4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells

The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designated as 4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs). PAIB-SAs are active in the submicromolar to low micromolar range showing selectivity toward CYP1A1-expressing MCF7 cells as compared to cells devoid of CYP1A1 activity such as MDA-MB-231 and HaCaT cells. The most potent, PAIB-SA 13, bearing a trimethoxyphenyl group on ring B blocks the cell cycle progression in G2/M phase, disrupts the microtubule dynamics and is biotransformed by CYP1A1 into CEU-638, its potent antimicrotuble counterpart. Structure-activity relationships related to PAIB-SOs and PAIB-SAs evidenced that PAIB-SOs and PAIB-SAs are true bioisosteric equivalents fully and selectively activatable by CYP1A-expressing cells into potent antimitotics.     

Weed biological control in the European Union: from serendipity to strategy

Biological control of weeds is a globally recognised approach to the management of some of the most troublesome invasive plants in the world. Accidental introductions of agents accounted for all weed biological control agent establishments in the European Union until 2010, but these examples include some current or emerging control successes both large and small, from the redistribution of the weevil Stenopelmus rufinasus Gyllenhal (Coleoptera: Curculionidae) for the control of small outbreaks of Azolla filiculoides Lam. (Azollaceae), to the large scale control provided by the cochineal insect Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae), used against some problematic prickly pears (Opuntia spp. (Cactaceae)), and the ragweed beetle Ophraella communa LeSage (Coleoptera: Chrysomelidae), against common ragweed, Ambrosia artemisiifolia L. (Asteraceae), which are providing benefits to an increasing number of Member States of the European Union. Recent programmes involving the intentional introduction of biological control agents against target weeds including Fallopia japonica (Hout.) Ronse Decr. (Polygonaceae), Impatiens glandulifera Royle (Balsaminaceae) and Acacia longifolia (Andrews) Willd (Fabaceae) show a shift from luck to judgement in the European Union. The inclusion of new weed targets on the European Invasive Species Regulation should lead to a growth in the profile and use of biological control which would be assisted by the publication of any successes from the few intentional introductions covered in this paper.     

AMD3100 is a potent antagonist at CXCR4(R334X) , a hyperfunctional mutant chemokine receptor and cause of WHIM syndrome

WHIM is an acronym for a rare immunodeficiency syndrome (OMIM #193670) caused by autosomal dominant mutations truncating the C-terminus of the chemokine receptor CXC chemokine receptor 4 (CXCR4). WHIM mutations may potentiate CXCR4 signalling, suggesting that the United States Food and Drug Administration (FDA)-approved CXCR4 antagonist AnorMED3100 (AMD3100) (also known as Plerixafor) may be beneficial in WHIM syndrome. We have tested this at the preclinical level by comparing Chinese hamster ovary (CHO) and K562 cell lines matched for expression of recombinant wild-type CXCR4 (CXCR4(WT)) and the most common WHIM variant of CXCR4 (CXCR4(R334X)), as well as leucocytes from a WHIM patient with the CXCR4(R334X) mutation versus healthy controls. We found that CXCR4(R334X) mediated modestly increased signalling (~2-fold) in all functional assays tested, but strongly resisted ligand-dependent down-regulation. AMD3100 was equipotent and equieffective as an antagonist at CXCR4(R334X) and CXCR4(WT) . Together, our data provide further evidence that CXCR4(R334X) is a gain-of-function mutation, and support clinical evaluation of AMD3100 as mechanism-based treatment in patients with WHIM syndrome.     

Genetically-engineered pig-to-baboon liver xenotransplantation: histopathology of xenografts and native organs

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4–7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4–7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.     

Methods of remote homology detection can be combined to increase coverage by 10% in the midnight zone

MOTIVATION: A recent development in sequence-based remote homologue detection is the introduction of profile-profile comparison methods. These are more powerful than previous technologies and can detect potentially homologous relationships missed by structural classifications such as CATH and SCOP. As structural classifications traditionally act as the gold standard of homology this poses a challenge in benchmarking them. RESULTS: We present a novel approach which allows an accurate benchmark of these methods against the CATH structural classification. We then apply this approach to assess the accuracy of a range of publicly available methods for remote homology detection including several profile-profile methods (COMPASS, HHSearch, PRC) from two perspectives. First, in distinguishing homologous domains from non-homologues and second, in annotating proteomes with structural domain families. PRC is shown to be the best method for distinguishing homologues. We show that SAM is the best practical method for annotating genomes, whilst using COMPASS for the most remote homologues would increase coverage. Finally, we introduce a simple approach to increase the sensitivity of remote homologue detection by up to 10%. This is achieved by combining multiple methods with a jury vote. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

pH dependence of the reduction-oxidation reaction of azurin with cytochrome c-551: role of histidine-35 of azurin in electron transfer

A fluorescence quenching experiment confirms that in the redox reaction between cytochrome c-551 and azurin, protein complexing is negligible. Azurin-pH indicator T-jump experiments show that Pseudomonas aeruginosa (Ps.) azurin exhibits a slow time constant, tau, in its return to pH equilibrium but Alcaligenes faecalis (Alc.) azurin does not. The decrease of l/tau with increasing pH shows that the rate-determining process is a slow transformation of the imidazolium form of histidine-35 from a conformation where it cannot ionize to one in which it can. The fast relaxation time constant of the redox reaction varies little with pH, but the slow time constant increased by a factor of approximately 2.5 increasing pH between pH 5 and pH 8. The corresponding amplitudes, especially the slow one, vary with pH. On the basis of all the present evidence it is concluded that, while some differences of redox reactivity do occur on protonation, these differences are not major. In general, the two proteins cyt c-551 and azurin react with each other with rates only weakly dependent upon pH. A classical pH titration was carried out on the reduced and oxidized form of Ps. and Alc. azurin with the result that two protons were released between pH 6 and pH 8, in the former from His-35 and -83 and in the latter from His-83 and Ala-1.     

Mechanism of reaction of peroxomethyl radicals with copper(II)(glycine)2 and copper(II)(glycylglycylglycine) in aqueous solutions

The reactions of O₂CH₃ radicals with Cu^II(glycine)₂ and Cu^II(GGG), GGG = glycylglycylglycine, in aqueous solutions were studied.The results demonstrate that the peroxyl radicals oxidize the copper complexes forming relatively stable intermediates of the type L_mCu^III-OOCH₃. These intermediates decompose via oxidation of the ligands glycine and GGG, respectively. Substituents on the alkyl of the peroxyl radical affect somewhat the kinetics of reaction but not the mechanism of oxidation. It is suggested that analogous reactions are probably contributing to the radical-induced deleterious biological processes.     

Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.     

Stoichiometry of LTbetaR binding to LIGHT

LTbetaR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTalpha1beta2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTbetaRIgG, and monomeric LTbetaR protein binding to both LIGHT and LTalpha1beta2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTalpha1beta2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTbetaRIgG and LTbetaR binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC-LS corresponded to a single LTbetaRIgG bound to a LIGHT trimer, or two LTbetaR bound per LIGHT. Sedimentation velocity of varied ratios of LTbetaR to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LTbetaR or LTbetaRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTbetaR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFbeta, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor-ligand complexation paradigm for the entire TNF family. The LTbetaR/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LTbetaR rather than trimerization as expected from the structural paradigm.