D. Swati

Comparative genome analysis of six malarial parasites using codon usage bias based tools.

Codon usage bias (CUB) is an omnipresent phenomenon, which occurs in nearly all organisms. Previous studies of codon bias in Plasmodium species were based on a limited dataset. This study uses whole genome datasets for comparative genome analysis of six Plasmodium species using CUB and other related methods for the first time. Codon usage bias, compositional variation in translated amino acid frequency, effective number of codons and optimal codons are analyzed for P.falciparum, P.vivax, P.knowlesi, P.berghei, P.chabaudii and P.yoelli. A plot of effective number of codons versus GC3 shows their differential codon usage pattern arises due to a combination of mutational and translational selection pressure. The increased relative usage of adenine and thymine ending optimal codons in highly expressed genes of P.falciparum is the result of higher composition biased pressure, and usage of guanine and cytosine bases at third codon position can be explained by translational selection pressure acting on them. While higher usage of adenine and thymine bases at third codon position in optimal codons of P.vivax highlights the role of translational selection pressure apart from composition biased mutation pressure in shaping their codon usage pattern. The frequency of those amino acids that are encoded by AT ending codons are significantly high in P.falciparum due to action of high composition biased mutational pressure compared with other Plasmodium species. The CUB variation in the three rodent parasites, P.berghei, P.chabaudii and P.yoelli is strikingly similar to that of P.falciparum. The simian and human malarial parasite, P.knowlesi shows a variation in codon usage bias similar to P.vivax but on closer study there are differences confirmed by the method of Principal Component Analysis (PCA). Abbreviations CDS - Coding sequences, GC1 - GC composition at first site of codon, GC2 - GC composition at second site of codon, GC3 - GC composition at third site of codon, Ala - Alanine, Arg - Arginine, Asn - Asparagine, Asp - Aspartic acid, Cys - Cysteine, Gln - Glutamine Glu - Glutamic acid Gly - Glycine His - Histidine Ile - Isoleucine Leu - Leucine Lys - Lysine Met - Methionine Phe - Phenylalanine Pro - Proline Ser - Serine Thr - Threonine Trp - Tryptophan Tyr - Tyrosine Val - Valine.

In Silico Study of Variable Surface Proteins in Plasmodium Species: Perspectives in Drug Design

The variable surface proteins expressed by P. falciparum and P. vivax are transported to the surface of infected erythrocyte and are exposed to the host immune system. The possibility of using variable surface proteins as a common drug target has been analyzed in both the Plasmodium species. Sequence analysis of variable surface proteins showed a low-level conservation within as well as between the species. Amino acid composition analysis revealed higher frequency of hydrophilic amino acids as compared with that of hydrophobic residues. In order to gain more insight into their diverse functional role, the three-dimensional structure was predicted using comparative modeling approach. These models were evaluated and validated by checking stereochemistry of underlying amino acids. Structural alignment of variable surface proteins by superimposing them shows less conservation. Due to differences at sequence as well as structural level, the variable surface proteins are expected to show difference in their degree of invasiveness. These differences were also cross-examined by evolutionary study, and the results obtained were in accordance with the aforesaid study. The existence of structural differences noticed in the present study showed that the variable surface proteins could not be used as a common drug target in both the malarial species. Therefore, species-specific strategy may be followed for drug targeting against variable surface proteins of P. falciparum and P. vivax.

A knowledge-based approach for identification of drugs against vivapain-2 protein of Plasmodium vivax through pharmacophore-based virtual screening with comparative modelling.

Malaria is one of the most infectious diseases in the world. Plasmodium vivax, the pathogen causing endemic malaria in humans worldwide, is responsible for extensive disease morbidity. Due to the emergence of resistance to common anti-malarial drugs, there is a continuous need to develop a new class of drugs for this pathogen. P. vivax cysteine protease, also known as vivapain-2, plays an important role in haemoglobin hydrolysis and is considered essential for the survival of the parasite. The three-dimensional (3D) structure of vivapain-2 is not predicted experimentally, so its structure is modelled by using comparative modelling approach and further validated by Qualitative Model Energy Analysis (QMEAN) and RAMPAGE tools. The potential binding site of selected vivapain-2 structure has been detected by grid-based function prediction method. Drug targets and their respective drugs similar to vivapain-2 have been identified using three publicly available databases: STITCH 3.1, DrugBank and Therapeutic Target Database (TTD). The second approach of this work focuses on docking study of selected drug E-64 against vivapain-2 protein. Docking reveals crucial information about key residues (Asn281, Cys283, Val396 and Asp398) that are responsible for holding the ligand in the active site. The similarity-search criterion is used for the preparation of our in-house database of drugs, obtained from filtering the drugs from the DrugBank database. A five-point 3D pharmacophore model is generated for the docked complex of vivapain-2 with E-64. This study of 3D pharmacophore-based virtual screening results in identifying three new drugs, amongst which one is approved and the other two are experimentally proved. The ADMET properties of these drugs are found to be in the desired range. These drugs with novel scaffolds may act as potent drugs for treating malaria caused by P. vivax.

Proteins Containing Leucine Rich Repeats as Potential Targets for Plasmodium knowlesi

In parasitic organisms, proteins containing repeats especially leucine-rich repeats (LRRs) play an important role in invasion and immune evasion. LRR containing domains are responsible for different types of protein-protein interactions. We have taken advantage of available computational methods to structurally and functionally analyse, and to facilitate multifaceted insights into Plasmodium knowlesi leucine-rich repeats containing proteins. We have identified thirteen leucine-rich repeat (LRR) containing protein sequences in P. knowlesi. The sequential and structural similarities of these repeats containing proteins were checked against their recently emerged human host. Among the thirteen identified leucine-rich repeats containing protein sequences of P. knowlesi, only two (B3L5R8 and B3L378) were found to be unique and thus, selected for in-depth study. The pathogenic and virulence properties of B3L5R8 and B3L378 proteins were studied along with their dissimilarity with the human host proteins, suggesting their important role in disease establishment. These two proteins may be used as a novel targets for the development of drugs and vaccines against the disease. The three dimensional structures of these proteins were modeled using threading approach. Ramachandran analysis was used to validate the modeled structures. Structurally, LRR domains of B3L5R8 and B3L378 consist of a structural motif that forms α/β horseshoe shape fold with the concave face consisting of parallel β-strands and the convex face representing helices. LRR domain in B3L5R8 and B3L378 proteins contains 12 and 17 coiled sub-structures respectively. The active site of the proteins is predicted to study their functionality. The modeled structure of B3L5R8 displays three functional sites which interact with different ligands: 2S8, peptides and nucleic acids. While the predicted structure of B3L378 interact with GAL, nucleic acid, RX8 and 2Z7XA03 at their four functional sites. The key amino acid residues involved in interaction with their respective ligand at the different active site of B3L5R8 and B3L378 proteins are responsible for stability of diverse ligands. These compounds along with the interacting amino acids may serve as a representative for designing novel inhibitors.

Hammerhead Ribozymes in Archaeal Genomes: A Computational Hunt

AbstractHammerhead ribozymes (HHRs) are small self-cleaving RNAs, first discovered in viroids and satellite RNAs of plant viruses. They are composed of a catalytic core of conserved nucleotides flanked by three helices. More recently, hammerhead-encoding sequences have been identified in the genomes of many eukaryotes, prokaryotes and other non-viral species regulating various functions. In this study we have explored the Archaeal domain to identify HHRs using three different bioinformatics approach. Our study reveals four putative hits of HHRs type I and type II in the group Thaumarchaeota and Euryarchaeota in the Archaeal domain, one of which is the instance of HHR 1 in C. symbiosum A, already identified in a previous study. These HHRs are very similar to those previously described in terms of the conservation of their catalytic core. Based on 3-D structure analysis and free energy, these instances were concluded as putative HHRs. Our findings reveal that the catalytic core contains the conserved motifs that are essential for cleavage activity, but there are some instances in which compensatory core variations are present. However, no instances of HHRs have been found in Crenarchaeota. This study reveals a very scarce presence of HHRs in Archaea which suggests the involvement of other ncRNA elements in gene regulatory system like RNase P which are abundantly found in the Archaeal domain.

Targeting Pyrimidine Pathway of Plasmodium knowlesi: New Strategies Towards Identification of Novel Antimalarial Chemotherapeutic Agents

AIM AND OBJECTIVE Plasmodium knowlesi has been recently recognized as a human malarial parasite, particularly in the region of south-east Asia. Unlike human host, P. knowlesi cannot salvage pyrimidine bases and relies solely on nucleotides synthesized from de novo pyrimidine pathway. The enzymes involved in this are also unique in terms of their structure and function to its human counterpart. Thus, targeting Dihydroorotase, an enzyme involved in the pyrimidine biosynthesis, provides a promising route for novel drug development. MATERIALS AND METHODS The 3D structure of P. knowlesi Dihydroorotase was predicted, refined and validated. Multiple docking was performed and the resultant complex was used for 3D structurebased pharmacophore modelling. A combinatorial library of 2,664,779 molecules was generated and used for structure based virtual screening. The stability of resultant compounds was checked using simulation studies. RESULTS The modelled 3D structure of P. knowlesi Dihydroorotase enzyme is relaxed by running an MD simulation of 20 ns, and structure is validated by using Ramachandran plot and G-factor analysis. A five point based pharmacophore model was created and used as a query for screening in house database. The stability of two negatively charged compounds was studied, and ZINC22066495-DHOase complex was more stable throughout the simulation. CONCLUSION The present study shows that ZINC22066495 compound has a high potential for disrupting P. knowlesi DHOase enzyme and may be used as a potential lead molecule for effective pyrimidine biosynthesis inhibition in P. knowlesi.