Mechanistic Studies of the Multiple Myeloma and Melanoma Cell-Selective Toxicity of the Rpn13-Binding Peptoid KDT-11.

We previously reported a peptoid ligand for the proteasomal ubiquitin receptor Rpn13 called KDT-11 and demonstrated that this compound is toxic to multiple myeloma cells, but not non-malignant cells. Here, we show that KDT-11 decreases the viability of a variety of cancer cell lines, especially melanomas and various blood cancers. The peptoid induces selective G1 cell-cycle arrest, resulting in eventual apoptosis.

Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190.

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13.

Antibody biomarker for de novo Parkinson disease: attempted validation.

Parkinson disease (PD) is a progressive neurodegenerative disease with motor symptoms that result from degeneration of midbrain dopaminergic neurons. Biomarker research seeks to identify the disease during the pre-symptomatic phase, which is a time when therapeutic intervention will be most helpful. Previously, we screened a combinatorial peptoid library to search for antibodies that are present at much higher levels in the serum of PD patients than in control subjects.

A cell based screening approach for identifying protein degradation regulators.

Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit.

Establishment of a suite of assays that support the discovery of proteasome stimulators.

Background: The proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing.

Methods: The cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators.

BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression.

Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4), a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV) to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences.

Discovery of Phosphorylated Peripherin as a Major Humoral Autoantigen in Type 1 Diabetes Mellitus.

A major goal in understanding autoimmune diseases is to define the antigens that elicit a self-destructive immune response, but this is a difficult endeavor. In an effort to discover autoantigens associated with type 1 diabetes (T1D), we used epitope surrogate technology that screens combinatorial libraries of synthetic molecules for compounds that could recognize disease-linked autoantibodies and enrich them from serum. Autoantibodies from one patient revealed a highly phosphorylated form of peripherin, a neuroendocrine filament protein, as a candidate T1D antigen.

Reliable diagnosis of murine type 1 diabetes using a panel of autoantigens and "antigen surrogates" mounted onto a liquid array.

Autoantibodies raised against β cell antigens are the most reliable preclinical biomarkers for predicting the imminent onset of type 1 diabetes mellitus (T1DM). The most current detection platforms are technically challenging or are run on clinically esoteric equipment. Here, we present a straightforward approach to detect autoantibody biomarkers that employs highly PEGylated microspheres onto which are mounted various capture agents that include affinity-tagged antigens or small molecule "antigen surrogates.

High affinity binding of conformationally constrained synthetic oligomers to an antigen-specific antibody: Discovery of a diagnostically useful synthetic ligand for murine Type 1 diabetes autoantibodies.

'Antigen surrogates' are synthetic, non-natural molecules that recognize the antigen-binding sites of antibodies. These molecules are of interest as replacements for native antigens as antibody 'capture agents' in ELISA-like assays of potential diagnostic utility, for example when the antibody is indicative of a disease state. Antigen surrogates for disease-related antibodies can be mined from one-bead one-compound (OBOC) libraries by first denuding the library of ligands for antibodies present in the serum of control patients or animals, followed by screening the remainder of the library against serum from individuals with a particular disease of interest.

A reversible and highly selective inhibitor of the proteasomal ubiquitin receptor rpn13 is toxic to multiple myeloma cells.

The proteasome is a multisubunit complex responsible for most nonlysosomal turnover of proteins in eukaryotic cells. Proteasome inhibitors are of great interest clinically, particularly for the treatment of multiple myeloma (MM). Unfortunately, resistance arises almost inevitably to these active site-targeted drugs.

Screening of cell cycle fusion proteins to identify kinase signaling networks.

Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation.

Discovery of native autoantigens via antigen surrogate technology: application to type 1 diabetes.

A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals.

Casein kinase 1δ-dependent Wee1 protein degradation.

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation.

Utility of redundant combinatorial libraries in distinguishing high and low quality screening hits.

Large one-bead one-compound (OBOC) combinatorial libraries can be constructed relatively easily by solid-phase split and pool synthesis. The use of resins with hydrophilic surfaces, such as TentaGel, allows the beads to be used directly in screens for compounds that bind selectively to labeled proteins, nucleic acids, or other biomolecules. However, we have found that this method, while useful, has a high false positive rate.

An ultra-high throughput cell-based screen for wee1 degradation inhibitors.

The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway.

Activation domain-dependent degradation of somatic Wee1 kinase.

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G(2) and mitosis to allow mitotic progression.

The anaphase promoting complex induces substrate degradation during neuronal differentiation.

The anaphase promoting complex (APC) is an E3 ubiquitin ligase required for the metaphase-to-anaphase transition and mitotic exit. However, APC also plays roles in G(1), where it is regulated by Cdh1, and APC activity has also been detected in differentiated and non-proliferating cells, suggesting that it may play roles outside the cell cycle. Here, we report that disrupting APC(Cdh1) activity inhibits neurite outgrowth of both PC12 pheochromocytoma cells and primary cerebellar granule cells.

Redundant ubiquitin ligase activities regulate wee1 degradation and mitotic entry.

The irreversible nature of mitotic entry is due to the activation of mitosis specific kinases such as cdk1/cyclin B. Cdk1/cyclin B induces activation of mitosis by promoting phosphatases while suppressing inhibitory factors such as the tyrosine kinase wee1. Since wee1 keeps cdk1/cyclin B inactive during the S and G2 phases, its activity must be down-regulated for mitotic progression to occur.

Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector.

Live, attenuated Salmonella strains can serve as vectors for the delivery of recombinant vaccine antigens for development of oral mucosal vaccines. Various vaccine parameters can affect the immune responses elicited by Salmonella vectors, including the expression level, location and timing of expressed antigens. We have previously established immunogenic Salmonella enterica serovar Typhimurium strains which cytoplasmically express hemagglutinin B (HagB) of Porphyromonas gingivalis, a putative periodontal pathogen.