A computational protocol for the discovery of lead molecules targeting DNA unique to pathogens.

With the rapid emergence of drug resistant pathogens, it has become imperative to develop alternative medications as well as find new drug targets to overcome this crisis. Hence, this has become prime focus of several academic laboratories and pharmaceutical companies. Here, we report a computational protocol for identifying unique DNA sequence(s) in the pathogen which is absent in human and related non-pathogenic strains of the microbe. In order to use the unique sequence as drug target, the protocol, in the second step, uses virtual screening against a million compound library to identify candidate small molecules which can bind to these unique DNA targets in the pathogen only. Theoretically the molecules identified after screening should not bind to human DNA. This methodology is demonstrated on Mycobacterium tuberculosis H37Rv, wherein a new octamer sequence present only in H37Rv has been identified and a few candidate small molecules as potential drug have been proposed. Being fast and cost effective, this protocol could be of importance in generating new potential drug candidates against infectious organisms for further experimental studies. This methodology is freely available at http://www.scfbio-iitd.res.in/PSDDF/.

Role of sequence encoded κB DNA geometry in gene regulation by Dorsal.

Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the 'activator/repressor' functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the 'sequence-encoded structure' of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the 'A-tract' core. The sixth nucleotide in the nonameric κB-motif, 'A' (A(6)) in the repressor motifs and 'T' (T(6)) in the activator motifs, is critical to confer this functional specificity as A(6) → T(6) mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that 'sequence encoded κB DNA-geometry' regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network.

Dynamic repositioning of dorsal to two different kappaB motifs controls its autoregulation during immune response in Drosophila.

Autoregulation is one of the mechanisms of imparting feedback control on gene expression. Positive autoregulatory feedback results in induction of a gene, and negative feedback leads to its suppression. Here, we report an interesting mechanism of autoregulation operating on Drosophila Rel gene dorsal that can activate as well as repress its expression. Using biochemical and genetic approaches, we show that upon immune challenge Dorsal regulates its activation as well as repression by dynamically binding to two different kappaB motifs, kappaB(I) (intronic kappaB) and kappaB(P) (promoter kappaB), present in the dorsal gene. Although the kappaB(I) motif functions as an enhancer, the kappaB(P) motif acts as a transcriptional repressor. Interestingly, Dorsal binding to these two motifs is dynamic; immediately upon immune challenge, Dorsal binds to the kappaB(I) leading to auto-activation, whereas at the terminal phase of the immune response, it is removed from the kappaB(I) and repositioned at the kappaB(P), resulting in its repression. Furthermore, we show that repression of Dorsal as well as its binding to the kappaB(P) depends on the transcription factor AP1. Depletion of AP1 by RNA interference resulted in constitutive expression of Dorsal. In conclusion, this study suggests that during acute phase response dorsal is regulated by following two subcircuits: (i) Dl-kappaB(I) for activation and (ii) Dl-AP1-kappaB(P) for repression. These two subcircuits are temporally delineated and bring about overall regulation of dorsal during immune response. These results suggest the presence of a previously unknown mechanism of Dorsal autoregulation in immune-challenged Drosophila.

Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes.

Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5'→3', 3' →5' or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically.Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm.

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

Intron loss is associated with gain of function in the evolution of the gloverin family of antibacterial genes in Bombyx mori.

Gene duplication is a characteristic feature of eukaryotic genomes. Here we investigated the role of gene duplication in the evolution of the gloverin family of antibacterial genes (Bmglv1, Bmglv2, Bmglv3, and Bmglv4) in Bombyx mori. We observed the following two significant changes during the first duplication event: (i) loss of intronV, located in the 3'-untranslated region (UTR) of the ancestral gene Bmglv1, and (ii) 12-bp deletion in exon3. We show that loss of intronV during Bmglv1 to Bmglv2 duplication was associated with embryonic expression of Bmglv2. Gel mobility shift, chromatin immunoprecipitation, and immunodepletion assays identified chorion factor 2, a zinc finger protein, as the repressor molecule that bound to a 10-bp regulatory motif in intronV of Bmglv1 and repressed its transcription. gloverin paralogs that lacked intronV were independent of chorion factor 2 regulation and expressed in embryo. These results suggest that change in cis-regulation because of intron loss resulted in embryonic expression of Bmglv2-4, a gain of function over Bmglv1. Studies on the significance of intron loss have focused on introns present within the coding sequences for their potential effect on the open reading frame, whereas introns present in the UTRs of the genes were not given due attention. This study emphasizes the regulatory function of the 3'-UTR intron. In addition, we also studied the genomic loss and show that "in-frame" deletion of 12 nucleotides led to loss of amino acids IHDF resulting in the generation of a prepro-processing site in BmGlv2. As a result, the N-terminal pro-part of BmGlv2, but not of BmGlv1, gets processed in an infection-dependent manner suggesting that prepro-processing is an evolved feature in Gloverins.