Hsp90 oligomers: A new outlook for the Hsp90 chaperone machineries
In September 2007 in its weekly epidemiological bulletin the Institut National de Veille Sanitaire (INVS) as announced that for the first time in France, cancer is the leading cause of death before cardiovascular diseases. Indeed, cancer incidence as doubled between 1980 and 2005. Therefore, the development of new innovative cancer treatments with new mechanism of action and new cancer targets are needed. As such, Hsp90 has emerged as a new therapeutic target in cancer and more recently in neurodegenerative diseases. Our general objective was to study Hsp90 function and screen new chemical inhibitors. Our work was divided in three parts. First, we studied Hsp90 oligomerization process and the influence of co-chaperones Aha1 (activator) and p23 (inhibitor) on it. Second, we produced new Hsp90 inhibitors derived from Novobiocin, these molecules were screened in regard of their solubility and their specific interaction with Hsp90. The third part of our work focused on the ability of Hsp90 to regulate amyloid association processes in vitro. Our studies bring a new look at Hsp90 function notably the implication of Hsp90 oligomers in the chaperoning cycle, we screened new Hsp90 potential inhibitors whose antitumoral activities are actually tested on cancer cell lines.
For the studies related to Hsp90 oligomerization process and the influence of co-chaperones, a pre-requisites was to optimized a chemical cross-linking protocol to stabilize complexes in order to determine stoichiometries of interaction. Once complexes stabilized, we combined biochemical and biophysical techniques such as analytical ultracentrifugation, size-exclusion chromatography coupled or not to multi-angle laser light scattering and high-mass spectrometry. The high-mass spectrometry technique was crucial to understand how complexes are formed. For the screening of Novobiocin derivative inhibitors, we elaborated a selection protocol based on several steps: their solubility in dimethylsulfoxyde, their solubility in aqueous solutions, their capacity to bind Hsp90 and specifically the C-terminal domain and we are now testing their anti proliferative activity on cancer line cells. For this work, we used biophysical techniques such as ultracentrifugation, spectrophotometry, spectrofluorimetry and we measure cellular growth to determine their half maximal inhibitory concentration (IC50). Concerning the third part of our work, we used biophysical techniques previously cited in association with electron microscopy to characterize amyloid peptides fibrillation processes and the effects of Hsp90 on it.
The originality of our project lies in the fact that we are interested in the role of Hsp90 oligomers. Indeed, we think that theses oligomers are the active forms as molecular chaperone and results obtained go in this way. Based on the interaction of Hsp90 with its co-chaperones Aha1 and p23, we propose a new model of Hsp90 chaperone cycle regulation. Concerning Hsp90 Novobiocin derivative inhibitors, from more than sixty molecules synthetized, 10 to 20 of them were selected and their anti proliferative activity are actually tested on cells. This new molecules are all potentially new drugs for cancer treatments. In the last part of our work, we demonstrated that the Hsp90, probably oligomers, are able to regulate amyloid fibrillation process. Thus, Hsp90 Novobiocin derivative inhibitors now appear as potential drugs for treatment of amyloid diseases too.
The optimization of complexes cross-linking protocol was published in “Analytical chemistry”. For Hsp90 oligomerization process and the influence of its co-chaperones two other publications are actually in revision in international journals: “BBA Proteins and Proteomics” and “Analytical chemistry”. Novobiocin derivative inhibitors were the objects of patents deposit by chemists’ team, compilation of obtained results with cellular tests in process will lead to the redaction of articles that will be published in international journals. The role of Hsp90 in amyloid fibrillation process is the subject of a publication in submission to “FEBS Journal”. Our studies have been presented in national and international congresses.
Chaperones are essential for cell life, as they are involved in the folding of newly synthesized proteins. Amongst them, the heat-shock protein of 90 kDa (Hsp90) plays a special role. The folding of many key proteins involved in cell cycle control or signal transduction is Hsp90-dependent. Under normal conditions, Hsp90 does not seem to directly participate in the folding of de novo synthesized proteins but, with the assistance of co-chaperones, helps specific client proteins to acquire their active conformation. Under stress conditions Hsp90 is over-expressed, suggesting a protective role. While Hsp90 is fundamental for the life of normal cells, a drawback of its activity is that it also protects mutated proteins against degradation, and thus promotes cancer cells survival. For this reason Hsp90 has emerged as a promising target for new cancer therapeutics, with the perspective of simultaneous disruption of a wide range of oncogenic pathways (Le Bras et al., 2007). Therefore, the understanding of the Hsp90 chaperoning cycle represents a major and competitive challenge. The cycle of Hsp90 is intimately associated with large conformational rearrangements. Our understanding of Hsp90 conformational changes derives mostly from structural information, which refers to the crystal states of either recombinant truncated Hsp90 or the prokaryotic homolog HtpG. Recently, we have determined the first nucleotide-free structures of the full-length eukaryotic Hsp90 (apo-Hsp90), along with its intrinsic flexibility. Although large structural rearrangements were previously attributed to the binding of nucleotides, we have shown that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Thus, taking into account the preponderant role of the dynamic nature of Hsp90's structure, we have reconsidered the Hsp90 ATPase cycle (Bron et al., 2008). Beside its dimeric state, Hsp90 oligomerizes in the presence of divalent cations and under heat stress, oligomers would then participate to the chaperone-cycle. To decipher a protein function mechanism, specific inhibitors of its activity are powerful tools. For Hsp90, two classes of inhibitors are known. The geldanamycin (GA) class is the most extensively studied contrary to the novobiocin (Nvb) one. Several studies have demonstrated that each class has a distinct effect on the Hsp90 chaperoning cycle (Keppler, 2006, Fan and Young 2006). Linking these inhibitors with nano-gold particles will allow us to create efficient molecular probes to localize Nvb binding site into Hsp90. The aims of this project is to characterize the molecular basis of the Hsp90 chaperoning cycle considering both dimer and oligomers implication, the Hsp90/co-chaperones function together with elucidation of the mode of action of novobiocin and its derivatives on this system. We plan to study the structure, dynamics and modulation of the Hsp90 chaperoning cycle by combining complementary expertise in medicinal chemistry and structural biology. Using state of the art structural approaches, such as X-Ray crystallography, cryo-electron microscopy, we will study Hsp90 homo and hetero-complexes formed with co-chaperones, client proteins and inhibitors to unravel the details of the Hsp90 cycle at the molecular level. The data generated during this program will have significant contribution to understanding of the Hsp90 oncogenic activity and should lead to the development of new or optimized Hsp90 inhibitors with potential activities in cancer therapy.
Monsieur Cyrille GARNIER (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE BRETAGNE ET PAYS- DE-LA-LOIRE) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UMR-CNRS 6026 CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE BRETAGNE ET PAYS- DE-LA-LOIRE
Help of the ANR 199,996 euros
Beginning and duration of the scientific project: - 48 Months