Subodh Kumar Mishra

G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins

Nucleic acid G-quadruplex structure (G4) Interacting Proteins DataBase (G4IPDB) is an important database that contains detailed information about proteins interacting with nucleic acids that forms G-quadruplex structures. G4IPDB is the first database that provides comprehensive information about this interaction at a single platform. This database contains more than 200 entries with details of interaction such as interacting protein name and their synonyms, their UniProt-ID, source organism, target name and its sequences, ∆Tm, binding/dissociation constants, protein gene name, protein FASTA sequence, interacting residue in protein, related PDB entries, interaction ID, graphical view, PMID, author’s name and techniques that were used to detect their interactions. G4IPDB also provides an efficient web-based “G-quadruplex predictor tool” that searches putative G-quadruplex forming sequences simultaneously in both sense and anti-sense strands of the query nucleotide sequence and provides the predicted G score. Studying the interaction between proteins and nucleic acids forming G-quadruplex structures could be of therapeutic significance for various diseases including cancer and neurological disease, therefore, having detail information about their interactions on a single platform would be helpful for the discovery and development of novel therapeutics. G4IPDB can be routinely updated (twice in year) and freely available on http://bsbe.iiti.ac.in/bsbe/ipdb/index.php.

NALDB: nucleic acid ligand database for small molecules targeting nucleic acid

Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php

Structural insight for the recognition of G-quadruplex structure at human c-myc promoter sequence by flavonoid Quercetin

Small molecule ligands that could stabilize G-quadruplex structure formed at the promoter region of human c-myc oncogene will regulate its expression in cancer cells. Flavonoids, a group of naturally available small molecule, have been known for their various promising effects on human health. In present study, we have performed detailed biophysical studies for the interaction of human c-myc G-quadruplex DNA with nine representative flavonoids: Luteolin, Quercetin, Rutin, Genistein, Kaempferol, Puerarin, Hesperidin, Myricetin and Daidzein. We found by using fluorescence titration that Quercetin interacts with c-myc G-quadruplex DNA sequence Pu24T with highest affinity. This interaction was further explored by using NMR spectroscopy and we have derived the first solution structure for the complex formed between Quercetin and biologically significant c-myc promoter DNA sequence forming G-quadruplex structure. In present solution structure, Quercetin stacks at 5′ and 3′ G-tetrads of Pu24T G-quadruplex structure and stabilize it via π-π stacking interactions. Furthermore, in vitro studies on HeLa cells suggested that Quercetin induces apoptosis-mediated cell death and down-regulated c-myc gene expression. This study emphasizes the potential of flavonoids as a promising candidate for targeting c-myc promoter region and thus, could act as a potential anti-cancer agent.

Emerging Methods for Structural Analysis of Protein Aggregation

Protein misfolding and aggregation is a key attribute of different neurodegenerative diseases. Misfolded and aggregated proteins are intrinsically disordered and rule out structure based drug design. The comprehensive characterization of misfolded proteins and associated aggregation pathway is prerequisite to develop therapeutics for neurodegenerative diseases caused due to the protein aggregation. Visible protein aggregates used to be the final stage during aggregation mechanism. The structural analysis of intermediate steps in such protein aggregates will help us to discern the conformational role and subsequently involved pathways. The structural analysis of protein aggregation using various biophysical methods may aid for improved therapeutics for protein misfolding and aggregation related neurodegenerative diseases. In this mini review, we have summarized different spectroscopic methods such as fluorescence spectroscopy, circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy for structural analysis of protein aggregation. We believe that the understanding of invisible intermediate of misfolded proteins and the key steps involved during protein aggregation mechanisms may advance the therapeutic approaches for targeting neurological diseases that are caused due to misfolded proteins.

G-quadruplex stabilization in the ions and maltose transporters inhibit Salmonella enterica growth and virulence.

The G-quadruplex structure forming motifs have recently emerged as a novel therapeutic drug target in various human pathogens. Herein, we report three highly conserved G-quadruplex motifs (SE-PGQ-1, 2, and3) in genome of all the 412 strains of Salmonella enterica. Bioinformatics analysis inferred the presence of SE-PGQ-1 in the regulatory region of mgtA, presence of SE-PGQ-2 in the open reading frame of entA and presence of SE-PGQ-3 in the promoter region of malE and malK genes. The products of mgtA and entA are involved in transport and homeostasis of Mg2+ and Fe3+ ion and thereby required for bacterial survival in the presence of reactive nitrogen/oxygen species produced by the host macrophages, whereas, malK and malE genes are involved in transport of maltose sugar, that is one of the major carbon source in the gastrointestinal tract of human. The formation of stable intramolecular G-quadruplex structures by SE-PGQs was confirmed by employing CD, EMSA and NMR spectroscopy. Cellular studies revealed the inhibitory effect of 9-amino acridine on Salmonella enterica growth. Next, CD melting analysis demonstrated the stabilizing effect of 9-amino acridine on SE-PGQs. Further, polymerase inhibition and RT-qPCR assays emphasize the biological relevance of predicted G-quadruplex in the expression of PGQ possessing genes and demonstrate the G-quadruplexes as a potential drug target for the devolping novel therapeutics for combating Salmonella enterica infection. Author Summary Since last several decades’ scientific community has witnessed a rapid increase in number of such human pathogenic bacterial species that acquired resistant to multiple antibacterial agents. Currently, emergence of multidrug-resistant strains remain a major public health concern for clinical investigators that rings a global alarm to search for novel and highly conserved drug targets. Recently, G-quadruplex structure forming nucleic acid sequences were endorsed as highly conserved Drug target for preventing infection of several human pathogens including viral and protozoan species. Therefore, here we explored the presence G-quadruplex forming motif in genome of Salmonella enterica bacteria that causes food poisoning, and enteric fever in human. The formation of intra molecular G-quadruplex structure in four genes (mgtA, entA, malE and malK) was confirmed by NMR, CD and EMSA. The 9-amino acridine, a known G-quadruplex binder has been shown to stabilize the predicted G-quadruplex motif and decreases the expressioin of G-quadruplex hourbouring genes using RT-PCR and cellular toxicity assay. This study concludes the presence of G-quadruplex motifs in essential genes of Salmonella enterica genome as a novel and conserved drug target and 9-amino acridine as candidate small molecule for preventing the infection of Salmonella enterica using a G4 mediated inhibition mechanism.

G-Quadruplex-Forming DNA Aptamers Inhibit the DNA-Binding Function of HupB and Mycobacterium tuberculosis Entry into Host Cells

The entry and survival of Mycobacterium tuberculosis (Mtb) within host cells is orchestrated partly by an essential histone-like protein HupB (Rv2986c). Despite being an essential drug target, the lack of structural information has impeded the development of inhibitors targeting the indispensable and multifunctional C-terminal domain (CTD) of HupB. To bypass the requirement for structural information in the classical drug discovery route, we generated a panel of DNA aptamers against HupB protein through systemic evolution of ligands by exponential (SELEX) enrichment. Two G-quadruplex-forming high-affinity aptamers (HupB-4T and HupB-13T) were identified, each of which bound two distinct sites on full-length HupB, with an estimated KD of ∼1.72 μM and ∼0.17 μM, respectively, for the high-affinity sites. While HupB-4T robustly inhibited DNA-binding activity of HupB in vitro, both the aptamers recognized surface-located HupB and significantly blocked Mtb entry into THP-1 monocytic cells (p < 0.0001). In summary, DNA aptamers generated in this study block DNA-binding activity of HupB, inhibit virulent Mtb infection in host cells, and demonstrate aptamers to be inhibitors of HupB functions. This study also illustrates the utility of SELEX in developing inhibitors against essential targets for whom structural information is not available.

SMMDB: a web-accessible database for small molecule modulators and their targets involved in neurological diseases

Abstract High-throughput screening and better understanding of small molecule’s structure–activity relationship (SAR) using computational biology techniques have greatly expanded the face of drug discovery process in better discovery of therapeutics for various disease. Small Molecule Modulators Database (SMMDB) includes >1100 small molecules that have been either approved by US Food and Drug Administration, are under investigation or were rejected in clinical trial for any kind of neurological diseases. The comprehensive information about small molecules includes the details about their molecular targets (such as protein or enzyme, DNA, RNA, antisense RNA etc.), pharmacokinetic and pharmacodynamic properties such as binding affinity to their targets (Kd, Ki, IC50 and EC50 if available), mode of action, log P-value, number of hydrogen bond donor and acceptors, their clinical trial status, their 2D and three-dimensional structures etc. To enrich the basic annotation of every small molecule entry present in SMMDB, it is hyperlinked to their description present in PubChem, DrugBank, PubMed and KEGG database. The annotation about their molecular targets was enriched by linking it with UniProt and GenBank and STRING database that can be utilized to study the interaction and relation between various targets involved in single neurological disease. All molecules present in the SMMDB are made available to download in single file and can be further used in establishing the SAR, structure-based drug designing as well as shape-based virtual screening for developing the novel therapeutics against neurological diseases. The scope of this database majorly covers the interest of scientific community and researchers who are engaged in putting their endeavor toward therapeutic development and investigating the pathogenic mechanism of various neurological diseases. The graphical user interface of the SMMDB is accessible on http://bsbe.iiti.ac.in/bsbe/smmdb.