ATP-binding Site Of Hsp90 Research Articles

Capsaicin binds the N-terminus of Hsp90, induces lysosomal degradation of Hsp70, and enhances the anti-tumor effects of 17-AAG (Tanespimycin)

Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.

Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90.

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N7/N9-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC50 values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay.

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.

Marine alkaloid oroidin analogues with antiviral potential: A novel class of synthetic compounds targeting the cellular chaperone Hsp90.

Marine organisms and their metabolites are a diverse source of scaffolds for potential pharmacological molecular probes and, less frequently, for pharmaceutical lead compounds. In this study, 157 synthetic analogues of marine sponge-derived alkaloids clathrodin and oroidin were screened against replicon models of two RNA viruses, hepatitis C (HCV) and Chikungunya virus (CHIKV) as part of a larger screening project. Four compounds were found to selectively inhibit the HCV replicon (IC50 1.6-4.6μm). These belong to the 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole class of compounds originally designed to target the ATP-binding site of bacterial DNA gyrase. The ATP-binding site of this bacterial protein has high structural similarity to the ATP-binding site of heat-shock protein 90 (Hsp90), a host cell chaperone universally required for viral replication, which led us to examine inhibition of Hsp90 as the compounds' potential mechanism of action. Binding of the four hit compounds to Hsp90 was evaluated through microscale thermophoresis and molecular modeling, which supported our hypothesis of interaction with Hsp90 (Kd 18-79μm) as basis for the compounds' antiviral activity. The presented novel structural class of small molecules that target the Hsp90 ATP-binding site has excellent potential for further antiviral drug development because of the compounds' low toxicity and synthetic accessibility.

Large scale free energy calculations for blind predictions of protein-ligand binding: the D3R Grand Challenge 2015.

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein-ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate binders from nonbinders in virtual screening and to more accurately predict the ligand binding modes prior to the more computationally expensive FEP calculations of binding affinity.

Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design.

Abstract 483: Docosahexaenoic acid inhibits heat shock protein 90 complex and client proteins by reducing intracellular ATP levels in breast and lung cancer

Abstract A major contributor to carcinogenesis is the molecular chaperone heat shock protein 90 (Hsp90). Hsp90 regulates the proper folding and stabilization of several client proteins, including human epidermal growth factor receptor 2 (ErbB2) and hypoxia-inducible factor 1α (HIF-1α). ErbB2 is a member of the epidermal growth factor receptor (EGFR) family of receptor-proteins, and is a clinical target for the treatment of metastatic cancer, while HIF-1α is often stabilized and overexpressed in several cancers. The ability for Hsp90 to become a mature complex and to properly fold client proteins is dependent on adenosine triphosphate (ATP) binding with the co-chaperone, p23. Specific Hsp90 inhibitors, like geldanamycin, target the Hsp90 ATP-binding site to prevent formation of the mature complex. Although supplementation with docosahexaenoic acid (DHA; C22:6 n-3) has not been shown to specifically target the Hsp90 ATP-binding site, the current study demonstrates a similar effect by DHA causing a reduction in available intracellular ATP levels. DHA is the longest, most unsaturated omega-3 polyunsaturated fatty acid (PUFA) existing in biological membranes and has been shown to possess anti-cancer activity in several cancer models. Studies from our laboratory have shown that DHA enrichment can reduce cancer cell metabolism and intracellular ATP levels in both breast and lung cancer models. In this study, a novel mechanism for the ability of DHA to inhibit the Hsp90 chaperone complex is defined. In vitro analyses of BT-474 human breast ductal carcinoma, as well as the A549 human lung adenocarcinoma cell lines were used to determine the impact of DHA-induced decreases of intracellular ATP levels. We found that the reduction in ATP levels by DHA treatment resulted in significant decreases in the association of Hsp90 and p23 in both cell lines. The decreased association of the Hsp90-p23 complex led to decreased levels of ErbB2 and HIF-1α client proteins, suggesting that DHA can modify Hsp90 chaperone function and attenuate client protein levels. Additionally, similar results were found when employing 2-deoxyglucose (2-DG), a glycolytic inhibitor, which confirms that DHA and 2-DG can disrupt Hsp90 molecular chaperone function by decreasing cellular ATP levels. Consistent with these observations, when using a dose of DHA that did not significantly reduce intracellular ATP levels, no change in the Hsp90-p23 chaperone complex or protein levels of ErbB2 and HIF-1α were seen. These results demonstrate a potential use for dietary intervention to improve cancer therapy in tumors that overexpress Hsp90 client proteins. Citation Format: Michael Mouradian, Irvin V. Ma, Erika D. Vicente, Keith D. Kikawa, Amy M. Chattin, Ronald S. Pardini. Docosahexaenoic acid inhibits heat shock protein 90 complex and client proteins by reducing intracellular ATP levels in breast and lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 483. doi:10.1158/1538-7445.AM2014-483

Hybrids of the Benzofuran Core from Natural Products and the 2,4-Dihydroxy-5-isopropylbenzene Fragment as Potent Hsp90 Inhibitors: Design, Synthesis and Bioevaluation.

Several chemical fragments have been confirmed as highly efficient cores for the design of Hsp90 inhibitors. Molecular hybridization of potent fragments has been widely used as a rational drug discovery strategy. In this study, a novel class of hybrids of benzofuran, a privileged core from natural products, and 2,4-dihydroxy-5-isopropyl phenyl, an efficient fragment in Hsp90 inhibitors, were designed and synthesized. Subsequent evaluation confirmed they inhibited cell proliferation and regulated the level of client proteins through Hsp90 inhibition. Some of the hybrids can serve as leads to obtain novel chemotypes of Hsp90 inhibitors. The methods reported here may expand the range of known structural types accommodated by the ATP binding site of Hsp90.

Synthesis and Evaluation of New Hsp90 Inhibitors Based on a 1,4,5-Trisubstituted 1,2,3-Triazole Scaffold

Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate.

Combining Solvent Thermodynamic Profiles with Functionality Maps of the Hsp90 Binding Site to Predict the Displacement of Water Molecules

Intermolecular interactions in the aqueous phase must compete with the interactions between the two binding partners and their solvating water molecules. In biological systems, water molecules in protein binding sites cluster at well-defined hydration sites and can form strong hydrogen-bonding interactions with backbone and side-chain atoms. Displacement of such water molecules is only favorable when the ligand can form strong compensating hydrogen bonds. Conversely, water molecules in hydrophobic regions of protein binding sites make only weak interactions, and the requirements for favorable displacement are less stringent. The propensity of water molecules for displacement can be identified using inhomogeneous fluid solvation theory (IFST), a statistical mechanical method that decomposes the solvation free energy of a solute into the contributions from different spatial regions and identifies potential binding hotspots. In this study, we employed IFST to study the displacement of water molecules from the ATP binding site of Hsp90, using a test set of 103 ligands. The predicted contribution of a hydration site to the hydration free energy was found to correlate well with the observed displacement. Additionally, we investigated if this correlation could be improved by using the energetic scores of favorable probe groups binding at the location of hydration sites, derived from a multiple copy simultaneous search (MCSS) method. The probe binding scores were not highly predictive of the observed displacement and did not improve the predictivity when used in combination with IFST-based hydration free energies. The results show that IFST alone can be used to reliably predict the observed displacement of water molecules in Hsp90. However, MCSS can augment IFST calculations by suggesting which functional groups should be used to replace highly displaceable water molecules. Such an approach could be very useful in improving the hit-to-lead process for new drug targets.

Targeting the hydrophobic region of Hsp90’s ATP binding pocket with novel 1,3,5-triazines

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in regulating the maturation and stabilization of many oncogenic proteins. In an attempt to discover a new class of Hsp90 inhibitors, a series of 1,3,5-triazine compounds were rationally designed, synthesized, and their biological activities were evaluated. Compound 3b was found to degrade Hsp90’s client proteins of Her2, Met and Akt and to induce the expression level of Hsp70. The binding mode of 3b in the ATP-binding site of Hsp90 was predicted by the molecular docking.

Simplified aminocoumarin analogues as anticancer agents: Amino isosteric replacement in the noviose moiety resulted in substantial enhancement of antiproliferative activity

Abstract Simplified aminocoumarin analogues, either noviosylated or simple basic heterocycle attached 3-amido-coumarin compounds, are known to be promising anticancer agents targeting the C-terminal ATP-binding site of Hsp90. In this study, 3′-amino isosteric replacement in the noviose moiety of two known noviose containing Hsp90 C-terminal inhibitors was synthetically realized for the first time. In vitro evaluation of these compounds suggested that the introduction of a basic amino group into the noviose subunit resulted in significant improvement of their cytotoxicities.

Synthesis and Biological Activities of a New Class of Heat Shock Protein 90 Inhibitors, Designed by Energy-Based Pharmacophore Virtual Screening

The design through energy-based pharmacophore virtual screening has led to aminocyanopyridine derivatives as efficacious new inhibitors of Hsp90. The synthesized compounds showed a good affinity for the Hsp90 ATP binding site in the competitive binding assay. Moreover, they showed an excellent antiproliferative activity against a large number of human tumor cell lines. Further biological studies on the derivative with the higher EC50 confirmed its specific influence on the cellular pathways involving Hsp90.

Dual inhibition of chaperoning process by taxifolin: Molecular dynamics simulation study

Hsp90 (heat shock protein 90), a molecular chaperone, stabilizes more than 200 mutated and over expressed oncogenic proteins in cancer development. Cdc37 (cell division cycle protein 37), a co-chaperone of Hsp90, has been found to facilitate the maturation of protein kinases by acting as an adaptor and load these kinases onto the Hsp90 complex. Taxifolin (a natural phytochemical) was found to bind at ATP-binding site of Hsp90 and stabilized the inactive “open” or “lid-up” conformation as evidenced by molecular dynamic simulation. Furthermore, taxifolin was found to bind to interface of Hsp90 and Cdc37 complex and disrupt the interaction of residues of both proteins which were essential for the formation of active super-chaperone complex. Thus, taxifolin was found to act as an inhibitor of chaperoning process and may play a potential role in the cancer chemotherapeutics.

A Compound That Inhibits the HOP–Hsp90 Complex Formation and Has Unique Killing Effects in Breast Cancer Cell Lines

The chaperone Hsp90 is required for the correct folding and maturation of certain "client proteins" within all cells. Hsp90-mediated folding is particularly important in cancer cells, because upregulated or mutant oncogenic proteins are often Hsp90 clients. Hsp90 inhibitors thus represent a route to anticancer agents that have the potential to be active against several different types of cancer. Currently, various Hsp90 inhibitors that bind to Hsp90 at its ATP-binding site are in preclinical and clinical trials. Some of the most promising Hsp90 ATP-binding site inhibitors are the well characterized geldanamycin derivative 17-AAG and the recently described compounds PU-H71 and NVP-AUY922. An undesirable characteristic of these compounds is the transcriptional upregulation of Hsp70 that has prosurvival effects. Here we characterize the activity of a new type of chaperone inhibitor, 1,6-dimethyl-3-propylpyrimido[5,4-e][1,2,4]triazine-5,7-dione (named C9 for simplicity). Using purified protein components in vitro, C9 prevents Hsp90 from interacting with the cochaperone HOP and is thus expected to impair the Hsp90-dependent folding pathway in vivo. We show that this compound is effective in killing various breast cancer cell lines including the highly metastatic MDA-MB-231. An important property of this compound is that it does not induce the transcriptional upregulation of Hsp70. Moreover, when cells are treated with a combination of C9 and either 17-AAG or NVP-AUY922, the overexpression of Hsp70 is counteracted considerably and C9's lethal-IC50 decreases compared to its value when added alone.

Features of theStreptomyces hygroscopicusHtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90

Much attention is focused on the benzoquinone ansamycins as anticancer agents, with several derivatives of the natural product geldanamycin (GdA) now in clinical trials. These drugs are selective inhibitors of Hsp90, a molecular chaperone vital for many of the activities that drive cancer progression. Mutational changes to their interaction site, the extremely conserved ATP binding site of Hsp90, would mostly be predicted to inactivate the chaperone. As a result, drug resistance should not arise readily this way. Nevertheless, Streptomyces hygroscopicus, the actinomycete that produces GdA, has evolved an Hsp90 family protein (HtpG) that lacks GdA binding. It is altered in certain of the highly conserved amino acids making contacts to this antibiotic in crystal structures of GdA bound to eukaryotic forms of Hsp90. Two of these amino acid changes, located on one side of the nucleotide-binding cleft, weakened GdA/Hsp90 binding and conferred partial GdA resistance when inserted into the endogenous Hsp90 of yeast cells. Crystal structures revealed their main effect to be a weakening of interactions with the C-12 methoxy group of the GdA ansamycin ring. This is the first study to demonstrate that partial GdA resistance is possible by mutation within the ATP binding pocket of Hsp90.

HSP90 Inhibitors: Multi-Targeted Antitumor Effects and Novel Combinatorial Therapeutic Approaches in Cancer Therapy

With the rapid rise of tumor resistance, combinatorial anticancer therapies have gained favor over single-molecule inhibition to maximize the suppression of oncogenic pathways. In this regard, HSP90 inhibitors have rapidly emerged as a class of promising drugs that can target multiple oncogenic pathways simultaneously. HSP90 is a highly conserved protein chaperone involved in essential cellular functions such as protein folding and cell signaling in both stressed and unstressed cells. In the last decade, a large number of oncogenic client proteins have been identified to associate with HSP90 and contribute to malignant transformation. Development of HSP90 inhibitors, derived from the natural compound geldanamycin that mimics the ATP binding site of HSP90, was designed to target HSP90 and allow degradation of these client proteins. Preclinical and clinical data with HSP90 inhibitors in various cancer models are promising, and evidences also hint at the potential for tumor-selective cytotoxicity as well as enhanced sensitization to chemo- and radiotherapy. This review will discuss the effects of HSP90 inhibition in cancer, the known mechanistic basis for the oncogenic toxicity and selectivity, as well as the current progress on single or combinatorial therapies with HSP90 inhibitors.

The HSP90 and DNA topoisomerase VI inhibitor radicicol also inhibits human type II DNA topoisomerase

Radicicol derivatives are currently investigated as promising antitumoral drugs because they inhibit the activity of the molecular chaperone heat shock protein (HSP90), causing the destabilization and eventual degradation of HSP90 client proteins that are often associated with tumor cells. These drugs interact with the ATP-binding site of HSP90 which is characterized by a structural element known as the Bergerat fold, also present in type II DNA topoisomerases (Topo II). We have previously shown that radicicol inhibits archaeal DNA topoisomerase VI, the prototype of Topo II of the B family (present in archaea, some bacteria and all the plants sequenced so far). We show here that radicicol also inhibits the human Topo II, a member of the A family (comprising the eukaryotic Topo II, bacterial gyrase, Topo IV and viral Topo II), which is a major target for antitumoral drugs. In addition, radicicol prevents in vitro induction of DNA cleavage by human Topo II in the presence of the antitumoral drug etoposide. The finding that radicicol can inhibit at least two different antitumoral drug targets in human, and interferes with drugs currently used in cancer treatment, could have implications in cancer therapy.

Structure-based discovery of a new class of Hsp90 inhibitors

Docking-based virtual screening identified 1-(2-phenol)-2-naphthol compounds as a new class of Hsp90 inhibitors of low to sub-micromolar potency. Here we report the binding affinities and cellular activities of several members of this class. A high resolution crystal structure of the most potent compound reveals its binding mode in the ATP binding site of Hsp90, providing a rationale for the observed activity of the series and suggesting strategies for developing compounds with improved properties.

Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol

Type II DNA topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA topoisomerase VI ‘Bergerat fold’, whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo.