Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET Signal.

Glycosyltransferase enzymes play diverse metabolic and regulatory roles by catalyzing the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors, and they are increasingly of interest as therapeutic targets in a number of diseases, including metabolic disorders, cancer, and infectious diseases. The glycosyltransferases are a challenging target class from an assay development perspective because of the diversity of both donor and acceptor substrates and the lack of suitable glycan detection methods. However, many glycosyltransferases use uridine 5'-diphosphate (UDP) sugars as donor substrates, and detection of the free UDP reaction product provides a generic approach for measuring the activity of those enzymes.

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis.

Identification and characterization of small molecule human papillomavirus E6 inhibitors.

Cervical cancer is the sixth most common cancer in women worldwide and the leading cause of women's death in developing countries. Nearly all cervical cancers are associated with infection of the human papillomavirus (HPV). This sexually transmitted pathogen disrupts the cell cycle via two oncoproteins: E6 and E7.

Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs.

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment.

New informatics and automated infrastructure to accelerate new leads discovery by high throughput screening (HTS).

The Lankenau Institute for Medical Research Chemical Genomics Center, Inc. has developed a new (patents issued and pending) Nanotube Automated Repository System (NARS) for dynamic storage of millions of 'single-shot' samples stored in a new monolithic microtiter-storage tube plate of our own design we call 'nanotubes.' We have integrated the NARS with customized software to efficiently access up to 10,000,000 samples stored continuously frozen (-20°C) in a dehumidified enclosure and sealed in a new microtiter NARS plate that is SBS compliant.

Identification and characterization of small molecule antagonists of pRb inactivation by viral oncoproteins.

The retinoblastoma protein pRb is essential for regulating many cellular activities through its binding and inhibition of E2F transcription activators, and pRb inactivation leads to many cancers. pRb activity can be perturbed by viral oncoproteins including human papillomavirus (HPV) that share an LxCxE motif. Because there are no treatments for existing HPV infection leading to nearly all cervical cancers and other cancers to a lesser extent, we screened for compounds that inhibit the ability of HPV-E7 to disrupt pRb/E2F complexes.

Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities.

Bin3 deletion causes cataracts and increased susceptibility to lymphoma during aging.

Bin3 encodes an evolutionarily conserved and ubiquitously expressed member of the BAR superfamily of curved membrane and GTPase-binding proteins, which includes the BAR, PCH/F-BAR, and I-BAR adapter proteins implicated in signal transduction and vesicular trafficking. In humans, Bin3 maps to chromosome 8p21.3, a region widely implicated in cancer suppression that is often deleted in non-Hodgkin's lymphomas and various epithelial tumors.

Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy.

Immune escape is a crucial feature of cancer progression about which little is known. Elevation of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) in tumor cells can facilitate immune escape. Not known is how IDO becomes elevated or whether IDO inhibitors will be useful for cancer treatment.

Targeted deletion of the suppressor gene bin1/amphiphysin2 accentuates the neoplastic character of transformed mouse fibroblasts.

The Bin1/Amphiphysin2 gene encodes several alternately spliced BAR adapter proteins that have been implicated in membrane-associated and nuclear processes. Bin1 expression is often attenuated during tumor progression and Bin1 splice isoforms that localize to the nucleus display tumor suppressor properties. While these properties may reflect the ability of these isoforms to interact with and suppress the cell transforming activity of c-Myc, the effects of Bin1 deletion on the oncogenicity of c-myc or other transforming genes has not been gauged directly.

Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation.

The mammalian Bin1/Amphiphysin II gene encodes an assortment of alternatively spliced adapter proteins that exhibit markedly divergent expression and subcellular localization profiles. Bin1 proteins have been implicated in a variety of different cellular processes, including endocytosis, actin cytoskeletal organization, transcription, and stress responses. To gain insight into the physiological functions of the Bin1 gene, we have disrupted it by homologous recombination in the mouse.

hob1+, the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress.

BAR (Bin/Amphiphysin/Rvs) adapter proteins have been suggested to regulate endocytosis, actin organization, apoptosis, and transcription, but their precise roles are obscure. There are at least five mammalian genes that encode BAR adapter proteins, including the evolutionarily conserved and ubiquitously expressed Bin1/Amphiphysin-II and Bin3 genes. Bin1 holds special interest as certain splice isoforms localize to the nucleus, interact with the c-Abl and c-Myc oncoproteins, and display tumor suppressor properties.

Increased transversions in a novel mutator colon cancer cell line.

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered.

Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability.

Colon cancer and an increasing number of other cancers have been found to exhibit instability of DNA microsatellite sequences. Such tumors have been designated as replication errors (RER) tumors. However, as microsatellites are only rarely found within coding regions of the genome, instability of these sequences cannot directly contribute to carcinogenesis.