Novel, Potent Small-Molecule Inhibitors of the Molecular Chaperone Hsp90 Discovered through Structure-Based Design

336

320

Pancreatic cancer is an aggressive disease with multiple biochemical and genetic alterations. Thus, a single agent to hit one molecular target may not be sufficient to treat this disease. The purpose of this study is to identify a novel Hsp90 inhibitor to disrupt protein-protein interactions of Hsp90 and its cochaperones for down-regulating many oncogenes simultaneously against pancreatic cancer cells. Here, we reported that celastrol disrupted Hsp90-Cdc37 interaction in the superchaperone complex to exhibit antitumor activity in vitro and in vivo. Molecular docking and molecular dynamic simulations showed that celastrol blocked the critical interaction of Glu 33 (Hsp90) and Arg 167 (Cdc37). Immunoprecipitation confirmed that celastrol (10 Mmol/L) disrupted the Hsp90-Cdc37 interaction in the pancreatic cancer cell line Panc-1. In contrast to classic Hsp90 inhibitor (geldanamycin), celastrol (0.1-100 Mmol/L) did not interfere with ATP binding to Hsp90. However, celastrol (1-5 Mmol/L) induced Hsp90 client protein degradation (Cdk4 and Akt) by 70% to 80% and increased Hsp70 expression by 12-fold. Celastrol induced apoptosis in vitro and significantly inhibited tumor growth in Panc-1 xenografts. Moreover, celastrol (3 mg/kg) effectively suppressed tumor metastasis by more than 80% in RIP1-Tag2 transgenic mouse model with pancreatic islet cell carcinogenesis. The data suggest that celastrol is a novel Hsp90 inhibitor to disrupt Hsp90-Cdc37 interaction against pancreatic cancer cells.

Section: Discussionmentioning

confidence: 99%

A novel Hsp90 inhibitor to disrupt Hsp90/Cdc37 complex against pancreatic cancer cells

Zhang

Hamza

Cao

et al. 2008

336

320

“…Compounds VER-49009 and VER-50589 were synthesized as described (29,30 Cell Culture Unless otherwise stated, cell lines were from the American Type Culture Collection (LGC Promochem, Middlesex, United Kingdom) and cultured as described (27). Penicillin (100 units/mL) and streptomycin (100 Ag/mL; Invitrogen, Paisley, United Kingdom) were added to the medium for human breast cancer cells.…”

Section: Methodsmentioning

confidence: 99%

“…1; ref. 29). Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole analogue VER-50589 (CCT0130024; Fig.…”

Section: Introductionmentioning

confidence: 99%

“…As seen from their chemical structures, VER-49009 and VER-50589 differ only in the pyrazole versus isoxazole heterocycle. The pyrazole to isoxazole switch does not affect the critical hydrogen bonding network exhibited by the pyrazole resorcinol unit that anchors this class of inhibitors to the HSP90 NH 2 -terminal ATP site (26,27,29), while also avoiding steric changes that would not be tolerated. We show here that the isoxazole has greater binding affinity than the corresponding pyrazole and also exhibits enhanced cellular uptake, leading to more potent HSP90 inhibition and antiproliferative Structural differences between the pyrazole VER-49009 (blue ) and the corresponding isoxazole VER-50589 (red ) are highlighted.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

Sharp

Prodromou

Boxall

et al. 2007

Self Cite

139

103

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K d ) of VER-50589 was 4.5 F 2.2 nmol/L compared with 78.0 F 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC 50 of 21 F 4 nmol/L for VER-50589 compared with 47 F 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI 50 values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 F 15 and 685 F 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and Pglycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI 50 for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as

“…In fact, the compound that was metabolized to the greatest extent in vitro exhibited the slowest clearance in vivo. Cassette dosing was subsequently applied in this project to identify compounds with optimal pharmacokinetic properties and, along with other assays, helped in lead optimization and progression toward compounds with appropriate properties for a preclinical development candidate (63).…”

Section: Dmpk In Drug Discoverymentioning

confidence: 99%

The application of cassette dosing for pharmacokinetic screening in small-molecule cancer drug discovery

Smith

Raynaud

Workman

2007

Self Cite

Pharmacokinetic evaluation is an essential component of drug discovery and should be conducted early in the process so that those compounds with the best chance of success are prioritized and progressed. However, pharmacokinetic analysis has become a serious bottleneck during the 'hit-to-lead' and lead optimization phases due to the availability of new targets and the large numbers of compounds resulting from advances in synthesis and screening technologies. Cassette dosing, which involves the simultaneous administration of several compounds to a single animal followed by rapid sample analysis by liquid chromatography/tandem mass spectrometry, was developed to increase the throughput of in vivo pharmacokinetic screening. Although cassette dosing is advantageous in terms of resources and throughput, there are possible complications associated with this approach, such as the potential for compound interactions. Following an overview of the cassette dosing literature, this article focuses on the application of the technique in anticancer drug discovery. Specific examples are discussed, including the evaluation of cassette dosing to assess pharmacokinetic properties in the development of cyclin-dependent kinase and heat shock protein 90 inhibitors. Subject to critical analysis and validation in each case, the use of cassette dosing is recommended in appropriate chemical series to enhance the efficiency of drug discovery and reduce animal usage. [Mol Cancer Ther 2007;6(2):428 -40]