A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks.

Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1.

Neurofibromatosis type 2 (NF2) is a dominantly inherited autosomal disease characterized by schwannomas of the 8th cranial nerve. The NF2 tumor suppressor gene encodes for Merlin, a protein implicated as a suppressor of multiple cellular signaling pathways. To identify potential drug targets in NF2-associated malignancies we assessed the consequences of inhibiting the tyrosine kinase receptor MET.

YAP Mediates Tumorigenesis in Neurofibromatosis Type 2 by Promoting Cell Survival and Proliferation through a COX-2-EGFR Signaling Axis.

The Hippo-YAP pathway has emerged as a major driver of tumorigenesis in many human cancers. YAP is a transcriptional coactivator and while details of YAP regulation are quickly emerging, it remains unknown what downstream targets are critical for the oncogenic functions of YAP. To determine the mechanisms involved and to identify disease-relevant targets, we examined the role of YAP in neurofibromatosis type 2 (NF2) using cell and animal models.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization.

FRAX597, a small molecule inhibitor of the p21-activated kinases, inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas.

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs.

Notch1 is required for Kras-induced lung adenocarcinoma and controls tumor cell survival via p53.

The Notch pathway has been implicated in a number of malignancies with different roles that are cell- and tissue-type dependent. Notch1 is a putative oncogene in non-small cell lung cancer (NSCLC) and activation of the pathway represents a negative prognostic factor. To establish the role of Notch1 in lung adenocarcinoma, we directly assessed its requirement in Kras-induced tumorigenesis in vivo using an autochthonous model of lung adenocarcinoma with concomitant expression of oncogenic Kras and deletion of Notch1.

PAZ6 cells constitute a representative model for human brown pre-adipocytes.

The role of brown adipose tissue (BAT) in human metabolism and its potential as an anti-obesity target organ have recently received much renewed attention. Following radiological detection of substantial amounts of BAT in adults by several independent research groups, an increasing number of studies are now dedicated to uncover BAT's genetic, developmental, and environmental determinants. In contrast to murine BAT, human BAT is not present as a single major fat depot in a well-defined location.

Direct binding of a hepatitis C virus inhibitor to the viral capsid protein.

Over 130 million people are infected chronically with hepatitis C virus (HCV), which, together with HBV, is the leading cause of liver disease. Novel small molecule inhibitors of Hepatitis C virus (HCV) are needed to complement or replace current treatments based on pegylated interferon and ribavirin, which are only partially successful and plagued with side-effects. Assembly of the virion is initiated by the oligomerization of core, the capsid protein, followed by the interaction with NS5A and other HCV proteins.

Potent inhibitors of hepatitis C core dimerization as new leads for anti-hepatitis C agents.

New indoline alkaloid-type compounds which inhibit HCV production by infected hepatoma cells have been identified. These compounds, dimeric-type compounds of previously known inhibitors, display double digit nanomolar IC(50) and EC(50) values, with cytotoxicity CC(50) indexes higher than 36 μM, thus providing ample therapeutic windows for further development of HCV drugs.

Core as a novel viral target for hepatitis C drugs.

Hepatitis C virus (HCV) infects over 130 million people worldwide and is a major cause of liver disease. No vaccine is available. Novel specific drugs for HCV are urgently required, since the standard-of-care treatment of pegylated interferon combined with ribavirin is poorly tolerated and cures less than half of the treated patients.

A time-resolved fluorescence-resonance energy transfer assay for identifying inhibitors of hepatitis C virus core dimerization.

Binding of hepatitis C virus (HCV) RNA to core, the capsid protein, results in the formation of the nucleocapsid, the first step in the assembly of the viral particle. A novel assay was developed to discover small molecule inhibitors of core dimerization. This assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-tag antibodies labeled with either europium cryptate (Eu) or allophycocyanin (XL-665).

New small molecule inhibitors of hepatitis C virus.

New small molecule inhibitors of HCV were discovered by screening a small library of indoline alkaloid-type compounds. An automated assay format was employed which allowed identification of dimerization inhibitors of core, the capsid protein of the virus. These compounds were subsequently shown to block production of infectious virus in hepatoma cells.

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors.

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site.

Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity.

Presence of tear lipocalin and other major proteins in lacrimal fluid of rabbits.

The lipocalins are a highly divergent, ubiquitous family of proteins that commonly function in binding lipophilic molecules. Although a specific tear lipocalin is a major component of lacrimal fluid and tears in many mammals, there has been no definitive identification of such a protein in rabbit tears. The goals of this project were to identify the major proteins in rabbit (Oryctolagus cuniculus) lacrimal fluid, so as to determine if they include a lipocalin and, if such a protein is present, to determine its source.