The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC50s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaca2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-Biotin pull-down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90, which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1–24 hour post treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

Electronic mail: dlosbor@sandia.gov (DLO), cataatj@sandia.gov (CAT).We have developed a multiplexed time-and photon-energy-resolved photoionization mass spectrometer for the study of the kinetics and isomeric product branching of gas phase, neutral chemical reactions. The instrument utilizes a side-sampled flow tube reactor, continuously tunable synchrotron radiation for photoionization, a multi-mass double-focusing mass spectrometer with 100% duty cycle, and a time-and positionsensitive detector for single ion counting. This approach enables multiplexed, universal detection of molecules with high sensitivity and selectivity. In addition to measurement of rate coefficients as a function of temperature and pressure, different structural isomers can be distinguished based on their photoionization efficiency curves, providing a more detailed probe of reaction mechanisms. The multiplexed 3-dimensional data structure

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotidefree Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.Heat shock protein 90 (Hsp90) 2 is a highly abundant and essential molecular chaperone in eukaryotic cells, accounting for as much as 1-2% of the cytosolic protein even under nonstressed conditions (1). Hsp90 protects cells not only through correcting the misfolded proteins under stress conditions, but also plays a key role under normal conditions in regulating the stability, maturation, and activation of a wide range of client substrates, including kinases, hormone receptors, and transcription factors (2). There is strong evidence that Hsp90 plays an important role in disease states, particularly in cancer. Hsp90 is expressed 2-10-fold higher in cancer cells compared with their normal counterparts, implying its crucial role in tumor cell growth or survival (3). The largest subset of Hsp90 clients is the protein kinase, many of which are mutated and/or overexpressed signaling proteins in cancers (4 -6). Furthermore, cancer cells are significantly more sensitive to Hsp90 inhibition than non-transformed cells (7). Therefore, Hsp90 has emerged as a promising target for cancer treatment.The crystal structure reveals that Hsp90 consists of three highly conserved domains: an N-terminal ATP-binding domain (25 kDa), a middle domain (35 kDa), and a C-terminal dimerization domain (12 kDa) (8 -10). Hsp90 exists as a homodimer (11). The N-terminal domain contains a specific ATP-binding pocket, which has been well characterized (9, 12). The middle domain is highly charged, and its major role is to distinguish various types of client proteins and adjust the molecular chaperone for proper substrate activation (13). The C-terminal domain strengthens the weak association between the two N-terminal domains of the Hsp90 dimer (10). A second ATP-binding site is located in the C terminus, which does not exhibit ATPase activity (14).Hsp90 chaperone function depends on the conformational changes driven by its ATPase activity (15). Numerous Hsp90 inhibitors, ranging from...

We previously reported the discovery of a class of spirooxindoles as potent and selective small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors). We report herein our efforts to improve their pharmacokinetic properties and in vivo antitumor activity. Our efforts led to the identification of 9 (MI-888) as a potent MDM2 inhibitor (Ki = 0.44 nM) with a superior pharmacokinetic profile and enhanced in vivo efficacy. Compound 9 is capable of achieving rapid, complete, and durable tumor regression in two types of xenograft models of human cancer with oral administration and represents the most potent and efficacious MDM2 inhibitor reported to date.

The tubular nanocomposite with well-dispersed distribution of small gold nanoparticles (AuNPs) assembled on the inside and outside surfaces of silica nanotubes (SNTs) was fabricated by combining the single capillary electrospinning technique and an in situ reduction approach. The AuNPs/SNTs nanocomposite exhibited a good catalytic activity for reduction of 4-nitrophenol (4-NP).