Shrikant Kukreti

Structural Diversity and Specific Recognition of Four Stranded G-Quadruplex DNA

Structural multitude of nucleic acids serves basis for its multiple merits and applications. During structural transitions, significant to perform respective cellular functions, these DNA forms can vary from the single stranded to multi-stranded species. Hence, beyond the image of a monotonous DNA double-helix, there is now increasing interest in other polymorphic/ multi-stranded forms, the roles they may play in vivo and their potential use in therapeutics. Distinct guanine-rich nucleic acid sequences readily form a structurally diverse four-stranded architecture called G-quadruplexes. In addition to their presence at physical ends of chromosomes called telomeres, occurrence of these structural motifs in the upstream promoter regions of a number of genes, oncogenes and near transcription start sites, highlights that G-quadruplexes are involved in regulation of gene expression. Cancer cells typically possess shorter telomeres and have telomerase activity greatly exceeding that of normal cells. These differences create an opportunity to use anticancer therapies targeting telomerase and telomeres. The ability of small molecules to interact with and presumably stabilize G-quadruplex structures as a means of inhibiting telomerase has been a major drug design effort. Ligands, capable of interacting with four-stranded G-quadruplex have been generated. The discovery of proteins including transcription factors, recognizing G-quadruplexes, and conferring stabilization or unfolding them in biological systems, again makes G-quadruplexes, biologically pertinent structures. This review is an attempt to summarize the rapidly evolving literature exploring the amazing polymorphism of G-quadruplexes, and understanding their structure-specific-recognition and biological relevance, keeping in mind that G-tetraplexes are not only important drug targets, but may also act as gene regulatory elements. A pertinent detail of the challenges towards the rational design of structure-specific novel drugs has also been discussed.

A bouquet of DNA structures: Emerging diversity

Structural polymorphism of DNA has constantly been evolving from the time of illustration of the double helical model of DNA by Watson and Crick. A variety of non-canonical DNA structures have constantly been documented across the globe. DNA attracted worldwide attention as a carrier of genetic information. In addition to the classical Watson–Crick duplex, DNA can actually adopt diverse structures during its active participation in cellular processes like replication, transcription, recombination and repair. Structures like hairpin, cruciform, triplex, G-triplex, quadruplex, i-motif and other alternative non-canonical DNA structures have been studied at length and have also shown their in vivo occurrence. This review mainly focuses on non-canonical structures adopted by DNA oligonucleotides which have certain prerequisites for their formation in terms of sequence, its length, number and orientation of strands along with varied solution conditions. This conformational polymorphism of DNA might be the basis of different functional properties of a specific set of DNA sequences, further giving some insights for various extremely complicated biological phenomena. Many of these structures have already shown their linkages with diseases like cancer and genetic disorders, hence making them an extremely striking target for structure-specific drug designing and therapeutic applications.

Structural polymorphism at LCR and its role in beta-globin gene regulation

Information on the secondary structures and conformational manifestations of eukaryotic DNA and their biological significance with reference to gene regulation and expression is limited. The human beta-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression, is a contiguous piece of DNA with five tissue-specific DNase I-hypersensitive sites (HSs). Since these HSs have a high density of transcription factor binding sites, structural interdependencies between HSs and different promoters may directly or indirectly regulate LCR functions. Mutations and SNPs may stabilize or destabilize the local secondary structures, affecting the gene expression by changes in the protein-DNA recognition patterns. Various palindromic or quasi-palindromic segments within LCR, could cause structural polymorphism and geometrical switching of DNA. This emphasizes the importance of understanding of the sequence-dependent variations of the DNA structure. Such structural motifs might act as regulatory elements. The local conformational variability of a DNA segment or action of a DNA specific protein is key to create and maintain active chromatin domains and affect transcription of various tissue specific beta-globin genes. We, summarize here the current status of beta-globin LCR structure and function. Further structural studies at molecular level and functional genomics might solve the regulatory puzzles that control the beta-globin gene locus.

Temperature induced hyperchromism exhibited by Hoechst 33258: evidence of drug aggregation from UV-melting method

UV-thermal denaturation is a simple optical method widely employed for determination of DNA stability and interaction with ligands. Thermal denaturation of DNA and DNA-ligand complex is usually monitored at 260 nm. These data are generally presented as a function of the absorption increase of DNA alone with no consideration of the temperature dependent hyperchromism of the free ligand. Since not every ligand has absorption at 260 nm, usually this property of the ligand is ignored. Here, we report the temperature dependent hyperchromicity exhibited by Hoechst 33258 in the presence and absence of DNA. The presence of Hoechst, added to the duplex (monophasic profile, T(m)=75 degrees C) in various ratios generates a new transition at lower temperature displaying biphasic thermal transition profiles. We attributed this new transition (hyperchromic), a mere contribution from Hoechst, which might exist in aggregated forms. The extent of drug aggregation/self-association is concentration dependent. We suggest that prior to UV-melting studies the thermal dependence of the free ligand should be investigated.

Protein engineering and de novo designing of a biocatalyst.

Proteins as a biomolecule have been recognized as a “molecule with manifold biological functions”. The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by‐products and their non‐hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf‐life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging.

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

6-Endo-dig-cyclization is an efficient method for the synthesis of 1,2-dihydroisoquinolines. We have synthesized few 1,2-dihydroisoquinolines having different functionality at the C-1, C-3, C-7, and N-2 positions for evaluation against HIV-1 integrase (HIV1-IN) inhibitory activity. A direct nitro-Mannich condensation of o-alkynylaldimines and dual activation of o-alkynyl aldehydes by inexpensive cobalt chloride yielded desired compounds. Out of 24 compounds, 4m and 6c came out as potent integrase inhibitors in in vitro strand transfer (ST) assay, with IC50 value of 0.7 and 0.8 μM, respectively. Molecular docking of these compounds in integrase revealed strong interaction between metal and ligands, which stabilizes the enzyme-inhibitor complex. The ten most active compounds were subjected to antiviral assay. Out of those, 6c reduced the level of p24 viral antigen by 91%, which is comparable to RAL in antiviral assay. Interestingly, these compounds showed similar ST inhibitory activity in G140S mutant, suggesting they can act against resistant strains.

Hairpin Formation In d-AAGCTTAAGCTT Under High Salt Conditions Shows Unusual Properties

The hairpin-duplex equilibria of the dodecamer d-AAGCTTAAGCTT and interaction of the duplex form with a pentapeptide, KGWGK, has been studied. UV thermal transitions are monophasic at low salt but biphasic at higher salt concentrations. At 10(-5) M or less oligomer concentration biphasic melting curves persist till 900 mM NaCl. The d(Tm)/d log(Na+) for the duplex form is 12 degrees C and for the hairpin is 18 degrees C. The delta H and delta S values for duplex formation are low (-25 K cal/mole and -59 Cal/mole respectively). KGWGK binds to the duplex form with a binding constant K = 3.4 x 10(5)M-1 measured from fluorescence quenching of tryptophan. These unusual results are markedly different from that reported for d-AGATCTAGATCT (Biochemistry 31, 6241-6245) and are discussed in terms of sequence dependence of loop folding and cruciform extrusion pathway of hairpin formation.

Comparative In Vitro Binding Studies of TiCl2(dpme)2, Ti(ada)2(bzac)2, and TiCl2(bzac)(bpme) Titanium Complexes with Calf-Thymus DNA

The binding of TiCl2(dpme)2 (1), (dpme = 6,6′-dimethyl-2,2′-bipyridine), Ti(ada)2(bzac)2 (2), (ada = adamantylamine; bzac = benzoylacetone), and TiCl2(bzac)(bpme) (3), (bpme = 4,4′-dimethyl-2,2′-bipyrdine) with calf thymus (ct) DNA has been studied by UV-visible spectroscopy, thermal denaturation, and circular dichroism spectroscopy. In UV-visible study complexes 1, 2, and 3 showed red, blue, and red shifts, respectively, upon the addition of ct-DNA along with a significant hyperchromism. The intrinsic binding constants (K b) calculated from UV-visible absorption studies were 2.3 × 103 M−1, 3.3 × 103 M−1 and, 7.1 × 103 M−1 for complexes 1, 2, and 3, respectively. The change in melting temperature (ΔT m) was calculated to be 2-3°C for each complex. Circular dichroism (CD) study showed blue shift for complex 2 and red shift for complexes 1 and 3 along with rise in molecular ellipticity upon the addition of complexes. Results suggest a binding mode of complex 2 different than 1 and 3.

Genetic contribution of CYP1A1 variant on treatment outcome in epilepsy patients: a functional and interethnic perspective.

CYP1A1 gene is involved in estrogen metabolism, and previously, we have reported association of variant rs2606345 with altered anti-epileptic drugs (AED) response in North Indian women with epilepsy (WWE). The present study aims to replicate the pharmacogenetic association, perform functional characterization and study its distribution within ethnically diverse Indian population. The variant was genotyped in 351 patients to assess the pharmacogenetic association and 552 healthy individuals belonging to 24 different ethnic groups to examine the distribution in Indian population. We observed significant overrepresentation of ‘A’ allele and ‘AA’ genotype in poor responders in WWE at Bonferroni-corrected significance levels. The recessive allele was found to lower the promoter activity by ~70–80% which was further substantiated by thermally less stable hairpin formed by it (ΔTm=7 °C). Among all ethnic groups, west Indo–European (IE-W-LP2) subpopulation showed highest genotypic frequency of the variant making women from this community more prone to poor AED response. Our results indicate that rs2606345 influences drug response in WWE by lowering CYP1A1 expression.

Synthesis, preclinical evaluation and molecular modelling of macrocyclic appended 1-(2-methoxyphenyl)piperazine for 5-HT1A neuroreceptor imaging

5-HT1A receptors are known to be implicit in a number of neuropsychiatric fluctuations related to mood and anxiety. Their visualization in the human brain using PET, SPECT or MRI is of great importance in the management and treatment of neurological disorders. The present work focuses on the metal complexes (Gd3+, Eu3+ and Ga3+) of DO3A-butyl-MPP to be used as brain (cerebral cortex, hippocampus, and amygdala) imaging agents using different modalities. Synthesis of 2,2′,2′′-(10-(2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DO3A-butyl-MPP) was achieved by conjugating the chloroacetylated derivative of 1-(2-methoxyphenyl)piperazine-butylamine with trisubstituted cyclen with subsequent cleavage with trifluoroacetic acid (TFA). The resulting compound was then labeled with GdCl3 and 68GaCl3 to perform MRI (relaxivity studies) and PET respectively. The longitudinal relaxivity (r1), and transverse relaxivity (r2), were determined to be 6.66 and 11.486 mM−1 s−1 respectively on 7 T at 21 °C. The SD (Sprague Dawley) rat brain uptake was 3.91% ID per g (percentage of the injected dose per gram) at 30 min post injection. Homology modeling and docking studies at the shallow antagonist binding pocket of 5-HT1A show a high G score of −12.132 that confers high binding of the ligand at the target receptor.