32 research outputs found
Biophysical characterization of the processes that drive ligand association with the minor groove of DNA
No full textEThOS - Electronic Theses Online ServiceGBUnited Kingdo
Quick and Sensitive UPLC-ESI-MS/MS Method for Simultaneous Estimation of Sofosbuvir and Its Metabolite in Human Plasma
No full textA simple, fast and highly sensitive RP-UPLC-MS/MS method was developed and validated for the simultaneous determination of sofosbuvir (SR) and its metabolite GS331007 in human plasma using ketotifen as an internal standard (IS). The separation was achieved on Acquity UPLC BEH C18 (50 × 2.1 mm, i.d. 1.7 µm, Waters, USA) column using acetonitrile:5 mM ammonium formate:0.1% formic acid (85:15:0.1% v/v/v) as a mobile phase at a flow rate of 0.35 mL/min in an isocratic elution. The Xevo TQD UPLC-MS/MS was operated under the multiple-reaction monitoring mode using positive electrospray ionization. Extraction with dichloromethane was used in the sample preparation. Method validation was performed as per the Food and Drug Administration (FDA) guidelines and the calibration curves of the proposed method were found to be linear in the range of 1–1000 ng/mL for SR and in the range of 10–1500 ng/mL for its metabolite (GS331007) with an elution time of 1.83 min. All validation parameters were within the acceptable range according to the bioanalytical methods validation guidelines. Furthermore, the obtained results of matrix effects indicate that ion suppression or enhancement from human plasma components was negligible under the optimized conditions. The proposed method can be applied in high-throughput analysis required for pharmacokinetic and bioequivalence studies in human samples
Ranking ligand affinity for the DNA minor groove by experiment and simulation
No full textThe structural and thermodynamic basis for the strength and selectivity of the interactions of minor-groove binders (MGBs) with DNA is not fully understood. In 2003 we reported the first example of a thiazole containing MGB that bound in a phase shifted pattern that spanned 6 base-pairs rather than the usual 4 (for tricyclic distamycin-like compounds). Since then, using DNA footprinting, nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and molecular dynamics, we have established that the flanking bases around the central 4 being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences
Rationalising sequence selection by ligand assemblies in the DNA minor groove : the case for thiazotropsin A
No full textDNA-sequence and structure dependence on the formation of minor groove complexes at 5′-XCTAGY-3′ by the short lexitropsin thiazotropsin A are explored based on NMR spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and qualitative molecular modeling. The structure and solution behaviour of the complexes are similar whether X = A, T, C or G and Z = T, A, I or C, CCTAGI being thermodynamically the most favoured (ΔG = -11.1 ± 0.1 kcal.mol-1). Binding site selectivity observed by NMR for ACTAGT in the presence of TCTAGA when both accessible sequences are concatenated in a 15-mer DNA duplex construct is consistent with thermodynamic parameters (ΙΔGΙACTAGT > ΙΔGΙTCTAGA) measured separately for the binding sites and with predictions from modeling studies. Steric bulk in the minor groove for Y = G causes unfavourable ligand-DNA interactions reflected in lower Gibbs free energy of binding (ΔG = -8.5 ± 0.01 kcal.mol-1). ITC and CD data establish that thiazotropsin A binds the ODNs with binding constants between 106 and 108 M-1 and reveal that binding is driven enthalpically through hydrogen bond formation and van der Waals interactions. The consequences of these findings are considered with respect to ligand self-association and the energetics responsible for driving DNA recognition by small molecule DNA minor groove binder
Quantitative determination of doxorubicin in the exosomes of A549/MCF-7 cancer cells and human plasma using ultra performance liquid chromatography-tandem mass spectrometry
No full textIn cancer therapy, exosomes efflux enhances resistance of cancer cells toward anticancer agents through mediating the transport of anticancer drugs outside the cells. In this study, a rapid, simple and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of Doxorubicin (DOX) in exosomes of cancer cells and human plasma using Ketotifen as an internal standard (IS). Plasma samples spiked with DOX and two cancer cell lines (A549 & MCF-7) were incubated with different concentrations of DOX and IS. The analytes were then extracted with methanol after protein precipitation and the chromatographic separation was carried out using a C18 column, with a mixture of acetonitrile–water- formic acid (85:15:0.1%, v/v/v) as mobile phase. Multiple reaction monitoring (MRM) was utilized to monitor the protonated precursor to product ion transitions of m/z 544.25 > 397.16 and m/z 310.08 > 96.97 for the quantification of DOX and IS, respectively. The method was linear over ranges of 1–1000 ng/mL for DOX in plasma and 2–1000 ng/mL for DOX in exosome samples. The lower limit of quantification of this method was 1 ng/mL, 2 ng/mL and 2 ng/mL in human plasma, A549 & MCF-7 cells respectively. Intra- and inter day precision of all quality control concentrations were less than 10.33% and the accuracy values ranged from −4.82 to 12.60%. The optimized UPLC-MS/MS method proved to be fast, specific, simple and highly sensitive and was successfully applied for the estimation of DOX in the exosomes of cancer cells and plasma. Keywords: Doxorubicin, Ultra performance liquid chromatography, Tandem mass spectrometry, Exosomes, Chemotherapy resistanc
Investigation of the Factors That Dictate the Preferred Orientation of Lexitropsins in the Minor Groove of DNA
No full textLexitropsins are small molecules that bind to the minor groove of DNA as antiparallel dimers in a specific orientation. These molecules have shown therapeutic potential in the treatment of several diseases; however, the development of these molecules to target particular genes requires revealing the factors that dictate their preferred orientation in the minor grooves, which to date have not been investigated. In this study, a distinct structure (thzC) was carefully designed as an analog of a well-characterized lexitropsin (thzA) to reveal the factors that dictate the preferred binding orientation. Comparative evaluations of the biophysical and molecular modeling results of both compounds showed that the position of the dimethylaminopropyl group and the orientation of the amide links of the ligand with respect to the 5'-3'-ends; dictate the preferred orientation of lexitropsins in the minor grooves. These findings could be useful in the design of novel lexitropsins to selectively target specific genes.</p
Structure-based drug design of DNA minor groove binders and evaluation of their antibacterial and anticancer properties
No full textAntimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.</p
Recognition of the DNA minor groove by thiazotropsin analogues
No full textSolution-phase self-association characteristics and DNA molecular recognition properties are reported for three close analogues of minor groove binding ligands from the thiazotropsin class of lexitropsin molecules, which incorporate isopropyl thiazole as a lipophilic building block. Thiazotropsin B (AcImPyiPrThDp) shows similar self-assembly characteristics to thiazotropsin A (FoPyPyiPrThDp) although being engineered, by incorporation of imidazole in place of N-methyl pyrrole, to swap its DNA recognition target from 5´-ACTAGT-3´ to 5´-ACGCGT-3´. Replacement of the formamide head-group in thiazotropsin A by nicotinamide in AIK-18/51 results in a measureable difference in solution phase self-assembly character and substantially enhanced DNA association characteristics. The structures and associated thermodynamic parameters of self-assembled ligand aggregates and their complexes with respective DNA targets are considered in the context of cluster targeting of DNA by minor groove complexes
Investigation of the Factors That Dictate the Preferred Orientation of Lexitropsins in the Minor Groove of DNA
No full textLexitropsins are
small molecules that bind to the minor groove
of DNA as antiparallel dimers in a specific orientation. These molecules
have shown therapeutic potential in the treatment of several diseases;
however, the development of these molecules to target particular genes
requires revealing the factors that dictate their preferred orientation
in the minor grooves, which to date have not been investigated. In
this study, a distinct structure (thzC) was carefully designed as
an analog of a well-characterized lexitropsin (thzA) to reveal the
factors that dictate the preferred binding orientation. Comparative
evaluations of the biophysical and molecular modeling results of both
compounds showed that the position of the dimethylaminopropyl group
and the orientation of the amide links of the ligand with respect
to the 5′–3′-ends; dictate the preferred orientation
of lexitropsins in the minor grooves. These findings could be useful
in the design of novel lexitropsins to selectively target specific
genes
Exploring novel thiazole-based minor groove binding agents as potential therapeutic agents against pathogenic Acanthamoeba castellanii
No full textDue to limited advances in diagnosis and targeted therapy, as well as poor understanding of pathophysiology, infections due to Acanthamoeba have remained a medical concern. With their ability to selectively bind to DNA sequences, minor groove binders have emerged as useful therapeutic agents against parasitic infections. Herein, 6 novel thiazole-based minor groove binders were synthesized. Purification of intermediate compounds was accomplished by utilising silica gel column chromatography, while thin-layer chromatography was utilised to monitor reactions. The purification of the final products was achieved using liquid chromatography. Confirmation of structures was achieved by NMR spectroscopy and mass spectrometry. All compounds were evaluated against pathogenic A. castellanii via in vitro assays. At micromolar concentrations, selected minor groove binder derivatives revealed potent effects against (i) A. castellanii trophozoites as observed using amoebicidal assays, (ii), against A. castellanii cysts as observed using excystation assays, and (iii) against A. castellanii-mediated host cell death utilising human cerebrovascular endothelial cells, but (iv) showed limited effects against host cells alone, using cytotoxicity assays. The binding interaction between minor groove binders and DNA was studied using isothermal titration calorimetry and molecular docking simulations to provide insights into their binding affinity and mode of interaction. The findings of our study underscore the therapeutic value of thiazole-based minor groove binders as potent agents against A. castellanii, demonstrating effective antiamoebic activity with a low propensity for human cell damage, thus supporting their further development as antiamoebic agents.</p
