DNA Conjugates as Tool Compounds for DEL Selections.

DEL selections are designed to discover novel small molecule compounds attached to DNA that bind to a target protein. A known small molecule or peptide ligand that binds to the target protein can be conjugated to DNA to mimic compounds contained in DEL libraries. The conjugate can be used as a ligand in preselection binding assays to validate a target protein and optimize selection methods and serve as a positive control in selection experiments. In this chapter, the design, synthesis, and use of DNA conjugate tool compounds are discussed. As an example, I describe the design of a DNA conjugate of a ligand to the CCR5 receptor and its use to optimize selection conditions and as a spike-in positive control in a DEL selection experiment. These methods are broadly applicable to both soluble protein targets and to targets that are displayed on the cell surface and to various types of compounds that can be conjugated to DNA.

Tunneling Nanotubes and Gap Junctions-Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communication and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediate a long-range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor.

Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.

An immunoglobulin fusion protein based on the gp120-CD4 receptor complex potently inhibits human immunodeficiency virus type 1 in vitro.

Fusion proteins containing immunoglobulin Fc domains attached to bioactive moieties have been developed as therapeutic agents against several diseases. Here, we describe the development and characteristics of a novel fusion protein (FLSC R/T-IgG1) that targets CCR5, the major coreceptor for HIV-1 during primary infection. FLSC R/T-IgG1 was expressed from a synthetic gene that linked a single chain gp120-CD4 complex containing an R5 gp120 sequence with the hinge-CH2-CH3 portion of human immunoglobulin gamma subtype 1. Purified FLSC R/T-IgG1 exhibited a molecular mass of 189 kDa under reducing conditions, which matched the expected size of one polypeptide chain. Chemically crosslinked or untreated FLSC R/T-IgG1 exhibited a mass of a 360-kDa polypeptide under reducing and nonreducing conditions, which indicated that the molecule adopts a disulfide-linked bivalent structure. The chimeric molecule bound specifically to CCR5-expressing cells and to peptides derived from the CCR5 N-terminus. Such binding was more efficient than what was obtained with a monomeric single chain gp120-CD4 complex. FLSC R/T-IgG1 binding to CCR5 was blocked by preincubation of coreceptor-expressing cells with CCR5 ligands and by antibody to the coreceptor binding domain of gp120. Conversely, FLSC R/T-IgG1 blocked the binding of chemokine to CCR5. However, FLSC R/T-IgG1 did not trigger intracellular Ca2+ mobilization in peripheral blood mononuclear cells. FLSC R/T-IgG1 potently neutralized primary R5 HIV-1 in both a PBMC-based assay and cell line-based assays but did not affect the replication of X4 viruses. These findings suggest that FLSC R/T-IgG1 might be used as a possible therapeutic agent against HIV.

Stimulation of enveloped virus infection by beta-amyloid fibrils.

Alzheimer's disease is characterized by deposition of beta-amyloid peptide (Abeta) into plaques in the brain, leading to neuronal toxicity and dementia. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system can also cause a dementia, and amyloid deposition in the central nervous system is significantly higher in HIV-1-infected individuals compared with uninfected controls. Here we report that Abeta fibrils stimulated, by 5-20-fold, infection of target cells expressing CD4 and an appropriate coreceptor by multiple HIV-1 isolates but did not permit infection of cells lacking these receptors. Abeta enhanced infection at the stage of virus attachment or entry into the cell. Abeta fibrils also stimulated infection by amphotrophic Moloney leukemia virus, herpes simplex virus, and viruses pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. Other synthetic fibril-forming peptides similarly enhanced viral infection and may be useful in gene delivery applications utilizing retroviral vectors. These data suggest that Abeta deposition may increase the vulnerability of the central nervous system to enveloped viral infection and that amyloidogenic peptides could be useful in enhancing gene transfer by enveloped viral vectors.