Synthesis and characterization of DNA minor groove binding alkylating agents.

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Characterization of DNA with an 8-oxoguanine modification.

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

Calorimetric and spectroscopic studies on the interaction of methimazole with bovine serum albumin.

The potential binding interaction(s) of the anti-thyroid drug methimazole (MMZ) with the protein bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) and UV-Visible, fluorescence and circular dichroism (CD) spectroscopic techniques. The binding of MMZ to BSA has been studied in both the presence and absence of added surfactants. Since, the ITC thermograms show the molar enthalpy of binding of MMZ and BSA to be zero within experimental error, either the enthalpy change of the binding interaction is zero or there is no binding occurring. The CD and the intrinsic fluorescence and life time spectra of BSA were unchanged by the addition of MMZ. This is also indicative of the absence of any significant interaction of MMZ with BSA.

Binding of naproxen and amitriptyline to bovine serum albumin: biophysical aspects.

Binding of the drugs naproxen (which is an anti-inflammatory) and amitriptyline (which is an anti-depressant) to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC), in combination with fluorescence and circular dichroism spectroscopies. Naproxen is observed to bind more strongly to BSA than amitriptyline. The temperature-dependent ITC results indicate the interaction of one molecule of naproxen with more than one protein molecule. On the other hand, amitriptyline binds to BSA with a reaction stoichiometry that varies from 1:1.2 to 1:2.9. The van't Hoff enthalpy, which is calculated from the temperature dependence of the binding constant, agrees well with the calorimetric enthalpy in the case of naproxen binding to BSA, indicating adherence to a two-state binding process. However, their disagreement in the case of amitriptyline indicates conformational changes in the protein upon ligand binding, as well as with the rise in temperature. The spectroscopic results did not suggest appreciable conformational changes as a result of binding; hence, the discrepancy could be attributed to the temperature-induced conformational changes. With increases in the ionic strength, a reduction in the binding affinity of naproxen to BSA is observed. This suggests the prevailing electrostatic interactions in the complexation process. The preponderance of the hydrophobic interactions in the binding of amitriptyline to BSA is indicated by the absence of any dependence of the ionic strength. A predominance of electrostatic interactions in the case of naproxen binding to BSA and that of hydrophobic interactions in the case of amitriptyline binding to BSA is further strengthened by the results of the binding experiments performed in the presence of ionic and nonionic surfactants. The binding parameters indicate that Triton X-100 blocks the hydrophobic binding sites on BSA, thereby altering the binding affinity of amitriptyline toward BSA. A partial overlap of the binding sites for these drugs is indicated by the binding parameters obtained in the titration of naproxen to the amitriptyline-BSA complex and vice versa. Thus, the results provide a quantitative understanding of the binding of naproxen and amitriptyline to BSA, which is important in understanding their effect as therapeutic agents individually and in combination therapy.

Elucidating the binding thermodynamics of 8-anilino-1-naphthalene sulfonic acid with the A-state of alpha-lactalbumin: an isothermal titration calorimetric investigation.

Isothermal titration calorimetry has been used to demonstrate that the heat profile associated with the binding of 8-anilino-1-naphthalene sulfonic acid (ANS) with the acid induced molten globule state (A-state) of alpha-lactalbumin (alpha-LA) is different from that with the native and denatured states of the protein. The results corroborate the spectroscopic observations that ANS binds more strongly to the partially folded states of the protein compared to that with the native and denatured states. ANS binds to the A-state of alpha-LA at two independent binding sites that remain nearly the same in the temperature range of 10-35 degrees C. The number of moles of ANS binding at site 1 at 10 degrees C is 14.0+/-0.2 and remains nearly the same with rise in temperature. However, the number of moles of ANS molecules binding at site 2 show an increase from 1.6+/-0.2 at 10 degrees C to 4.1+/-0.1 at 35 degrees C. The deviation of the slope of enthalpy-entropy compensation plot from unity and nonadherence to van't Hoff dictates implies that the binding sites on the A-state of alpha-LA for ANS are not well defined and specific; rather, these binding sites are formed due to greater exposure of hydrophobic clusters in the A-state of the protein. The results for the first time demonstrate the use of isothermal titration calorimetry in characterizing the A-state of alpha-LA both qualitatively and quantitatively.

Thermodynamic insights into the binding of Triton X-100 to globular proteins: a calorimetric and spectroscopic investigation.

The interaction of the nonionic surfactant Triton X-100 (TX-100) with two proteins (bovine serum albumin (BSA) and alpha-lactalbumin (alpha-LA)) has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry, and fluorescence and circular dichroism spectroscopies. All of the calorimetric transitions in BSA were partially reversible, while being two-state and reversible in the case of alpha-LA. TX-100 molecules do not reduce the thermal stability of the protein in the monomeric form. However, in the micellar form the protein might become thermally destabilized by the micelles depending upon the nature of the protein. Isothermal titration calorimetry has been used to demonstrate that TX-100 binds to BSA at two sets of sites with 4:1 stoichiometry in each case. The van't Hoff enthalpy calculated from the temperature dependence of the binding constant did not match with the calorimetric enthalpy indicating conformational change in the protein upon surfactant binding. The surfactant binds to alpha-LA with one class of binding site, and the thermal unfolding results indicate it to be a stronger destabilizer than BSA. The fluorescence, circular dichroism, and differential scanning calorimetric results corroborate well with each other. The effect of ionic strength on the binding parameters suggests that TX-100 can bind to the protein surface via both hydrophobic and polar interactions depending upon the nature of the protein. The physical chemistry underlying the interactions between TX-100 and proteins has been presented. The mode of interaction of TX-100 with proteins is via direct binding, which has been discussed quantitatively in this work.