Prospects of multitarget drug designing strategies by linking molecular docking and molecular dynamics to explore the protein-ligand recognition process.

The designing of drugs that can simultaneously affect different protein targets is one novel and promising way to treat complex diseases. Multitarget drugs act on multiple protein receptors each implicated in the same disease state, and may be considered to be more beneficial than conventional drug therapies. For example, these drugs can have improved therapeutic potency due to synergistic effects on multiple targets, as well as improved safety and resistance profiles due to the combined regulation of potential primary therapeutic targets and compensatory elements and lower dosage typically required. This review analyzes in-silico methods that facilitate multitarget drug design that facilitate the discovery and development of novel therapeutic agents. Here presented is a summary of the progress in structure-based drug discovery techniques that study the process of molecular recognition of targets and ligands, moving from static molecular docking to improved molecular dynamics approaches in multitarget drug design, and the advantages and limitations of each.

Host Factor Nucleophosmin 1 (NPM1/B23) Exerts Antiviral Effects against Chikungunya Virus by Its Interaction with Viral Nonstructural Protein 3.

Chikungunya virus (CHIKV) hijacks host cell machinery to support its replication. Nucleophosmin 1 (NPM1/B23), a nucleolar phosphoprotein, is one of the host proteins known to restrict CHIKV infection; however, the mechanistic details of the antiviral role of NPM1 are not elucidated. It was seen in our experiments that the level of NPM1 expression affected the expression levels of interferon-stimulated genes (ISGs) that play antiviral roles in CHIKV infection, such as IRF1, IRF7, OAS3, and IFIT1, indicating that one of the antiviral mechanisms could be through modulation of interferon-mediated pathways. Our experiments also identified that for CHIKV restriction, NPM1 must move from the nucleus to the cytoplasm. A deletion of the nuclear export signal (NES), which confines NPM1 within the nucleus, abolishes its anti-CHIKV action. We observed that NPM1 binds CHIKV nonstructural protein 3 (nsP3) strongly via its macrodomain, thereby exerting a direct interaction with viral proteins to limit infection. Based on site-directed mutagenesis and coimmunoprecipitation studies, it was also observed that amino acid residues N24 and Y114 of the CHIKV nsP3 macrodomain, known to be involved in virus virulence, bind ADP-ribosylated NPM1 to inhibit infection. Overall, the results show a key role of NPM1 in CHIKV restriction and indicate it as a promising host target for developing antiviral strategies against CHIKV. IMPORTANCE Chikungunya, a recently reemerged mosquito-borne infection caused by a positive-sense, single-stranded RNA virus, has caused explosive epidemics in tropical regions. Unlike the classical symptoms of acute fever and debilitating arthralgia, incidences of neurological complications and mortality were reported. Currently there are no antivirals or commercial vaccines available against chikungunya. Like all viruses, CHIKV uses host cellular machinery for establishment of infection and successful replication. To counter this, the host cell activates several restriction factors and innate immune response mediators. Understanding these host-virus interactions helps to develop host-targeted antivirals against the disease. Here, we report the antiviral role of the multifunctional host protein NPM1 against CHIKV. The significant inhibitory effect of this protein against CHIKV involves its increased expression and movement from its natural location within the nucleus to the cytoplasm. There, it interacts with functional domains of key viral proteins. Our results support ongoing efforts toward development of host-directed antivirals against CHIKV and other alphaviruses.

Novel small synthetic HIV-1 V3 crown variants: CCR5 targeting ligands.

The CC chemokine receptor 5 (CCR5) antagonism represents a promising pharmacological strategy for therapeutic intervention as it plays a significant role in reducing the severity and progression of a wide range of pathological conditions. Here we designed and generated peptide ligands targeting the chemokine receptor, CCR5, that were derived from the critical interaction sites of the V3 crown domain of envelope protein glycoprotein gp120 (TRKSIHIGPGRAFYTTGEI) of HIV-1 using computational biology approach and the peptide sequence corresponding to this region was taken as the template peptide, designated as TMP-1. The peptide variants were synthesized by employing Fmoc chemistry using polymer support and were labelled with rhodamine B to study their interaction with the CCR5 receptor expressed on various cells. TMP-1 and TMP-2 were selected as the high-affinity ligands from in vitro receptor-binding assays. Specific receptor-binding experiments in activated peripheral blood mononuclear cells and HOS.CCR5 cells indicated that TMP-1 and TMP-2 had significant CCR5 specificity. Further, the functional analysis of TMP peptides using chemotactic migration assay showed that both peptides did not mediate the migration of responsive cells. Thus, template TMP-1 and TMP-2 represent promising CCR5 targeting peptide candidates.

Genomic attributes differ between Vibrio parahaemolyticus environmental and clinical isolates including pathotypes.

Vibrio parahaemolyticus is a marine bacterium and causes opportunistic gastroenteritis in humans. Clinical strains of V. parahaemolyticus contain haemolysin and type III secretion systems (T3SS) that define their pathotype. A growing number of strains isolated recently from the environment have acquired these virulence genes constituting a pool of potential pathogens. This study used comparative genomics to identify genetic factors that delineate environmental and clinical V. parahaemolyticus population and understand the similarities and differences between the T3SS2 phylotypes. The comparative analysis revealed the presence of a cluster of genes belonging to bacterial cellulose synthesis (bcs) in isolates of environmental origin. This cluster, previously unreported in V. parahaemolyticus, exhibit significant similarity to that of Aliivibrio fischeri, and might dictate a potentially new mechanism of its environmental adaptation and persistence. The study also identified many genes predicted in silico to be T3SS effectors that are unique to T3SS2ß of tdh- trh+ and tdh+ trh+ pathotype and having no identifiable homologue in tdh+ trh- T3SS2α. Overall, these findings highlight the importance of understanding the genes and strategies V. parahaemolyticus utilize for the myriad interactions with its hosts, either marine invertebrates or humans.

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2.

BACKGROUND: Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian beta-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. RESULTS: MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. CONCLUSION: The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.

Pax6 modulates intra-retinal axon guidance and fasciculation of retinal ganglion cells during retinogenesis.

Intra-retinal axon guidance involves a coordinated expression of transcription factors, axon guidance genes, and secretory molecules within the retina. Pax6, the master regulator gene, has a spatio-temporal expression typically restricted till neurogenesis and fate-specification. However, our observation of persistent expression of Pax6 in mature RGCs led us to hypothesize that Pax6 could play a major role in axon guidance after fate specification. Here, we found significant alteration in intra-retinal axon guidance and fasciculation upon knocking out of Pax6 in E15.5 retina. Through unbiased transcriptome profiling between Pax6fl/fl and Pax6-/- retinas, we revealed the mechanistic insight of its role in axon guidance. Our results showed a significant increase in the expression of extracellular matrix molecules and decreased expression of retinal fate specification and neuron projection guidance molecules. Additionally, we found that EphB1 and Sema5B are directly regulated by Pax6 owing to the guidance defects and improper fasciculation of axons. We conclude that Pax6 expression post fate specification of RGCs is necessary for regulating the expression of axon guidance genes and most importantly for maintaining a conducive ECM through which the nascent axons get guided and fasciculate to reach the optic disc.

miR Cluster 143/145 Directly Targets Nrl and Regulates Rod Photoreceptor Development.

Retinal histogenesis requires coordinated and temporal functioning of factors by which different cell types are generated from multipotent progenitors. Development of rod photoreceptors is regulated by multiple transcription factors, and Nrl is one of the major factors involved in their fate specification. Presence or absence of Nrl at the postnatal stages decides the generation of cone photoreceptors or other later retinal cells. This suggests the need for regulated expression of Nrl in order to accelerate the generation of other cell types during retinal development. We found that miR cluster 143/145, comprising miR-143 and miR-145, targets and imparts a posttranscriptional inhibition of Nrl. Expression of both miRNAs was differentially regulated during retinal development and showed least expression at PN1 stage in which most of the rod photoreceptors are generated. Downregulation of rod photoreceptor regulators and markers upon miR cluster 143/145 overexpression demonstrated that this cluster indeed negatively regulates rod photoreceptors. Further, we prove that Nrl positively regulates miR cluster 143/145, thus establishing a feedback loop regulatory mechanism. This may be one possible mechanism by which Nrl is posttranscriptionally regulated to facilitate the generation of other cell types in retina.

Correlation of phylogenetic clade diversification and in vitro infectivity differences among Cosmopolitan genotype strains of Chikungunya virus.

Cosmopolitan genotypes of Chikungunya virus caused the large-scale febrile disease outbreaks in the last decade in Asian and African continents. Molecular analyses of these strains had revealed significant genetic diversification and occurrence of novel mosquito-adaptive mutations. In the present study we looked into whether the genetic diversification has implications in the infectivity phenotype. A detailed sequence and phylogenetic analyses of these virus strains of Indian Ocean lineage from Kerala, South India from the years 2008 to 2013 identified three distinct genetic clades (I, II and III), which had presence of clade-specific amino acid changes. The E2 envelope protein of the strains from the years 2012 to 2013 had a K252Q or a novel K252H change. This site is reported to affect mosquito cell infectivity. Most of these strains also had the E2 G82R mutation, a mutation previously identified to increase mammalian cell infectivity, and a novel mutation E2 N72S. Positive selection was identified in four sites in the envelope proteins (E1 K211E, A226V and V291I; E2 K252Q/H). In infectivity analysis, we found that strains from clade III had enhanced cytopathogenicity in HEK293 and Vero cells than by strains representing other two clades. These two strains formed smaller sized plaques and had distinctly higher viral protein expression, infectious virus production and apoptosis induction in HEK293 cells. They had novel mutations R171Q in the nsP1; I539S in nsP2; N409T in nsP3; and N72S in E2. Our study identifies a correlation between phylogenetic clade diversification and differences in mammalian cell infectivity phenotype among Cosmopolitan genotype CHIKV strains.

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR.

Rv3334 protein of Mycobacterium tuberculosis belongs to the MerR family of transcriptional regulators and is upregulated during hypoxia and other stress conditions. Employing GFP reporter constructs, mobility shift assays and ChIP assays, we demonstrate that Rv3334 binds to its own promoter and acts as an autorepressor. We were able to locate a 22 bp palindrome in its promoter that we show to be the cognate binding sequence of Rv3334. Using chase experiments, we could conclusively prove the requirement of this palindrome for Rv3334 binding. Recombinant Rv3334 readily formed homodimers in vitro, which could be necessary for its transcriptional regulatory role in vivo. Although the DNA-binding activity of the protein was abrogated by the presence of certain divalent metal cations, the homodimer formation remained unaffected. In silico predictions and subsequent assays using GFP reporter constructs and mobility shift assays revealed that the expression of ketosteroid regulator gene (kstR), involved in lipid catabolism, is positively regulated by Rv3334. ChIP assays with aerobically grown M. tuberculosis as well as dormant bacteria unambiguously prove that Rv3334 specifically upregulates expression of kstR during dormancy. Our study throws light on the possible role of Rv3334 as a master regulator of lipid catabolism during hypoxia-induced dormancy.

A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways.

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs.