Valproic Acid-Induced Upregulation of Multidrug Efflux Transporter ABCG2/BCRP via PPARα-Dependent Mechanism in Human Brain Endothelial Cells.

Despite the progress made in the development of new antiepileptic drugs (AEDs), poor response to them is a rising concern in epilepsy treatment. Of several hypotheses explaining AED treatment failure, the most promising theory is the overexpression of multidrug transporters belonging to ATP-binding cassette (ABC) transporter family at blood-brain barrier. Previous data show that AEDs themselves can induce these transporters, in turn affecting their own brain bioavailability. Presently, this induction and the underlying regulatory mechanism involved at human blood-brain barrier is not well elucidated. Herein, we sought to explore the effect of most prescribed first- and second-line AEDs on multidrug transporters in human cerebral microvascular endothelial cells, hCMEC/D3. Our work demonstrated that exposure of these cells to valproic acid (VPA) induced mRNA, protein, and functional activity of breast cancer resistance protein (BCRP/ABCG2). On examining the substrate interaction status of AEDs with BCRP, VPA, phenytoin, and lamotrigine were found to be potential BCRP substrates. Furthermore, we observed that siRNA-mediated knockdown of peroxisome proliferator-activated receptor alpha (PPARα) or use of PPARα antagonist, resulted in attenuation of VPA-induced BCRP expression and transporter activity. VPA was found to increase PPARα expression and trigger its translocation from cytoplasm to nucleus. Findings from chromatin immunoprecipitation and luciferase assays showed that VPA enhances the binding of PPARα to its response element in the ABCG2 promoter, resulting in elevated ABCG2 transcriptional activity. Taken together, these in vitro findings highlight PPARα as the potential molecular target to prevent VPA-mediated BCRP induction, which may have important implications in VPA pharmacoresistance. SIGNIFICANCE STATEMENT: Induction of multidrug transporters at blood-brain barrier can largely affect the bioavailability of the substrate antiepileptic drugs in the brains of patients with epilepsy, thus affecting their therapeutic efficacy. The present study reports a mechanistic pathway of breast cancer resistance protein (BCRP/ABCG2) upregulation by valproic acid in human brain endothelial cells via peroxisome proliferator-activated receptor alpha involvement, thereby providing a potential strategy to prevent valproic acid pharmacoresistance in epilepsy.

Lipid mediated brain disorders: A perspective.

The brain, one of the most resilient organs of the body is highly enriched in lipid content, suggesting the essential role of lipids in brain physiological activities. Lipids constitute an important structural part of the brain and act as a rich source of metabolic energy. Besides, lipids in their bioactive form (known as bioactive lipids) play an essential signaling and regulatory role, facilitating neurogenesis, synaptogenesis, and cell-cell communication. Brain lipid metabolism is thus a tightly regulated process. Any alteration/dysregulation of lipid metabolism greatly impact brain health and activity. Moreover, since central nervous system (CNS) is the most metabolically active system and lacks an efficient antioxidative defence system, it acts as a hub for the production of reactive oxygen species (ROS) and subsequent lipid peroxidation. These peroxidation events are reported during pathological changes such as neuronal tissue injury and inflammation. Present review is a modest attempt to gain insights into the role of dysregulated bioactive lipid levels and lipid oxidation status in the pathogenesis and progression of neurodegenerative disorders. This may open up new avenues exploiting lipids as the therapeutic targets for improving brain health, and treatment of nervous system disorders.

Porphyrin induced structural destabilization of a parallel DNA G-quadruplex in human MRP1 gene promoter.

Porphyrins are among the first ligands that have been tested for their quadruplex binding and stabilization potential. We report the differential interaction of the positional cationic porphyrin isomers TMPyP3 and TMPyP4 with a parallel G-quadruplex (GQ) formed by 33-mer (TP) regulatory sequence present in the promoter region of the human multidrug resistance protein 1 (MRP1) transporter gene. This GQ element encompasses the three evolutionary conserved SP1 transcription factor binding sites. Taking into account that SP1 binds to a non-canonical GQ motif with higher affinity than to a canonical duplex DNA consensus motif, it is suggestive that GQ distortion by cationic porphyrin will have important implications in the regulation of MRP1 expression. Herein, we employed biophysical analysis using circular dichroism, visible absorption, UV-thermal melting and steady-state fluorescence spectroscopy, reporting destabilization of MRP1 GQ by cationic porphyrins. Results suggest that TMPyP4 and TMPyP3 interact with GQ with a binding affinity of 106 to 107 M-1 . Thermodynamic analysis indicated a significant decrease in melting temperature of GQ (ΔTm of 15.5°C-23.5°C), in the presence of 2 times excess of porphyrins. This study provides the biophysical evidence indicating the destabilisation of a parallel DNA G-quadruplex by cationic porphyrins.

Multidrug efflux transporter ABCG2: expression and regulation.

The adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) was originally discovered in a multidrug-resistant breast cancer cell line. Studies in the past have expanded the understanding of its role in physiology, disease pathology and drug resistance. With a widely distributed expression across different cell types, ABCG2 plays a central role in ATP-dependent efflux of a vast range of endogenous and exogenous molecules, thereby maintaining cellular homeostasis and providing tissue protection against xenobiotic insults. However, ABCG2 expression is subjected to alterations under various pathophysiological conditions such as inflammation, infection, tissue injury, disease pathology and in response to xenobiotics and endobiotics. These changes may interfere with the bioavailability of therapeutic substrate drugs conferring drug resistance and in certain cases worsen the pathophysiological state aggravating its severity. Considering the crucial role of ABCG2 in normal physiology, therapeutic interventions directly targeting the transporter function may produce serious side effects. Therefore, modulation of transporter regulation instead of inhibiting the transporter itself will allow subtle changes in ABCG2 activity. This requires a thorough comprehension of diverse factors and complex signaling pathways (Kinases, Wnt/ß-catenin, Sonic hedgehog) operating at multiple regulatory levels dictating ABCG2 expression and activity. This review features a background on the physiological role of transporter, factors that modulate ABCG2 levels and highlights various signaling pathways, molecular mechanisms and genetic polymorphisms in ABCG2 regulation. This understanding will aid in identifying potential molecular targets for therapeutic interventions to overcome ABCG2-mediated multidrug resistance (MDR) and to manage ABCG2-related pathophysiology.

Mechanistic Insight into Oxidative Stress-Triggered Signaling Pathways and Type 2 Diabetes.

Oxidative stress (OS) is a metabolic dysfunction mediated by the imbalance between the biochemical processes leading to elevated production of reactive oxygen species (ROS) and the antioxidant defense system of the body. It has a ubiquitous role in the development of numerous noncommunicable maladies including cardiovascular diseases, cancers, neurodegenerative diseases, aging and respiratory diseases. Diseases associated with metabolic dysfunction may be influenced by changes in the redox balance. Lately, there has been increasing awareness and evidence that diabetes mellitus (DM), particularly type 2 diabetes, is significantly modulated by oxidative stress. DM is a state of impaired metabolism characterized by hyperglycemia, resulting from defects in insulin secretion or action, or both. ROS such as hydrogen peroxide and the superoxide anion introduce chemical changes virtually in all cellular components, causing deleterious effects on the islets of ß-cells, in turn affecting insulin production. Under hyperglycemic conditions, various signaling pathways such as nuclear factor-κß (NF-κß) and protein kinase C (PKC) are also activated by ROS. All of these can be linked to a hindrance in insulin signaling pathways, leading to insulin resistance. Hyperglycemia-induced oxidative stress plays a substantial role in complications including diabetic nephropathy. DM patients are more prone to microvascular as well as atherosclerotic macrovascular diseases. This systemic disease affects most countries around the world, owing to population explosion, aging, urbanization, obesity, lifestyle, etc. However, some modulators, with their free radical scavenging properties, can play a prospective role in overcoming the debilitating effects of OS. This review is a modest approach to summarizing the basics and interlinkages of oxidative stress, its modulators and diabetes mellitus. It may add to the understanding of and insight into the pathophysiology of diabetes and the crucial role of antioxidants to weaken the complications and morbidity resulting from this chronic disease.

Insights into Structural Modifications of Valproic Acid and Their Pharmacological Profile.

Valproic acid (VPA) is a well-established anticonvulsant drug discovered serendipitously and marketed for the treatment of epilepsy, migraine, bipolar disorder and neuropathic pain. Apart from this, VPA has potential therapeutic applications in other central nervous system (CNS) disorders and in various cancer types. Since the discovery of its anticonvulsant activity, substantial efforts have been made to develop structural analogues and derivatives in an attempt to increase potency and decrease adverse side effects, the most significant being teratogenicity and hepatotoxicity. Most of these compounds have shown reduced toxicity with improved potency. The simple structure of VPA offers a great advantage to its modification. This review briefly discusses the pharmacology and molecular targets of VPA. The article then elaborates on the structural modifications in VPA including amide-derivatives, acid and cyclic analogues, urea derivatives and pro-drugs, and compares their pharmacological profile with that of the parent molecule. The current challenges for the clinical use of these derivatives are also discussed. The review is expected to provide necessary knowledgebase for the further development of VPA-derived compounds.

Oxidative Stress: Role and Response of Short Guanine Tracts at Genomic Locations.

Over the decades, oxidative stress has emerged as a major concern to biological researchers. It is involved in the pathogenesis of various lifestyle-related diseases such as hypertension, diabetes, atherosclerosis, and neurodegenerative diseases. The connection between oxidative stress and telomere shortening via oxidative guanine lesion is well documented. Telomeres are confined to guanine rich ends of chromosomes. Owing to its self-association properties, it adopts G-quadruplex structures and hampers the overexpression of telomerase in the cancer cells. Guanine, being the most oxidation prone nucleobase, when structured in G-quadruplex entity, is found to respond peculiarly towards oxidative stress. Interestingly, this non-Watson-Crick structural feature exists abundantly in promoters of various oncogenes, exons and other genomic locations. The involvement of G-quadruplex architecture in oncogene promoters is well recognized in gene regulation processes. Development of small molecules aimed to target G-quadruplex structures, have found to alter the overexpression of oncogenes. The interaction may lead to the obstruction of diseased cell having elevated level of reactive oxygen species (ROS). Thus, presence of short guanine tracts (Gn) forming G-quadruplexes suggests its critical role in oxidative genome damage. Present review is a modest attempt to gain insight on the association of oxidative stress and G-quadruplexes, in various biological processes.

Oxidative Stress: A Key Modulator in Neurodegenerative Diseases.

Oxidative stress is proposed as a regulatory element in ageing and various neurological disorders. The excess of oxidants causes a reduction of antioxidants, which in turn produce an oxidation-reduction imbalance in organisms. Paucity of the antioxidant system generates oxidative-stress, characterized by elevated levels of reactive species (oxygen, hydroxyl free radical, and so on). Mitochondria play a key role in ATP supply to cells via oxidative phosphorylation, as well as synthesis of essential biological molecules. Various redox reactions catalyzed by enzymes take place in the oxidative phosphorylation process. An inefficient oxidative phosphorylation may generate reactive oxygen species (ROS), leading to mitochondrial dysfunction. Mitochondrial redox metabolism, phospholipid metabolism, and proteolytic pathways are found to be the major and potential source of free radicals. A lower concentration of ROS is essential for normal cellular signaling, whereas the higher concentration and long-time exposure of ROS cause damage to cellular macromolecules such as DNA, lipids and proteins, ultimately resulting in necrosis and apoptotic cell death. Normal and proper functioning of the central nervous system (CNS) is entirely dependent on the chemical integrity of brain. It is well established that the brain consumes a large amount of oxygen and is highly rich in lipid content, becoming prone to oxidative stress. A high consumption of oxygen leads to excessive production of ROS. Apart from this, the neuronal membranes are found to be rich in polyunsaturated fatty acids, which are highly susceptible to ROS. Various neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), among others, can be the result of biochemical alteration (due to oxidative stress) in bimolecular components. There is a need to understand the processes and role of oxidative stress in neurodegenerative diseases. This review is an effort towards improving our understanding of the pivotal role played by OS in neurodegenerative disorders.

Formation of G-wires, bimolecular and tetramolecular quadruplex: Cation-induced structural polymorphs of G-rich DNA sequence of human SYTX gene.

An exceptional property of auto-folding into a range of intra- as well as intermolecular quadruplexes by guanine-rich oligomers (GROs) of promoters, telomeres and various other genomic locations is still one of the most attractive areas of research at present times. The main reason for this attention is due to their established in vivo existence and biological relevance. Herein, the structural status of a 20-nt long G-rich sequence with two G5 stretches (SG20) is investigated using various biophysical and biochemical techniques. Bioinformatics analysis suggested the presence of a 17-nt stretch of this SG20 sequence in the intronic region of human SYTX (Synaptotagmin 10) gene. The SYTX gene helps in sensing out the Ca2+ ion, causing its intake in the pre-synaptic neuron. A range of various topologies like bimolecular, tetramolecular and guanine-wires (nano-wires) was exhibited by the studied sequence, as a function of cations (Na+ /K+ ) concentration. UV-thermal denaturation, gel electrophoresis, and circular dichroism (CD) spectroscopy showed correlations and established a cation-dependent structural switch. The G-wire formation, in the presence of K+ , may further be explored for its possible relevance in nano-biotechnological applications.

A topological transition from bimolecular quadruplex to G-triplex/tri-G-quadruplex exhibited by truncated double repeats of human telomere.

Human telomeric G-rich sequences can fold back into various conformations depending upon the salt (Na+ or K+) at physiological pH. On the basis of results obtained by native PAGE electrophoresis, circular dichroism, and UV-melting experiments, we report here that truncated sequences of human telomere (d-GGGTTAGGG; GM9, d-AGGGTTAGGG; GM10, d-TAGGGTTAGGG; GM11) adopt a varied range of quadruplex conformations as a function of the cation present. By correlating CD and gel electrophoresis experiments; it was concluded that the GM9 oligonucleotide can self-associate to form a tetramer quadruplex (antiparallel; AP) in Na+ solution and a mixture of G-triplex (AP) or tri-G-quadruplex (parallel; P) along with a tetramer G-quadruplex structure (AP) in K+. The GM10 oligonucleotide formed a bimolecular G-quadruplex in both Na+ and K+ solutions, while GM11 associated to form a bimolecular G-quadruplex (AP) structure in Na+ solution and a mixture of bimolecular G-quadruplex (AP) and bimolecular G-quadruplex (P) along with parallel G-triplex or antiparallel tri-G-quadruplex in K+. All the UV-melting profiles, thermal difference spectra, and CD melting curves suggested the formation of a variety of G-quadruplex conformations by the DNA sequences studied in Na+ and K+ ions. Hypothetical models for different conformations adopted by these DNA molecules have also been proposed, which may further enhance our knowledge about the divergent topologies of guanine quadruplexes.

Magnesium and molecular crowding of the cosolutes stabilize the i-motif structure at physiological pH.

Most of the important genomic regions, especially the G,C rich gene promoters, consist of sequences with potential to form G,C-tetraplexes on both the DNA strands. In this study, we used three C-rich oligonucleotides (11Py, 21Py, and HTPy), of which 11Py and 21Py are located at various transcriptional regulatory elements of the human genome while HTPy sequence is a C-rich strand of human telomere sequence. These C-rich oligonucleotides formed i-motif structures, verified by Circular Dichroism (CD), UV absorption melting experiments, and native gel electrophoresis. The CD spectra revealed that 11Py and 21Py form i-motif structures at acidic pH values of 4.5 and 5.7 in the presence of 100 mM NaCl but remain unstructured at pH 7.0. However, 21Py can form stable i-motif structure even at neutral pH in presence of 1 mM MgCl2 . UV-thermal melting studies showed stabilization of 21Py i-motif at pH 5.7 in the presence of Na+ or K+ with increasing concentration of MgCl2 or CaCl2 from 1 to 10 mM. Significant shift in the CD peak of HTPy sequence was observed as the positive peak from 286 nm shifted to 276 nm while the negative peak from 265 to 254 nm. Further, inevitable necessity of 1 mM Mg2+ to form i-motif structure at neutral pH was observed. Under similar ionic conditions and neutral pH, all the three C-rich sequences were able to form stable i-motif structures (11Py, 21Py) or altered i-motif/homoduplex structures (HTPy) in the presence of MgCl2 and cell mimicking molecular crowding conditions of 40 wt% PEG 200. It is concluded that presence of Mg2+ ions and molecular crowding agents induce and stabilize i-motif structures at physiological solution environment.

Luminescence, circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin.

Chalcones possess various biological properties, for example, antimicrobial, anti-inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using 1 H NMR 13 C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein-drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non-covalent binding interactions in the protein-ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site-specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.

Effect of Oxidative Stress on ABC Transporters: Contribution to Epilepsy Pharmacoresistance.

Epilepsy is a neurological disorder affecting around 1%-2% of population worldwide and its treatment includes use of antiepileptic drugs to control seizures. Failure to respond to antiepileptic drug therapy is a major clinical problem and over expression of ATP-binding cassette transporters is considered one of the major reasons for pharmacoresistance. In this review, we have summarized the regulation of ABC transporters in response to oxidative stress due to disease and antiepileptic drugs. Further, ketogenic diet and antioxidants were examined for their role in pharmacoresistance. The understanding of signalling pathways and mechanism involved may help in identifying potential therapeutic targets and improving drug response.

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. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter-relationships. Copyright © 2016 John Wiley & Sons, Ltd.

Biotinidase Resistant 68Gallium-Radioligand Based on Biotin/Avidin Interaction for Pretargeting: Synthesis and Preclinical Evaluation.

A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly 68Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled with 68Ga(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC50 of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. µPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

Prospecting the cancer therapeutic edge of chitosan-based gold nanoparticles through conformation selective binding to the parallel G-quadruplex formed by short telomeric DNA sequence: A multi-spectroscopic approach.

The biological relevance of G4 structures formed in telomere & oncogenes promoters make them extremely crucial therapeutic target for cancer treatment. Herein, we have synthesized chitosan-based gold nanoparticles (CH-Au NPs) through green method and have investigated their interaction with G4 structures formed by short telomeric sequences to evaluate their potential for targeting G4 structures. Firstly, we have characterized morphological/physical attributes of synthesized CH-Au NPs and salt dependent structural aspects of model G-rich DNA sequence, 12-mer d(T2G4)2 [TETRA] using spectroscopic and biophysical techniques. The molecular interactions between CH-Au NPs and parallel/antiparallel TETRA G4 structures were evaluated using UV-Visible, CD, Fluorescence, CD melting, DLS and Zeta potential studies. The experimental data indicated that CH-Au NPs showed strong binding interactions with Parallel TETRA G4 and provided thermal stabilization to the structure, whereas their interactions with Antiparallel TETRA G4 DNA and Ct-DNA (DNA duplex) were found to be negligible. Further, CH-Au NPs were also investigated for their selectivity aptitude for different G4 structures formed by human telomeric sequences; d(T2AG3)3 [HUM-12] and d(T2AG3)4T [HUM-25]. Our findings suggested that CH-Au NPs exhibited topology specific binding aptitude towards G4 structure, which can be utilized to inhibit/modulate crucial biological functions for potential anticancer activity.

Alkali cation-mediated topology displayed by an exonic G-rich sequence of TRPA1 gene.

G-rich sequences are intrinsic parts of the genome, widespread in promoters, telomeres, or other regulatory regions. The in vivo existence and biological significance have established the functional aspect of G-quadruplex structures and thus have developed immense interest in exploring their therapeutic aspects. Herein, using biophysical methods, we examined the structural status and comprehensive cation-dependence of a 17-bp G-rich genomic sequence (SKGT17) located in the coding region of the human TRPA1 gene, known to be associated with various neurovascular, cardiovascular, and respiratory conditions. TRPA1 is primarily seen as a therapeutic target for the development of novel analgesics. Bioinformatics analysis has suggested that 17-bp quadruplex motif is a binding site for transcription factor 'Sp1'. The formation and recognition of SKGT17 G-quadruplex might impact its regulatory functioning. Biophysical studies confirmed that the presence of alkali metal ions facilitated the formation of G-quadruplex in parallel topology. Native gel further substantiated the formation of a biomolecular species. Circular dichroism (CD), UV-thermal melting (Tm), and CD melting confirmed the formation of parallel G-quadruplex with metal ion-dependent stability. The stability of the G-quadruplex formed is found to be significantly high in the presence of K+ ions than that of other ions. Intriguingly, we have also established that this segment of the TRAP1 gene favors G-quadruplex formation over its participation in the corresponding duplex formation under K+ ions conditions. This study attempts to explain the rationale for the stabilization of G-quadruplex in the presence of alkali metal ions and may add to a better understanding and insights into DNA-metal ions interactions.

Nanomaterials and DNA multistranded structures: a treasure hunt for targeting specific biomedical applications.

The advent in nanoscience and nanotechnology has enabled the successful synthesis and characterization of different nanomaterials with unique electrical, optical, magnetic and catalytic activities. However, with respect to sensing applications, nanomaterials intrinsically lack target recognition ability to selectively bind with the analyte. DNA, an important genetic material carrying biopolymer is polymorphic in nature and shows structural polymorphism, forming secondary/multistranded structures like hairpin, cruciform, pseudoknot, duplex, triplex, G-quadruplex and i-motif. Studies reported so far have suggested that these polymorphic structures have been targeted specifically for the treatment or diagnosis of various diseases. DNA is widely used in conjugation with nanomaterials for the development of nanoarchitectures due to its rigidity, sequence programmability and specific molecular recognition, which makes this biomolecule a treasure for designing of DNA based frameworks. These two entities (DNA and nanomaterials) can be used in association with each other, as their alliance can result into creation of novel assay platforms for different purposes, ranging from imaging, sensing and diagnostics to targeted delivery. In this review, we have discussed about the recent reports on association of various mutistranded/ polymorphic forms of DNA with nanomaterials. Furthermore, different applications using this versatile DNA-nanomaterial assembly has also been elaborated at length. This review aims to target the interests of scientists from various interdisciplinary fields, including biologists, chemists and nanotechnologists, who wish to gain an understanding of nano-fabrications using a plethora of DNA polymorphic forms.Communicated by Ramaswamy H. Sarma.

G-quadruplex formation at human DAT1 gene promoter: Effect of cytosine methylation.

The dopamine transporter gene (DAT1), a recognized genetic risk factor for attention deficit hyperactivity disorder (ADHD) is principally responsible for the regulation of dopamine synaptic levels and serves as a key target in many psychostimulants drugs. DAT1 gene methylation has been considered an epigenetic marker in ADHD. The identification of G-rich sequence motifs potential to form G-quadruplexes is correlated with functionally important genomic regions. Herein, biophysical and biochemical techniques are employed to investigate the structural polymorphism along with the effect of cytosine methylation on a 26-nt G-rich sequence present in the promoter region of the DAT1 gene. The gel electrophoresis, circular dichroism spectroscopy, and UV-thermal melting data are well correlated and conclude the formation of a parallel (bimolecular), as well as antiparallel (tetramolecular) G-quadruplex in Na+ solution. Interestingly, the existence of uni-, bi-, tri-, and tetramolecular quadruplex structures in K+ solution exhibited only the parallel type G-quadruplex. The results demonstrate that in presence of either cation (Na+ or K+) the cytosine methylation reserved the structural topologies unaltered. However, methylation lowers the thermal stability of G-quadruplexes and the duplex structures, as well. These findings provide insights to understand the regulatory mechanisms underlying the formation of the G-quadruplex structure induced by DNA methylation.

Comparative assessment of 9-bromo noscapine ionic liquid and noscapine: Synthesis, in-vitro studies plus computational & biophysical evaluation with human hemoglobin.

Noscapine is a proficient anticancer drug active against wide variety of tumors including lung cancer. Over time, several noscapine analogues have been assessed to maximize the efficiency of the drug, amongst which 9-bromo noscapine remains one of the most potent analogues till date. In the present work, we have synthesized 9-bromo noscapine ionic liquid [9-Br-Nos]IBr2, an active pharmaceutical ingredient based ionic liquid (API-IL) to address the existing issues of solubility and targeted drug delivery in the parent alkaloid as well as the synthesized analogues. We have devised a novel two-step synthesis route (first-ever ionic to ionic bromination) to obtain the desired [9-Br-Nos]IBr2 which is advantageous to its organic analogue in terms of increased solubility, lesser reaction time and better yield. Furthermore, we have compared 9-bromo noscapine ionic liquid with noscapine based on its binding interaction with human hemoglobin (Hb) studied via computational along with spectroscopic studies, and bioactivity against non-small cell lung cancer. We inferred formation of a complex between [9-Br-Nos]IBr2 and Hb in the stoichiometric ratio of 1:1, similar to noscapine. At 298 K, [9-Br-Nos]IBr2-Hb binding was found to exhibit Kb and ∆G of 36,307 M-1 and -11.5 KJmol-1, respectively, as compared to 159 M-1 and -12.5 KJmol-1 during Noscapine-Hb binding. This indicates a more stronger and viable interaction between [9-Br-Nos]IBr2 and Hb than the parent compound. From computational studies, the observed higher stability of [9-Br-Nos]I and better binding affinity with Hb with a binding energy of -91.75 kcalmol-1 supported the experimental observations. In the same light, novel [9-Br-Nos]IBr2 was found to exhibit an IC50 = 95.02 ± 6.32 µM compared to IC50 = 128.82 ± 2.87 µM for noscapine on A549 (non-small lung cancer) cell line at 48 h. Also, the desired ionic liquid proved to be more cytotoxic inducing a mortality rate of 87 % relative to 66 % evoked by noscapine at concentrations of 200 µM after 72 h.