Structure-based virtual screening against multiple Plasmodium falciparum kinases reveals antimalarial compounds.

The continuous emergence of resistance against most frontline antimalarial drugs has led to countless deaths in malaria-endemic countries, counting 619,000 deaths in 2021, with mutation in drug targets being the sole cause. As mutation is correlated frequently with fitness cost, the likelihood of mutation emergence in multiple targets at a time is extremely low. Hence, multitargeting compounds may seem promising to address drug resistance issues with additional benefits like increased efficacy, improved safety profile, and the requirement of fewer pills compared to traditional single and combinational drugs. In this study, we attempted to use the High Throughput Virtual Screening approach to predict multitarget inhibitors against six chemically validated Plasmodium falciparum (Pf) kinases (PfPKG, PfMAP2, PfCDPK4, PfTMK, PfPK5, PfPI4K), resulting in 21 multitargeting hits. The molecular dynamic simulation of the top six complexes (Myricetin-MAP2, Quercetin-CDPK4, Myricetin-TMK, Quercetin-PKG, Salidroside-PK5, and Salidroside-PI4K) showed stable interactions. Moreover, hierarchical clustering reveals the structural divergence of the compounds from the existing antimalarials, indicating less chance of cross-resistance. Additionally, the top three hits were validated through parasite growth inhibition assays, with quercetin and myricetin exhibiting an IC50 value of 1.84 and 3.93 µM, respectively.

Azo-Stilbene and Pyridine-Amine Hybrid Multifunctional Molecules to Target Metal-Mediated Neurotoxicity and Amyloid-ß Aggregation in Alzheimer's Disease.

Neurodegenerative diseases such as Alzheimer's disease (AD) are associated with progressive neuronal cell death, and they are commonly correlated with aberrant protein misfolding and aggregation of Aß peptides. Transition metal ions (Cu, Fe, and Zn) have been shown to promote aggregation and oxidative stress through formation of Aß-metal complexes. In this context, integrating molecular scaffolds rationally is used here to generate multifunctional molecules as modulators for metal-induced abnormalities. This work encompasses two azo-stilbene (AS)-derived compounds (AS-HL1 and AS-HL2), the rationale behind the design, their synthesis, characterization, and metal chelation ability [Cu(II) and Zn(II)]. The molecular frameworks of the designed compounds consist of stilbene as an Aß-interacting moiety, whereas N,N,O and N,N,N,O donor atoms are linked to generate the metal chelation moiety. Furthermore, we went on exploring their multifunctionality with respect to (w.r.t.) (i) their metal chelating capacities and (ii) their utility to modulate the aggregation pathways of both metal-free and metal-bound amyloid-ß, (iii) scavenge free radicals, and (iv) inhibit the activity of acetylcholinesterase and (v) cytotoxicity. Moreover, the compounds were able to sequester Cu2+ from the Aß-Cu complex as studied by the UV-visible spectroscopic assay. Molecular docking studies were also performed with Aß and acetylcholinesterase enzyme. Overall, the studies presented here qualify these molecules as promising candidates for further investigation in the quest for finding a treatment for Alzheimer's disease.

Synthesis and inhibition studies towards the discovery of benzodiazepines as potential antimalarial compounds.

The target-based discovery of therapeutics against apicoplast, an all-important organelle is an overriding perspective. MEP pathway, an accredited drug target provides an insight into the importance of apicoplast in the survival of the parasite. In this study, we present the rational design strategy employing sustainable catalysis for the synthesis of benzodiazepine (BDZ) conformers followed by their biological evaluation as prospective inhibitors against the potential target of the IPP pathway, 1-deoxy-D-xylulose-5-phosphatereductoisomerase (DXR). The study reported the inhibitory profile of 8c and 6d against the quintessential step of the only drug target in the erythrocytic stages of parasite development. The potential compounds were identified to represent a novel class of inhibitors that serve as the lead molecules to impede the pathway and further affect the survival of the parasite.

Cancer/testis antigens as molecular drug targets using network pharmacology.

Present chemotherapeutic drugs have limited efficacy and severe side effects. Considering the complexity of cancer, an effective strategy is necessary to discover multiple new drug targets. Cancer/testis antigens are vital for cancer cell progression. We have performed a computational network analysis of cancer/testis antigens and assessed these antigens as drug targets. During this analysis, protein interaction network of 700 human CT antigens was investigated. CT antigen network consisted of eight independent components. Four major hubs and two minor hubs were identified that play nodal role in the flow of information across the largest network. We have predicted 30 potential drug targets by analysing several topological parameters such as betweenness centrality, cluster coefficient and probable protein complexes. Structural and functional roles of potential drug targets have also been anatomized. Analysis of the CT antigen network enables us to pinpoint a set of candidate proteins that if targeted could be detrimental for cancerous cell without affecting any normal cell. The list of putative proteins is a starting point for experimental validation and may help further in the discovery of new anticancer drug targets.

Identification of key genes involved in root development of tomato using expressed sequence tag analysis.

Root system of plants are actually fascinating structures, not only critical for plant development, but also important for storage and conduction. Due to its agronomic importance, identification of genes involved in root development has been a subject of intense study. Tomato is the one of the most consumed vegetables in the world. Tomato has been used as model system for dicot plants because of its small genome, well-established transformation techniques and well-constructed physical map. The present study is targeted to identify of root specific genes expressed temporally and also gene(s) involved in lateral root and profuse root development. A total of 890 ESTs were identified from five EST libraries constructed using SSH approach which included temporal gene regulation (early and late) and genes involved in morphogenetic traits (lateral and profuse rooting). One hundred sixty-one unique ESTs identified from various libraries were categorized based on their putative functions and deposited in NCBI-dbEST database. In addition, 36 ESTs were selected for validation of their expression by RT-PCR. The present findings will help in shedding light to the unexplored developmental process of root growth in tomato and plant in general.

Exploring malaria parasite surface proteins to devise highly immunogenic multi-epitope subunit vaccine for Plasmodium falciparum.

BACKGROUND: Malaria has remained a major health concern for decades among people living in tropical and sub-tropical countries. Plasmodium falciparum is one of the critical species that cause severe malaria and is responsible for major mortality. Moreover, the parasite has generated resistance against all WHO recommended drugs and therapies. Therefore, there is an urgent need for preventive measures in the form of reliable vaccines to achieve the target of a malaria-free world. Surface proteins are the preferable choice for subunit vaccine development because they are rapidly detected and engaged by host immune cells and vaccination-induced antibodies. Additionally, abundant surface or membrane proteins may contribute to the opsonization of pathogens by vaccine-induced antibodies. RESULTS: In our study, we have listed all those surface proteins from the literature that could be functionally important and essential for infection and immune evasion of the malaria parasite. Eight Plasmodium surface and membrane proteins from the pre-erythrocyte and erythrocyte stages were shortlisted. Thirty-seven epitopes (B-cell, CTL, and HTL epitopes) from these proteins were predicted using immune-informatic tools and joined with suitable peptide linkers to design a vaccine construct. A TLR-4 agonist peptide adjuvant was added at the N-terminus of the multi-epitope series, followed by the PADRE sequence and EAAAK linker. The TLR-4 receptor was docked with the construct's anticipated model structure. The complex of vaccine and TLR-4, with the lowest energy -1514, was found to be stable under simulated physiological settings. CONCLUSION: This study has provided a novel multi-epitope construct that may be exploited further for the development of an efficient vaccine for malaria.

Malaria therapeutics: are we close enough?

Malaria is a vector-borne parasitic disease caused by the apicomplexan protozoan parasite Plasmodium. Malaria is a significant health problem and the leading cause of socioeconomic losses in developing countries. WHO approved several antimalarials in the last 2 decades, but the growing resistance against the available drugs has worsened the scenario. Drug resistance and diversity among Plasmodium strains hinder the path of eradicating malaria leading to the use of new technologies and strategies to develop effective vaccines and drugs. A timely and accurate diagnosis is crucial for any disease, including malaria. The available diagnostic methods for malaria include microscopy, RDT, PCR, and non-invasive diagnosis. Recently, there have been several developments in detecting malaria, with improvements leading to achieving an accurate, quick, cost-effective, and non-invasive diagnostic tool for malaria. Several vaccine candidates with new methods and antigens are under investigation and moving forward to be considered for clinical trials. This article concisely reviews basic malaria biology, the parasite's life cycle, approved drugs, vaccine candidates, and available diagnostic approaches. It emphasizes new avenues of therapeutics for malaria.

Drug repurposing based novel anti-leishmanial drug screening using in-silico and in-vitro approaches.

Visceral leishmaniasis is a neglected tropical disease and is mainly caused by L. donovani in the Indian subcontinent. The mitochondria genome replication in Leishmania spp. is having a very specific mechanism, and it is initiated by a key enzyme called mitochondrial primase. This enzyme is essential for the onset of the replication process and growth of the parasite. Therefore, we focused on the primase protein as a potential therapeutic target for combating leishmaniasis diseases. We started our studies molecular modeling and followed by docking of the FDA-approved drug library into the binding site of the primase protein. The top 30 selected compounds were subjected for molecular dynamics studies. Also, the target protein was cloned, purified, and tested experimentally (primase activity assays and inhibition assays). Some compounds were very effective against the Leishmania cell culture. All these approaches helped us to identify few possible novel anti-leishmanial drugs such as Pioglitazone and Mupirocin. These drugs are effectively involved in inhibiting the promastigote of L. donovani, and it can be utilized in the next level of clinical trials. Communicated by Ramaswamy H. Sarma.

Ligand-bound structures provide atomic snapshots for the catalytic mechanism of D-amino acid deacylase.

D-tyrosyl-tRNA(Tyr) deacylase (DTD) is an editing enzyme that removes D-amino acids from mischarged tRNAs. We describe an in-depth analysis of the malaria parasite Plasmodium falciparum DTD here. Our data provide structural insights into DTD complexes with adenosine and D-amino acids. Bound adenosine is proximal to the DTD catalysis site, and it represents the authentic terminal adenosine of charged tRNA. DTD-bound D-amino acids cluster at three different subsites within the overall active site pocket. These subsites, called transition, active, and exit subsites allow docking, re-orientation, chiral selection, catalysis, and exit of the free D-amino acid from DTD. Our studies reveal variable modes of D-amino acid recognition by DTDs, suggesting an inherent plasticity that can accommodate all D-amino acids. An in-depth analysis of native, ADP-bound, and D-amino acid-complexed DTD structures provide the first atomic snapshots of ligand recognition and subsequent catalysis by this enzyme family. We have mapped sites for the deacylation reaction and mark possible routes for entry and egress of all substrates and products. We have also performed structure-based inhibitor discovery and tested lead compounds against the malaria parasite P. falciparum using growth inhibition assays. Our studies provide a comprehensive structural basis for the catalytic mechanism of DTD enzymes and have implications for inhibition of this enzyme in P. falciparum as a route to inhibiting the parasite.

Structure of D-tyrosyl-tRNATyr deacylase using home-source Cu Kalpha and moderate-quality iodide-SAD data: structural polymorphism and HEPES-bound enzyme states.

D-Tyrosyl-tRNA(Tyr) deacylase (DTD) is an editing enzyme that removes D-amino acids from mischarged tRNAs. The crystal structure of Plasmodium falciparum DTD (PfDTD) was determined using the iodide-SAD phasing method. Iodide-derivatized PfDTD crystals were obtained using the quick cryo-soaking procedure in which native crystals were soaked for a short period of 10-30 s in cryoprotectant solution containing 0.2-1 M NaI. Iodide-SAD data sets were collected to 3.3 and 2.74 A resolution from PfDTD crystals that belonged to two different space groups, P4(3) and P1, using an in-house X-ray copper-anode source. This is the first report to detail structure solution using low iodide anomalous signal, modest resolution and redundancy and average solvent content for SAD phasing of 984 and 1312 amino acids in the triclinic P1 and tetragonal P4(3) space groups, respectively. A total of 85% and 56% of the residues were automatically built into the iodide-phased electron-density maps using PHENIX AutoBuild. The structure of HEPES-bound PfDTD was subsequently determined by molecular replacement and refined to 2.83 A resolution. The crystals obtained from various batches of crystallization trials of PfDTD exhibited polymorphism in terms of belonging to different crystal forms and space groups. Even within a given crystal system the unit-cell parameters showed high non-isomorphism. These packing variations were exploited in order to conduct a systematic study of conformational changes in PfDTD. It is shown that the disposition of a ten-residue insertion loop affects packing within the PfDTD crystals and seems to determine the non-isomorphism in unit-cell parameters. By tracking the changes in PfDTD unit cells, it was possible to map conformational differences within PfDTD that may be of significance for enzyme activity.

A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum.

BACKGROUND: Plasmodium parasites are causative agents of malaria which affects >500 million people and claims approximately 2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism. RESULTS: Using various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i) fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii) 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv) several PfaaRSs posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs. CONCLUSION: We have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

Author Correction: Genome-wide identification and expression analysis of E2 ubiquitin-conjugating enzymes in tomato.

A correction has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Novel Immunoinformatics Approaches to Design Multi-epitope Subunit Vaccine for Malaria by Investigating Anopheles Salivary Protein.

Malaria fever has been pervasive for quite a while in tropical developing regions causing high morbidity and mortality. The causal organism is a protozoan parasite of genus Plasmodium which spreads to the human host by the bite of hitherto infected female Anopheles mosquito. In the course of biting, a salivary protein of Anopheles helps in blood feeding behavior and having the ability to elicit the host immune response. This study represents a series of immunoinformatics approaches to design multi-epitope subunit vaccine using Anopheles mosquito salivary proteins. Designed subunit vaccine was evaluated for its immunogenicity, allergenicity and physiochemical parameters. To enhance the stability of vaccine protein, disulfide engineering was performed in a region of high mobility. Codon adaptation and in silico cloning was also performed to ensure the higher expression of designed subunit vaccine in E. coli K12 expression system. Finally, molecular docking and simulation study was performed for the vaccine protein and TLR-4 receptor, to determine the binding free energy and complex stability. Moreover, the designed subunit vaccine was found to induce anti-salivary immunity which may have the ability to prevent the entry of Plasmodium sporozoites into the human host.

Relict plastidic metabolic process as a potential therapeutic target.

The alignment of the evolutionary history of parasites with that of plants provides a different panorama in the drug development process. The housing of different metabolic processes, essential for parasite survival, adds to the indispensability of the apicoplast. The different pathways responsible for fueling the apicoplast and parasite offer a myriad of proteins responsible for the apicoplast function. The studies emphasizing the target-based approaches might help in the discovery of antimalarials. The different putative drug targets and their roles are highlighted. In addition, the origin of the apicoplast and metabolic processes are reviewed and the different drugs acting upon the enzymes of the apicoplast are discussed.

Development of multi-epitope driven subunit vaccine in secretory and membrane protein of Plasmodium falciparum to convey protection against malaria infection.

Malaria infection is the severe health concern for a long time. As per the WHO reports, the malarial infection causes huge mortality all around the world and is incomparable with any other infectious diseases. The absence of effective treatment options and increasing drug resistance to the available therapeutics like artemisinin and other derivatives demand an efficient alternative to overcome this death burden. Here, we performed the literature survey and sorted the Plasmodium falciparum secretory and membrane proteins to design multi-epitope subunit vaccine using an adjuvant, B-cell- and T-cell epitopes. Every helper T-lymphocyte (HTL) epitope was IFN-γ positive and IL-4 non-inducer. The physicochemical properties, allergenicity, and antigenicity of designed vaccine were analyzed for the safety concern. Homology modeling and refinement were performed to obtain the functional tertiary structure of vaccine protein followed by its molecular docking with the toll-like receptor-4 (TLR-4) immune receptor. Molecular dynamics simulation was performed to check the interaction and stability of the receptor-ligand complex. Lastly, in silico cloning was performed to generate the restriction clone of designed vaccine for the futuristic expression in a microbial expression system. This way, we designed the multi-epitope subunit vaccine to serve the people living in the global endemic zone.

Chemical system biology based molecular interactions to identify inhibitors against Q151M mutant of HIV-1 reverse transcriptase.

The emergence of mutations leading to drug resistance is the main cause of therapeutic failure in the human HIV infection. Chemical system biology approach has drawn great attention to discover new antiretroviral hits with high efficacy and negligible toxicity, which can be used as a prerequisite for HIV drug resistance global action plan 2017-21. To discover potential hits, we docked 49 antiretroviral analogs (n = 6294) against HIV-1 reverse transcriptase Q151M mutant & its wild-type form and narrow downed their number in three sequential modes of docking using Schrödinger suite. Later on, 80 ligands having better docking score than reference ligands (tenofovir and lamivudine) were screened for ADME, toxicity prediction, and binding energy estimation. Simultaneously, the area under the curve (AUC) was estimated using receiver operating characteristics (ROC) curve analysis to validate docking protocols. Finally, single point energy and molecular dynamics simulation approaches were performed for best two ligands (L3 and L14). This study reveals the antiretroviral efficacy of obtained two best ligands and delivers the hits against HIV-1 reverse transcriptase Q151M mutant.

Genome-wide identification and expression analysis of E2 ubiquitin-conjugating enzymes in tomato.

The ubiquitin-proteasomal degradation mechanism has gained the attention over the past decade. The E2 ubiquitin conjugating enzymes are the crucial part of ubiquitination mechanism and they are believed to hold imperative association for plant development. It accepts ubiquitin from the E1 enzyme and interacts with the E3 ligase to transfer ubiquitin or directly transfers ubiquitin to the substrate. The functional aspects of E2 ubiquitin enzymes in plant systems are unclear. Tomato is being used as a model plant and rarely explored to study E2 ubiquitin enzyme. We have utilized in-silico methods to analyze E2 enzymes in Solanum lycopersicum and 59 genes were identified with UBC family domains. The physio-chemical properties, chromosomal localization, structural organization, gene duplication, promoter analysis, gene ontology and conserved motifs were investigated along with phylogenetic analysis of tomato E2 genes exploring evolutionary relations. The gene expression analysis of RNA sequencing data revealed expression profile of tomato E2 genes in seedling, root, leaf, seed, fruit, and flower tissues. Our study aid in the understanding of distribution, expansion, evolutionary relation and probable participation in plant biological processes of tomato E2 enzymes that will facilitate strong base for future research on ubiquitin-mediated regulations in tomato and other plant systems.

Plasmodium falciparum apicoplast and its transcriptional regulation through calcium signaling.

Malaria has been present since ancient time and remains a major global health problem in developing countries. Plasmodium falciparum belongs to the phylum Apicomplexan, largely contain disease-causing parasites and characterized by the presence of apicoplast. It is a very essential organelle of P. falciparum responsible for the synthesis of key molecules required for the growth of the parasite. Indispensable nature of apicoplast makes it a potential drug target. Calcium signaling is important in the establishment of malaria parasite inside the host. It has been involved in invasion and egress of merozoites during the asexual life cycle of the parasite. Calcium signaling also regulates apicoplast metabolism. Therefore, in this review, we will focus on the role of apicoplast in malaria biology and its metabolic regulation through Ca++ signaling.

Molecular modeling, in silico screening and molecular dynamics of PfPRL-PTP of P. falciparum for identification of potential anti-malarials.

Millions of deaths occur every year due to malaria. Growing resistance against existing drugs for treatment of malaria has exaggerated the problem further. There is an intense demand of identifying drug targets in malaria parasite. PfPRL-PTP protein is PRL group of phosphatase, and one of the interesting drug targets being involved in three important pathways of malaria parasite (secretion, phosphorylation, and prenylation). Therefore, in this study, we have modeled three-dimensional structure of PfPRL-PTP followed by validation of 3D structure using RAMPAGE, verify3D, and other structure validation tools. We could identify 12 potential inhibitory compounds using in silico screening of NCI library against PfPRL-PTP with Glide. The molecular dynamics simulation was also performed using GROMACS on PfPRL-PTP model alone and PfPRL-PTP-inhibitor complex. This study of identifying potential drug-like molecules would add up to the process of drug discovery against malaria parasite.