Mohammad Tabish

Studying non-covalent drug–DNA interactions

Drug-DNA interactions have been extensively studied in the recent past. Various techniques have been employed to decipher these interactions. DNA is a major target for a wide range of drugs that may specifically or non-specifically interact with DNA and affect its functions. Interaction between small molecules and DNA are of two types, covalent interactions and non-covalent interactions. Three major modes of non-covalent interactions are electrostatic interactions, groove binding and intercalative binding. This review primarily focuses on discussing various techniques used to study non-covalent interactions that occur between drugs and DNA. Additionally, we report several techniques that may be employed to analyse the binding mode of a drug with DNA. These techniques provide data that are reliable and simple to interpret.

Interaction of 6 Mercaptopurine with Calf Thymus DNA – Deciphering the Binding Mode and Photoinduced DNA Damage

DNA is one of the major intracellular targets for a wide range of anticancer and antibiotic drugs. Elucidating the binding between small molecules and DNA provides great help in understanding drug-DNA interactions and in designing of new and promising drugs for clinical use. The ability of small molecules to bind and interfere with DNA replication and transcription provides further insight into how the drugs control the expression of genes. Interaction of an antimetabolite anticancer drug 6mercaptopurine (6MP) with calf thymus DNA was studied using various approaches like UV-visible spectroscopy, fluorescence spectroscopy, CD, viscosity and molecular docking. UV-visible spectroscopy confirmed 6MP-DNA interaction. Steady state fluorescence experiments revealed a moderate binding constant of 7.48×103 M−1 which was consistent with an external binding mode. Competitive displacement assays further confirmed a non-intercalative binding mode of 6MP which was further confirmed by CD and viscosity experiments. Molecular docking further revealed the minimum energy conformation (−119.67 kJ/mole) of the complex formed between DNA and 6MP. Hence, the biophysical techniques and in-silico molecular docking approaches confirmed the groove binding/electrostatic mode of interaction between 6MP and DNA. Further, photo induced generation of ROS by 6MP was studied spectrophotometrically and DNA damage was assessed by plasmid nicking and comet assay. There was a significant increase in ROS generation and consequent DNA damage in the presence of light.

Interaction of coumarin with calf thymus DNA: deciphering the mode of binding by in vitro studies.

DNA is the major target for a wide range of therapeutic substances. Thus, there has been considerable interest in the binding studies of small molecules with DNA. Interaction between small molecules and DNA provides a structural guideline in rational drug designing and in the synthesis of new and improved drugs with enhanced selective activity and greater clinical efficacy. Plant derived polyphenolic compounds have a large number of biological and pharmacological properties. Coumarin is a polyphenolic compound which has been extensively studied for its diverse pharmacological properties. However, its mode of interaction with DNA has not been elucidated. In the present study, we have attempted to ascertain the mode of binding of coumarin with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of UV-visible absorbance spectra and fluorescence spectra indicates the formation of complex between coumarin and Ct-DNA. Several other experiments such as effect of ionic strength, iodide induced quenching, competitive binding assay with ethidium bromide, acridine orange and Hoechst 33258 reflected that coumarin possibly binds to the minor groove of the Ct-DNA. These observations were further supported by CD spectral analysis, viscosity measurements, DNA melting studies and in silico molecular docking.

The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics.

Here we describe the phenotypic characterization of the cta4 + gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast. The cta4Δ mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased. This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. These defects are likely due to a general imbalance in cation homeostasis. Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein–tagged Cta4 localizes to the ER. Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for Ca2+ indicated that Cta4p regulates the cellular Ca2+ concentration. Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint Ca2+ as a key ion in controlling these processes.

Naproxen intercalates with DNA and causes photocleavage through ROS generation

Naproxen is an important non‐steroidal anti‐inflammatory drug with many pharmacological and biological properties. In this study, we have attempted to ascertain the mode of action and the mechanism of binding of naproxen to DNA. We have also demonstrated that, upon irradiation with white light, naproxen generates reactive oxygen species, causing DNA cleavage. Generation of reactive oxygen species from photo‐irradiated naproxen as determined spectrophotometrically was found to lead to nicking of plasmid DNA as analyzed by agarose gel electrophoresis. Without photo‐irradiation, naproxen binds to DNA and forms drug–DNA complexes as revealed by spectroscopic techniques. Several experiments such as determination of the effect of urea, iodide‐induced quenching and a competitive binding assay with ethidium bromide showed that naproxen binds to DNA primarily in an intercalative manner. These observations were further supported by CD analysis, viscosity measurements and molecular docking. Using DNA as a template, fluorescence resonance energy transfer between naproxen and ethidium bromide was also observed, further strengthening the evidence for intercalative binding of naproxen with DNA.

Deciphering the interactions between chlorambucil and calf thymus DNA: a multi-spectroscopic and molecular docking study.

Non-covalent interactions of chlorambucil with calf thymus DNA was investigated using multi-spectroscopic techniques and molecular docking study. Binding constant calculated was found to be 1.54×10(4)M(-1) at 290K, significantly lower than various known intercalators. Quenching process was found to be static as evident by biomolecular quenching constant. Thermodynamic parameters revealed the involvement of hydrophobic interactions and hydrogen bonds in the binding. Chlorambucil was found to interact via external binding mode and follow groove binding as it replaces Hoechst (a typical groove binder) from the groove of DNA but does not replace intercalating dyes including ethidium bromide and acridine orange from the DNA helix. These results were further supported by KI quenching experiments, DNA melting studies, CD spectroscopy and molecular docking.

Spectroscopic and molecular docking evidence of aspirin and diflunisal binding to DNA: a comparative study

Aspirin and diflunisal belong to the salicylate class of non-steroidal anti-inflammatory drugs with diverse pharmacological and biological activities. Deciphering the interaction of drugs with DNA not only offers insights for the rational design of novel and more efficient drugs targeted to DNA, but also gives an opportunity for developing effective therapeutic agents for the control of gene expression. A series of spectroscopic studies were performed to ascertain the binding mode of aspirin and diflunisal with calf thymus DNA. UV-visible spectroscopy confirmed aspirin and diflunisal interaction with DNA. Steady state fluorescence experiments revealed a binding constant of 2.3 × 104 L mol−1 for aspirin and 7.9 × 103 L mol−1 for diflunisal. In addition, their binding modes with calf thymus DNA were established by a series of experiments including competitive displacement assays, urea denaturation, iodide quenching, viscosity measurements, DNA melting studies and CD analysis. They corroborated the intercalative binding of aspirin and groove binding of diflunisal with calf thymus DNA. The effect of ionic strength established the role of electrostatic interaction in aspirin–DNA binding processes. Molecular docking studies further complemented the experimental results.

Determination of the cationic amphiphilic drug-DNA binding mode and DNA-assisted fluorescence resonance energy transfer amplification.

Understanding the mechanism of drug-DNA binding is crucial for predicting the potential genotoxicity of drugs. Agarose gel electrophoresis, absorption, steady state fluorescence, and circular dichroism have been used in exploring the interaction of cationic amphiphilic drugs (CADs) such as amitriptyline hydrochloride (AMT), imipramine hydrochloride (IMP), and promethazine hydrochloride (PMT) with calf thymus or pUC19 DNA. Agarose gel electrophoresis assay, along with absorption and steady state fluorescence studies, reveal interaction between the CADs and DNA. A comparative study of the drugs with respect to the effect of urea, iodide induced quenching, and ethidium bromide (EB) exclusion assay reflects binding of CADs to the DNA primarily in an intercalative fashion. Circular dichroism data also support the intercalative mode of binding. Besides quenching, there is fluorescence exchange energy transfer (FRET) in between CADs and EB using DNA as a template.

Caffeic acid binds to the minor groove of calf thymus DNA: A multi-spectroscopic, thermodynamics and molecular modelling study

Caffeic acid (CA) is a plant polyphenol which acts as an antioxidant and has various pharmacological effects. DNA is one of the major cellular targets of therapeutic molecules. Thus, studying the interaction of small molecules with DNA is of great importance. In the current article, we have studied the mode of binding of CA with calf thymus DNA (Ct-DNA) using a series of biophysical techniques. Formation of complex between CA and Ct-DNA is ascertained by analyzing the UV-vis absorbance and fluorescence emission spectra of CA upon successive addition of Ct-DNA. Binding constants of CA with Ct-DNA obtained using multiple experiments was in the order of 103 M-1 which is consistent with known groove binders. Analysis of thermodynamic parameters suggest that hydrogen bonding and van der Waals forces played major role in the binding process. Competitive displacement studies confirmed that CA binds to the minor groove of Ct-DNA. These observations were further validated by KI quenching experiment, DNA melting studies, CD and viscosity measurements. In silico molecular docking further provided insight into the mode of binding of CA with Ct-DNA. Through in vitro experiments and in silico molecular docking studies, it was concluded that CA binds to the minor groove of Ct-DNA.

Redox cycling of Cu(II) by 6-mercaptopurine leads to ROS generation and DNA breakage: possible mechanism of anticancer activity

Abstract6-Mercaptopurine (6MP) is a well-known purine antimetabolite used to treat childhood acute lymphoblastic leukemia and other diseases. Cancer cells as compared to normal cells are under increased oxidative stress and show high copper level. These differences between cancer cells and normal cells can be targeted to develop effective cancer therapy. Pro-oxidant property of 6MP in the presence of metal ions is not well documented. Redox cycling of Cu(II) to Cu(I) was found to be efficiently mediated by 6MP. We have performed a series of in vitro experiments to demonstrate the pro-oxidant property of 6MP in the presence of Cu(II). Studies on human lymphocytes confirmed the DNA damaging ability of 6MP in the presence of Cu(II). Since 6MP possesses DNA damaging ability by producing reactive oxygen species (ROS) in the presence of Cu(II), it may also possess apoptosis-inducing activity by involving endogenous copper ions. Essentially, this would be an alternative and copper-dependent pathway for anticancer activity of 6MP.