A molecular docking study of estrogenically active compounds with 1,2-diarylethane and 1,2-diarylethene pharmacophores

Numerous selective estrogen receptor modulators (SERMs) have been synthesized and assayed in recent years. The focus of this study is to apply coarse-grain molecular docking procedures coupled with fine-grain all-atom force field optimization strategies to shed light on the binding mechanisms of currently available estrogen receptor-active compounds. Although the mechanics of ligand binding in estrogen receptors is generally well understood, there is room for surprises. In this paper computational evidence corroborating the experimentally observed type I agonistic binding mode for estradiol (E2) and diethylstilbesterol (DES) and the type II antagonistic binding mode for 4-hydroxytamoxifen and raloxifen is presented. Included in this type I agonistic mode are the DES derivatives, transstilbene and 1,2-diaryldiaminoethane. In addition, a novel ‘type II agonistic’ binding mode for 2,3-diarylimidazolines, 4,5-diarylimidazoles, 2,3-diarylpiperazines is introduced. This mode is stabilized by suggesting alternative hydrogen bond anchor points in the ligand binding domain as potential leads for future drug design.     

Bicyclic peptidomimetic tetrahydrofuro[3,2-b]pyrrol-3-one and hexahydrofuro[3,2-b]pyridine-3-one based scaffolds: synthesis and cysteinyl proteinase inhibition

A stereoselective synthesis of (3aS,6aR)-tetrahydrofuro[3,2-b]pyrrol-3-ones and (3aS,7aR)-hexahydrofuro[3,2-b]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13. A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis-fused geometry in comparison to that of the corresponding trans-fused. Since the bridgehead stereocentre situated beta to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10, 45a-e, 11 and 46. Both series were chirally stable with 5,5-series 10 and 45a-e exhibiting potent in vitro activity against a range of CAC1 cysteinyl proteinases. Compound 10, a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance

We present a novel method for the comparison of multiple protein alignments with assessment of statistical significance (COMPASS). The method derives numerical profiles from alignments, constructs optimal local profile-profile alignments and analytically estimates E-values for the detected similarities. The scoring system and E-value calculation are based on a generalization of the PSI-BLAST approach to profile-sequence comparison, which is adapted for the profile-profile case. Tested along with existing methods for profile-sequence (PSI-BLAST) and profile-profile (prof_sim) comparison, COMPASS shows increased abilities for sensitive and selective detection of remote sequence similarities, as well as improved quality of local alignments. The method allows prediction of relationships between protein families in the PFAM database beyond the range of conventional methods. Two predicted relations with high significance are similarities between various Rossmann-type folds and between various helix-turn-helix-containing families. The potential value of COMPASS for structure/function predictions is illustrated by the detection of an intricate homology between the DNA-binding domain of the CTF/NFI family and the MH1 domain of the Smad family.     

The speed of soil carbon throughput in an upland grassland is increased by liming

In situ (13)C pulse labelling was used to measure the temporal and spatial carbon flow through an upland grassland. The label was delivered as (13)C-CO(2) to vegetation in three replicate plots in each of two treatments: control and lime addition. Harvests occurred over a two month period and samples were taken along transects away from the label delivery area. The (13)C concentration of shoot, root, bulk soil, and soil-respired CO(2) was measured. There was no difference in the biomass and (13)C concentration of shoot and root material for the control and lime treatments meaning that the amount of (13)C-CO(2) assimilated by the vegetation and translocated below ground was the same in both treatments. The (13)C concentration of the bulk soil was lower in the lime treatment than in the control and, conversely, the (13)C concentration of the soil-respired CO(2) was higher in the lime. Unlike the difference in bulk soil (13)C concentration between treatments, the difference in the (13)C concentration of the soil-respired CO(2) was obvious only at the delivery site and primarily within 1 d after labelling. An observed increase in the abundance of mycorrhizal fungi in the lime treatment was a possible cause for this faster carbon throughput. The potential key role of mycorrhizas in the soil carbon cycle is discussed. The importance of a better understanding of soil processes, especially biological ones, in relation to the global carbon cycle and environmental change is highlighted.     

Either of the CD45RB and CD45RO isoforms are effective in restoring T cell,but not B cell,development and function in CD45-null mice

The protein tyrosine phosphatase CD45 is expressed as a series of isoforms whose tissue and differentiation stage specificity is broadly conserved in evolution. CD45 has been shown to be an important regulator of a variety of functions in many different hemopoietic lineages. We have chosen an in vivo genetic complementation strategy to investigate the differential functions between isoforms. In this study, we report the characterization of transgenic mice which express the isoforms CD45RO or CD45RB as their only CD45 molecules, at a variety of expression levels and in the majority of hemopoietic lineages. Both CD45RO and CD45RB isoforms reconstitute thymocyte development in a CD45-null mouse background when expressed above a threshold level. The resulting mature T cells populate the peripheral lymphoid organs where they are found at normal frequency. Both CD45RO and CD45RB isoforms also permit T cell function in the periphery, although the threshold for normal function here appears to be set higher than in the thymus. In contrast, neither isoform is capable of fully restoring peripheral B cell maturation, even at levels approaching those in heterozygous CD45(+/-) mice in which maturation is normal. In vitro activation of B cells by Ag-receptor stimulation is only minimally complemented by these CD45RO and CD45RB transgenes. Our results suggest that CD45 isoforms play unique roles which differ between the T and B lineages.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.     

Structural characterization of a human cytosolic NMN/NaMN adenylyltransferase and implication in human NAD biosynthesis

Pyridine dinucleotides (NAD and NADP) are ubiquitous cofactors involved in hundreds of redox reactions essential for the energy transduction and metabolism in all living cells. In addition, NAD also serves as a substrate for ADP-ribosylation of a number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing regulation, and for cyclic ADP ribose (cADPR)-dependent Ca(2+) signaling. Pyridine nucleotide adenylyltransferase (PNAT) is an indispensable central enzyme in the NAD biosynthesis pathways catalyzing the condensation of pyridine mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or NaAD). Here we report the identification and structural characterization of a novel human PNAT (hsPNAT-3) that is located in the cytoplasm and mitochondria. Its subcellular localization and tissue distribution are distinct from the previously identified human nuclear PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the catalytic mechanism of the enzyme. The characterization of the cytosolic human PNAT-3 provided compelling evidence that the final steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and mitochondria, potentially contributing to their NAD/NADP pool.