Tuesday, August 18, 2009

Heat Shock Protein Inhibitors

Heat shock protein inhibitors bind to the ATP pocket in the N-terminal domain of the heat shock protein (HSP90) molecular chaperone. Inhibition of the ATPase activity results in the depletion of HSP90 client proteins, particularly kinases that are involved in signal transduction.

Benzoquinone Ansamycin
Geldanamycin are naturally occurring benzoquinone ansamycin antibiotics that can be isolated through fermentation of Streptomyces hygroscopicus. In addition, analogue compounds, such as the geldanamycin derivative 17 allylamino-17-demethoxygeldanamycin (17-AAG), have been shown to possess antitumor activity in several human xenograft models, including colon, breast, and prostate cancer and have been used in patient clinical trials. These compounds bind to the ATP/ADP binding pocket in the NH2-terminal domain with high affinity, inhibiting Hsp90, and thus are potent inhibitors of cancer cell growth and the malignant phenotype in a number of cancer cell types1.

Radicicol (RDC), isolated from Diheterospora chlamydosporia, has proven to have many downstream oncogenic effects through Hsp90 inhibition. A novel radicicol oxime derivative, radicicol 6-oxime (KF25706), has been shown to be potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model2.

Isolation of the structurally similar pochonin family of natural products from Pochonia chlamydosporia has also shown promise in Hsp90 inhibition, particularly with pochonin A and pochonin D. Pochonin A and D have been shown to directly inhibit Hsp90. Pochonins A–F, while themselves displaying cytotoxicity in the micromolar range3.

Novobiocin, Coumermycin A1, and Clorobiocin
The coumermycin family of antibiotics, isolated from Streptomyces spheroids, has long been used clinically for antimicrobial purposes. Novobiocin in particular has been shown to display anti-cancer properties, and has been used in the clinic for many years. Ground breaking work by Neckers and co-workers demonstrated that this activity could be ascribed to novobiocin's Hsp90 inhibitory activity4.

Mode of action
Benzoquinone ansamycins, such as geldanamycin exhibit anticancer activity by binding to heat shock protein 90 (Hsp90), a molecular chaperone. In this interaction, geldanamycin competes with adenosine triphosphate at the N-terminal-binding site of Hsp90. The interaction results in the proteosome-mediated degradation of several important oncogenic proteins, including Raf-1, c-ErbB2, and mutant (but not wild-type) p53 (also known as TP53). Clearly, this molecular profile offers considerable potential for antitumor activity. Further investigation proved that GDA specifically inhibited the Src-Hsp90 heteroprotein complex, facilitating degradation of the client protein and thus attributing to its antitumor properties1.

To date, radicicol (RDC) is the most potent natural product inhibitor of Hsp90,. Its mechanism of action is similar to that of GDA, in that it binds to the N-terminal ATP-binding pocket of Hsp90.

Mechanistically, they bind the ATP binding pocket of DNA gyrase, another member of the GHKL superfamily, thus preventing ATP hydrolysis2.

Inhibition of Heat Shock Protein 90 Function Down-Regulates Akt Kinase - The phosphatidylinositol 3'-kinase/Akt pathway is activated frequently in human cancer, and has been implicated in tumor proliferation, cell survival, and resistance to apoptotic stimuli. Akt forms a complex with heat shock protein (Hsp) 90 and Cdc37, and inhibitors of Hsp90 cause Akt degradation. 17-AAG inhibits Akt activation and expression in tumors, and has antitumor activity in breast cancer xenografts. The combination of 17-AAG and Taxol is synergistic, and 17-AAG sensitizes tumor cells to Taxol-induced apoptosis in a schedule-dependent manner. Enhancement was schedule-dependent and maximal when Taxol and 17-AAG were administered on the same day. These results suggest that Hsp90 inhibitors can effectively suppress Akt activity in animal models of human cancer at nontoxic doses, thus sensitizing tumor cells to proapoptotic stimuli5.

Heat Shock Protein 90 Inhibitors Protect and Restore Pulmonary Endothelial Barrier Function - Exposure of confluent bovine pulmonary arterial endothelial cell (BPAEC) monolayer to TGF 1, thrombin, bacterial LPS or VEGF, increased BPAEC permeability, as revealed by decreased transendothelial electrical resistance (TER). Treatment of injured endothelium with hsp90 inhibitors completely restored TER of BPAEC. Similarly, pre-incubation of BPAEC with hsp90 inhibitors prevented the decline in TER induced by the exposure to thrombin, LPS, VEGF or TGFb1. Additionally, hsp90 inhibitors restored the EC barrier function after nocodazole- induced hyperpermeability. These effects of the hsp90 inhibitors were associated with the restoration of TGFb1- or nocodazole- induced decrease in VE-cadherin and b-catenin expression at EC junctions. The protective effect of hsp90 inhibitors on TGFb1-induced hyper-permeability was critically dependent upon preservation of F-actin cytoskeleton and was associated with the inhibition of agonist-induced myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, F-actin stress fibers formation, microtubule disassembly, increase in hsp27 phosphorylation, and association of hsp90 with hsp27. This implies that hsp90 inhibitors exert barrier protective effects on BPAEC, via inhibition of hsp27-mediated, agonist-induced cytoskeletal-rearrangement.

1.Schulte TW, Neckers LM (1998). The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer. Chemother. Pharmacol, 42, 273-9.
2.Soga S, Neckers LM, Schulte TW, Shiotsu Y, Akasaka K, Narumi H, Agatsuma T, Ikuina Y, Murakata C, Tamaoki T, Akinaga S (1999). KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules. Cancer Res., 59(12), 2931-8.
3.Moulin E, Barluenga S, Winssinger N (2005). Concise synthesis of pochnin A, an HSP90 inhibitor. Org. Lett., 8, 7(25), 5637-9.
4.Burlison JA, Neckers L, Smith AB, Maxwell A, Blagg BS (2006). Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90. J. Am. Chem. Soc., 128 (48), 15529-36.
5.David B. Solit, Andrea D. Basso, Adam B. Olshen, Howard I. Scher and Neal Rosen (2003). Inhibition of Heat Shock Protein 90 Function Down-Regulates Akt Kinase and Sensitizes Tumors to Taxol. Cancer Research 63, 2139-2144.
6.Antonov A, Snead C, Gorshkov B, Antonova GN, Verin AD and Catravas JD (2008). Heat Shock Protein 90 Inhibitors Protect and Restore Pulmonary Endothelial Barrier Function. Am. J. Respir. Cell Mol. Biol., 39 (5), 551-559.

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