The diketopiperazine NPI-2358 is a synthetic analog of NPI-2350, a natural product isolated from Aspergillus sp., which depolymerizes microtubules in A549 human lung carcinoma cells. Although structurally different from the colchicine-binding site agents reported to date, NPI-2358 binds to the colchicine-binding site of tubulin. NPI-2358 has potent in-vitro anti-tumor activity against various human tumor cell lines and maintains activity against tumor cell lines with various multidrug-resistant (MDR) profiles. In addition, when evaluated in proliferating human umbilical vein endothelial cells (HUVECs), concentrations as low as 10 nmol/l NPI-2358 induced tubulin depolymerization within 30 min. Furthermore, NPI-2358 dose dependently increases HUVEC monolayer permeability--an in-vitro model of tumor vascular collapse. NPI-2358 was compared with three tubulin-depolymerizing agents with vascular-disrupting activity: colchicine, vincristine and combretastatin A-4 (CA4). Results showed that the activity of NPI-2358 in HUVECs was more potent than either colchicine or vincristine; the profile of CA4 approached that of NPI-2358. Altogether, our data show that NPI-2358 is a potent anti-tumor agent which is active in MDR tumor cell lines, and is able to rapidly induce tubulin depolymerization and monolayer permeability in HUVECs. These data warrant further evaluation of NPI-2358 as a vascular-disrupting agent in vivo. Currently, NPI-2358 is in preclinical development for the treatment of cancer.

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.

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Abstract. Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP~/S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGAsepharose--depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein-containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae. T HE nuclear pore is responsible for establishing the distinct nuclear and cytoplasmic compartments of the eukaryotic cell. The pore is a highly selective channel through which macromolecular traffic must pass to enter or exit the nucleus. Although ions and other small molecules freely diffuse through the pore, larger molecules require a specific targeting signal for transit (for reviews, see Goldfarb and Michaud,.1991;Forbes, 1992;Gerace, 1992;Osborne and Silver, 1993;Powers and Forbes, 1994).The nuclear pore complex is a large and elaborate structure of ~120 million daltons and is comprised of N1,000 proteins. Structurally, it appears to consist of three ringsThe first two authors, Eva Meier and Brian R. Miller, contributed equally to the results presented here.Please address all correspondence to Douglass J. Forbes, Department of Biology, University of California at San Diego, La,lolla, CA 92093-0347. Tel.: (619) 534-0555....

(2011) A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity, mAbs, 3:3, 273-288,