Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue.

BACKGROUND: Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. RESULTS: Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1) inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. CONCLUSION: The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

Cloning and pharmacological characterization of CCR7, CCL21 and CCL19 from Macaca fascicularis.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play an important role in lymphocyte homing and have also been associated with inflammatory, allergic and lung disorders. Cloning of the cynomolgus monkey genes encoding CCR7, CCL19 and CCL21 revealed 93-97% sequence identity of the deduced proteins with their respective human homologs. In chemotaxis assays, B300-19 cells transfected with the cynomolgus (c) CCR7 receptor migrated in response to cCCL19 and cCCL21 in a dose-dependent manner with EC(50) values of 324+/-188nM and 247+/-29nM, respectively. cCCL19 and cCCL21 also elicited calcium responses in stable cell CHO-K1 lines expressing the cCCR7 receptor with EC(50) values of 227+/-4nM and 484+/-163nM, respectively. Although both human (h) CCL19 and hCCL21 elicited increases in intracellular calcium at the cCCR7 receptor, hCCL19 almost completely inhibited subsequent stimulation by hCCL21 whilst hCCL21 failed to inhibit subsequent stimulation by hCCL19. These results identify novel cynomolgus monkey genes and provide a model system for pre-clinical studies of potential drug candidates.

Improved expression of kinases in Baculovirus-infected insect cells upon addition of specific kinase inhibitors to the culture helpful for structural studies.

As exemplified by three cases, we show that the addition of a small molecular weight inhibitor to the culture of Baculovirus-infected insect cells can dramatically improve the expression of a recombinant kinase. The expression of the tyrosine kinase KDR was sevenfold higher and mainly in a soluble form, when the KDR inhibitor PTK/ZK was added to the culture at the time of Baculovirus infection. The expression of the catalytic domain of the serine/threonine kinase PKCtheta, which is otherwise not possible with the Baculovirus expression system, was expressed mainly soluble at 120mg/L by the addition of the PKC inhibitor BIM XI to the culture of Baculovirus-infected insect cells. For Abl kinase, the expression could also be significantly increased by the addition of the Abl kinase inhibitor STI571 to the culture. For all three kinases, this method had previously been applied by us for the improved production of kinase/inhibitor complex protein, leading to the co-crystal structures. It is shown here at the cases KDR-PTK/ZK and PKCtheta-BIM XI, that the stimulatory effect of an inhibitor on kinase expression is applicable under many culture conditions. The presented method represents a valuable tool to obtain structural knowledge on kinase-inhibitor complexes.

A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system.

The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%.