4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and geldanamycin

Heat shock protein (Hsp)90 is emerging as an important therapeutic target for the treatment of cancer. Two analogues of the Hsp90 inhibitor geldanamycin are currently in clinical trials. Geldanamycin (GA) and its analogues have been reported to bind purified Hsp90 with low micromolar potency, in stark contrast to their low nanomolar antiproliferative activity in cell culture and their potent antitumor activity in animal models. Several models have been proposed to account for the approximately 100-fold-greater potency in cell culture, including that GA analogues bind with greater affinity to a five-protein Hsp90 complex than to Hsp90 alone. We have determined that GA and the fluorescent analogue BODIPY-GA (BDGA) both demonstrate slow, tight binding to purified Hsp90. BDGA, used to characterize the kinetics of ligand-Hsp90 interactions, was found to bind Hsp90alpha with k(off) = 2.5 x 10(-3) min(-1), t(1/2) = 4.6 h, and Ki* = 10 nM. It was found that BDGA binds to a functional multiprotein Hsp90 complex with kinetics and affinity identical to that of Hsp90 alone. Also, BDGA binds to Hsp90 from multiple cell lysates in a time-dependent manner with similar kinetics. Therefore, our results indicate that the high potency of GA in cell culture and in vivo can be accounted for by its time-dependent, tight binding to Hsp90 alone. In the broader context, these studies highlight the essentiality of detailed biochemical characterization of drug-target interactions for the effective translation of in vitro pharmacology to cellular and in vivo efficacy.

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Boron Compounds; Cells, Cultured; Fluorescent Dyes; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Protein Binding; Quinones; Recombinant Proteins

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90alpha (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90alpha with high affinity. Even at low Hsp90alpha concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z' values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90alpha with EC(50)s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.

Topics: Benzoquinones; Boron Compounds; Fluorescence Polarization; Fluorescent Dyes; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Quinones