Inhibitors of Molecular Chaperones as Therapeutic Agents / Edition 1 available in Hardcover
Inhibitors of Molecular Chaperones as Therapeutic Agents / Edition 1
- ISBN-10:
- 1849736669
- ISBN-13:
- 9781849736664
- Pub. Date:
- 10/30/2013
- Publisher:
- RSC
Inhibitors of Molecular Chaperones as Therapeutic Agents / Edition 1
Hardcover
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$224.00Overview
Arranged into three main sections the book covers structure and function, small molecule inhibitors and concludes with a section discussing clinical perspectives. With specific chapters covering the discovery of key molecules such as, BIIB028, STA-9090, Serenex Hsp90 inhibitor, NVP-AUY922 and NVP-HSP990, this comprehensive text is an essential treatise for researchers working in academia and industry.
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Inhibitors of Molecular Chaperones as Therapeutic Agents
By Timothy D Machajewski, Zhenhai Gao
The Royal Society of Chemistry
Copyright © 2014 The Royal Society of ChemistryAll rights reserved.
ISBN: 978-1-84973-666-4
CHAPTER 1
Overview of Molecular Chaperones in Health and Disease
TAI WANG, PABLO C. ECHEVERRÍA AND DIDIER PICARD
Département de Biologie Cellulaire, Université de Genève, Sciences III, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland
1.1 Proteostasis and the Central Role of Molecular Chaperones
After being synthesized on ribosomes as linear amino acid chains, proteins need to be folded into their native states, a dynamic equilibrium of closely related three-dimensional structures. In addition to this initial process, cells also need protein quality control and the maintenance of proteome homeostasis (known as proteostasis), both of which are crucial for cellular and organismal health. That is why many diseases appear to be caused by misregulation of protein maintenance. Examples of this are the loss-of-function diseases such as cystic fibrosis and the gain-of-toxic-function diseases such as Alzheimer's, Parkinson's and Huntington's diseases. Proteostasis is maintained by a complex regulatory network, which comprises proteins, cofactors and processes that control protein synthesis, folding, trafficking, aggregation, disaggregation and degradation. Molecular chaperones and their regulators are central players of the proteostatic network.
Molecular chaperones interact with their targets to provide a temporary stabilization, which facilitates folding into a functionally active conformation, unfolding for degradation, or assembly or disassembly of multi-component complexes. Typically, molecular chaperones are not associated with their target proteins once these have acquired their final functional conformation. Recent findings show that there are exceptions. It was recently demonstrated that the glucorticoid receptor (GR) translocates into the nucleus as an Hsp90 heterocomplex upon stimulation by glucocorticoids before dissociating within the nucleus. Moreover, the intranuclear dynamics of GR is still Hsp90dependent, which indicates that some molecular chaperones may escort their clients during their entire lifespan.
Molecular chaperones further ensure proteostasis and prevent proteotoxicity by promoting protein degradation and disposal through multiple pathways. For example, Hsp70, Hsp90 and their co-chaperones target unfolded proteins for degradation via the ubiquitin–proteasome system. They also assist autophagy. Recent studies show the contribution of Hsp70 to this type of removal of pathogenic proteins. Hsp70 is involved in a certain type of autophagy involving late endosomes known as endosomal microautophagy, and in a form of macroautophagy mediating the degradation of protein aggregates known as chaperone-assisted selective autophagy. The more selective chaperone-mediated autophagy (CMA) requires that cytosolic proteins that contain the pentapeptide targeting motif KFERQ are recognized by heat-shock cognate 70 (Hsc70) and delivered to the surface of lysosomes to be translocated and degraded by lysosomal proteases. A fraction of Hsp90 is present at lysosomes, bound to the luminal side of the lysosomal membrane, and it can either increase or decrease CMA activity depending on the cell type.
1.2 The Major Classes of Molecular Chaperones
Molecular chaperones are classified into five families according to their molecular size, namely Hsp100, Hsp90, Hsp70 and J proteins, chaperonins and small heat-shock proteins (sHsp).
1.2.1 Hsp100
Hsp100 chaperones are members of a large superfamily of AAA+ ATPases. They form oligomeric rings involved in protein refolding, disaggregation and degradation. They are found in bacteria, yeast and plants but not in animal cells. Most members of this family use the energy derived from ATP to unfold substrates and to translocate them for degradation to a protease subunit that can be associated with them. Other chaperone machines such as the Hsp70 and J proteins cooperate with the protein disaggregation activity of Hsp100 proteins.
1.2.2 Hsp90
This molecular chaperone is highly abundant in the cytosol of bacterial and eukaryotic cells under physiological conditions, and can be further up-regulated by cellular stress. The Hsp90 family in mammalian cells is composed of four major homologs: Hsp90α (inducible form) and Hsp90β (constitutive form) are cytosolic isoforms; the 94 kDa glucose-regulated protein (GRP94) is localized in the endoplasmic reticulum, and TRAP1 resides in the mitochondrial matrix. The cytosolic forms of Hsp90 bind proteins in a metastable native-like state, which they may have acquired with the help of other chaperone machines. Hsp90 acts with a group of co-chaperones that modulate its client recognition, ATPase cycle and chaperone function. Due to the nature of its clients, its proteostatic functions affect several essential cellular activities such as development, transcription, cell cycle, intracellular signaling, apoptosis, protein degradation and innate and adaptive immunity.
1.2.3 Hsp70 and J Proteins
Hsp70 proteins are highly conserved, present both as constitutively expressed and stress-inducible cytosolic isoforms, and isoforms localized to other cellular compartments such as the endoplasmic reticulum and mitochondria. They are important for de novo folding, but also for other functions, including protein trafficking, unfolding and degradation of misfolded proteins. More generally speaking, Hsp70s together with a group of essential cofactors, the J proteins of the Hsp40 family and the large nucleotide-exchange factors (NEFs), are involved in ATP-regulated binding and release of non-native proteins including nascent polypeptide chains. Binding and release by Hsp70 is achieved through the allosteric coupling of a conserved N-terminal ATPase domain with a separate substrate binding domain. Hydrolysis of ATP to ADP is strongly accelerated by Hsp40 proteins. Hsp40s also interact directly with unfolded polypeptides and can recruit Hsp70 to protein substrates. Binding of Hsp70 to non-native substrates impedes aggregation by rapidly protecting exposed hydrophobic segments thereby reducing the presence of species tending to aggregate. Recently, it was described that the nucleotide exchange factor Hsp110, an Hsp70 homolog in eukaryotes, cooperates with the conventional Hsp70-Hsp40 machinery to disaggregate and to refold aggregated proteins. Hsp70 machines act in concert with yet other molecular chaperone machines; for example, they often act upstream of chaperonins and the Hsp90 chaperone machine. Hsp70s are especially important under stress by preventing the aggregation of unfolded proteins and by refolding aggregated proteins. This and different features described in Table 1.3 confer the capacity to Hsp70 to act as a survival factor, which is particularly relevant to provide resistance to apoptosis and autophagy in cancer cells.
1.2.4 Chaperonins
Chaperonins are ring-shaped multi-subunit chaperones that encapsulate nonnative proteins in an ATP-dependent manner. In bacteria, the GroE machinery consists of a multi-subunit structure of the two proteins GroEL and GroES. The closely related proteins of eukaryotic mitochondria are called Hsp60 and Hsp10, respectively. The non-native protein is trapped in the cavity of GroEL, which becomes a highly hydrophilic environment with a net negative charge where the protein is free to fold after the open structure is capped by GroES binding. The eukaryotic cytosolic chaperonin TRiC is independent of Hsp10. Instead, it contains finger-like projections in its apical domain, which act as a lid and replace the Hsp10/GroES functions.
1.2.5 Small Hsps
These are ATP-independent molecular chaperones that interact with large numbers of partially folded target proteins to prevent their aggregation upon stress. They act as a depository for unfolded proteins, which will later be refolded by other chaperone machines like Hsp70 and Hsp100. In their native state, they form ring-like oligomers of 12–32 subunits with internal spaces in symmetrically blocked dimeric subunits.
1.3 Miscellaneous Other Molecular Chaperones
The following are molecular chaperones that are not part of the major chaperone families, but are nevertheless worth thinking about as potential drug targets.
ADCK3 (Chaperone activity of bc1 complex-like, mitochondrial): this chaperone-like protein kinase is essential for the proper conformation and functioning of protein complexes in the respiratory chain. Its absence produces a decrease of the Coenzyme Q10 (CoQ10), and increased ROS production and oxidation of lipids and proteins. ADCK3 mutations were detected in patients with cerebellar ataxia.
AHSP (Alpha-hemoglobin-stabilizing protein): a molecular chaperone that is important to prevent the harmful aggregation of free α-hemoglobin during normal erythroid cell development and in β-thalassemic erythroid precursor cells. Gene knockout studies in mice confirmed that AHSP is required for normal erythropoiesis. AHSP knockout mice exhibit anemia, decreased hematocrit and high levels of ROS, consistent with the presence of unstable α-globin.
ANKRD13 (Ankyrin repeat domain-containing protein 13): acts as a molecular chaperone for G protein-coupled receptors, controlling their biogenesis and exit from the endoplasmic reticulum. It also regulates the rapid internalization of ligand-activated EGFR.
Histones chaperones: a group of proteins interacting with histones from their synthesis, during import into the nucleus, and for association with target DNA throughout DNA replication, repair or transcription.
CLU (clusterin): functions as extracellular chaperone that prevents aggregation of non-native proteins. Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones. In Alzheimer's disease, CLU contributes to limit amyloidogenic Aβ species misfolding and facilitates their clearance from the extracellular space.
TOR1A (Dystonia 1 protein): TorsinA is a member of the AAA-ATPase family of molecular chaperones, assisting in the proper folding of secreted and/or membrane proteins. Defects in TOR1A are the cause of dystonia type 1.
HYPK (Huntingtin-interacting protein K): it has a molecular chaperone activity that prevents polyglutamine (polyQ) aggregation of the huntingtin protein.52
1.3 Molecular Chaperones in Health and Disease
Molecular chaperones are sensitive hubs of the proteostasis network. As a consequence, genetic alterations of the expression or sequence of members of this network may cause disease. Table 1.1 summarizes the disorders associated with mutations of genes encoding members of the molecular chaperones families.
In addition, the phenotypes of the genetic ablation of different members of the molecular chaperone families in mouse models further emphasize the importance of these genes at the organismic level. Table 1.2 displays a complete compilation of the annotated data concerning the knockout of several molecular chaperones and their co-chaperones present in the PhenomicDB.
Tables 1.1 and 1.2 clearly show that genetic polymorphisms, mutations or the complete ablation of a member of any molecular chaperone machine have an impact on organisms, often with catastrophic consequences. The more or less severely perturbed proteostasis can affect multiple organs and physiological processes, including aging. In accordance with their key hub function, knockout mouse models are often embryonically lethal or have severe complications in development.
It is noteworthy that the presence (or disproportionate presence) of molecular chaperones is not always beneficial. Even when their participation in the protection against apoptosis could be favorable in the context of some disorders, it can unfortunately also promote the initiation and progression of cancer. In addition, several "clients" of these chaperone machines are oncoproteins. These relationships are summarized in Table 1.3.
This overview provides valuable information in view of the use of inhibitors of selected molecular chaperones for therapeutic interventions, for example against cancer or neurodegenerative diseases. It highlights the huge therapeutic potential, but it also gives a flavor of the extremes of the adverse effects that may have to be expected. To emphasize this point further, we have attempted to model the effect of removing, i.e. inhibiting, Hsp90 as one of the key hubs of proteostasis. Figure 1.1 illustrates the dramatic impact that such a treatment can have on part of the proteome.
1.4 Strategies for Modulating Chaperone Activities
The next chapters present various strategies aimed at modulating chaperone activities. In the interest of space, we decided to focus on a few major chaperone machines. This is not a serious limitation since a limited number of molecular chaperones have received almost all of the attention of developers. Other molecular chaperones have yet to be explored as drug targets altogether. Most of the "strategies" reported to date are based on small organic molecules that inhibit molecular chaperones, but more diversity in terms of types of molecules, approaches and impact on molecular chaperones can be expected from future efforts. And to the best of our knowledge and despite very high hopes, "molecular chaperone drugs" have yet to make it into the clinic. Nevertheless, the emerging diversity attests to the huge interest that this class of proteins has attracted over the last few years.
1.5.1 Hsp90 Inhibitors
The functionality of Hsp90 requires highly complex conformational rearrangements regulated by a large spectrum of co-chaperones in which ATP hydrolysis plays a fundamental role in driving the chaperone machinery. Hsp90 features a conserved GHKL-type (gyrase, Hsp90, Histidine Kinase, MutL) ATPase domain at the N-terminus. Therefore, designing competitive inhibitors that target the N-terminal ATP binding pocket of Hsp90 has been the main strategy to block Hsp90. The following paragraphs first present these inhibitors before giving an overview of inhibitors that target other Hsp90 domains and Hsp90 co-chaperones (see Table 1.4).
1.5.1.1 Inhibitors Targeting the N-Terminal ATP Binding Pocket
These types of inhibitors arrest the chaperone cycle by trapping Hsp90 in a conformation reminiscent of the ADP-bound conformation. This leads to an early release of immature client proteins followed by their degradation through the ubiquitin-proteasome pathway. Among the clients of Hsp90, many are oncogenic such as B-Raf, v-Src, HER2, Akt, mutant p53, HIF-1α and Bcr-Abl, which exemplifies the central role of Hsp90 in maintaining the homeostasis of cancer cells. As a result of Hsp90 inhibition and degradation of these and other clients, cancer cells stop proliferating and even undergo apoptosis. A corollary of the inhibition of Hsp90 is the induction of Hsp70 expression through the proteasomal stress response and by derepression of the heat-shock factor 1 (HSF-1).
Several classes of N-terminally targeted Hsp90 inhibitors have emerged from both the pharmaceutical industry and the academic world. Based on the natural and prototypical inhibitor geldanamycin, tanespimycin (17-AAG) and 17-DMAG were identified as improved derivatives with less toxicity and better water solubility. However, the reduction of the benzoquinone core by NQO1/ DT-diaphorase is required for these inhibitors to exert full efficacy. This prompted the development of IPI-504 (Infinity Pharmaceuticals), a tanespimycin derivative bearing a stabilized hydroquinone ring as an attempt to reduce or to abolish the dependence on the NQO1-mediated reduction. This inhibitor is currently in clinical trials and demonstrates promising potency against non-small-cell lung cancer. However, despite an excellent specificity for Hsp90, many benzoquinone ansamycin-based molecules have intrinsic hepatotoxicity.
The discovery of the purine-based inhibitor PU3 brought a new impetus for the development of synthetic inhibitors. PU3 stabilizes the N-terminal ATPase pocket of Hsp90 in a conformation characterized by the formation of an a-helix between Leu107 and Gly114. In comparison with the ADP-bound form, this structural rearrangement creates a secondary hydrophobic binding pocket where appropriate hydrophobic decorations of ligands can be accommodated. Apart from the ansamycin family of inhibitors, most of the subsequently developed inhibitors take advantage of this secondary site.
The purine-based inhibitors mimic the adenosine moiety of the natural lig- and ATP, which ensures recognition by Hsp90. The more recently reported aminopyri(mi)dines, thienopyrimidines (from Astex Pharmaceuticals, Evotec, Abbott or Vernalis), the azaindole (from Sanofi-Aventis) and the benzamide (SNX-5422, SNX-2112, XL888) derivatives actually share the same or very similar but more cryptic bioisosteres that were inspired by the adenosine moiety.
Resorcinol derivatives constitute another important group of the Nterminally targeted Hsp90 inhibitors. Their dihydroxyl benzene group is present in radicicol, a natural antibiotic that binds tightly to Hsp90. The binding mode of the resorcinol motif appears to be very stable and conserved in Hsp90s and therefore provides an excellent "starting point" for further drug design by both structure-based and fragment-based approaches.
1.5.1.2 Inhibitors Targeting Other Surfaces of the N-Terminal Domain of Hsp90
Gambogic acid
Gambogic acid binds to the N-terminal domain of Hsp90 and impedes the association with Hsp70 and Cdc37. This leads to the degradation of client proteins and up-regulation of Hsp70 and Hsp90. It induces apoptosis through inactivation of the TNF-α/NFκB pathway. The binding of gambogic acid to the N-terminal domain of Hsp90 is not affected by the presence of geldanamycin, suggesting that the drug may bind to a site distinct from the ATP binding pocket.
Shepherdin (Peptide)
Shepherdin is a fragment of a peptide from the Hsp90 client survivin. Initially designed to block its interaction with Hsp90, the peptide was shown to bind to the N-terminal domain of Hsp90 and to disrupt the Hsp90 ATPase activity. It induces apoptosis in tumor cells following the degradation of Hsp90 clients such as Akt, CDK-4, CDK-6 and survivin without affecting the levels of Hsp70.
Sansalvamide A Derivatives (Peptide)
Sansalvamide A is a cyclic pentapeptide that is extracted from a marine fungus of the genus Fusarium sp. The peptides were demonstrated to bind to the junction between the N-terminal and middle domains of Hsp90 and to inhibit the Hsp90 cycle. The mechanism is most likely an allosteric regulation, which is supported by the fact that the peptides preferentially bind to the closed con- formation of yeast Hsc82 stabilized by the non-hydrolyzable ATP derivative AMPPNP. Further biological characterization showed that Sansalvamide A derivatives induce caspase-dependent apoptosis in cells. Some of them impair the recruitment of clients or co-chaperones including IP6K2, FKBP38, FKBP52 and HOP. Like 17-AAG, these compounds also elicit the up-regulation of Hsp70. In contrast, one of the compounds causes also cytotoxic effects and caspase-mediated apoptosis while it does not affect the interaction between the co-chaperone/clients and Hsp90, suggesting that it may be used as a unique chemical tool that inhibits Hsp90 without altering its binding to clients or co-chaperones.
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