falciparum, human, and 15 negative control species, i.e., species not infected by P. falciparum. We aligned human and control structures to the parasite structures using Foldseek. ResultsWe identified molecular mimicry in three proteins that have been previously proposed as mediators of Plasmodium-human interactions. By extending this approach to all P. falciparum proteins, we identified an additional 41 potential mimics that are supported by additional experimental data. DiscussionOur findings demonstrate a valuable application of AlphaFold-derived tertiary structural models, and we discuss key considerations for its effective use in other host-parasite systems.

A mixed methods study assessing the adoption potential of a saliva-based malaria rapid test in the Democratic Republic of Congo

BackgroundThe reliance on blood for thin and thick blood smear microscopy—using a relatively invasive procedure has presented challenges to the use of reliable diagnostic tests in non-clinical settings at the point-of-need (PON). To improve the capacity of non-blood-based rapid diagnostic tests to confirm subclinical infections, and thereby identify and quantify the human reservoir at the PON, a cross-sectoral collaboration between university researchers and commercial partners produced an innovative, non-invasive saliva-based RDT capable of identifying novel, non-hrp2/3 parasite biomarkers. While this new saliva-based malaria asymptomatic and asexual rapid test (SMAART-1) shows increased detection sensitivity and precision potential by identifying a new P. falciparum protein marker (PSSP17), appraising its utility in the field—particularly with respect to its adoption potential with children and adults in high risk, endemic regions—is necessary to warrant its continued development.MethodsThe purpose of this study was to assess the acceptability and adoption potential of the SMAART-1 at select PON sites in the Kinshasa Province. Teachers, community health workers, nurses, and laboratory technicians participated in data collection at three distinct community sites in Kinshasa Province, Democratic Republic of the Congo. Three data collection methods were utilized in this mixed methods study to provide an overarching acceptability evaluation of the SMAART-1 at PON field sites: observation checklists of SMAART-1 implementation, focus group discussions, and surveys with local health care practitioners—particularly teachers and community health workers.ResultsFindings indicate participants were interested in and supportive of the SMAART-1 protocol, with approximately 99% of the participants surveyed indicating that they either “agreed” or “strongly agreed” with the statement that they “would use the saliva-based malaria asymptomatic rapid test as part of a community malaria detection and treatment programme.” Data also suggest that the protocol was broadly appealing for its testing sensitivity and ease of use.ConclusionsThe SMAART-1 protocol’s clinically reliable results demonstrate a promising new level of sensitivity and precision for detecting parasite biomarkers. This study’s mixed-methods assessment of the protocol’s utility and adoption potential in the field, with a target user audience, advances its development and points to opportunities to formalize and expand evaluation efforts.

Screening Carica Papaya Compounds as an Antimalarial Agent: In Silico Study

Malaria is a highly prevalent infectious disease caused by the Plasmodium parasite transmitted through Anopheles mosquitoes, which poses a significant public health challenge worldwide, including in Indonesia. Therefore, a study was conducted to identify potential drug compounds from the Carica papaya plant that could inhibit various antimalarial proteins or receptors, such as Plasmodium falciparum DXR reductase complex with fosmidomycin, Plasmepsin V Plasmodium vivax, P. falciparum dihydroorotate dehydrogenase, P. falciparum hexose transporter, P. falciparum protein kinase 5, and P. falciparum dihydrofolate reductase-thymidylate synthase. Theresearchers used the Pyrx application to dock the C. papaya compounds with the targeted antimalarial proteins to determine the binding affinity values. Additionally, they used the Yasara dynamics application to conduct molecular dynamics simulation to ensure the stability of the bonds formed between the ligands and proteins. The results showed that 14 compounds found in C. papaya, particularly flavonoids and terpenoids, had the potential to inhibit the six antimalarial proteins with the lowest binding affinity values. Furthermore, the molecular dynamics simulation on 6M20 and 1V0P proteins indicated that the compounds effectively inhibited Plasmodium proteins, as they had an RMSD value below 2.5 Angstrom. The study suggests that C. papaya could be a potential source of antimalarial compounds, which could be developed into new drugs to combat this disease.&nbsp

Epitope specific antibodies to N- and C cytoplasmic domains of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) differentiate native and post-translationally modified variant

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined.

Plasmodium falciparum has evolved multiple mechanisms to hijack human immunoglobulin M

Plasmodium falciparum causes the most severe malaria in humans. Immunoglobulin M (IgM) serves as the first line of humoral defense against infection and potently activates the complement pathway to facilitate P. falciparum clearance. A number of P. falciparum proteins bind IgM, leading to immune evasion and severe disease. However, the underlying molecular mechanisms remain unknown. Here, using high-resolution cryo-electron microscopy, we delineate how P. falciparum proteins VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2 target IgM. Each protein binds IgM in a different manner, and together they present a variety of Duffy-binding-like domain-IgM interaction modes. We further show that these proteins interfere directly with IgM-mediated complement activation in vitro, with VAR2CSA exhibiting the most potent inhibitory effect. These results underscore the importance of IgM for human adaptation of P. falciparum and provide critical insights into its immune evasion mechanism.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Naphthyl bearing 1,3,4-thiadiazoleacetamides targeting the parasitic folate pathway as anti-infectious agents: in silico, synthesis, and biological approach.

Malaria is still a complex and lethal parasitic infectious disease, despite the availability of effective antimalarial drugs. Resistance of malaria parasites to current treatments necessitates new antimalarials targeting P. falciparum proteins. The present study reported the design and synthesis of a series of a 2-(4-substituted piperazin-1-yl)-N-(5-((naphthalen-2-yloxy)methyl)-1,3,4-thiadiazol-2-yl)acetamide hybrids for the inhibition of Plasmodium falciparum dihydrofolate reductase (PfDHFR) using computational biology tools followed by chemical synthesis, structural characterization, and functional analysis. The synthesized compounds were evaluated for their in vitro antimalarial activity against CQ-sensitive PfNF54 and CQ-resistant PfW2 strain. Compounds T5 and T6 are the most active compounds having anti-plasmodial activity against PfNF54 with IC50 values of 0.94 and 3.46 μM respectively. Compound T8 is the most active against the PfW2 strain having an IC50 of 3.91 μM. Further, these active hybrids (T5, T6, and T8) were also evaluated for enzyme inhibition assay against PfDHFR. All the tested compounds were non-toxic against the Hek293 cell line with good selectivity indices. Hemolysis assay also showed non-toxicity of these compounds on normal uninfected human RBCs. In silico molecular docking studies were carried out in the binding pocket of both the wild-type and quadruple mutant Pf-DHFR-TS to gain further insights into probable modes of action of active compounds. ADME prediction and physiochemical properties support their drug-likeness. Additionally, they were screened for antileishmanial activity against L. donovani promastigotes to explore broader applications. Thus, this study provides molecular frameworks for developing potent antimalarials and antileishmanial agents.

Evaluation of Antimalarial Activity of Ethanolic Extract of Annona muricata L.: An in vivo and an in silico Approach.

In Nigeria, Annona muricata L. has been used to treat a variety of ailments. The mechanism of the antimalarial activity of ethanolic leaf extract of Annona muricata (EEAML) was investigated using both an in vivo and an in silico approach. The experimental mice were divided into five groups: A-F. The mice in groups B-F were inoculated with Plasmodium berghei NK-65 and treated accordingly. Groups A and B are the negative and positive controls (infected and untreated), respectively. Group C received 10 mg/kg chloroquine (standard drug), whereas groups D-F received 100, 200, and 300 mg/kg body weight of the extract orally respectively. The mice were euthanized eight days after infection, and their liver and blood were collected and used in biochemical tests. Molecular docking was performed using the extract's HPLC compounds and Plasmodium falciparum proteins. In the suppressive, prophylactic, and curative tests, there was a significant decrease (p < 0.05) in parasitemia levels in groups treated with the extract compared to the positive control and standard drug. When compared to the positive control, there was a significant (p < 0.05) reduction in liver MDA, total cholesterol, and total triglyceride levels. The binding energies of luteolin and apigenin-pfprotein complexes were significantly (p < 0.05) higher compared to their respective references. The anti-plasmodial activity of the extract may result from its hypolipidemic effect, which deprives the parasite of essential lipid molecules needed for parasite growth, as well as from the inhibitory effects of apigenin and luteolin on specific proteins required for the Plasmodium metabolic pathway.

Persistence of Plasmodium falciparum HRP-2 antigenaemia after artemisinin combination therapy is not associated with gametocytes

BackgroundIn some settings, sensitive field diagnostic tools may be needed to achieve elimination of falciparum malaria. To this end, rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum protein HRP-2 are being developed with increasingly lower limits of detection. However, it is currently unclear how parasite stages that are unaffected by standard drug treatments may contribute to HRP-2 detectability and potentially confound RDT results even after clearance of blood stage infection. This study assessed the detectability of HRP-2 in periods of post-treatment residual gametocytaemia.MethodsA cohort of 100 P.falciparum infected, gametocyte positive individuals were treated with or without the gametocytocidal drug primaquine (PQ), alongside standard artemisinin-based combination therapy (ACT), in the context of a randomised clinical trial in Ouelessebougou, Mali. A quantitative ELISA was used to measure levels of HRP-2, and compared time to test negativity using a standard and ultra-sensitive RDT (uRDT) between residual gametocyte positive and negative groups.ResultsTime to test negativity was longest by uRDT, followed by ELISA and then standard RDT. No significant difference in time to negativity was found between the treatment groups with and without residual gametocytes: uRDT (HR 0.79 [95% CI 0.52–1.21], p = 0.28), RDT (HR 0.77 [95% CI 0.51–1.15], p = 0.20) or ELISA (HR 0.88 [95% CI 0.59–1.32], p = 0.53). Similarly, no difference was observed when adjusting for baseline asexual parasite density. Quantified levels of HRP-2 over time were similar between groups, with differences attributable to asexual parasite densities. Furthermore, no difference in levels of HRP-2 was found between individuals who were or were not infectious to mosquitoes (OR 1.19 [95% CI 0.98–1.46], p = 0.077).ConclusionsSurviving sexual stage parasites after standard ACT treatment do not contribute to the persistence of HRP-2 antigenaemia, and appear to have little impact on RDT results.

Disulfide bond and crosslinking analyses reveal inter-domain interactions that contribute to the rigidity of placental malaria VAR2CSA structure and formation of CSA binding channel

VAR2CSA, a multidomain Plasmodium falciparum protein, mediates the adherence of parasite-infected red blood cells to chondroitin 4-sulfate (C4S) in the placenta, contributing to placental malaria. Therefore, detailed understanding of VAR2CSA structure likely help developing strategies to treat placental malaria. The VAR2CSA ectodomain consists of an N-terminal segment (NTS), six Duffy binding-like (DBL) domains, and three interdomains (IDs) present in sequence NTS-DBL1x-ID1-DBL2x-ID2-DBL3x-DBL4ε-ID3-DBL5ε-DBL6ε. Recent electron microscopy studies showed that VAR2CSA is compactly organized into a globular structure containing C4S-binding channel, and that DBL5ε-DBL6ε arm is attached to the NTS-ID3 core structure. However, the structural elements involved in inter-domain interactions that stabilize the VAR2CSA structure remain largely not understood. Here, limited proteolysis and peptide mapping by mass spectrometry showed that VAR2CSA contains several inter-domain disulfide bonds that stabilize its compact structure. Chemical crosslinking-mass spectrometry showed that all IDs interact with DBL4ε; additionally, IDs interact with other DBL domains, demonstrating that IDs are the key structural scaffolds that shape the functional NTS-ID3 core. Ligand binding analysis suggested that NTS considerably restricts the C4S binding. Overall, our study revealed that inter-domain disulfide bonds and interactions between IDs and DBL domains contribute to the stability of VAR2CSA structural architecture and formation of C4S-binding channel.

Transcriptomic complexity of the human malaria parasite Plasmodium falciparum revealed by long-read sequencing.

The Plasmodium falciparum human malaria parasite genome is incompletely annotated and does not accurately represent the transcriptomic diversity of this species. To address this need, we performed long-read transcriptomic sequencing. 5' capped mRNA was enriched from samples of total and nuclear-fractionated RNA from intra-erythrocytic stages and converted to cDNA library. The cDNA libraries were sequenced on PacBio and Nanopore long-read platforms. 12,495 novel isoforms were annotated from the data. Alternative 5' and 3' ends represent the majority of isoform events among the novel isoforms, with retained introns being the next most common event. The majority of alternative 5' ends correspond to genomic regions with features similar to those of the reference transcript 5' ends. However, a minority of alternative 5' ends showed markedly different features, including locations within protein-coding regions. Alternative 3' ends showed similar features to the reference transcript 3' ends, notably adenine-rich termination signals. Distinguishing features of retained introns could not be observed, except for a tendency towards shorter length and greater GC content compared with spliced introns. Expression of antisense and retained intron isoforms was detected at different intra-erythrocytic stages, suggesting developmental regulation of these isoform events. To gain insights into the possible functions of the novel isoforms, their protein-coding potential was assessed. Variants of P. falciparum proteins and novel proteins encoded by alternative open reading frames suggest that P. falciparum has a greater proteomic repertoire than the current annotation. We provide a catalog of annotated transcripts and encoded alternative proteins to support further studies on gene and protein regulation of this pathogen.

A non-traditional crystal-based compound screening method targeting the ATP binding site of Plasmodium falciparum GRP78 for identification of novel nucleoside analogues.

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight ‘hit’ compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti-Plasmodium specific compounds.

IN SILICO AND IN VITRO STUDIES OF ACETAZOLAMIDE AS A GAMETOCIDAL AGENT

&#x0D; &#x0D; &#x0D; &#x0D; .&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; Malaria remains the leading cause of mortality around the world and is the most common disease in Africa and some countries in Asia with the highest number of indigenous cases with Plasmodium falciparum responsible for the majority of the disease burden. Plasmodium has a complex life cycle consisting of multiple stages in two hosts. During the erythrocytic life cycle, it follows one of two developmental fates: terminal sexual differentiation into gametocytes or asexual propagation. Transmission from an infected human host to a susceptible mosquito is done through highly specialized sexual stages gametocytes because they are the only form of malaria parasite that are transmissible to mosquito vector, hence the discovery of new antimalarials with transmission blocking activity remains a key issue in efforts to control malaria and eventually eradicate the disease. In this study the in silico and in vitro gametocidal properties of one of the Sulfonamide drugs Acetazolamide had been checked in order to find new antiplasmodial agent. In vitro cultivation of the RKL-9 (Chloroquine resistant) strain of P. falciparum was continued to develop sexual gametocytic early stages. The drug acetazolamide was screened for gametocidal assay in order to check its transmission blocking activity by targeting early sexual stages of the parasites. Three Plasmodium falciparum proteins Pfg27, Pfs230 and PUF1were selected for molecular docking with the acetazolamide as ligand. HEX 8.0.0 server was used as docking tool and the results obtained were analysed in Discovery studio visualizer. Our findings of in vitro studies revealed that the selected drug acetazolamide possess promising gametocidal activity with IC50 = 4.812µg/ml. Hence the study can be used further for drug formulations as antimalarials targeting Gametocytic stages. Based on the in silico analysis the good binding scores -196.76, -173.87 and -193.29 (Kcal/mol) were obtained for all three selected Plasmodium falciparum proteins Pfg27, Pfs230 and PUF1 with the ligand acetazolamide respectively. Thus the docking energy results of in silico studies also supported our in vitro results that the acetazolamide can be used as an antiplasmodial drug against the gametocytes. Further studies for the mechanism of action of the acetazolamide as a gametocidal agent can be done for preventing the transmission of malaria.