A real challenge of leadership today is to encourage the citizens of nation states to understand the extent to which their imagined control over events and their sovereignty has to be shared with ...others in order to be meaningful and effective. That is far from easy in a world where identity politics still dominates so many societies. But the problems that crowd in on nation states are beyond their capacity to deal with on their own. Terrorism, organised crime, epidemic disease and especially climate change all demand a multilateral approach. We need to co-operate at a more profound, wider and deeper level than ever before.
P-glycoprotein (P-gp) is a transmembrane efflux transporter which possesses many important functions in drug absorption, disposition, metabolism, and toxicity. The ultimate goal of investigating drug ...interactions between P-gp and drug molecules in early drug discovery is to understand the contribution of P-gp to the pharmacokinetic and pharmacodynamic properties of drug candidates and to project drug-drug interaction (DDI) potentials in humans. Understanding species differences in P-gp activities further helps the prediction of P-gp-mediated drug disposition and DDI in humans from preclinical pharmacokinetics data. The objective of the present study is to investigate the species difference in P-gp activities, via P-gp ATPase assays, using rhesus monkey Mdr1, beagle dog Mdr1, and human MDR1 expressed insect cell membranes. Twenty-one compounds with diverse chemical structures and different P-gp binding sites were chosen for the ATPase assays. P-gp ATPase binding affinities (alphaKa) and fold increases in P-gp ATPase activities (beta) of P-gp substrates were determined. Consistent with the gene and amino acid similarity, the binding affinities of test compounds to rhesus monkey P-gp were much closer to those of human P-gp than beagle dog P-gp. This is the first study which investigates the ligand affinities of P-gp from three different species. The result of this study provides an example of how to use membrane P-gp ATPase assays to evaluate interspecies P-gp differences.
Acetaminophen (APAP), a commonly used analgesic, is catalyzed by cytochrome P450 (P450) enzymes to a toxic intermediate which can be trapped by glutathione. Using this approach, involvement of ...enzymes in the activation of APAP and their kinetics were studied. With human liver microsomes, there were three apparent Km values (approximately 10,474, and 13,000 microM) for the oxidation of APAP to its glutathione conjugate. With rat liver microsomes (control and ethanol induced) the kinetic data were best fit to a two-Km model (approximately 30 and 1100 microM). Liver microsomes from dexamethasone (DEX)-treated female rats showed a single Km of 56 microM and a Vmax of 7500 pmol of product formed/(min.mg of protein). Antibodies specific for rat P450s 2E1 and 1A2 each inhibited approximately 40% of the APAP metabolism in control male rat microsomes. Only slight inhibition was observed with the P450 3A1/2 antibodies in control male or female rat liver microsomes. Antibodies against rat P450s 3A1/2 inhibited the activity in DEX microsomes by 80%. Antibodies inhibitory to human P450 3A4 inhibited 38% of the activity in human liver microsome sample HL107 and 76% in human microsome sample HL110. Human P450s (2A6, 2E1, 1A2, 3A4, 3A5, 3A3, 2D6, 2F1, 2C8, 2B6, and 2C9) expressed in Hep G2 cells using a vaccinia virus expression system were each tested for APAP metabolism. Of these, P450 2E1, 1A2, and 3A4 showed substantial activity, with respective Km and Vmax values of 680 microM and 330 pmol/(min.mg) for P450 2E1 (with added cytochrome b5), 3430 microM and 74 pmol/(min.mg) for P450 1A2, and 280 microM and 130 pmol/(min.mg) for P450 3A4.
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors ...binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the ...endogenous
-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (
), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.
THE RISE OF CHINA Patten, Chris
The RUSI journal,
06/2010, Volume:
155, Issue:
3
Journal Article
Peer reviewed
Open access
China is often described as a rising economic giant. Former Governor of Hong Kong Chris Patten argues that China has long been a global powerhouse, and that coming to terms with its spectacular and ...ongoing growth requires a coherent approach that will most successfully be led by a more unified European perspective.
•BET Bromodomain inhibition reduces dendritic cell maturation and cytokine responses in mouse and human cells.•BET Bromodomain inhibition specifically enhances the generation of regulatory T cells ...that possess suppressive capacity.•BET Bromodomains regulate the potential of human dendritic cells to enhance proliferation of antigen specific T cells.
Transcription of inflammatory genes is tightly regulated by acetylation and deacetylation of histone tails. An inhibitor of the acetylated-lysine reader bromodomain and extra-terminal domain (BET) proteins, I-BET151, is known to counteract the induction of expression of inflammatory genes in macrophages. We have investigated the effects of I-BET151 on dendritic cell function, including expression of co-stimulatory molecules and cytokines, and capacity for T cell activation.
Treatment of mouse bone marrow derived dendritic cells (BMDC) and human monocyte derived DCs (mdDC) with I-BET151 reduced LPS-induced expression of co-stimulatory molecules, as well as the production of multiple cyokines and chemokines. Most strikingly, secretion of IL-6, IL-12 and IL-10 was significantly reduced to 89.7%, 99.9% and 98.6% respectively of that produced by control cells.
I-BET151-treated mdDC showed a reduced ability to stimulate proliferation of autologous Revaxis-specific T cells. Moreover, while I-BET151 treatment of BMDC did not affect their ability to polarise ovalbumin specific CD4+ CD62L+ naive T cells towards Th1, Th2, or Th17 phenotypes, an increase in Foxp3 expressing Tregs secreting higher IL-10 levels was observed. Suppression assays demonstrated that Tregs generated in response to I-BET151-treated BMDC displayed anti-proliferative capacity. Finally, evidence that I-BET151 treatment can ameliorate inflammation in vivo in a T cell dependent colitis model is shown.
Overall, these results demonstrate marked effects of BET inhibition on DC maturation, reducing their capacity for pro-inflammatory cytokine secretion and T cell activation and enhancing the potential of DC to induce Foxp3 expressing Treg with suppressive properties.
A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted ...with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.
Chris Patten was European commissioner for external affairs from 2000 to 2004 and the last British governor of Hong Kong from 1992 to 1997. In an interview, Patten comments on Iran's pursuit of ...enriching uranium, the likelihood that China would support the US's policy toward China in a UN vote, whether Iran's objective is to ultimately obtain a nuclear weapon, and the need to toughen the non-proliferation treaty.
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