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Efficient Inhibition (efficient + inhibition)
Selected AbstractsBisubstrate Inhibitors of the Enzyme Catechol O -Methyltransferase (COMT): Efficient Inhibition Despite the Lack of a Nitro GroupCHEMBIOCHEM, Issue 9 2004Ralph Paulini A new generation of bisubstrate inhibitors for the S -adenosylmethionine- and magnesium ion-dependent enzyme catechol O -methyltransferase (COMT), feature binding affinities (IC50 values) in the double-digit nanomolar range despite the lack of a nitro group on the catechol moiety. Inhibitor potency does not directly correlate with the pKa value of the catechol HO groups and is strongly enhanced by hydrophobic aromatic substituents attached in a biaryl-type fashion to position 5 of the catechol ring. [source] Crystal structure of the parasite inhibitor chagasin in complex with papain allows identification of structural requirements for broad reactivity and specificity determinants for target proteasesFEBS JOURNAL, Issue 3 2009Izabela Redzynia A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and papain, a classic family C1 cysteine protease, has been crystallized. Kinetic studies revealed that inactivation of papain by chagasin is very fast (kon = 1.5 × 106 m,1·s,1), and results in the formation of a very tight, reversible complex (Ki = 36 pm), with similar or better rate and equilibrium constants than those for cathepsins L and B. The high-resolution crystal structure shows an inhibitory wedge comprising three loops, which forms a number of contacts responsible for the high-affinity binding. Comparison with the structure of papain in complex with human cystatin B reveals that, despite entirely different folding, the two inhibitors utilize very similar atomic interactions, leading to essentially identical affinities for the enzyme. Comparisons of the chagasin,papain complex with high-resolution structures of chagasin in complexes with cathepsin L, cathepsin B and falcipain allowed the creation of a consensus map of the structural features that are important for efficient inhibition of papain-like enzymes. The comparisons also revealed a number of unique interactions that can be used to design enzyme-specific inhibitors. As papain exhibits high structural similarity to the catalytic domain of the T. cruzi enzyme cruzipain, the present chagasin,papain complex provides a reliable model of chagasin,cruzipain interactions. Such information, coupled with our identification of specificity-conferring interactions, should be important for the development of drugs for treatment of the devastating Chagas disease caused by this parasite. [source] Inhibition of factor VIII with a partially inhibitory human recombinant monoclonal antibody prevents thrombotic events in a transgenic model of type II HBS antithrombin deficiency in miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2004M. Dewerchin Summary., Venous thromboembolic disease is a major cause of morbidity and mortality, necessitating antithrombotic therapy. A human monoclonal anti-factor (F)VIII antibody, LCL-mAb-LE2E9, produced by a lymphoblastoid cell line derived from a hemophilia A patient with inhibitor to wild-type but not mutant self FVIII, was previously reported to achieve efficient inhibition of thrombosis in an experimental vena cava thrombosis model in mice. Here, the antithrombotic efficacy of a recombinant DNA-derived version of this anti-FVIII antibody (rec-mAb-LE2E9) was tested in mice which carry a type II heparin binding site antithrombin deficiency mutation and display spontaneous chronic thrombosis in several sites including the penile vein of sexually active males. The recombinant anti-FVIII antibody (100 µg, repeated after 3 days) prevented thrombotic priapism in all treated males, whereas all control animals treated with saline (group of four animals) developed priapism within 6 days after mating (P < 0.05 for treated vs. saline). The rec-mAb-LE2E9 and the original LCL-mAb-LE2E9 were equally effective (five and seven males/group, respectively). These results confirm that FVIII inhibition represents a potent antithrombotic strategy, and show that both LCL-mAb-LE2E9 and rec-mAb-LE2E9 efficiently prevent thrombosis in a physiological model representative of thrombosis in patients with a severe prothrombotic risk. [source] BI-69A11-mediated inhibition of AKT leads to effective regression of xenograft melanomaPIGMENT CELL & MELANOMA RESEARCH, Issue 2 2009Supriya Gaitonde Summary The AKT/PKB pathway plays a central role in tumor development and progression and is often up-regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI-69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI-69A11 was performed in melanoma cells, a tumor type that commonly exhibits up-regulation of AKT. Treatment of the UACC903 human melanoma cells, harboring the PTEN mutation, with BI-69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI-69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP-90. BI-69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI-69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra-peritoneal injection of BI-69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI-69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors. [source] Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitorsPROTEIN SCIENCE, Issue 3 2010Susmita Khamrui Abstract For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed "mini-proteins," containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECIL -WCIS, ETIL -WCIS, and STIL -WCIS, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECIL -WCIS and STIL -WCIS behave like good inhibitors, ETIL -WCIS behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETIL -WCIS shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL -WCIS leading to a loss of canonical conformation, explaining its substrate-like behavior. Incorporation of this barrier back in ETIL -WCIS through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop-scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design. [source] Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fc, receptor I,directed immunotoxinARTHRITIS & RHEUMATISM, Issue 5 2003Joel A. G. Van Roon Objective To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin-conjugated antibody CD64,ricin A (CD64-RiA) directed toward the high-affinity receptor for IgG (Fc,RI), exploiting the capacity of Fc,RI to efficiently endocytose antibody which it has bound. Methods Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64-RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light-scatter patterns and CD14 and Fc,RI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64-RiA,induced cell death of macrophages affected their capacity to stimulate antigen-induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64-RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated. Results Inflammatory macrophages from RA SF expressed elevated levels of Fc,RI and were selectively eliminated by CD64-RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of Fc,RI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen-induced lymphocyte proliferation and a reduction in tumor necrosis factor , (TNF,) release. Consistent with these effects on SF macrophages, CD64-RiA also inhibited TNF, production, interleukin-1, production, and cartilage-degrading activity of RA synovial tissue explants. Conclusion Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through Fc,RI-directed immunotoxins could be a novel approach to the treatment of RA. [source] Down regulation of BRCA2 causes radio-sensitization of human tumor cells in vitro and in vivoCANCER SCIENCE, Issue 4 2008Dong Yu In order to study the role of BRCA2 protein in homologous recombination repair and radio-sensitization, we utilized RNA interference strategy in vitro and in vivo with human tumor cells. HeLa cells transfected with small-interfering BRCA2 NA (BRCA2 siRNA) (Qiagen) as well as negative-control siRNA for 48 h were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock- or negative-control siRNA transfected cells. DNA double strand break repair as measured by constant field gel-electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno-staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the non-homologous end joining proteins nor the phosphorylation of DNA-dependent protein kinase catalytic subunit was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in xenograft model with HeLa cells showed an efficient inhibition of in vivo tumor growth. Our results demonstrate down-regulation of BRCA2 leads to radio-sensitization mainly through the inhibition of homologous recombination repair type double-strand break repair; a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise. (Cancer Sci 2008; 99: 810,815) [source] | |||||||||||||