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Brian S J Blagg

Publications and source records attributed to Brian S J Blagg.

At least 19 recordsLinked to original sources
PubMed2007

High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase.

Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor=0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microg/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <10 microM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.

Cell Division↗
PubMed2006

Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.

Binding Sites↗
PubMed2006

Synthesis and evaluation of coumermycin A1 analogues that inhibit the Hsp90 protein folding machinery.

[structure: see text] The coumarin antibiotics are not only potent inhibitors of DNA gyrase but also represent the most effective C-terminal inhibitors of 90 kDa heat shock proteins (Hsp90) reported thus far. In contrast to the N-terminal ATP-binding site, little is known about the Hsp90 C-terminus. In addition, very limited structure-activity relationships exist between this class of natural products and Hsp90. In this letter, the syntheses of dimeric coumarin analogues are presented along with their inhibitory values in breast cancer cell lines.

Aminocoumarins↗
PubMed2006

Design, synthesis, and structure--activity relationships for chimeric inhibitors of Hsp90.

Inhibition of the 90 kDa heat shock protein (Hsp90) family of molecular chaperones represents a promising new chemotherapeutic approach toward the treatment of several cancers. Previous studies have demonstrated that the natural products, radicicol and geldanamycin, are potent inhibitors of the Hsp90 N-terminal ATP binding site. The cocrystal structures of these molecules bound to Hsp90 have been determined, and through molecular modeling and superimposition of these ligands, hybrids of radicicol and geldanamycin have been designed. A series of macrocylic chimeras of radicicol and geldanamycin and the corresponding seco-agents have been prepared and evaluated for both antiproliferative activity and their ability to induce Hsp90-dependent client protein degradation.

Antineoplastic Agents↗
PubMed2006

A new approach for enhancing differential selectivity of drugs to cancer cells.

The degree to which anticancer agents selectively target cancer cells is a key determinant in successful therapeutic outcomes. Inhibitors of the Hsp90 molecular chaperone represent an important new class of anticancer agents. We propose here a novel mechanism by which physiochemical properties of Hsp90 inhibitors can be optimized to increase selectivity towards cancer cells. The basis for this approach relies on differential intracellular pH gradients that have been shown to exist between normal and transformed cells. Five Hsp90 inhibitors containing basic or neutral properties were evaluated in antiproliferation assays using cells with variable lysosomal pH. Inhibitors with basic functionalities had reduced activity in cells with normal (low) lysosomal pH but showed significantly greater activity in cells with abnormally elevated lysosomal pH (similar to what has been recorded in many types of cancer cells). Conversely, such selectivity enhancement was not observed for neutral inhibitors. The mechanistic basis for the observed selectivity was demonstrated quantitatively by determining the concentration of inhibitors within relevant intracellular compartments. Collectively, these findings suggest that Hsp90 inhibitors with optimal basicity and physicochemical properties have enhanced selectivity toward cancer cells than their neutral counterparts. It is anticipated that these findings may be applicable to other classes of anticancer agents for improvement of differential selectivity.

Antineoplastic Agents↗
PubMed2006

High-throughput screening for Hsp90 ATPase inhibitors.

Recently, we reported a useful assay for the determination of yeast Hsp90 ATPase activity. Using this assay, high-throughput screening of approximately 10,000 compounds was performed to determine the feasibility of this assay on large scale. Results from high-throughput screening indicated that the assay was reproducible (av Z-factor = 0.80) and identified 0.57% of the compounds as Hsp90 inhibitors that exhibited IC50s less than 20 microM. The structures of several of these inhibitory scaffolds are reported along with their IC50 values.

Adenosine Triphosphatases↗
PubMed2006

A versatile approach toward the ansamycin antibiotics.

The ansamycin antibiotics contain metacyclophanic macrolactams, many of which possess potent antitumor activity. Only a few total syntheses of this family of natural products have been reported, and modifications to increase potency have not been described. Therefore, a method was developed to prepare the trienomycin A core via resin-bound triphenylphosphonium salts, which serve as both a reagent and a traceless linker to afford olefinic products that undergo ring-closing metathesis (RCM) to give macrocyclic scaffolds of varying ring sizes.

Anti-Bacterial Agents↗
PubMed2006

Radanamycin, a macrocyclic chimera of radicicol and geldanamycin.

Radicicol and geldanamycin are potent inhibitors of the Hsp90 protein folding machinery, which is an emerging target for the development of cancer chemotherapeutics. However, radicicol is inactive in vivo and geldanamycin suffers from its redox-active behavior that produces toxicity unrelated to Hsp90 inhibition. It was proposed that a chimeric molecule containing the resorcinol ring of radicicol and the quinone of geldanamycin could provide an opportunity to elucidate structure-activity relationships for both natural products and serve as a starting point for the development of more potent inhibitors. Synthesis of the macrocyclic chimera named radanamycin is reported along with the biological activity exhibited by this compound in MCF-7 breast cancer cells.

Benzoquinones↗
PubMed2006

Hsp90 inhibitors: small molecules that transform the Hsp90 protein folding machinery into a catalyst for protein degradation.

The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides and the renaturation of denatured proteins. In transformed cells, numerous mutated and overexpressed proteins rely on the Hsp90 protein folding machinery for tumor progression. The Hsp90-mediated protein folding process is dependent upon ATP, and when inhibitors of ATP are present, the Hsp90 machinery is unable to fold client proteins into their biologically active form, which results in the degradation of protein substrates via the ubiquitin-proteasome pathway. Consequently, Hsp90 has evolved into a promising anti-cancer target because multiple oncogenic proteins can be simultaneously degraded as a consequence of Hsp90 inhibition. This review serves to explain the Hsp90 protein folding process, the impact of Hsp90 inhibition, the identification of natural product inhibitors, and the development of rationally designed inhibitors of the Hsp90 protein folding machinery.

Benzoquinones↗
PubMed2006

Geldanamycin, radicicol, and chimeric inhibitors of the Hsp90 N-terminal ATP binding site.

Natural products have continued to drive the development of new chemotherapeutics and elucidation of new biological targets for the treatment of disease. Since Whitesell and Neckers' original discovery that geldanamycin does not directly inhibit v-Src, but instead manifests its biological activity through inhibition of the Hsp90 molecular chaperone, additional natural products and natural product derivatives have been identified and developed to inhibit the Hsp90 protein folding machinery. 17-AAG, a geldanamycin analogue, is currently in clinical trials for the treatment of several types of cancer. Recent work has produced improved radicicol analogues that show promising Hsp90 inhibitory activity in vitro. In addition, chimeric molecules of these two natural products are active in vitro and represent a novel class of Hsp90 inhibitors for cancer treatment. In addition to their chemotherapeutic uses, natural product inhibitors and their derivatives have been utilized to probe the biological mechanisms by which Hsp90 inhibition regulates tumor cell growth. As a consequence of these studies, the molecular chaperones have emerged as an exciting new class of therapeutic targets. This review will highlight the utility of the natural products, geldanamycin and radicicol, as well as improved analogues and the activities exhibited by these compounds against various cancer cell lines.

Adenosine Triphosphate↗
PubMed2005

Development and optimization of a useful assay for determining Hsp90's inherent ATPase activity.

The Hsp90 molecular chaperone is responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Inhibition of Hsp90 represents a promising approach towards the treatment of cancer because numerous signaling cascades can be simultaneously targeted by disruption of the Hsp90-mediated process. Hsp90's ATPase activity is essential to the Hsp90-mediated protein folding process, consequently, a coupled assay was developed and optimized for determination of Hsp90's inherent ATPase activity. Using maltose phosphorylase, glucose oxidase, and horseradish peroxidase as components of this assay, a highly reproducible assay with a Z-factor of 0.87 has been produced.

Adenosine Triphosphatases↗
PubMed2005

Hsp90 inhibitors identified from a library of novobiocin analogues.

Novobiocin is a C-terminal inhibitor of the Hsp90 protein folding machinery, which is responsible for the conformational maturation of numerous proteins involved in cancer growth and survival. Due to novobiocin's poor inhibitory activity ( approximately 700 muM), very little attention has been paid toward the development of novobiocin analogues for Hsp90 inhibition. In this study, a parallel library of 20 novobiocin derivatives was prepared and the biological activity of each evaluated by Western blot analysis of Hsp90 client proteins. A4 was found to be a potent inhibitor of Hsp90 as determined by its ability to cause the degradation of several Hsp90 client proteins in both breast and prostate cancer cell lines. In the presence of 1 muM A4, several Hsp90 client proteins were degraded, including AKT, Her2, Hif-1alpha, and the androgen receptor.

Cell Line, Tumor↗
PubMed2005

Synthesis of mono- and dihydroxylated furanoses, pyranoses, and an oxepanose for the preparation of natural product analogue libraries.

[structures: see text] Numerous biologically active natural products contain furanoses and pyranoses with mono- and dihydroxylated substituents. However, much of the structure-activity studies on such molecules is gathered on the aglycons without attention to the corresponding carbohydrate components. Consequently, there are few synthetic procedures that enable the rapid preparation of mono- and dihydroxyfuranoses and mono- and dihydroxypyranoses and no report for a 3,4-dihydroxyoxepanose. In this article we report the practical synthesis of orthogonally protected five-, six-, and seven-membered carbohydrate derivatives. The succinct manner in which these molecules were synthesized allows the rapid preparation of analogues aimed at discovering the role of ring size and individual hydroxyl moieties on the pyranose skeleton.

Biological Factors↗
PubMed2005

Radester, a novel inhibitor of the Hsp90 protein folding machinery.

[reaction: see text]. The antitumor antibiotics radicicol and geldanamycin are potent inhibitors of the Hsp90 protein folding machinery. Radester is a hybrid composed of radicicol's resorcinol ring and geldanamycin's quinone through an isopropyl ester. Radester was prepared, and the cytotoxicity of it and the corresponding hydroquinone were determined in MCF-7 breast cancer cells to be 13.9 and 7.1 microM, respectively. Protein degradation assays were performed on Hsp90-dependent client proteins, Her-2 and Raf, to correlate Hsp90 inhibition to cytotoxicity.

Antineoplastic Agents↗
PubMed2004

Syntheses of photolabile novobiocin analogues.

Novobiocin was recently shown to inhibit Hsp90 through a previously unrecognized C-terminal ATP binding site. Although the N-terminal region of Hsp90 has been solved by X-ray crystallography, the C-terminal region has not. In an effort to elucidate the C-terminal binding site of Hsp90, four photolabile analogues of novobiocin were prepared.

Binding Sites↗
PubMed2004

Design, synthesis, and evaluation of a radicicol and geldanamycin chimera, radamide.

A chimera composed of the natural products radicicol and geldanamycin has been prepared through an amide linkage connecting the resorcinol moiety of radicicol to the quinone ring of geldanamycin. The inhibitory activity of these compounds was determined by their ability to inhibit Hsp90's inherent ATPase activity along with degradation of the Hsp90 client protein, HER-2 in MCF-7 breast cancer cells. [reaction: see text]

Acetanilides↗