Small-molecule modulators of the NF-kappaB pathway newly identified by a translocation-based cellular assay.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor. Aberrant regulation of the NF-kappaB pathway is frequently observed in a number of major ailments such as cancer and inflammatory diseases. Hence NF-kappaB modulators have been intensely pursued for their potential therapeutic applications. Numerous reviews have described recent progress in the development of these agents. More recently, a variety of structurally and functionally novel small molecules, identified through high-throughput screens conducted within the Molecular Libraries Screening Center Network (MLSCN) of the NIH Roadmap for Medical Research, have been added to the current list of NF-kappaB regulators. This review will discuss the inhibitors and activators newly discovered by Columbia's Molecular Libraries Screening Center (MLSC) using a well-designed and stable cellular assay.

Physiological and pathological role of alpha-synuclein in Parkinson's disease through iron mediated oxidative stress; the role of a putative iron-responsive element.

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 (alpha-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 alpha-syn oligomerization during PD pathology. Also, alpha-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.

Discovery of potent non-urea inhibitors of soluble epoxide hydrolase.

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.

Potent inhibitors of Huntingtin protein aggregation in a cell-based assay.

A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small Molecule Repository (MLSMR). A structure and activity study yielded leads with submicromolar potency.

Discovery of novel small molecule cell type-specific enhancers of NF-kappaB nuclear translocation.

An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.

Discovery of a novel submicromolar inhibitor of the lymphoid specific tyrosine phosphatase.

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.

Identification of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating NFkappaB activity within two separate high-throughput screens of NFkappaB activation.

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.

Cell-based assays using primary endothelial cells to study multiple steps in inflammation.

Cell-based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell-based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell-based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor alpha (TNFalpha)-induced translocation of the transcription factor NF-kappaB from the cytoplasm into the nucleus 20 min after stimulation with TNFalpha. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFalpha. Indirect immunofluorescence and high-throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z' values between 0.7 and 0.8 make each of the three assays well suited for use in high-throughput screening for drug or probe discovery.

Protease-induced release of functional peptides from bioplexes.

Linking peptide functions directly to nucleic acids can be used to improve transfection. We have previously demonstrated this by sequence-specific hybridization of a bifunctional peptide nucleic acid (PNA) consisting of a nucleic acid binding moiety conjugated to a peptide. The resulting biological complex of PNA/DNA is called a Bioplex. The bifunctional PNA is continuously synthesized with one or more functional entities. For certain applications, it might be preferable to eliminate a functional entity after it has served its purpose. We have addressed this issue by adding a specific protease cleavage site to the construct. In this first approach, cathepsin L was used to cleave a linker sequence including a cathepsin L site: afrsaaq, thereby releasing the tri-peptide Arg-Gly-Asp (RGD) from the PNA anchor. In vitro and in vivo experiments showed an efficient cleavage of the peptide. Moreover, bifunctional PNA constructs were shown to retain activity of the second entity following removal of the first function. Since cathepsin L is ubiquitously expressed in eukaryotic cells and becomes active as the endosomal pH drops, inclusion of cathepsin sites makes it possible to remove functional entities in late endosomes/early lysosomes.

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras.

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins.

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.

Human cytomegalovirus protein pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR alpha-chain.

Human cytomegalovirus (HCMV) has developed multiple strategies to escape immune recognition. Here, we demonstrate that HCMV down-regulates HLA-DR expression in infected interferon gamma (IFN-gamma)-stimulated fibroblasts at 1 day after infection. Decreased HLA-DR expression was not observed on cells infected with an HCMV strain lacking the pp65 gene (RVAD65), but was observed on cells transfected with the pp65 gene. HLA-DR expression accumulated in vacuoles near the nucleus in HCMV-infected, but not in uninfected or RVAD65-infected cells. In addition, the HLA-DR alpha-chain, but not the beta-chain or HLA-DM, was degraded in HCMV-infected but not in RVAD65-infected cells. Thus, the HCMV protein pp65 mediates decreased expression of HLA-DR, by mediating an accumulation of HLA class II molecules in lysosomes that results in degradation of the HLA-DR alpha-chain.

Expression profiling in transformed human B cells: influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays.

We have used both Clontech Atlas Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein-Barr virus (EBV)-transformed B cells and genes regulated by Bruton's tyrosine kinase (Btk). We compared EBV-transformed wild-type (WT) B cells from a healthy individual, WT1 and an X-linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were > or =1.9-fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down-regulated in XLA2, indicating their biological role. Higher-fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real-time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell-specific OCT binding factor-1 (OBF-1) were induced > or =1.9-fold in both XLA1 and XLA2 analyzed by Atlas filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses.