We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA-NLS peptide. The PNA-NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA-NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA-PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

Abstract:Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer's disease (AD) and represents a large health burden to society. Genetic and oxidative risk factors have been proposed as possible causes, but their relative contribution remains unclear. Dysfunction of alpha-synuclein (α-syn) has been associated with PD due to its increased presence, together with iron, in Lewy bodies. Brain oxidative damage caused by iron may be partly mediated by α-syn oligomerization during PD pathology. Also, α-syn gene dosage can cause familial PD and inhibition of its gene expression by blocking translation via a newly identified Iron Responsive Element-like RNA sequence in its 5'-untranslated region may provide a new PD drug target.

We report here a class of thiazolidine-2,4-diones and 2-thioxothiazolidin-4-ones as potent inhibitors of the lymphoid specific tyrosine phosphatase (Lyp) identified from high throughput screens. Chemical modification by incorporating the known phosphotyrosine (pTyr) mimics led to the discovery of a salicylate-based inhibitor with submicromolar potency.

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Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC 50 s of the most potent compounds range from micromolar to low nanomolar.© 2008 Elsevier Ltd. All rights reserved. *Corresponding author. Tel: +1 212 3055839; Fax: +1 212 3053475; dwl1@columbia.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To date, the most successful sEH inhibitors are 1,3-disubstituted ureas, which display anti-hypertension and anti-inflammatory effects through inhibition of EET hydrolysis. However, urea-based inhibitors often suffer from poor solubility and bioavailability 4 and new scaffolds are needed for therapeutic applications. Here we describe the HTS, design and synthesis of a series of potent non-urea sEH inhibitors. NIH Public AccessA fluorescent assay 5 was employed for HTS using recombinant human sEH and a water soluble α-cyanocarobonate epoxide (PHOME) as the substrate. As shown in Figure 1, sEH-catalyzed hydrolysis of the non-fluorescent substrate is followed by spontaneous cyclization to a cyanohydrin that under basic conditions, rapidly decomposes to a highly fluorescent naphthaldehyde. Fluorescence with excitation at 320nm and emission at 460nm was recorded at the endpoint of the reaction cascade. From the compound collection provided by the NIH Roadmap project, a variety of hits were identified with low micromolar to nanomolar potency. 6 A large proportion of hits were ureas, but several non-urea compounds showed substantial activities. The most potent compound among these non-ureas was the sulfonyl isonipecotamide 1, a nanomolar inhibitor (IC 50 =20.0nm) with some structural similarity to the previously reported piperidine-containing urea AMAU (Figure 2). 7A secondary library based on 1 was prepared by modifying either the amide head group or the sulfonamide tail group. The synthesis of the sulfonamide-modified analogs is outlined Scheme 1. Methyl isonipecotate 2 was first protected with benzyl chloroformate, and then converted to the acid chloride 4 by hydrolyzing the methyl ester and then treating with oxalyl chloride. Coupling of 4 with 2,4-dichlorobenzylamine followed by palladium catalyzed hydrogenation afforded amine 5, which was reacted with a variety of sulfonyl chlorides, carbonyl chlorides and chloroformates to yield products 6-1 to 6-37.

No abstract

We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFkappaB pathway identified from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were confirmed in both primary and secondary assays of NFkappaB activation and expanded upon through analogue synthesis. The series exhibited potencies in the cell-based assays at as low as 0.6 microM, and several indications suggest that the targeted activity lies within a common region of the NFkappaB pathway.

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.