Relative Binding Order Research Articles

Evaluation of Sorption Coefficients for Pyroxasulfone, s-Metolachlor, and Dimethenamid-p

Sorption coefficients were evaluated for pyroxasulfone, s-metolachlor, and dimethenamid- p across 25 soil types with different chemical and physical properties to better understand how soil properties influence the binding of pyroxasulfone in different soils. Sorption coefficients were determined using a batch equilibrium method to evaluate relative differences in binding among the three herbicides. Based on water solubility values, we would have expected the relative order of binding to be dimethenamid- p (1450 mg L−1 at 20°C), s-metolachlor (530 mg L−1 at 20°C), and pyroxasulfone (3.49 mg L−1 at 20°C) in order of increasing binding. However, when sorption coefficients were calculated, we observed the order of pyroxasulfone = dimethenamid- p < s-metolachlor in the order of increasing binding. The average Kd(sorption coefficient) values were 1.7, 2.3, and 4.0 L kg−1 for pyroxasulfone, dimethenamid- p, and s-metolachlor, respectively. Although dimethenamid- p has water solubility values that are over 400 times greater than pyroxasulfone, there was no statistical difference in binding between pyroxasulfone and dimethenamid- p. s-Metolachlor binding was statistically greater than both pyroxasulfone and dimethenamid- p. Across all soil chemical and physical properties, sorption coefficients for all three herbicides were highly and statistically correlated to soil organic matter. Sand and silt were also statistically correlated to binding, although these correlations could be explained by the high correlation of organic matter to these properties. Evaluation of sorption coefficients indicates that pyroxasulfone is most prevalent in the soil solution where herbicides are available for plant uptake. Reduced soil binding and greater activity at the target site could contribute to comparable weed control efficacies of pyroxasulfone even when applied at lower use rates compared to either s-metolachlor or dimethenamid- p.

The thermochemical studies of protonated amine–crown ether complexes: Extension of the kinetic method

We have devised a new approach that extends the kinetic method to systems where the competitive dissociation (the cornerstone of the kinetic method) will occur not at one binding center but at two spatially different places. This approach was tested on dissociation of complexes of two symmetric doubly protonated diamines with seven different crown ethers resulting in the relative binding order of alkylammonium cation (half of the protonated diamine dication) to the crown ethers. For the experiments with 1,10-diaminodecane, leading to the n-alkylammonium binding ladder, the order was dicyclohexano-18-crown-6>dibenzo-21-crown-7>18-crown-6>dibenzo-24-crown-8>dibenzo-18-crown-6>15-crown-5>12-crown-4. When N,N′-dimethyl-1,8-octanediamine was used, the experiments gave rise to the relative binding of N-methyl-n-alkylammonium cation, which was dibenzo-24-crown-8>dicyclohexano-18-crown-6>dibenzo-21-crown-7>dibenzo-18-crown-6>18-crown-6>15-crown-5>12-crown-4. The observed binding trends were explained in terms of size of the alkylammonium cation, size of the cavity of the crown, flexibility of the crown substituents, and overall crown polarizability. Our data qualitatively correlate well to the literature gas-phase values for these systems as well as to the solution data (where available).

Competitive binding comparison of endocrine‐disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human

Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor alpha (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols.

Unusual covalent bond-breaking reactions of beta-cyclodextrin inclusion complexes of nucleobases/nucleosides and related guest molecules.

Host-guest complexes between nucleobases or nucleosides and beta-cyclodextrin can be observed by electrospray ionization mass spectrometry (ESI-MS) and their relative abundances appear to correlate with the condensed-phase binding order. Using Fourier transform ion cyclotron resonance mass spectrometry, the extent of the interactions between the host oligosaccharide and guest species have also been examined for permethylated beta-cyclodextrin : adenine/deoxyadenosine and permethylated maltoheptaose : adenine/deoxyadenosine using gas-phase exchange reactions with the gaseous amines, n-propylamine and ethylenediamine. The ease of guest exchange in the gas-phase follows the order : deoxyadenosine > adenine > deoxycytidine > cytosine, which is in contrast to their relative binding order in solution. Collision-induced dissociation (CID) has been used to probe the fragmentation behavior of oligosaccharide : nucleobase/nucleoside complexes. Under these conditions the inclusion complexes either (a) dissociate, (b) result in cleavage of the host oligosaccharide or (c) result in cleavage of the guest molecule. This study has shown that the preferred dissociation pathway of these complexes depends on the structures of both the cyclodextrin and guest molecule.

Development of a high-throughput fluorescence polarization assay for Bcl-x L

Antiapoptotic protein Bcl-x L has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x L inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x L and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x L with a K d of 21.48 nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC 50 values of 0.048 and 1.14 μM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x L, did not compete. The relative binding order of the peptides (Bad > Bak > Bcl-2 > Bax > mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z ′ factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x L–peptide interaction, but also for high-throughput screening of Bcl-x L inhibitors.

DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives.

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.

Solution and solid-state structures of phosphine adducts of monomeric zinc bisphenoxide complexes. Importance of these derivatives in CO2/epoxide copolymerization processes.

Phosphine derivatives of the monomeric zinc phenoxide complexes, (phenoxide)2ZnLn, where phenoxide equals 2,6-di-tert-butylphenoxide, 2,4,6-tri-tert-butylphenoxide, and 2,6-diphenylphenoxide and n = 1 or 2, have been synthesized from the reaction of Zn[N(SiMe3)2]2 and the corresponding phenol followed by the addition of phosphine. The complexes have been characterized in solution by 31P NMR spectroscopy and in selected instances in the solid-state by X-ray crystallography. The small, basic phosphine, PMe3, provided the only case of an isolated complex possessing two phosphine ligands (i.e., n = 2). For all other larger phosphines only the monophosphine adducts were obtained. Furthermore, only fairly basic phosphines were found to bind to zinc, e.g., whereas PPh3 (pKa = 2.73) was ineffective, PPh2Me (pKa = 4.57) did form a strong bond to zinc. The solid-state structures of the monophosphine adducts consist of a near-trigonal planar geometry about the zinc center, where the average P-Zn-O angles are larger than the O-Zn-O angles. On the other hand, the bisphosphine adduct, Zn(O-2,4,6-tBu3C6H2)(2).2PMe3, is a distorted tetrahedral structure with O-Zn-O and P-Zn-P bond angles of 108.8(2) degrees and 107.1(9) degrees, respectively. Competitive phosphine binding studies monitored by 31P NMR spectroscopy provided a relative binding order of PPh3 approximately PtBu3 << PPh2Me < PCy3 < PMe2Ph < PnBu3 < PEt3 < PMe3. Hence, the relative binding of basic phosphine ligands at these congested zinc sites is largely determined by their steric requirements. All phosphine adducts, with the exception of PMe2Ph and PMe3, were found to undergo slow self-exchange (< 600 s-1) with free phosphine by 31P NMR spectroscopy. However, the two small phosphines, PMe2Ph (cone angle = 122 degrees) and PMe3 (cone angle = 118 degrees), were shown to undergo rapid exchange presumably via an associative mechanism. Although there was no kinetic preferences for PCy3 binding to cadmium vs zinc, cadmium was thermodynamically favored by about a factor of 2.5. The addition of up to 3 equiv of PCy3 to the Zn(O-2,6-tBu2C6H3)2 or Zn(O-2,4,6-tBu3C6H2)2 derivatives did not significantly alter the reactivity of these catalysts for the copolymerization of cyclohexene oxide (CHO) and CO2 to high-molecular weight poly(cyclohexene carbonate). However, the presence of PCy3 greatly retarded their ability to homopolymerize CHO to polyether or to afford polyether linkages during the copolymerization of CHO/CO2.

Charged analogues of chlorpromazine as dopamine antagonists.

Chlorpromazine (1, CPZ) is a potent dopamine antagonist that has been used widely as an antipsychotic agent. Since dopaminergic antagonists, like dopaminergic agonists, exist in solution as the charged and uncharged molecular species, it is not clear which form of the amine is most important for interaction with the dopamine receptor. Previous work from our laboratory has indicated that a variety of permanently charged species could replace the amine/ammonium moiety of dopamine and retain dopamine agonist activity. This paper describes the synthesis and dopamine antagonist activity of both the trimethylammonium iodide and the dimethylsulfonium iodide analogues of chlorpromazine. The permanently uncharged methyl sulfide analogue was also synthesized; however, due to its lack of aqueous solubility, its pharmacological activity could not be evaluated. Binding of both the dimethylsulfonium iodide and the trimethylammonium iodide analogues to D-2 dopamine receptors of rat striatal tissue was observed. The observed relative order of binding was CPZ greater than CPZ sulfonium analogue greater than CPZ ammonium analogue. These compounds had a similar order of activity in antagonizing the apomorphine-induced inhibition of potassium-induced release of [3H]acetylcholine from mouse striatal slices.

In vitro binding of 14c-misonidazole to hepatocytes and hepatoma cells debora

The binding rate of 14C-Misonidazole was determined for freshly isolated mouse hepatocytes, mouse hepatoma cells, EMT-6 tumor cells, and V79 Chinese hamster lung fibroblasts. At 10 μM drug concentration, the four different cell lines bound 14C-Misonidazole at rates of 12.4, 29.9, 51.6, and 13.5 pmoles/106 cells/hr, respectively. This relative order of binding was observed over a drug concentration range of 10–100 μM. These data indicate that in extreme hypoxia, mouse hepatocytes do not bind 14C-Misonidazole at a uniquely high rate in vitro, compared to other normal and tumor cell lines, This observation suggests that the increased binding to liver in vivo observed by other investigators is due to the liver existing at a reduced oxygen tension, compared to other normal tissues.

Fluorescence polarization studies of the interactions between protamine and enzymes in aqueous solutions

Fluorescence polarization has been used to study the interaction of dansylated protamine with the enzymes: pepsin, α-chymotrypsin, alkaline phosphatase and invertase. These interactions have been compared with those between dansylated protamine and polyacrylate, or polyvinylsulphate. Each of the various complexes was found to be dissociated by the addition of sodium nitrate and a critical electrolyte concentration (CEC) was determined for each system, to allow assessment of the relative order of binding to the dansylated protamine. This order was: polyvinylsulphate >pepsin >polyacrylate >alkaline phosphatase >α-chymotrypsin. The strength of binding was also assessed by determination of a binding constant, K a . The values of K a showed the same relative order of binding as the CEC values. Invertase behaved similarly to the other enzymes, but it was not possible to obtain an unambiguous assessment of the comparative strength of binding. In each case, the stoichiometry of the complex was also determined.

Physical association of oestrogens and other steroids with DNA

14C-Labelled mestranol, 3-O-methyl oestrone and cholesterol-5α,6α-oxide have been prepared. Along with the natural oestrogens, E1 and E2, and other steroids, these compounds have been used to determine the extent of their physical association with DNA. Analysis of binding both by equilibrium solubilization and by caesium chloride density gradient centrifugation showed the same relative order of binding: mestranol > 3-O-methyl oestrone > oestrone > cholic acid > cholesterol-5α,6α-oxide > progesterone, testosterone > oestradiol. DNA from Micrococcus lysodeikticus showed a higher affinity for cholic acid than did calf thymus DNA, while pretreatment of the latter with proteinase K somewhat reduced the level of physical binding of oestrone.

Metal-ligand complexes involved in rheumatoid arthritis—III: Bovine serum albumin -copper(II), -zinc(II) and -lead(II) interactions investigated using potentiometric analysis and molecular filtration

The interactions between BSA and Cu 2+, Zn 2+ and Pb 2+ have been studied in aqueous solution (37°, I = 150 mmol dm −3) using potentiometric analysis and molecular filtration. Formation constants for BSA/20-protonation and zinc complexing were determined. The relative binding order for the metal ions studied was Cu > Zn > Pb. The magnitude of the BSA/20-Zn β values were verified by molecular filtration as an independent method.

Mercury(II) binding to s4U in E. coli tRNA(Val).

The accessibility of the s(4)U base in native tRNA(Val) from E.coli was monitored by studying the binding of various mercurials. The relative binding order HgBr(2)[unk]HgCl(2)>>CH(3)HgOAc[unk]CH(3)HgCl[unk]PCMB parallels approximately the steric requirements of linear HgX(2) or RHgX compounds for S(N)2 displacement by sulfur, although other factors are operative. Para-chloromercuri-benzoate (PCMB) does not bind the thiolated nucleotide unless the tertiary structure of the tRNA is opened up by removal of Mg(2+) ions and heating to 40 degrees . Under these conditions, equilibrium dialysis measurements using (14)C-labeled PCMB showed one binding site (n = 0.93) with an association constant, K(1), of 9 x 10(4)M(-1).