A High-Throughput Screen for Inhibitors of the Hsp90-Chaperone Machine.

Hsp90 has emerged as a key chemotherapeutic target for the development of drugs for the treatment of cancer and neurodegenerative diseases. The shortcomings of many of the Hsp90 inhibitors that have made it to clinical trials have bolstered the need to identify new lead compounds with superior properties. Here, we describe a high-throughput screen for the identification of Hsp90 inhibitors based on the refolding of thermally denatured firefly luciferase.

Immunodominant West Nile Virus T Cell Epitopes Are Fewer in Number and Fashionably Late.

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.

High-throughput screen of natural product libraries for hsp90 inhibitors.

Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine.

Gambogic acid, a natural product inhibitor of Hsp90.

A high-throughput screening of natural product libraries identified (-)-gambogic acid (1), a component of the exudate of Garcinia harburyi, as a potential Hsp90 inhibitor, in addition to the known Hsp90 inhibitor celastrol (2). Subsequent testing established that 1 inhibited cell proliferation, brought about the degradation of Hsp90 client proteins in cultured cells, and induced the expression of Hsp70 and Hsp90, which are hallmarks of Hsp90 inhibition. Gambogic acid also disrupted the interaction of Hsp90, Hsp70, and Cdc37 with the heme-regulated eIF2α kinase (HRI, an Hsp90-dependent client) and blocked the maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that 1 bound to the N-terminal domain of Hsp90 with a low micromolar Kd, in a manner that was not competitive with the Hsp90 inhibitor geldanamycin (3). Molecular docking experiments supported the posit that 1 binds Hsp90 at a site distinct from Hsp90s ATP binding pocket. The data obtained have firmly established 1 as a novel Hsp90 inhibitor and have provided evidence of a new site that can be targeted for the development of improved Hsp90 inhibitors.

Synthesis and evaluation of Hsp90 inhibitors that contain the 1,4-naphthoquinone scaffold.

High-throughput screening of a library of diverse molecules has identified the 1,4-naphthoquinone scaffold as a new class of Hsp90 inhibitors. The synthesis and evaluation of a rationally-designed series of analogues containing the naphthoquinone core scaffold has provided key structure-activity relationships for these compounds. The most active inhibitors exhibited potent in vitro activity with low micromolar IC(50) values in anti-proliferation and Her2 degradation assays. In addition, 3g, 12, and 13a induced the degradation of oncogenic Hsp90 client proteins, a hallmark of Hsp90 inhibition. The identification of these naphthoquinones as Hsp90 inhibitors provides a new scaffold upon which improved Hsp90 inhibitors can be developed.

Dietary alkaloids and the development of androconial organs in Estigmene acrea.

Male salt marsh moths, Estigmene acrea (Lepidoptera: Arctiidae), possess inflatable androconial organs called coremata. Prior to mating males form aggregations and inflate their coremata en masse. The communal display attracts additional males and females for the purpose of mating. The coremata are known to carry the plant-derived dihydropyrrolizine, hydroxydanaidal. This pheromonal substance is derived from secondary plant chemicals called pyrrolizidine alkaloids found in the larval diet. When E. acrea larvae were raised on semi-synthetic diets containing different levels of the pyrrolizidine alkaloid precursors the alkaloids triggered a pronounced morphogenetic effect. Adult males that fed on high levels of the pyrrolizidine alkaloid monocrotaline N-oxide (2500 microg) developed the largest coremata. Males that fed on lower levels of monocrotaline N-oxide (500 microg) or no alkaloid, while normal in body weight, had coremata that were progressively smaller and less robust. The size of the coremata and their commensurate pheromonal charge may have behavioral consequences in the unusual mating system of this species.