The binding of berenil to Escherichia coli ribosome.

The binding of the nonintercalating dye berenil to the 70S ribosome of Escherichia coli has been demonstrated by spectrophotometric measurements and gel filtration through Biogel P100 column. The berenil spectrum is gradually shifted towards the red region with the increasing amount of ribosome added, the isosbestic point being at 375 nm. There is positive cooperativity in the binding of berenil to the ribosome as demonstrated by the equilibrium dialysis. On binding with berenil, the ribosome is degraded faster by RNase I especially at low Mg++ concentration and its capacity to inhibit RNase I catalysed hydrolysis of ribopolymers is decreased. These indicate the unfolding of the structure of the ribosome.     

Irreversible inhibition of putrescine-stimulated S-adenosyl-L-methionine decarboxylase by berenil and pentamidine.

The putrescine-stimulated S-adenosyl-L-methionine decarboxylases from rat liver and yeast were strongly inhibited by Berenil and to a lesser extent by Pentamidine. Ten times greater drug concentrations were needed to achieve a similar level of inhibition of a Mg2+-stimulated bacterial enzyme. The inhibition was irreversible in that extensive dialyses or precipitation with (NH4)2SO4 did not restore enzyme activity. Putrescine did not protect the enzyme against Berenil, but adenosylmethionine either alone or with putrescine partially protected the irreversible action of Berenil. The compound 4,4'-diamidinodiphenylamine, which differs from Berenil only in lacking the azo group between benzene rings, was a weaker inhibitor than Berenil, and its inhibition was reversible. Berenil also inhibited the activity of adenosylmethionine decarboxylase in vivo, by depressing the activity of the enzyme in normal rat liver, for at least 24 h after a single injection (50 mg/kg body wt.) of the drug.     

Heterogeneous DNA binding modes of berenil.

Isothermal titration calorimetry (ITC) profiles of berenil bound to different DNAs show that, despite the strong preference of berenil for AT-rich regions in DNA, it can bind to other DNA sequences significantly. The ITC results were used to quantify the binding of berenil, and the thermodynamic profiles were obtained using natural DNAs as well as synthetic polynucleotides. ITC binding isotherms cannot be simply described when a single set of identical binding sites is considered, except for poly[d(A-T)2]. Ultraviolet melting of DNA and differential scanning calorimetry were also used to quantify several aspects of the binding of berenil to salmon testes DNA. We present evidence for secondary binding sites for berenil in DNA, corresponding to G+C rich sites. Berenil binding to poly[d(G-C)2] is also observed. Circular dichroism experiments showed that binding to GC-rich sites involves drug intercalation. Using a molecular modeling approach we demonstrate that intercalation of berenil into CpG steps is sterically feasible.     

Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins

The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV–blood protein were determined from fluorescence quenching studies. Stern–Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow’s site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11 % in free HHb to 38.34 % in STV-HHb, and from 66.44 % in free HSA to 52.26 % in STV–HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV–HHb and STV–HSA, respectively based on Föster’s non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8 %.     

Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins

Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process.     

In vitro trypanocidal activity of the anti-helminthic drug niclosamide

Only a few drugs are available for chemotherapy of African trypanosomiasis and there is an urgent need for the development of new anti-trypanosomal agents. In this study, the anti-helminthic drug niclosamide was tested for its trypanocidal activity in vitro using culture-adapted bloodstream forms of Trypanosoma brucei brucei and Trypanosoma congolense. The concentrations of niclosamide to reduce the growth rate by 50% and to kill all cells were in the low- and mid micromolar ranges for T. b. brucei and T. congolense, respectively. The very low toxicity of niclosamide for mammals makes the compound interesting for drug development for African trypanosomiasis.     

Comparison of binding sites in DNA for berenil, netropsin and distamycin. A footprinting study

Techniques of DNase I and micrococcal nuclease footprinting have been used to compare the binding sites for berenil, netropsin and distamycin on two different DNA fragments. Each ligand binds to the A + T-rich zones which contain clusters of at least four A.T base pairs. Neither guanosine nor cytidine nucleotides appear to be allowed within the A + T-rich runs which constitute the preferred binding sites, although they are sometimes protected from DNase I cleavage in neighbouring regions. Berenil and netropsin share with distamycin the property of causing enhanced rates of cleavage at certain sequences flanking their binding sites. There are significant differences in the concentrations of each ligand required to produce defined patterns of protection, seemingly dependent upon the nature (and possibly the gross base composition) of the piece of DNA being used in the experiment.     

Netropsin, distamycin and berenil interact differentially with a high-affinity binding site for the high mobility group protein HMG-I

Netropsin, distamycin, berenil and the chromosomal protein HMG-I share the ability to bind preferentially to AT-rich regions of DNA. We studied the binding behaviour of the chemical agents towards a high-affinity binding site for HMG-I by DNase I and MPE footprinting and analyzed their ability to challenge HMG-I-DNA complexes by competition experiments. Significant differences in the binding affinities and in the efficiencies to abolish HMG-I-DNA complexes were observed for the three drugs. Netropsin proved to be the most avidly binding compound and the most efficient competitor raising the interesting possibility that netropsin affects cell growth by interfering with HMG-I-DNA interaction.     

Comparative survey of blood thyroxine binding proteins in turtles

The nature of plasma thyroxine (T4) binding activity was surveyed in turtles; binding to [125I]T4 was measured on polyacrylamide gel electrophoresis--PAGE--and on minicolumns of Sephadex G-25. An electrophoretically distinct T4 binding protein was identified in all 8 species of Pseudemys studied and in 3 other genera (Chrysemys, Deirochelys, and Emyoidea) of the same family, Emydidae. Levels of this binding activity were highly variable among individuals, but they consistently showed a similar low relative mobility (Rf) compared to albumin, and a relatively low capacity was indicated by displacement with unlabeled T4. Two emydids (Terrapene, Clemmys) showed a similar slow migrating binding peak, but binding activity was low and not as easily displaced by unlabeled T4. T4 binding to albumins was minimal in most of these emydid species, even when binding to the higher affinity, low capacity component was low or displaced by unlabeled T4 (2.5 micrograms/ml). In contrast, there was no clear evidence for a similar high affinity, low capacity binding protein in any of the other 19 species representing 13 genera of 8 families from two suborders. In these species, binding activity on Sephadex G-25 was typically low and binding on PAGE was associated largely with albumin; binding levels for albumins were highly variable. In several nonemydids (from distant lineages), binding activity on Sephadex was elevated and PAGE showed a second binding protein distinct from albumin, but it had high capacity (not readily saturable). Thus, an evolutionary divergence in T4 transport proteins is suggested within Chelonia.     

Irreversible alterations in the hemoglobin structure affect oxygen binding in human packed red blood cells

The ability of hemoglobin (Hb) to transport respiratory gases is directly linked to its quaternary structure properties and reversible changes between T (tense) and R (relax) state. In this study we demonstrated that packed red blood cells (pRBCs) storage resulted in a gradual increase in the irreversible changes in the secondary and quaternary structures of Hb, with subsequent impairment of the T↔R transition. Such alteration was associated with the presence of irreversibly settled in the relaxed form, quaternary structure of Hb, which we termed R′. On the secondary structure level, disordered protein organization involved formation of β-sheets and a decrease in α-helices related to the aggregation process stabilized by strong intermolecular hydrogen bonding. Compensatory changes in RBCs metabolism launched to preserve reductive microenvironment were disclosed as an activation of nicotinamide adenine dinucleotide phosphate (NADPH) production and increased reduced to oxidized glutathione (GSH/GSSG) ratio. For the first time we showed the relationship between secondary structure changes and the occurrence of newly discovered R′, which through an artificial increase in oxyhemoglobin level altered Hb ability to bind and release oxygen.     

In vitro triiodothyronine binding to cytoplasmic proteins from human red blood cells.

In vitro incubations of cytosol proteins from human red blood cells with [¹²⁵I] labelled L-3,5,3′ triiodothyronine demonstrated the existence of high affinity and limited capacity binding sites for T₃. At 4°C, the rate constant of association was 3 × 10⁷ M^?1h^?1, and the rate constant of dissociation was 9.10^?3h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 3 and 7.10^?10M. The maximum binding capacity was 1.4 f moles of L-3,5,3′ triiodothyronine per mg cytosol proteins. A close parallel between the biological pontency of the analogs of L-T₃ was observed.     

Effects of drug administration on gonadotropins, sex steroid hormones and binding proteins in humans

The possible mechanisms by which the administration of drugs may alter the gonadal function in humans are considered in this review. Based on personal data, and on data published in the literature, the following events may occur: (1) blockade of gonadal steroidogenesis; (2) interaction of drug(s) with the steroid-binding protein system in plasma, and (3) interference of drug(s) at the level of the feedback control of gonadotropin secretion. Representative examples of the above mechanisms are as following: (1) Ketoconazole possesses inhibitory effects in vitro on cytochrome P-450. When given in adult males, it decreased the plasma concentrations of testosterone (T) and androstenedione and increased 17 alpha-hydroxyprogesterone levels, suggesting that this drug acts in vivo on gonadal steroidogenesis by blocking the 17,20-lyase. (2) Danazol is a progestagen with high affinity for sex steroid-binding protein (SBP); when given in high dosages in normal males, it increased rapidly the dialyzable fraction (percent protein unbound or free fraction) of T. This suggests that by interacting with the binding sites of SBP, danazol and/or its metabolites displace the fraction of T bound to SBP. However, in males as well as in females, the long-term administration of danazol decreased also the binding capacity of SBP, and consequently increased the free fraction of sex steroid hormones. (3) Dihydrotestosterone (DHT), the most active androgen in many target cells, given at therapeutic dosages to adult males, resulted in a decrease in plasma concentrations of luteinizing hormone (LH) and T, without any significant change in the percent of free T, even though the affinity of DHT for SBP is higher than that of T. This suggests that the main effect of DHT is to inhibit gonadotropin secretion at the central level. (4) Flutamide, a nonsteroidal antiandrogen, increased both LH and T levels, demonstrating its pure antiandrogenic activity on gonadotropin secretion. The consequence(s) of the effects of such drugs on the production, the metabolic clearance rate and the bioavailability of sex steroid hormones are discussed.