Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

Anti-nicotine vaccines may aid smoking cessation via the induction of anti-nicotine antibodies (Ab) which reduce nicotine entering the brain, and hence the associated reward. Ab function depends on both the quantity (titer) and the quality (affinity) of the Ab. Anti-nicotine vaccines tested previously in clinical studies had poor efficacy despite high Ab titer, and this may be due to inadequate function if Ab of low affinity were induced. In this study, we designed and synthesized a series of novel nicotine-like haptens which were all linked to diphtheria toxoid (DT) as carrier, but which differed in the site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. The resulting hapten conjugates were evaluated in a mouse model, using CpG (a TLR9 agonist) and aluminum hydroxide (Al(OH)3) as adjuvants, whereby Ab titers, affinity and function were evaluated using a radiolabeled nicotine challenge model. A series of additional linkers varying in length, rigidity and polarity were used with a single hapten to generate additional DT-conjugates, which were also tested in mice. Conjugates made with different haptens resulted in various titers of anti-nicotine Ab. Several haptens gave similarly high Ab titers, but among these, Ab affinity and hence function varied considerably. Linker also influenced Ab titer, affinity and function. These results demonstrate that immune responses induced in mice by nicotine-conjugate antigens are greatly influenced by hapten design including site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. While both Ab titer and affinity contributed to function, affinity was more sensitive to antigen differences.

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Natural products continue to be a robust source of novel therapeutics, with ~50% of currently approved anticancer drugs being natural products or their derivatives. 1 The luminacin family of natural products, discovered from the fermentation broth of the soil bacterium Streptomyces sp., 2 contains several members that have shown promising anticancer activity in multiple assays and cell lines. Two members of this family, luminacin D (1a), and luminacin C2 (1b, also known as UCS15A), have been shown to be potent inhibitors of angiogenesis in several in vitro assays. 3 Luminacin D was also shown to inhibit the proliferation of several cancer cell lines. 3 Additional studies with Luminacin C2 have shown it to be a protein-protein interaction inhibitor that targets Src signal transduction by inhibiting the SH3 domain-mediated interactions of Src kinase with its targets, thus preventing the src-specific tyrosine phosphorylation of numerous proteins. 4 Src kinases play a key role in the signaling and regulation of multiple processes associated with cancer, such as cell migration, cell adhesion, extracellular matrix sensing, cell cycle timing, as well as several poorly understood events necessary for angiogenesis.Luminacin C2 was further demonstrated to inhibit the invasion and metastasis of model breast cancer cell lines in vitro, by inhibition of the protein-protein interaction of the src-homology domain of cortactin with AMAP1. 5 The recent report that two structurally related compounds, named migracin A and B (1c), inhibit the migration of a breast cancer cell line, 6 provides further evidence for the anticancer potential of this molecule, or its derivatives.There is comparatively little information about the mode of action or biological function of luminacin D. Given that it is the most potent member of this family in several of the originally reported assays, 3 there is significant potential and need for an approach that enables the synthesis of sufficient quantities of this molecule to enable further research into its cellular mode of action.There are a few syntheses of the luminacins reported, 7 however each with shortcomings in terms of length and/or unselective reaction steps. A particular concern is the epoxide introduction, with three total syntheses featuring a late stage epoxidation step with very low, or undesired selectivity. 7b-d,8 Because of this, we sought to develop a synthetic approach in which an enantiopure epoxide intermediate is assembled first, to then utilise its stereochemistry for diastereoselective completion of the aliphatic portion, from which the luminacins and the migracins can be synthesised.Herein we report a successful total synthesis of (-)-luminacin D using this strategy, and report on the excellent diastereocontrol possible by allylation of aldehydes having α-oxygenated centres, including quaternary centres, under 1,3-chelation conditions. We also unambiguously show that this type of aldehyde addition is consistent with the Cornforth-Evans (CE) model of stereoinduction. Scheme 1....

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A heterotrifunctional template was developed that utilizes thiol-maleimide and click chemistries (both copper-free and copper-mediated) to effect sequential biomolecule conjugations in a one-pot process. The breadth of compatible substrates was illustrated through highly efficient conjugations of protein, peptide, sugar, lipid, fluoroalkane, biotin and fluorophore molecules. This template should be useful for the creation of chemically-enhanced/enabled biotherapeutics, especially through the expression of discontinuous (and heterogeneous) epitopes.

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