Structural Insight into Groove Binding of Yohimbine with Calf Thymus DNA: A Spectroscopic, Calorimetric, and Computational Approach.

A variety of anticancer and antibacterial drugs target DNA as one of their primary intracellular targets. Understanding ligand-DNA interactions and developing new, promising bioactive molecules for clinical use are greatly aided by elucidating the interaction between small molecules and natural polymeric DNAs. Small molecules' ability to attach to and inhibit DNA replication and transcription provides more information on how drugs impact the expression of genes. Yohimbine has been broadly studied in pharmacological properties, while its binding mode to DNA has not been explicated so far. In this study, an attempt was made to explore the interaction between Yohimbine (YH) and calf thymus (CT-DNA) by using varying thermodynamics and in silico approaches. Minor hypochromic and bathochromic shifts of fluorescence intensity were observed, suggesting the binding of YH to CT-DNA. The Scatchard plot analysis using the McGhee-von Hipple method revealed noncooperative binding and affinities in the range of 105 M-1. The binding stoichiometry value is 2:1 (2 molecules of YH were span by 1 base pair) and was determined by Job's plot. The thermodynamic parameters suggested exothermic binding, which was favored by negative enthalpy and positive entropy changes from both isothermal titration calorimetry and temperature-dependent fluorescence experiment. Salt-dependent fluorescence suggested that the interaction between the ligand and DNA was governed by nonpolyelectrolytic forces. Kinetics experiment confirmed the static type of quenching. The results of iodide quenching, urea denaturation assay, dye displacement, DNA melting, and in silico molecular docking (MD) suggested groove binding of YH to CT-DNA. Circular dichroism spectra confirmed minimal perturbation of CT-DNA with YH binding via groove region. Therefore, the groove binding mechanism of interaction was validated by biophysical techniques and in silico, MD approaches. The findings supported here may contribute to the development of new YH therapeutics possessing better efficacy and lesser side effects.

Biophysical and molecular modeling evidences for the binding of sulfa molecules with hemoglobin.

The molecular mechanism of the heme protein, hemoglobin (Hb) interaction with sulfa molecule, sulfadiazine (SDZ) has been investigated through spectroscopic, neutron scattering and molecular modeling techniques. Absorption and emission spectroscopic studies showed that SDZ molecules were bound to Hb protein, non-cooperatively. The binding affinityof SDZ-Hb complex at standard experimental condition was evaluated to be around (4.2 ± 0.07) ×104, M-1with 1:1 stoichiometry. Drug induced structural perturbation of the 3 D protein moiety was confirmed through circular dichroism (CD), synchronous fluorescence and small angle neutron scattering methods. From the temperature dependent spectrofluorometric studies, the negative standard molar Gibbs energy change suggested the spontaneity of the reaction. The negative enthalpy and positive entropy change(s) indicated towards the involvement of both electrostatic and hydrophobic forces during the association process. Salt dependent fluorescence study revealed major contributions from non-poly-electrolytic forces. Molecular modeling studies determined the probable binding sites, types of interaction involved and the conformational alteration of the compactness of the Hb structure upon interaction with SDZ molecule. Overall, the study provides detailed insights into the binding mechanism of SDZ antibiotics to Hb protein.Communicated by Ramaswamy H. Sarma.

Application of Drug Aggregation to Solubilize Antimicrobial Compound and Enhancing its Bioavailability.

With the progress and advancement in discovery of novel antimicrobial drugs, efficient solubility plays an important component for a drug to express its out-turn effectively. A biocompatible neutral/non-ionic surfactant, Triton X-100 (Tx-100), was successfully employed to solubilize an antibiotic drug, sulfamethazine (SMZ), through micellization process. The association process of Tx-100 toward SMZ was confirmed through the characteristic spectral change in absorption and emission spectroscopy. The morphological behavior of the complex was studied from small angle neutron scattering (SANS). Changes in size(s) and charge(s) of the micelles were monitored using zeta (z) potential technique. This present study emphasized the molecular mechanism and characteristics of Tx-100 as an effective drug solubilizing and carrier agent. Thus, the drug-loaded micellar system can enhance cellular uptake and increase the antibacterial effects of drugs in the biological system(s). Schematic illustration of drug-surfactant micelle formation and target release of drug at the targeted site.

Heme Protein Binding of Sulfonamide Compounds: A Correlation Study by Spectroscopic, Calorimetric, and Computational Methods.

Protein-ligand interaction studies are useful to determine the molecular mechanism of the binding phenomenon, leading to the establishment of the structure-function relationship. Here, we report the binding of well-known antibiotic sulfonamide drugs (sulfamethazine, SMZ; and sulfadiazine, SDZ) with heme protein myoglobin (Mb) using spectroscopic, calorimetric, ζ potential, and computational methods. Formation of a 1:1 complex between the ligand and Mb through well-defined equilibrium was observed. The binding constants obtained between Mb and SMZ/SDZ drugs were on the order of 104 M-1. SMZ with two additional methyl (-CH3) substitutions has higher affinity than SDZ. Upon drug binding, a notable loss in the helicity (via circular dichroism) and perturbation of the three-dimensional (3D) protein structure (via infrared and synchronous fluorescence experiments) were observed. The binding also indicated the dominance of non-polyelectrolytic forces between the amino acid residues of the protein and the drugs. The ligand-protein binding distance signified high probability of energy transfer between them. Destabilization of the protein structure upon binding was evident from differential scanning calorimetry results and ζ potential analyses. Molecular docking presented the best probable binding sites of the drugs inside protein pockets. Thus, the present study explores the potential binding characteristics of two sulfonamide drugs (with different substitutions) with myoglobin, correlating the structural and energetic aspects.

Serum levels of interleukin 1 and tumor necrosis factor alpha correlate with peritoneal adhesion grades in humans after major abdominal surgery.

Peritoneal adhesions are a leading cause of potential morbidity and mortality. We undertook this prospective study to determine the clinical relevance of interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) levels as biological markers for peritoneal adhesion formation in humans. Fifteen patients who had previous colectomies and were undergoing re-exploration for an elective vascular procedure were studied. Blood samples were collected from each patient preoperatively and 30 minutes after the abdominal incision was made. Serum levels of IL-1 and TNF-alpha were determined using enzyme-linked immunosorbent assay kits. Adhesions were graded using an adhesion scale of 0 (none), 1 (mild), 2 (moderate), and 3 (extensive, dense). Preoperative levels of IL-1 and TNF-alpha did not differ significantly among all patients (IL-1 level was 60 +/- 14 pg/mL, and TNF-alpha level was 45 +/- 11 pg/mL; mean +/- standard deviation). Significant correlation was observed between grades of adhesions and early intraoperative levels of IL-1 [101 +/- 36 pg/mL for grade 1 (n = 8) vs 298 +/- 73 pg/mL for grade 3 (n = 6); P < 0.01] and TNF-alpha (88 +/- 23 pg/mL for grade 1 vs 261 +/- 88 mL for grade 3; P < 0.02). We conclude that early elevations of IL-1 and TNF-alpha are reliable biological markers for postoperative adhesions in humans. Studies utilizing cytokines antibodies to these markers may further elucidate the efficacy of this method for prevention of peritoneal adhesions.