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Inhibitors and chemical probes for molecular chaperone networks

  • Jason E. Gestwicki
    Correspondence
    To whom correspondence should be addressed: UCSF, Sandler Center, 675 Nelson Rising Ln., San Francisco, CA 94158. Tel.:415-502-7121;
    Affiliations
    Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California San Francisco, San Francisco, California 94158
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  • Hao Shao
    Affiliations
    Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California San Francisco, San Francisco, California 94158
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  • Author Footnotes
    3 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
Open AccessPublished:September 13, 2018DOI:https://doi.org/10.1074/jbc.TM118.002813
      The molecular chaperones are central mediators of protein homeostasis. In that role, they engage in widespread protein–protein interactions (PPIs) with each other and with their “client” proteins. Together, these PPIs form the backbone of a network that ensures proper vigilance over the processes of protein folding, trafficking, quality control, and degradation. The core chaperones, such as the heat shock proteins Hsp60, Hsp70, and Hsp90, are widely expressed in most tissues, yet there is growing evidence that the PPIs among them may be re-wired in disease conditions. This possibility suggests that these PPIs, and perhaps not the individual chaperones themselves, could be compelling drug targets. Indeed, recent efforts have yielded small molecules that inhibit (or promote) a subset of inter-chaperone PPIs. These chemical probes are being used to study chaperone networks in a range of models, and the successes with these approaches have inspired a community-wide objective to produce inhibitors for a broader set of targets. In this Review, we discuss progress toward that goal and point out some of the challenges ahead.

      Introduction

      Molecular chaperones help ensure protein homeostasis (i.e. proteostasis), playing essential roles in the folding, trafficking, sequestration, and turnover of proteins (
      • Hartl F.U.
      • Bracher A.
      • Hayer-Hartl M.
      Molecular chaperones in protein folding and proteostasis.
      ). There are ∼150 genes for molecular chaperones in the human genome, including the heat shock proteins Hsp110, Hsp90, Hsp70, Hsp60, Hsp27, etc. and the associated proteins such as co-chaperones, TCP-1 ring complex (TRiC),
      The abbreviations used are:
      TRiC
      TCP-1 ring complex
      PPI
      protein–protein interaction
      BSA
      buried surface area
      ACD
      α-crystallin domain
      PDB
      Protein Data Bank
      NBD
      nucleotide-binding domain
      SBD
      substrate-binding domain
      JDP
      J-domain containing protein
      NEF
      nucleotide exchange factor
      PDI
      protein-disulfide isomerase
      PPIase
      peptidyl-prolyl cis-trans isomerase
      sHSP
      small heat-hock protein
      TPR
      tetratricopeptide repeat
      MoA
      mechanism-of-action
      SAR
      structure–activity relationship.
      protein-disulfide isomerases (PDIs), peptidyl-prolyl cis-trans isomerases (PPIases), calnexin/calreticulin, and more (
      • Hageman J.
      • Kampinga H.H.
      Computational analysis of the human HSPH/HSPA/DNAJ family and cloning of a human HSPH/HSPA/DNAJ expression library.
      ,
      • Kampinga H.H.
      • Hageman J.
      • Vos M.J.
      • Kubota H.
      • Tanguay R.M.
      • Bruford E.A.
      • Cheetham M.E.
      • Chen B.
      • Hightower L.E.
      Guidelines for the nomenclature of the human heat shock proteins.
      ). Together, the coordinated activity of these factors serves to balance proteostasis and protect cells from protein misfolding and/or aggregation. Other articles in this Review Series cover the structure and function of the individual chaperone families in more detail. Here, we focus on the roles played by chemical probes in understanding their activity (
      • Brandvold K.R.
      • Morimoto R.I.
      The chemical biology of molecular chaperones–implications for modulation of proteostasis.
      ,
      • Powers E.T.
      • Morimoto R.I.
      • Dillin A.
      • Kelly J.W.
      • Balch W.E.
      Biological and chemical approaches to diseases of proteostasis deficiency.
      ). For example, our knowledge of Hsp90 biology has benefitted from the availability of chemical inhibitors, which can be applied to cells or organisms to ask how Hsp90 might be involved in a process. In this Review, we briefly introduce how chemical probes are developed and then outline how these ideas are being applied to chaperones.

      What is a chemical probe?

      One simple definition of a chemical probe is as follows: a small molecule that, at a given concentration, selectively inhibits the function of a biological target (
      • Frye S.V.
      The art of the chemical probe.
      ). It is essential that a chemical probe be selective for the intended target. Otherwise, it is difficult to ascribe its activity in cells or organisms to the function of the intended protein (
      • Hu Y.
      • Gupta-Ostermann D.
      • Bajorath J.
      Exploring compound promiscuity patterns and multi-target activity spaces.
      ). Accordingly, the community of chemists and chemical biologists has developed an intuitive, experimental workflow that can be used to understand whether a molecule might be sufficiently selective to be considered a chemical probe. In 2010, Frye (
      • Frye S.V.
      The art of the chemical probe.
      ) published an influential commentary that coalesced many of these emerging ideas, and this concept has been expanded and extended by others (
      • Blagg J.
      • Workman P.
      Choose and use your chemical probe wisely to explore cancer biology.
      ,
      • Shortt J.
      • Ott C.J.
      • Johnstone R.W.
      • Bradner J.E.
      A chemical probe toolbox for dissecting the cancer epigenome.
      ). From a pragmatic perspective, a good chemical probe is typically evaluated through a combination of chemical, biochemical, and genetic experiments (Table 1). Often, this process starts with discovery of an active molecule in a high-throughput screen. Then, a medicinal chemistry campaign is used to create analogs that reveal the relationship between the compound's chemical structure and its activity in vitro (e.g. binding and/or functional assays) and in cells (e.g. cell growth, gene expression, or another phenotype). This correlation is typically referred to as a structure–activity relationship (SAR). An important (and sometimes overlooked) product of an SAR campaign is the selection of a negative control molecule, which is structurally similar to the active molecules but does not bind to the target. Finally, this process is often coupled with determination of the solubility, metabolism, permeability, and lifetime of key analogs. Together, these studies provide a chemical and pharmacological basis for understanding how much active compound is present and whether it would be expected to bind to the intended target under those conditions.
      Table 1Select criteria for consideration of a molecule as a high-quality chemical probe
      Chemical
      • Test the relationship between compound structure and function in multiple in vitro and cell-based assays
      • Key molecules were examined for metabolic stability, reactivity, solubility, pharmacokinetics and other pharmacological properties
      • Compound is active at the expected concentrations in cellular and animal models
      Biochemical
      • Immobilized compound will “pull down” the target; this interaction is competitive with free compound but not with related controls
      • Compound, but not control, will stabilize the target in vitro (thermal shift assays) and in cells (CETSA)
      • Evidence of inactivity is against closely related family members
      • Compound affects known biomarkers
      Genetic
      • Treatment with compound resembles phenotype of knockdown or mutagenesis of the target
      • Overexpression or knockdown of the target changes the potency of the molecule (short hairpin RNA or CRISPR screens)
      • Resistance to the compound occurs by mutation of the target or target pathway (drug-resistance screening)
      From this starting point, the putative probe and its controls are then evaluated in a series of cell-based experiments that are intended to establish confidence that, at a given concentration, it will primarily bind to the intended target and not others. A classic method to assess selectivity is to immobilize the compound on a bead and determine whether it will preferentially “pull down” the intended target from cell lysates (see Table 1). Often, this experiment is supported by a combination of other assays, including cellular thermal shift assays (
      • Martinez Molina D.
      • Nordlund P.
      The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies.
      ), drug-resistance screens (
      • Kapoor T.M.
      • Miller R.M.
      Leveraging chemotype-specific resistance for drug target identification and chemical biology.
      ), and/or testing of the compound in cells in which the putative target has been knocked down, knocked out, or removed by CRISPRi (
      • Kampmann M.
      Elucidating drug targets and mechanisms of action by genetic screens in mammalian cells.
      ). Together, the results of these experiments are used to assess whether a molecule is selective enough to be considered a chemical probe. Because so many different experiments are needed to understand selectivity, the evaluation process often takes years and involves multiple independent laboratories (typically in both academics and industry). Thus, many groups continue using a probe during its evaluation period, often confirming any results with independent methods. It is also important to note that the criteria needed to define a chemical probe are different from those needed to define a drug/therapeutic, i.e. selectivity is relatively more important for probes, whereas safety is of high value for drugs (
      • Garbaccio R.M.
      • Parmee E.R.
      The impact of chemical probes in drug discovery: a pharmaceutical industry perspective.
      ,
      • Jackson M.R.
      Chemical probe development versus drug development.
      ).
      Where does one learn whether an existing compound is considered a good chemical probe? On-line resources that collate this information, such as Chemical Probes (www.chemicalprobes.org)
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      and Probe Miner (https://probeminer.icr.ac.uk/#/),3 are places to start. In addition, reference tools are available for rooting out the worst, most promiscuous molecules, such as pan-assay interference molecules (
      • Baell J.B.
      • Nissink J.W.
      Seven year itch: pan-assay interference compounds (PAINS) in 2017-utility and limitations.
      ) and protein aggregators (
      • Irwin J.J.
      • Duan D.
      • Torosyan H.
      • Doak A.K.
      • Ziebart K.T.
      • Sterling T.
      • Tumanian G.
      • Shoichet B.K.
      An aggregation advisor for ligand discovery.
      ). PubChem (https://pubchem.ncbi.hlm.nih.gov/) provides another guide, as it can be used to determine other assays in which a molecule has been found to be active (
      • Wang Y.
      • Suzek T.
      • Zhang J.
      • Wang J.
      • He S.
      • Cheng T.
      • Shoemaker B.A.
      • Gindulyte A.
      • Bryant S.H.
      PubChem BioAssay: 2014 update.
      ). Finally, Open Science Probes (https://www.sgc.ffm.uni-frankfurt.de/)3 describes a collection of industry-derived tool molecules that have already undergone extensive validation (
      • Müller S.
      • Ackloo S.
      • Arrowsmith C.H.
      • Bauser M.
      • Baryza J.L.
      • Blagg J.
      • Böttcher J.
      • Bountra C.
      • Brown P.J.
      • Bunnage M.E.
      • Carter A.J.
      • Damerell D.
      • Dötsch V.
      • Drewry D.H.
      • Edwards A.M.
      • et al.
      Donated chemical probes for open science.
      ). Together, these resources make it easier for the casual user to become quickly informed about a molecule's suitability for his/her experiment, including using it at the proper concentrations.

      Categories of chemical probes

      In the case of targets in the chaperone network, it is worth considering two major classes of chemical probes: (i) those that inhibit the enzyme activity of a chaperone, and (ii) those that inhibit protein–protein interactions (PPIs) at the connection between two chaperones. These designations are somewhat arbitrary, but the methods for finding and improving them can be quite different, so a brief review of their characteristics is warranted.

      Inhibitors of enzyme activity

      The simplest case of a chemical probe is a molecule that binds at an enzyme's active site. The structure of these inhibitors is often based on the enzyme's substrate or product; thus, in a cellular context, it must bind tight enough to compete for the natural ligand (e.g. ATP). In contrast, allosteric inhibitors bind to a distal pocket (i.e. away from the active site) and only indirectly disrupt enzyme function, so they might not be competitive binders. The major target enzymes in the chaperone network are the ATPases, including TriC, Hsp70, Hsp90, and Hsp60. These chaperones use ATP hydrolysis to power conformational motions that are coupled to their function (albeit not directly (
      • Koldewey P.
      • Horowitz S.
      • Bardwell J.C.
      Chaperone-client interactions: non-specificity engenders multifunctionality.
      ,
      • Chang L.
      • Thompson A.D.
      • Ung P.
      • Carlson H.A.
      • Gestwicki J.E.
      Mutagenesis reveals the complex relationships between ATPase rate and the chaperone activities of Escherichia coli heat shock protein 70 (Hsp70/DnaK).
      )). Thus, compounds that inhibit nucleotide binding in these proteins would be expected to block chaperone activity.

      Inhibitors of PPIs

      Not all of the chaperones have enzymatic activity; for example, small heat shock proteins (sHSPs), Spy, trigger factor, clusterin, and prefoldin, are nonenzymatic chaperones that seem to specialize in limiting protein aggregation (
      • Haslbeck M.
      • Franzmann T.
      • Weinfurtner D.
      • Buchner J.
      Some like it hot: the structure and function of small heat shock proteins.
      • Horowitz S.
      • Koldewey P.
      • Stull F.
      • Bardwell J.C.
      Folding while bound to chaperones.
      ,
      • Narayan P.
      • Orte A.
      • Clarke R.W.
      • Bolognesi B.
      • Hook S.
      • Ganzinger K.A.
      • Meehan S.
      • Wilson M.R.
      • Dobson C.M.
      • Klenerman D.
      The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β(1–40) peptide.
      ,
      • Sörgjerd K.M.
      • Zako T.
      • Sakono M.
      • Stirling P.C.
      • Leroux M.R.
      • Saito T.
      • Nilsson P.
      • Sekimoto M.
      • Saido T.C.
      • Maeda M.
      Human prefoldin inhibits amyloid-β (Aβ) fibrillation and contributes to formation of nontoxic Aβ aggregates.
      • Wruck F.
      • Avellaneda M.J.
      • Koers E.J.
      • Minde D.P.
      • Mayer M.P.
      • Kramer G.
      • Mashaghi A.
      • Tans S.J.
      Protein folding mediated by trigger factor and Hsp70: new insights from single-molecule approaches.
      ). Moreover, even the ATP-utilizing chaperones are assisted by non-enzymatic co-chaperones, which serve as critical adapters between different categories of chaperones (
      • Rauch J.N.
      • Tse E.
      • Freilich R.
      • Mok S.A.
      • Makley L.N.
      • Southworth D.R.
      • Gestwicki J.E.
      BAG3 is a modular, scaffolding protein that physically links heat shock protein 70 (Hsp70) to the small heat shock proteins.
      ,
      • Hishiya A.
      • Salman M.N.
      • Carra S.
      • Kampinga H.H.
      • Takayama S.
      BAG3 directly interacts with mutated αB-crystallin to suppress its aggregation and toxicity.
      ) and between chaperones and other proteostasis pathways. Thus, PPIs are another possible source of targets for chemical probes.
      PPIs are potentially interesting targets because they are often less well conserved than active sites (
      • Ma B.
      • Elkayam T.
      • Wolfson H.
      • Nussinov R.
      Protein–protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces.
      ); thus, selectivity may be easier to achieve (
      • Arkin M.R.
      • Whitty A.
      The road less traveled: modulating signal transduction enzymes by inhibiting their protein–protein interactions.
      ). PPIs also tend to be associated with “tuning” activity rather than switching it off, which could be useful when considering the housekeeping roles of some chaperones. Together, these features seem, on the surface, to generate significant opportunities for probe development (
      • Arkin M.R.
      • Wells J.A.
      Small-molecule inhibitors of protein–protein interactions: progressing towards the dream.
      ,
      • Dougherty P.G.
      • Qian Z.
      • Pei D.
      Macrocycles as protein–protein interaction inhibitors.
      ). However, targeting PPIs also comes with a number of important technical hurdles, namely these contacts tend to have a larger buried surface area (BSA) than enzyme-active sites, making it more difficult to identify small, drug-like molecules (less than 500 Da) that are able to block them. Indeed, recent retrospective analyses of ∼200 successful PPI inhibitors have shown that a majority of the most potent ones act on PPIs with relatively small BSA values (<2,000 to 4,000 Å2) (
      • Thompson A.D.
      • Dugan A.
      • Gestwicki J.E.
      • Mapp A.K.
      Fine-tuning multiprotein complexes using small molecules.
      ,
      • Cesa L.C.
      • Mapp A.K.
      • Gestwicki J.E.
      Direct and propagated effects of small molecules on protein–protein interaction networks.
      • Ran X.
      • Gestwicki J.E.
      Inhibitors of protein–protein interactions (PPIs): an analysis of scaffold choices and buried surface area.
      ). Moreover, the most “druggable” PPIs also tend to be those with tight affinity (Kd <500 nm), likely because those contacts involve a closely spaced combination of hydrophobic and polar residues that facilitates tight inhibitor binding. Thus, not all PPIs are considered equally “druggable.” If PPI targets are placed into four quadrants based on their BSA and affinity values, then those with weak affinity and large BSA values are usually the most difficult. Conversely, targets with small BSA values and/or tight affinity tend to be more tractable.

      Targets in the chaperone network: nodes and edges

      The chaperones and co-chaperones are physically linked to each other through a series of protein–protein interactions, existing as a PPI network (
      • Freilich R.
      • Arhar T.
      • Abrams J.L.
      • Gestwicki J.E.
      Protein–protein interactions in the molecular chaperone network.
      ,
      • Rizzolo K.
      • Huen J.
      • Kumar A.
      • Phanse S.
      • Vlasblom J.
      • Kakihara Y.
      • Zeineddine H.A.
      • Minic Z.
      • Snider J.
      • Wang W.
      • Pons C.
      • Seraphim T.V.
      • Boczek E.E.
      • Alberti S.
      • Costanzo M.
      • et al.
      Features of the chaperone cellular network revealed through systematic interaction mapping.
      ). In this parlance and borrowing from the systems biology lexicon, we term the major chaperones (i.e. Hsp70, Hsp90, Hsp60, and TRiC) as “nodes.” In turn, these nodes are connected by a series of “edges” that represent the PPIs. As will be detailed below, we find these designations useful when considering chemical probes of the chaperone network; specifically, enzyme inhibitors target the ATP-utilizing enzymes of the nodes, whereas PPI inhibitors target the edges.
      However, one caution in this nomenclature is that the edges should not be considered equivalent. Indeed, the physical connections between chaperones come in a great variety of shapes and sizes. For example, a short peptide sequence from the C terminus of Hsp70, EEVD, binds to the tetratricopeptide repeat (TPR) domain that is present in a family of co-chaperones (Fig. 1), such as CHIP and HOP (
      • Assimon V.A.
      • Southworth D.R.
      • Gestwicki J.E.
      Specific binding of tetratricopeptide repeat proteins to heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) is regulated by affinity and phosphorylation.
      ,
      • D'Andrea L.D.
      • Regan L.
      TPR proteins: the versatile helix.
      ). The EEVD–TPR interaction is of relatively tight affinity (Kd ∼ 0.5 μm), and it involves a small surface area (BSA ∼1,100 Å2) (Fig. 2) (
      • Zhang H.
      • Amick J.
      • Chakravarti R.
      • Santarriaga S.
      • Schlanger S.
      • McGlone C.
      • Dare M.
      • Nix J.C.
      • Scaglione K.M.
      • Stuehr D.J.
      • Misra S.
      • Page R.C.
      A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.
      ,
      • Xu Z.
      • Devlin K.I.
      • Ford M.G.
      • Nix J.C.
      • Qin J.
      • Misra S.
      Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein.
      ). By comparison, the interaction between Hsp60 and Hsp10 is weaker (Kd ∼7 μm; Figure 1., Figure 2.A) and involves a 5-fold bigger contact surface area (BSA ∼5,500 Å2) (
      • Parnas A.
      • Nisemblat S.
      • Weiss C.
      • Levy-Rimler G.
      • Pri-Or A.
      • Zor T.
      • Lund P.A.
      • Bross P.
      • Azem A.
      Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-chaperonin.
      ). More globally, we have shown a subset of chaperone PPI structures in Fig. 1 and collated the BSA values from available PDB-deposited structures of chaperone complexes and matched these to measured Kd values in Fig. 2A. Together, this information, although certainly not inclusive, drives home the point that inter-chaperone contacts (“edges”) have quite distinct topologies. For example, the measured Kd values range nearly 6 orders-of-magnitude (from 0.04 to >100 μm), whereas the BSA values can be compact (∼700 Å2 for the Hsp27 system) or large (>20,000 Å2 for the Hsp90–Cdc37 contact).
      Figure thumbnail gr1
      Figure 1.Diversity of PPIs between molecular chaperones. Representative structures of PPIs between chaperones are shown. Hsp70 refers to the nucleotide-binding domain of either the prokaryotic or eukaryotic protein, and ACD is the α-crystallin domain of a small heat shock protein. Monomers of Hsp60 are shown in blue and orange. Please see the citations and PDB codes for information on the exact constructs used: Hsp70-J domain (5NRO); Hsp70–BAG (1HX1); TPR–EEVD (4KBQ); Hsp90–Aha1 (1USU); Hsp90–p23 (2CG9); Hsp90–Cdc37–Cdk4 (5FWP); Hsp60–Hsp10 (4PJ1); TRiC (5GW4); α-crystallin ACD–ACD (2WJ7); and Hsp27 ACD–IPV (4MJH).
      Figure thumbnail gr2
      Figure 2.PPIs between chaperones and their binding partners. A, table of BSA and affinity values for PPIs between chaperones. B, categorization of PPIs based on BSA and affinity values. Based on retrospective analyses of PPI inhibitors, certain quadrants are comparatively easier (green), challenging (gray), or difficult (red) to inhibit with drug-like small molecules.
      Based on this analysis, some of the chaperone PPIs are expected to be relatively more difficult to inhibit. For example, PPIs with weak affinity (>500 nm) and large BSA values (>4,000 Å2), including Hsp90–p23, Hsp60–Hsp10, and Hsp90–Cdc37, are predicted to be particularly challenging (Fig. 2B). Other contacts, such as the ones between Hsp70–BAG1 and Hsp70-HOP, are predicted to be relatively tractable. Recent examinations of published PPI inhibitors have shown that small molecules (<500 Da) can often be used to inhibit a subset of PPIs, whereas the ones with larger BSA values typically need larger molecules, such as peptides or macrocycles (
      • Ran X.
      • Gestwicki J.E.
      Inhibitors of protein–protein interactions (PPIs): an analysis of scaffold choices and buried surface area.
      ). Therefore, it is reasonable to speculate that many different types of chemical scaffolds may be needed to inhibit the full suite of chaperone PPIs. In the following sections, we discuss a few examples of PPI systems that have been successfully targeted, with a focus on Hsp70, Hsp90, Hsp60, and sHSPs. In this discussion, we also comment on the current status of each molecule's ongoing evaluation as a chemical probe, according to the criteria in Table 1.

      Inhibitors of the Hsp70 sub-network

      Hsp70 is called the “triage” chaperone (
      • Wickner S.
      • Maurizi M.R.
      • Gottesman S.
      Posttranslational quality control: folding, refolding, and degrading proteins.
      ) because it plays keys roles in both protein folding and turnover (
      • Mayer M.P.
      Hsp70 chaperone dynamics and molecular mechanism.
      ,
      • Zuiderweg E.R.
      • Hightower L.E.
      • Gestwicki J.E.
      The remarkable multivalency of the Hsp70 chaperones.
      ). Hsp70s are composed of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) (
      • Mayer M.P.
      • Bukau B.
      Hsp70 chaperones: cellular functions and molecular mechanism.
      ). ATP binds in the NBD, and the misfolded clients interact with the SBD. This chaperone is assisted by co-chaperones, including the J-domain containing proteins (JDPs) and nucleotide exchange factors (NEFs). Accordingly, there are at least two conceptual ways of targeting Hsp70: block its ATPase activity or change its PPIs with co-chaperones (
      • Assimon V.A.
      • Gillies A.T.
      • Rauch J.N.
      • Gestwicki J.E.
      Hsp70 protein complexes as drug targets.
      ). Molecules targeting the ATP-binding cleft include VER-155008 and apoptozole (Fig. 3) (
      • Macias A.T.
      • Williamson D.S.
      • Allen N.
      • Borgognoni J.
      • Clay A.
      • Daniels Z.
      • Dokurno P.
      • Drysdale M.J.
      • Francis G.L.
      • Graham C.J.
      • Howes R.
      • Matassova N.
      • Murray J.B.
      • Parsons R.
      • Shaw T.
      • et al.
      Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.
      ,
      • Ko S.K.
      • Kim J.
      • Na D.C.
      • Park S.
      • Park S.H.
      • Hyun J.Y.
      • Baek K.H.
      • Kim N.D.
      • Kim N.K.
      • Park Y.N.
      • Song K.
      • Shin I.
      A small molecule inhibitor of ATPase activity of HSP70 induces apoptosis and has antitumor activities.
      ), which have been recently reviewed (
      • Evans C.G.
      • Chang L.
      • Gestwicki J.E.
      Heat shock protein 70 (hsp70) as an emerging drug target.
      ). VER-155008 competes with nucleotide for binding, as shown by crystallography (
      • Macias A.T.
      • Williamson D.S.
      • Allen N.
      • Borgognoni J.
      • Clay A.
      • Daniels Z.
      • Dokurno P.
      • Drysdale M.J.
      • Francis G.L.
      • Graham C.J.
      • Howes R.
      • Matassova N.
      • Murray J.B.
      • Parsons R.
      • Shaw T.
      • et al.
      Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.
      ), and this molecule has been shown to have the anti-proliferative activity expected of an Hsp70 inhibitor in HCT116 cells (
      • Williamson D.S.
      • Borgognoni J.
      • Clay A.
      • Daniels Z.
      • Dokurno P.
      • Drysdale M.J.
      • Foloppe N.
      • Francis G.L.
      • Graham C.J.
      • Howes R.
      • Macias A.T.
      • Murray J.B.
      • Parsons R.
      • Shaw T.
      • Surgenor A.E.
      • et al.
      Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design.
      ). Similarly, immobilized apoptozole will pull down Hsp70 from A549 (adenocarcinoma) cells (
      • Ko S.K.
      • Kim J.
      • Na D.C.
      • Park S.
      • Park S.H.
      • Hyun J.Y.
      • Baek K.H.
      • Kim N.D.
      • Kim N.K.
      • Park Y.N.
      • Song K.
      • Shin I.
      A small molecule inhibitor of ATPase activity of HSP70 induces apoptosis and has antitumor activities.
      ), and the molecule induces apoptosis in that system. However, Hsp70s, as compared with kinases or Hsp90s, have an unusually tight affinity for ATP (Kd ∼100–500 nm), such that competition for cellular ATP (∼1–10 mm) creates a significant challenge. Covalent versions of VER-155008 have recently been developed (
      • Pettinger J.
      • Le Bihan Y.V.
      • Widya M.
      • van Montfort R.L.
      • Jones K.
      • Cheeseman M.D.
      An irreversible inhibitor of HSP72 that unexpectedly targets lysine-56.
      ), which might circumvent this challenge. The next step for these compounds is evaluation in a greater number of biological systems, using the chemical, genetic, and biochemical validation assays in Table 1.
      Figure thumbnail gr3
      Figure 3.Selected chemical probes for molecular chaperones. See text for citations and details.
      The other way to inhibit Hsp70 is by targeting its PPIs, and one of the first chemical series found to do this were the dihydropyrimidines (Fig. 3; Table 2) (
      • Fewell S.W.
      • Smith C.M.
      • Lyon M.A.
      • Dumitrescu T.P.
      • Wipf P.
      • Day B.W.
      • Brodsky J.L.
      Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity.
      ,
      • Huryn D.M.
      • Brodsky J.L.
      • Brummond K.M.
      • Chambers P.G.
      • Eyer B.
      • Ireland A.W.
      • Kawasumi M.
      • Laporte M.G.
      • Lloyd K.
      • Manteau B.
      • Nghiem P.
      • Quade B.
      • Seguin S.P.
      • Wipf P.
      Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators.
      ). These molecules were inspired by the natural product spergualin, and they were found to bind at an interface between bacterial Hsp70 and the JDPs (
      • Kampinga H.H.
      • Craig E.A.
      The HSP70 chaperone machinery: J proteins as drivers of functional specificity.
      ). Limited medicinal chemistry efforts (
      • Fewell S.W.
      • Smith C.M.
      • Lyon M.A.
      • Dumitrescu T.P.
      • Wipf P.
      • Day B.W.
      • Brodsky J.L.
      Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity.
      ,
      • Wisén S.
      • Androsavich J.
      • Evans C.G.
      • Chang L.
      • Gestwicki J.E.
      Chemical modulators of heat shock protein 70 (Hsp70) by sequential, microwave-accelerated reactions on solid phase.
      ) showed that, depending on their individual chemical substitution patterns, the dihydropyrimidines either promote or inhibit this PPI (
      • Wisén S.
      • Bertelsen E.B.
      • Thompson A.D.
      • Patury S.
      • Ung P.
      • Chang L.
      • Evans C.G.
      • Walter G.M.
      • Wipf P.
      • Carlson H.A.
      • Brodsky J.L.
      • Zuiderweg E.R.
      • Gestwicki J.E.
      Binding of a small molecule at a protein–protein interface regulates the chaperone activity of hsp70–hsp40.
      ). A recent crystal structure of Hsp70 bound to a J domain (
      • Kityk R.
      • Kopp J.
      • Mayer M.P.
      Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones.
      ) (see Fig. 1) is expected to increase our understanding of how these compounds might work. Still, although the potency and pharmacokinetics of this chemical series remain un-optimized (i.e. EC50 ∼ micromolar), there is reason to be optimistic. For example, immobilized dihydropyrimidines pull down Hsp70 from cell lysates (
      • Wisén S.
      • Bertelsen E.B.
      • Thompson A.D.
      • Patury S.
      • Ung P.
      • Chang L.
      • Evans C.G.
      • Walter G.M.
      • Wipf P.
      • Carlson H.A.
      • Brodsky J.L.
      • Zuiderweg E.R.
      • Gestwicki J.E.
      Binding of a small molecule at a protein–protein interface regulates the chaperone activity of hsp70–hsp40.
      ), and treatment with analogs, such as MAL3-101 and MAL1-27, has been shown to induce known Hsp70 biomarkers (
      • Sabnis A.J.
      • Guerriero C.J.
      • Olivas V.
      • Sayana A.
      • Shue J.
      • Flanagan J.
      • Asthana S.
      • Paton A.W.
      • Paton J.C.
      • Gestwicki J.E.
      • Walter P.
      • Weissman J.S.
      • Wipf P.
      • Brodsky J.L.
      • Bivona T.G.
      Combined chemical-genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma.
      ). Additional evidence for target engagement comes from studies in which yeast treated with an agonist, SW02, was partially protected from genetic deletion of a JDP (
      • Wisén S.
      • Bertelsen E.B.
      • Thompson A.D.
      • Patury S.
      • Ung P.
      • Chang L.
      • Evans C.G.
      • Walter G.M.
      • Wipf P.
      • Carlson H.A.
      • Brodsky J.L.
      • Zuiderweg E.R.
      • Gestwicki J.E.
      Binding of a small molecule at a protein–protein interface regulates the chaperone activity of hsp70–hsp40.
      ). Moreover, treatment with MAL1-27 (also called 115-7c) protects against polyglutamine (polyQ) aggregation in multiple models, which mirrors what happens when Hsp70 is overexpressed (
      • Walter G.M.
      • Smith M.C.
      • Wisén S.
      • Basrur V.
      • Elenitoba-Johnson K.S.
      • Duennwald M.L.
      • Kumar A.
      • Gestwicki J.E.
      Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes.
      ,
      • Chafekar S.M.
      • Wisén S.
      • Thompson A.D.
      • Echeverria A.
      • Walter G.M.
      • Evans C.G.
      • Makley L.N.
      • Gestwicki J.E.
      • Duennwald M.L.
      Pharmacological tuning of heat shock protein 70 modulates polyglutamine toxicity and aggregation.
      ). Finally, acquired resistance to MAL3-101 in rhabdocarcinoma cells was mapped to an hsp70 gene (
      • Sabnis A.J.
      • Guerriero C.J.
      • Olivas V.
      • Sayana A.
      • Shue J.
      • Flanagan J.
      • Asthana S.
      • Paton A.W.
      • Paton J.C.
      • Gestwicki J.E.
      • Walter P.
      • Weissman J.S.
      • Wipf P.
      • Brodsky J.L.
      • Bivona T.G.
      Combined chemical-genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma.
      ). Together, these results provide support for selectivity in cells. The next steps for these molecules include expanded medicinal chemistry efforts to increase their potency and identification of additional negative controls. Given the importance of JDPs to Hsp70 biology (
      • Wruck F.
      • Avellaneda M.J.
      • Koers E.J.
      • Minde D.P.
      • Mayer M.P.
      • Kramer G.
      • Mashaghi A.
      • Tans S.J.
      Protein folding mediated by trigger factor and Hsp70: new insights from single-molecule approaches.
      ,
      • Kampinga H.H.
      • Craig E.A.
      The HSP70 chaperone machinery: J proteins as drivers of functional specificity.
      ), this chemical series seems worth careful exploration.
      Table 2Subset of reported chaperone inhibitors along with a summary of ongoing studies that have evaluated their suitability as chemical probes
      TargetChemical seriesKDMuta-genesisBiochemical assay, IC50Cellular assay, EC50PulldownBiomarkerIn vivo efficacy
      Hsp70
          Hsp70–JDPsDihydropyrimidines MAL3-101 (
      • Fewell S.W.
      • Smith C.M.
      • Lyon M.A.
      • Dumitrescu T.P.
      • Wipf P.
      • Day B.W.
      • Brodsky J.L.
      Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity.
      ,
      • Wisén S.
      • Androsavich J.
      • Evans C.G.
      • Chang L.
      • Gestwicki J.E.
      Chemical modulators of heat shock protein 70 (Hsp70) by sequential, microwave-accelerated reactions on solid phase.
      ,
      • Wisén S.
      • Bertelsen E.B.
      • Thompson A.D.
      • Patury S.
      • Ung P.
      • Chang L.
      • Evans C.G.
      • Walter G.M.
      • Wipf P.
      • Carlson H.A.
      • Brodsky J.L.
      • Zuiderweg E.R.
      • Gestwicki J.E.
      Binding of a small molecule at a protein–protein interface regulates the chaperone activity of hsp70–hsp40.
      ,
      • Sabnis A.J.
      • Guerriero C.J.
      • Olivas V.
      • Sayana A.
      • Shue J.
      • Flanagan J.
      • Asthana S.
      • Paton A.W.
      • Paton J.C.
      • Gestwicki J.E.
      • Walter P.
      • Weissman J.S.
      • Wipf P.
      • Brodsky J.L.
      • Bivona T.G.
      Combined chemical-genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma.
      )
      AllostericMid to high μmYesHigh μmMid to high μmYesUPR, protein aggregationYes
          Hsp70–nucleotide-exchange factorsBenzothiazole-rhodacyanines JG-231 (
      • Li X.
      • Srinivasan S.R.
      • Connarn J.
      • Ahmad A.
      • Young Z.T.
      • Kabza A.M.
      • Zuiderweg E.R.
      • Sun D.
      • Gestwicki J.E.
      Analogs of the allosteric heat shock protein 70 (Hsp70) inhibitor, MKT-077, as anti-cancer agents.
      ,
      • Shao H.
      • Li X.
      • Moses M.A.
      • Gilbert L.A.
      • Kalyanaraman C.
      • Young Z.T.
      • Chernova M.
      • Journey S.N.
      • Weissman J.S.
      • Hann B.
      • Jacobson M.P.
      • Neckers L.
      • Gestwicki J.E.
      Exploration of benzothiazole-rhodacyanines as allosteric inhibitors of protein–protein interactions with heat shock protein 70 (Hsp70).
      )
      AllostericLow μmYesLow μmMid nm to low μmYesXIAP, Akt, IAP, CDK4, Raf-1Yes (
      • Colvin T.A.
      • Gabai V.L.
      • Gong J.
      • Calderwood S.K.
      • Li H.
      • Gummuluru S.
      • Matchuk O.N.
      • Smirnova S.G.
      • Orlova N.V.
      • Zamulaeva I.A.
      • Garcia-Marcos M.
      • Li X.
      • Young Z.T.
      • Rauch J.N.
      • Gestwicki J.E.
      • et al.
      Hsp70–Bag3 interactions regulate cancer-related signaling networks.
      ,
      • Moses M.A.
      • Kim Y.S.
      • Rivera-Marquez G.M.
      • Oshima N.
      • Watson M.J.
      • Beebe K.E.
      • Wells C.
      • Lee S.
      • Zuehlke A.D.
      • Shao H.
      • Bingman 3rd., W.E.
      • Kumar V.
      • Malhotra S.V.
      • Weigel N.L.
      • Gestwicki J.E.
      • et al.
      Targeting the Hsp40/Hsp70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer.
      )
          Hsp70 NBDYK5 and its analogs (
      • Taldone T.
      • Kang Y.
      • Patel H.J.
      • Patel M.R.
      • Patel P.D.
      • Rodina A.
      • Patel Y.
      • Gozman A.
      • Maharaj R.
      • Clement C.C.
      • Lu A.
      • Young J.C.
      • Chiosis G.
      Heat shock protein 70 inhibitors. 2. 2,5′-Thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.
      ,
      • Kang Y.
      • Taldone T.
      • Patel H.J.
      • Patel P.D.
      • Rodina A.
      • Gozman A.
      • Maharaj R.
      • Clement C.C.
      • Patel M.R.
      • Brodsky J.L.
      • Young J.C.
      • Chiosis G.
      Heat shock protein 70 inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides as irreversible binders to an allosteric site on heat shock protein 70.
      )
      AllostericHigh μmYes∼7 μmLow to mid μmYesHer2, Raf-1, AktNA
          Hsp70 NBDHS-72 (
      • Howe M.K.
      • Bodoor K.
      • Carlson D.A.
      • Hughes P.F.
      • Alwarawrah Y.
      • Loiselle D.R.
      • Jaeger A.M.
      • Darr D.B.
      • Jordan J.L.
      • Hunter L.M.
      • Molzberger E.T.
      • Gobillot T.A.
      • Thiele D.J.
      • Brodsky J.L.
      • Spector N.L.
      • Haystead T.A.
      Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.
      )
      AllostericNANANAMid μmYesHer2, AktYes
      Hsp90
          C terminusNovobiocin and its analogs (
      • Anyika M.
      • McMullen M.
      • Forsberg L.K.
      • Dobrowsky R.T.
      • Blagg B.S.
      Development of noviomimetics as C-terminal Hsp90 inhibitors.
      ,
      • Zhao H.
      • Donnelly A.C.
      • Kusuma B.R.
      • Brandt G.E.
      • Brown D.
      • Rajewski R.A.
      • Vielhauer G.
      • Holzbeierlein J.
      • Cohen M.S.
      • Blagg B.S.
      Engineering an antibiotic to fight cancer: optimization of the novobiocin scaffold to produce anti-proliferative agents.
      ,
      • Ghosh S.
      • Liu Y.
      • Garg G.
      • Anyika M.
      • McPherson N.T.
      • Ma J.
      • Dobrowsky R.T.
      • Blagg B.S.
      Diverging novobiocin anti-cancer activity from neuroprotective activity through modification of the amide tail.
      ,
      • Burlison J.A.
      • Neckers L.
      • Smith A.B.
      • Maxwell A.
      • Blagg B.S.
      Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.
      ,
      • Yu X.M.
      • Shen G.
      • Neckers L.
      • Blake H.
      • Holzbeierlein J.
      • Cronk B.
      • Blagg B.S.
      Hsp90 inhibitors identified from a library of novobiocin analogues.
      ,
      • Allan R.K.
      • Mok D.
      • Ward B.K.
      • Ratajczak T.
      Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization.
      ,
      • Yun B.G.
      • Huang W.
      • Leach N.
      • Hartson S.D.
      • Matts R.L.
      Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90–cochaperone–client interactions.
      ,
      • Ghosh S.
      • Shinogle H.E.
      • Garg G.
      • Vielhauer G.A.
      • Holzbeierlein J.M.
      • Dobrowsky R.T.
      • Blagg B.S.
      Hsp90 C-terminal inhibitors exhibit antimigratory activity by disrupting the Hsp90α/Aha1 complex in PC3-MM2 cells.
      )
      AllostericNAYesMid μmMid to high μmYesErbB2, mutant p53 (
      • Marcu M.G.
      • Chadli A.
      • Bouhouche I.
      • Catelli M.
      • Neckers L.M.
      The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone.
      ), Her2, Akt, Raf-1 (
      • Zhao H.
      • Donnelly A.C.
      • Kusuma B.R.
      • Brandt G.E.
      • Brown D.
      • Rajewski R.A.
      • Vielhauer G.
      • Holzbeierlein J.
      • Cohen M.S.
      • Blagg B.S.
      Engineering an antibiotic to fight cancer: optimization of the novobiocin scaffold to produce anti-proliferative agents.
      )
      NA
          Hsp90–cdc37Celastrol (
      • Hieronymus H.
      • Lamb J.
      • Ross K.N.
      • Peng X.P.
      • Clement C.
      • Rodina A.
      • Nieto M.
      • Du J.
      • Stegmaier K.
      • Raj S.M.
      • Maloney K.N.
      • Clardy J.
      • Hahn W.C.
      • Chiosis G.
      • Golub T.R.
      Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators.
      ,
      • Zhang T.
      • Li Y.
      • Yu Y.
      • Zou P.
      • Jiang Y.
      • Sun D.
      Characterization of celastrol to inhibit hsp90 and cdc37 interaction.
      ,
      • Zhang T.
      • Hamza A.
      • Cao X.
      • Wang B.
      • Yu S.
      • Zhan C.G.
      • Sun D.
      A novel Hsp90 inhibitor to disrupt Hsp90/Cdc37 complex against pancreatic cancer cells.
      )
      Allosteric1 μmYes∼10 μmMid μmNoAR, FLT3, EGFR, BCR-ABL, Akt, CDK4Yes
      Hsp60
      Epolactaene (
      • Nagumo Y.
      • Kakeya H.
      • Shoji M.
      • Hayashi Y.
      • Dohmae N.
      • Osada H.
      Epolactaene binds human Hsp60 Cys442 resulting in the inhibition of chaperone activity.
      )
      NANAYes>7 μmMid μmYesNANA
      o-Carboranyl phenoxyacetanilide (
      • Ban H.S.
      • Shimizu K.
      • Minegishi H.
      • Nakamura H.
      Identification of HSP60 as a primary target of o-carboranylphenoxyacetanilide, an HIF-1α inhibitor.
      )
      NANANALow μmMid μmYesNANA
      Gold(III)–porphyrin complexes (
      • Hu D.
      • Liu Y.
      • Lai Y.T.
      • Tong K.C.
      • Fung Y.M.
      • Lok C.N.
      • Che C.M.
      Anticancer gold(III) porphyrins target mitochondrial chaperone Hsp60.
      )
      NA∼3.7 μmNALow μmMid μmYesNANA
      Myrtucommulone (
      • Wiechmann K.
      • Müller H.
      • König S.
      • Wielsch N.
      • Svatoš A.
      • Jauch J.
      • Werz O.
      Mitochondrial chaperonin HSP60 is the apoptosis-related target for myrtucommulone.
      )
      NANANA∼10 μmMid μmYesNANA
      KHS-101 (
      • Polson E.S.
      • Kuchler V.B.
      • Abbosh C.
      • Ross E.M.
      • Mathew R.K.
      • Beard H.A.
      • da Silva B.
      • Holding A.N.
      • Ballereau S.
      • Chuntharpursat-Bon E.
      • Williams J.
      • Griffiths H.B.S.
      • Shao H.
      • Patel A.
      • Davies A.J.
      • et al.
      KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice.
      )
      NANANA14 μmMid μmYesYesYes
      sHsp
          Hsp27Peptide aptamers (
      • Rérole A.L.
      • Gobbo J.
      • De Thonel A.
      • Schmitt E.
      • Pais de Barros J.P.
      • Hammann A.
      • Lanneau D.
      • Fourmaux E.
      • Deminov O.N.
      • Micheau O.
      • Lagrost L.
      • Colas P.
      • Kroemer G.
      • Garrido C.
      Peptides and aptamers targeting HSP70: a novel approach for anticancer chemotherapy.
      )
      NANANonenzymeNAYesp21–Waf1Yes
          CryABOxysterols (
      • Makley L.N.
      • McMenimen K.A.
      • DeVree B.T.
      • Goldman J.W.
      • McGlasson B.N.
      • Rajagopal P.
      • Dunyak B.M.
      • McQuade T.J.
      • Thompson A.D.
      • Sunahara R.
      • Klevit R.E.
      • Andley U.P.
      • Gestwicki J.E.
      Pharmacological chaperone for α-crystallin partially restores transparency in cataract models.
      )
      Stabilizer∼10 μmYesNonenzymeNANoProtein aggregationYes
      The other major category of Hsp70 PPIs is the one with the NEFs, including the BAG family of proteins that bind to the NBD through a conserved BAG domain (
      • Bracher A.
      • Verghese J.
      The nucleotide exchange factors of Hsp70 molecular chaperones.
      ). The NEFs are important mediators of Hsp70 function because they control the release of clients from the complex (
      • Gowda N.K.C.
      • Kaimal J.M.
      • Kityk R.
      • Daniel C.
      • Liebau J.
      • Öhman M.
      • Mayer M.P.
      • Andréasson C.
      Nucleotide exchange factors Fes1 and HspBP1 mimic substrate to release misfolded proteins from Hsp70.
      ,
      • Young Z.T.
      • Rauch J.N.
      • Assimon V.A.
      • Jinwal U.K.
      • Ahn M.
      • Li X.
      • Dunyak B.M.
      • Ahmad A.
      • Carlson G.A.
      • Srinivasan S.R.
      • Zuiderweg E.R.
      • Dickey C.A.
      • Gestwicki J.E.
      Stabilizing the Hsp70–Tau complex promotes turnover in models of tauopathy.
      ). Thus, blocking NEF binding to Hsp70 would be expected to increase the dwell time of clients in the chaperone complex, favoring their degradation in some cases (
      • Young Z.T.
      • Rauch J.N.
      • Assimon V.A.
      • Jinwal U.K.
      • Ahn M.
      • Li X.
      • Dunyak B.M.
      • Ahmad A.
      • Carlson G.A.
      • Srinivasan S.R.
      • Zuiderweg E.R.
      • Dickey C.A.
      • Gestwicki J.E.
      Stabilizing the Hsp70–Tau complex promotes turnover in models of tauopathy.
      ). A series of rhodacyanine-benzothiazoles (Fig. 3; Table 2) have been identified that inhibit this PPI. These molecules were first described by Wadhwa et al. (
      • Wadhwa R.
      • Sugihara T.
      • Yoshida A.
      • Nomura H.
      • Reddel R.R.
      • Simpson R.
      • Maruta H.
      • Kaul S.C.
      Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function.
      ) in phenotypic anticancer screens and only later were they found to bind to cytoplasmic and mitochondrial Hsp70 family members in pulldowns. NMR studies showed that the compounds of this series bind in a deep, allosteric pocket on Hsp70 (
      • Young Z.T.
      • Rauch J.N.
      • Assimon V.A.
      • Jinwal U.K.
      • Ahn M.
      • Li X.
      • Dunyak B.M.
      • Ahmad A.
      • Carlson G.A.
      • Srinivasan S.R.
      • Zuiderweg E.R.
      • Dickey C.A.
      • Gestwicki J.E.
      Stabilizing the Hsp70–Tau complex promotes turnover in models of tauopathy.
      ,
      • Rousaki A.
      • Miyata Y.
      • Jinwal U.K.
      • Dickey C.A.
      • Gestwicki J.E.
      • Zuiderweg E.R.
      Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones.
      ). Binding to this site favors the ADP-bound form of Hsp70 and disrupts binding to BAG proteins through a conformational change. As expected from the natural role of the NEFs, treatment of cells with these compounds induces degradation of particularly sensitive “client” proteins, such as FoxM1 (
      • Colvin T.A.
      • Gabai V.L.
      • Gong J.
      • Calderwood S.K.
      • Li H.
      • Gummuluru S.
      • Matchuk O.N.
      • Smirnova S.G.
      • Orlova N.V.
      • Zamulaeva I.A.
      • Garcia-Marcos M.
      • Li X.
      • Young Z.T.
      • Rauch J.N.
      • Gestwicki J.E.
      • et al.
      Hsp70–Bag3 interactions regulate cancer-related signaling networks.
      ), Akt (
      • Koren 3rd., J.
      • Jinwal U.K.
      • Jin Y.
      • O'Leary J.
      • Jones J.R.
      • Johnson A.G.
      • Blair L.J.
      • Abisambra J.F.
      • Chang L.
      • Miyata Y.
      • Cheng A.M.
      • Guo J.
      • Cheng J.Q.
      • Gestwicki J.E.
      • Dickey C.A.
      Facilitating Akt clearance via manipulation of Hsp70 activity and levels.
      ), RIP1 (
      • Srinivasan S.R.
      • Cesa L.C.
      • Li X.
      • Julien O.
      • Zhuang M.
      • Shao H.
      • Chung J.
      • Maillard I.
      • Wells J.A.
      • Duckett C.S.
      • Gestwicki J.E.
      Heat shock protein 70 (Hsp70) suppresses RIP1-dependent apoptotic and necroptotic cascades.
      ), and inhibitor of apoptosis proteins (
      • Cesa L.C.
      • Shao H.
      • Srinivasan S.R.
      • Tse E.
      • Jain C.
      • Zuiderweg E.R.P.
      • Southworth D.R.
      • Mapp A.K.
      • Gestwicki J.E.
      X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition.
      ). Medicinal chemistry campaigns (>400 analogs) produced more potent molecules (EC50 ∼30 nm) and inactive controls (JG-258) and allowed initial correlation between in vitro activity and cellular functions (
      • Li X.
      • Srinivasan S.R.
      • Connarn J.
      • Ahmad A.
      • Young Z.T.
      • Kabza A.M.
      • Zuiderweg E.R.
      • Sun D.
      • Gestwicki J.E.
      Analogs of the allosteric heat shock protein 70 (Hsp70) inhibitor, MKT-077, as anti-cancer agents.
      ,
      • Shao H.
      • Li X.
      • Moses M.A.
      • Gilbert L.A.
      • Kalyanaraman C.
      • Young Z.T.
      • Chernova M.
      • Journey S.N.
      • Weissman J.S.
      • Hann B.
      • Jacobson M.P.
      • Neckers L.
      • Gestwicki J.E.
      Exploration of benzothiazole-rhodacyanines as allosteric inhibitors of protein–protein interactions with heat shock protein 70 (Hsp70).
      ). Target engagement in cells and animals has also been explored using genetic approaches; for example, overexpression of a point mutant of BAG3 (R480A) that cannot bind to Hsp70s gives a similar phenotype to compound treatment in breast cancer cells (
      • Colvin T.A.
      • Gabai V.L.
      • Gong J.
      • Calderwood S.K.
      • Li H.
      • Gummuluru S.
      • Matchuk O.N.
      • Smirnova S.G.
      • Orlova N.V.
      • Zamulaeva I.A.
      • Garcia-Marcos M.
      • Li X.
      • Young Z.T.
      • Rauch J.N.
      • Gestwicki J.E.
      • et al.
      Hsp70–Bag3 interactions regulate cancer-related signaling networks.
      ). In addition, whole-genome CRISPRi studies revealed that knockdown of Hsp70 family members gives rise to compound sensitivity (
      • Shao H.
      • Li X.
      • Moses M.A.
      • Gilbert L.A.
      • Kalyanaraman C.
      • Young Z.T.
      • Chernova M.
      • Journey S.N.
      • Weissman J.S.
      • Hann B.
      • Jacobson M.P.
      • Neckers L.
      • Gestwicki J.E.
      Exploration of benzothiazole-rhodacyanines as allosteric inhibitors of protein–protein interactions with heat shock protein 70 (Hsp70).
      ). Most recently, JG-231 and other analogs have been characterized in vitro for liver microsome stability and in mice for maximal-tolerated dose and pharmacokinetics (
      • Shao H.
      • Li X.
      • Moses M.A.
      • Gilbert L.A.
      • Kalyanaraman C.
      • Young Z.T.
      • Chernova M.
      • Journey S.N.
      • Weissman J.S.
      • Hann B.
      • Jacobson M.P.
      • Neckers L.
      • Gestwicki J.E.
      Exploration of benzothiazole-rhodacyanines as allosteric inhibitors of protein–protein interactions with heat shock protein 70 (Hsp70).
      ). This pharmacological information enables use of the compounds in some animal and tissue models. For example, they were used to identify a role for Hsp70–BAG in breast cancer initiation (
      • Colvin T.A.
      • Gabai V.L.
      • Gong J.
      • Calderwood S.K.
      • Li H.
      • Gummuluru S.
      • Matchuk O.N.
      • Smirnova S.G.
      • Orlova N.V.
      • Zamulaeva I.A.
      • Garcia-Marcos M.
      • Li X.
      • Young Z.T.
      • Rauch J.N.
      • Gestwicki J.E.
      • et al.
      Hsp70–Bag3 interactions regulate cancer-related signaling networks.
      ), tau homeostasis (
      • Abisambra J.
      • Jinwal U.K.
      • Miyata Y.
      • Rogers J.
      • Blair L.
      • Li X.
      • Seguin S.P.
      • Wang L.
      • Jin Y.
      • Bacon J.
      • Brady S.
      • Cockman M.
      • Guidi C.
      • Zhang J.
      • Koren J.
      • et al.
      Allosteric heat shock protein 70 inhibitors rapidly rescue synaptic plasticity deficits by reducing aberrant τ.
      ), Dengue viral replication (
      • Taguwa S.
      • Maringer K.
      • Li X.
      • Bernal-Rubio D.
      • Rauch J.N.
      • Gestwicki J.E.
      • Andino R.
      • Fernandez-Sesma A.
      • Frydman J.
      Defining Hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection.
      ), and castration-resistant prostate cancer (
      • Moses M.A.
      • Kim Y.S.
      • Rivera-Marquez G.M.
      • Oshima N.
      • Watson M.J.
      • Beebe K.E.
      • Wells C.
      • Lee S.
      • Zuehlke A.D.
      • Shao H.
      • Bingman 3rd., W.E.
      • Kumar V.
      • Malhotra S.V.
      • Weigel N.L.
      • Gestwicki J.E.
      • et al.
      Targeting the Hsp40/Hsp70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer.
      ). Together, this type of data, acquired in different laboratories and in different model systems, begins to build confidence in the suitability of the inhibitors as chemical probes. With that being said, the pharmacophore has chemical liabilities that limit its use, including its poor solubility and photosensitivity, so further optimization is needed.
      Additional Hsp70 inhibitors are at a comparatively early stage in their evaluation as chemical probes (Fig. 3; Table 2). For example, the compound YK-5 and its analogs were designed to bind to a distinct, allosteric site in Hsp70, and this series has been explored in a series of medicinal chemistry campaigns (
      • Taldone T.
      • Kang Y.
      • Patel H.J.
      • Patel M.R.
      • Patel P.D.
      • Rodina A.
      • Patel Y.
      • Gozman A.
      • Maharaj R.
      • Clement C.C.
      • Lu A.
      • Young J.C.
      • Chiosis G.
      Heat shock protein 70 inhibitors. 2. 2,5′-Thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.
      ,
      • Kang Y.
      • Taldone T.
      • Patel H.J.
      • Patel P.D.
      • Rodina A.
      • Gozman A.
      • Maharaj R.
      • Clement C.C.
      • Patel M.R.
      • Brodsky J.L.
      • Young J.C.
      • Chiosis G.
      Heat shock protein 70 inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides as irreversible binders to an allosteric site on heat shock protein 70.
      ). These molecules have clear SAR; they pull down Hsp70 from lysates, and they have promising anti-proliferative activity in breast cancer models, providing a strong basis for further evaluation. In a quite different approach, the compound HS-72 was discovered in a screen for nucleotide-binding molecules (
      • Howe M.K.
      • Bodoor K.
      • Carlson D.A.
      • Hughes P.F.
      • Alwarawrah Y.
      • Loiselle D.R.
      • Jaeger A.M.
      • Darr D.B.
      • Jordan J.L.
      • Hunter L.M.
      • Molzberger E.T.
      • Gobillot T.A.
      • Thiele D.J.
      • Brodsky J.L.
      • Spector N.L.
      • Haystead T.A.
      Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.
      ). In follow-up studies, binding to Hsp70 was confirmed in vitro and by using pulldowns. Finally, phenotypic screens have identified PES (
      • Leu J.I.
      • Pimkina J.
      • Frank A.
      • Murphy M.E.
      • George D.L.
      A small molecule inhibitor of inducible heat shock protein 70.
      ) and novolactone (
      • Hassan A.Q.
      • Kirby C.A.
      • Zhou W.
      • Schuhmann T.
      • Kityk R.
      • Kipp D.R.
      • Baird J.
      • Chen J.
      • Chen Y.
      • Chung F.
      • Hoepfner D.
      • Movva N.R.
      • Pagliarini R.
      • Petersen F.
      • Quinn C.
      • et al.
      The novolactone natural product disrupts the allosteric regulation of hsp70.
      ) as inhibitors of Hsp70. Both of these compounds were found to bind at different allosteric sites in the SBD by structural approaches, and in both cases, the site was confirmed by mutagenesis of the interacting residues. Each of these chemical series (i.e. TK5, HS-72, PES, and novoloactone) is at a different stage of evaluation as a chemical probe, but each holds promise due to their different binding sites and mechanisms-of-action (MoAs) (
      • Li X.
      • Shao H.
      • Taylor I.R.
      • Gestwicki J.E.
      Targeting allosteric control mechanisms in heat shock protein 70 (Hsp70).
      ).

      Inhibitors of the Hsp90 sub-network

      Hsp90 is a dimeric chaperone composed of three domains: an N-terminal ATPase domain, a middle region, and a C-terminal dimerization motif. In addition, this protein binds to a number of co-chaperones, including Aha1, p23, and Cdc37 (see Fig. 1). The best-known Hsp90 inhibitors are enzyme inhibitors that bind in the N-terminal domain, such as geldanamycin and its analogs (e.g. 17-AAG) (
      • Shrestha L.
      • Patel H.J.
      • Chiosis G.
      Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease.
      ). Some of these molecules are clinical candidates (
      • Pacey S.
      • Wilson R.H.
      • Walton M.
      • Eatock M.M.
      • Hardcastle A.
      • Zetterlund A.
      • Arkenau H.T.
      • Moreno-Farre J.
      • Banerji U.
      • Roels B.
      • Peachey H.
      • Aherne W.
      • de Bono J.S.
      • Raynaud F.
      • Workman P.
      • Judson I.
      A phase I study of the heat shock protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced solid tumors.
      ,
      • Powers M.V.
      • Workman P.
      Inhibitors of the heat shock response: biology and pharmacology.
      ), and they have been extensively explored for selectivity, including screening against a panel of ATP-binding proteins (
      • Nordin B.E.
      • Liu Y.
      • Aban A.
      • Brown H.E.
      • Wu J.
      • Hainley A.K.
      • Rosenblum J.S.
      • Nomanbhoy T.K.
      • Kozarich J.W.
      ATP acyl phosphate reactivity reveals native conformations of Hsp90 paralogs and inhibitor target engagement.
      ), so they are generally considered to be good chemical probes (
      • Neckers L.
      • Blagg B.
      • Haystead T.
      • Trepel J.B.
      • Whitesell L.
      • Picard D.
      Methods to validate Hsp90 inhibitor specificity, to identify off-target effects, and to rethink approaches for further clinical development.
      ). Indeed, these compounds have been crucial in expanding our knowledge of Hsp90 function, including being used to identify its clients. Molecules of this type have been extensively reviewed (
      • Shrestha L.
      • Patel H.J.
      • Chiosis G.
      Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease.
      ), so they will not be discussed further.
      Alternative ways of inhibiting Hsp90 have also been explored. For example, the natural products novobiocin/coumermycin (
      • Anyika M.
      • McMullen M.
      • Forsberg L.K.
      • Dobrowsky R.T.
      • Blagg B.S.
      Development of noviomimetics as C-terminal Hsp90 inhibitors.
      ,
      • Zhao H.
      • Donnelly A.C.
      • Kusuma B.R.
      • Brandt G.E.
      • Brown D.
      • Rajewski R.A.
      • Vielhauer G.
      • Holzbeierlein J.
      • Cohen M.S.
      • Blagg B.S.
      Engineering an antibiotic to fight cancer: optimization of the novobiocin scaffold to produce anti-proliferative agents.
      ) and sansalvamide A (
      • Vasko R.C.
      • Rodriguez R.A.
      • Cunningham C.N.
      • Ardi V.C.
      • Agard D.A.
      • McAlpine S.R.
      Mechanistic studies of Sansalvamide A-amide: an allosteric modulator of Hsp90.
      ) served as inspiration for the development of inhibitors directed against the C-terminal domain (Fig. 3; Table 2). For example, Blagg and co-workers (
      • Ghosh S.
      • Liu Y.
      • Garg G.
      • Anyika M.
      • McPherson N.T.
      • Ma J.
      • Dobrowsky R.T.
      • Blagg B.S.
      Diverging novobiocin anti-cancer activity from neuroprotective activity through modification of the amide tail.
      ,
      • Burlison J.A.
      • Neckers L.
      • Smith A.B.
      • Maxwell A.
      • Blagg B.S.
      Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.
      • Yu X.M.
      • Shen G.
      • Neckers L.
      • Blake H.
      • Holzbeierlein J.
      • Cronk B.
      • Blagg B.S.
      Hsp90 inhibitors identified from a library of novobiocin analogues.
      ) and others (
      • Matts R.L.
      • Dixit A.
      • Peterson L.B.
      • Sun L.
      • Voruganti S.
      • Kalyanaraman P.
      • Hartson S.D.
      • Verkhivker G.M.
      • Blagg B.S.
      Elucidation of the Hsp90 C-terminal inhibitor binding site.
      ,
      • Marcu M.G.
      • Chadli A.
      • Bouhouche I.
      • Catelli M.
      • Neckers L.M.
      The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone.
      ) have synthesized analogs of novobiocin, complete with negative controls (i.e. inactive molecules), and information about the binding site. Treatment with these compounds induces degradation of Hsp90 clients in multiple cancer cell types. In addition, they may disrupt Hsp90 dimerization (
      • Allan R.K.
      • Mok D.
      • Ward B.K.
      • Ratajczak T.
      Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization.
      ) and co-chaperone interactions (
      • Yun B.G.
      • Huang W.
      • Leach N.
      • Hartson S.D.
      • Matts R.L.
      Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90–cochaperone–client interactions.
      ,
      • Ghosh S.
      • Shinogle H.E.
      • Garg G.
      • Vielhauer G.A.
      • Holzbeierlein J.M.
      • Dobrowsky R.T.
      • Blagg B.S.
      Hsp90 C-terminal inhibitors exhibit antimigratory activity by disrupting the Hsp90α/Aha1 complex in PC3-MM2 cells.
      ), suggesting that they are bona fide Hsp90 PPI inhibitors. Similarly, McAlpine and co-workers (
      • Vasko R.C.
      • Rodriguez R.A.
      • Cunningham C.N.
      • Ardi V.C.
      • Agard D.A.
      • McAlpine S.R.
      Mechanistic studies of Sansalvamide A-amide: an allosteric modulator of Hsp90.
      ) have produced biotinylated analogs of sansalvamide A and shown that they pull down Hsp90 from cells and disrupt binding to some client proteins. In this way, molecules from these two series are progressing toward becoming chemical probes. Interestingly, treatment with certain analogs of novobiocin and sansalvamide A does not induce a stress response in cells, which is considered a hallmark biomarker of the canonical, competitive Hsp90 inhibitors (
      • Neckers L.
      • Blagg B.
      • Haystead T.
      • Trepel J.B.
      • Whitesell L.
      • Picard D.
      Methods to validate Hsp90 inhibitor specificity, to identify off-target effects, and to rethink approaches for further clinical development.
      ,
      • Wang Y.
      • McAlpine S.R.
      N-terminal and C-terminal modulation of Hsp90 produce dissimilar phenotypes.
      ). This finding highlights the importance of using multiple assays for assessing selectivity (see Table 1), as molecules with different MoAs might not always share the same biomarkers.
      In addition to these chemical series, a number of other reports have introduced leads toward potential Hsp90 PPI inhibitors (
      • Zierer B.K.
      • Weiwad M.
      • Rübbelke M.
      • Freiburger L.
      • Fischer G.
      • Lorenz O.R.
      • Sattler M.
      • Richter K.
      • Buchner J.
      Artificial accelerators of the molecular chaperone Hsp90 facilitate rate-limiting conformational transitions.
      • Sreeramulu S.
      • Gande S.L.
      • Göbel M.
      • Schwalbe H.
      Molecular mechanism of inhibition of the human protein complex Hsp90–Cdc37, a kinome chaperone-cochaperone, by triterpene celastrol.
      ,
      • Hieronymus H.
      • Lamb J.
      • Ross K.N.
      • Peng X.P.
      • Clement C.
      • Rodina A.
      • Nieto M.
      • Du J.
      • Stegmaier K.
      • Raj S.M.
      • Maloney K.N.
      • Clardy J.
      • Hahn W.C.
      • Chiosis G.
      • Golub T.R.
      Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators.
      ,
      • Zhang T.
      • Li Y.
      • Yu Y.
      • Zou P.
      • Jiang Y.
      • Sun D.
      Characterization of celastrol to inhibit hsp90 and cdc37 interaction.
      • Zhang T.
      • Hamza A.
      • Cao X.
      • Wang B.
      • Yu S.
      • Zhan C.G.
      • Sun D.
      A novel Hsp90 inhibitor to disrupt Hsp90/Cdc37 complex against pancreatic cancer cells.
      ). Although these chemical series, such as celasterol, are relatively early in their analysis as chemical probes, further work may expand the suite of available chemical series for the Hsp90 sub-network.

      Inhibitors of the Hsp60 sub-network

      Hsp60–Hsp10 and its prokaryotic ortholog, GroEL–GroES, play important roles in protein folding (
      • Bukau B.
      • Weissman J.
      • Horwich A.
      Molecular chaperones and protein quality control.
      ). Hsp60 is thought to be located in the mitochondria of eukaryotes, where it helps stabilize client proteins. In addition to their ATPase activity, these systems involve multiple types of PPIs, including interactions between Hsp60 protomers and those between Hsp60 and the regulatory component (i.e. Hsp10; see Fig. 1). This system also likely interacts with the Hsp70 sub-network through direct PPIs. Most of the Hsp60 inhibitors that have been identified thus far originated in phenotypic screens, and only later did the pulldown studies suggest Hsp60 as a potential target. It is not yet clear whether these compounds are enzyme inhibitors (i.e. targeting “nodes”) or whether they disrupt PPIs (i.e. act on “edges”).
      The chemical series described as Hsp60 inhibitors thus far are structurally diverse (Fig. 3; Table 2) and include 2-phenothiazole-pyrimidine-2,4-diamines, such as KHS101 (
      • Polson E.S.
      • Kuchler V.B.
      • Abbosh C.
      • Ross E.M.
      • Mathew R.K.
      • Beard H.A.
      • da Silva B.
      • Holding A.N.
      • Ballereau S.
      • Chuntharpursat-Bon E.
      • Williams J.
      • Griffiths H.B.S.
      • Shao H.
      • Patel A.
      • Davies A.J.
      • et al.
      KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice.
      ), gold porphyrins (
      • Hu D.
      • Liu Y.
      • Lai Y.T.
      • Tong K.C.
      • Fung Y.M.
      • Lok C.N.
      • Che C.M.
      Anticancer gold(III) porphyrins target mitochondrial chaperone Hsp60.
      ), pyrazolo-pyridazines (
      • Alagramam K.N.
      • Gopal S.R.
      • Geng R.
      • Chen D.H.
      • Nemet I.
      • Lee R.
      • Tian G.
      • Miyagi M.
      • Malagu K.F.
      • Lock C.J.
      • Esmieu W.R.
      • Owens A.P.
      • Lindsay N.A.
      • Ouwehand K.
      • Albertus F.
      • et al.
      A small molecule mitigates hearing loss in a mouse model of Usher syndrome III.
      ), phenoxyacetanilides (
      • Ban H.S.
      • Shimizu K.
      • Minegishi H.
      • Nakamura H.
      Identification of HSP60 as a primary target of o-carboranylphenoxyacetanilide, an HIF-1α inhibitor.
      ), and the natural products suvanine (
      • Cassiano C.
      • Monti M.C.
      • Festa C.
      • Zampella A.
      • Riccio R.
      • Casapullo A.
      Chemical proteomics reveals heat shock protein 60 to be the main cellular target of the marine bioactive sesterterpene suvanine.
      ), epolactaene (
      • Nagumo Y.
      • Kakeya H.
      • Shoji M.
      • Hayashi Y.
      • Dohmae N.
      • Osada H.
      Epolactaene binds human Hsp60 Cys442 resulting in the inhibition of chaperone activity.
      ), and myrtucommulone (
      • Wiechmann K.
      • Müller H.
      • König S.
      • Wielsch N.
      • Svatoš A.
      • Jauch J.
      • Werz O.
      Mitochondrial chaperonin HSP60 is the apoptosis-related target for myrtucommulone.
      ). Although more work remains to verify the selectivity of these molecules in cells, the striking lack of similarity in these chemical structures is suggestive of different binding sites or MoAs. However, none of these putative Hsp60 inhibitors has yet been subject to extensive medicinal chemistry or the full spectrum of analyses that are needed to give great confidence in their use as probes (see Table 1). Overall, given the central role of Hsp60–Hsp10 in mitochondrial protein quality control (
      • Cappello F.
      • Conway de Macario E.
      • Marasà L.
      • Zummo G.
      • Macario A.J.
      Hsp60 expression, new locations, functions and perspectives for cancer diagnosis and therapy.
      ), it seems worth a greater investment in chemical probe discovery for this system. For example, KHS101 has been shown to disrupt energy metabolism in glioblastoma (
      • Polson E.S.
      • Kuchler V.B.
      • Abbosh C.
      • Ross E.M.
      • Mathew R.K.
      • Beard H.A.
      • da Silva B.
      • Holding A.N.
      • Ballereau S.
      • Chuntharpursat-Bon E.
      • Williams J.
      • Griffiths H.B.S.
      • Shao H.
      • Patel A.
      • Davies A.J.
      • et al.
      KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice.
      ), suggesting a cancer-specific role for Hsp60 in mitochondrial function. These efforts might also benefit from screens focused on finding inhibitors of the folding activity of the prokaryotic GroEL–GroES system using in vitro assays (
      • Chapman E.
      • Farr G.W.
      • Furtak K.
      • Horwich A.L.
      A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL.
      ).

      Inhibitors of the sHSPs

      The sHSPs are chaperones that lack enzymatic function; rather, they seem to operate by binding directly to each other and to their client proteins and co-chaperones, such as BAG3 (
      • Rauch J.N.
      • Tse E.
      • Freilich R.
      • Mok S.A.
      • Makley L.N.
      • Southworth D.R.
      • Gestwicki J.E.
      BAG3 is a modular, scaffolding protein that physically links heat shock protein 70 (Hsp70) to the small heat shock proteins.
      ). Thus, the only way to inhibit these systems is to target their PPIs. The sHSPs engage in a number of distinct interactions, such as the one between conserved α-crystallin domains (ACDs) that are known to stabilize sHsp dimers (see Fig. 1). Another, nonoverlapping interaction is the one between the ACD and the IXI motif that is found in the C terminus of some sHSPs (
      • Delbecq S.P.
      • Jehle S.
      • Klevit R.
      Binding determinants of the small heat shock protein, αB-crystallin: recognition of the 'IxI' motif.
      ). Finally, the N-terminal domain of some sHSPs also seems to make interactions within larger oligomers (
      • Mainz A.
      • Peschek J.
      • Stavropoulou M.
      • Back K.C.
      • Bardiaux B.
      • Asami S.
      • Prade E.
      • Peters C.
      • Weinkauf S.
      • Buchner J.
      • Reif B.
      The chaperone αB-crystallin uses different interfaces to capture an amorphous and an amyloid client.
      ). Thus, sHSPs are a rich source of PPIs, which could become targets for chemical probes. However, the structural complexity of the system has hindered development of such molecules. Aptamers directed at Hsp27 (HSPB1) (
      • Rérole A.L.
      • Gobbo J.
      • De Thonel A.
      • Schmitt E.
      • Pais de Barros J.P.
      • Hammann A.
      • Lanneau D.
      • Fourmaux E.
      • Deminov O.N.
      • Micheau O.
      • Lagrost L.
      • Colas P.
      • Kroemer G.
      • Garrido C.
      Peptides and aptamers targeting HSP70: a novel approach for anticancer chemotherapy.
      ), diterpenes that seem to bind to Hsp27 (
      • Faiella L.
      • Piaz F.D.
      • Bisio A.
      • Tosco A.
      • De Tommasi N.
      A chemical proteomics approach reveals Hsp27 as a target for proapoptotic clerodane diterpenes.
      ), and oxysterols, such as compound 29, that bind to the ACD of α-crystallin (HSPB5) (
      • Makley L.N.
      • McMenimen K.A.
      • DeVree B.T.
      • Goldman J.W.
      • McGlasson B.N.
      • Rajagopal P.
      • Dunyak B.M.
      • McQuade T.J.
      • Thompson A.D.
      • Sunahara R.
      • Klevit R.E.
      • Andley U.P.
      • Gestwicki J.E.
      Pharmacological chaperone for α-crystallin partially restores transparency in cataract models.
      ) have been identified (Fig. 3; Table 2), but their selectivity in cells has not been extensively explored.

      Other inhibitors

      We have focused this discussion on molecules that target a handful of heat shock proteins (i.e. Hsp70, Hsp90, Hsp60, and sHSPs). However, the proteostasis system includes other chaperones that are not classified as heat shock proteins but could be important targets. For example, Kelly and co-workers (
      • Plate L.
      • Cooley C.B.
      • Chen J.J.
      • Paxman R.J.
      • Gallagher C.M.
      • Madoux F.
      • Genereux J.C.
      • Dobbs W.
      • Garza D.
      • Spicer T.P.
      • Scampavia L.
      • Brown S.J.
      • Rosen H.
      • Powers E.T.
      • Walter P.
      • et al.
      Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.
      ) have recently described inhibitors of PDIs, including information on target validation, MoA, and medicinal chemistry. These molecules activate the unfolded protein response, so they have promise in improving quality control in the endoplasmic reticulum. Recent efforts are also producing new inhibitors of the FK506-binding protein (FKBP) family of PPIases (
      • Holt D.A.
      • Luengo J.I.
      • Yamashita D.S.
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      ,
      • De Leon J.T.
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      • Fletterick R.J.
      • Guy R.K.
      • et al.
      Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells.
      ), including the first selective inhibitors of FKBP51 (
      • Gaali S.
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      • Sippel C.
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      Rapid, structure-based exploration of pipecolic acid amides as novel selective antagonists of the FK506-binding protein 51.
      ). Such molecules might be especially good probes of steroid hormone receptor biology (
      • De Leon J.T.
      • Iwai A.
      • Feau C.
      • Garcia Y.
      • Balsiger H.A.
      • Storer C.L.
      • Suro R.M.
      • Garza K.M.
      • Lee S.
      • Kim Y.S.
      • Chen Y.
      • Ning Y.M.
      • Riggs D.L.
      • Fletterick R.J.
      • Guy R.K.
      • et al.
      Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells.
      ). The broader protein quality control field also benefits from the availability of chemical probes that inhibit proteins that are not widely considered to be chaperones, including VCP/p97 (
      • Anderson D.J.
      • Le Moigne R.
      • Djakovic S.
      • Kumar B.
      • Rice J.
      • Wong S.
      • Wang J.
      • Yao B.
      • Valle E.
      • Kiss von Soly S.
      • Madriaga A.
      • Soriano F.
      • Menon M.K.
      • Wu Z.Y.
      • Kampmann M.
      • et al.
      Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis.
      ), the proteasome (
      • Crawford L.J.
      • Walker B.
      • Irvine A.E.
      Proteasome inhibitors in cancer therapy.
      ), the Sec61 channel (
      • Mackinnon A.L.
      • Paavilainen V.O.
      • Sharma A.
      • Hegde R.S.
      • Taunton J.
      An allosteric Sec61 inhibitor traps nascent transmembrane helices at the lateral gate.
      ), and the integrated stress response (
      • Sidrauski C.
      • Tsai J.C.
      • Kampmann M.
      • Hearn B.R.
      • Vedantham P.
      • Jaishankar P.
      • Sokabe M.
      • Mendez A.S.
      • Newton B.W.
      • Tang E.L.
      • Verschueren E.
      • Johnson J.R.
      • Krogan N.J.
      • Fraser C.S.
      • Weissman J.S.
      • et al.
      Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response.
      ). Although we lack the space to adequately describe these molecules or evaluate their validation as chemical probes (see Table 1), they collectively serve to provide a wider chemical toolbox for studying proteostasis.

      Outlook for the future

      Chemical probes are powerful tools for studying and perturbing the chaperone network. Despite the production of probes for a handful of chaperone systems, such as Hsp70 and Hsp90, there is much more work to be done. For example, there are no validated probes for major nodes, such as TRiC. Likewise, hundreds of PPIs (“edges”) lack chemical tools. An optimistic vision for the future is one in which each chaperone node and edge has a well-validated inhibitor. Although this goal is certainly ambitious, there is legitimate reason for hope. New technologies, such as CRISPRi, high content screening, cryo-EM (
      • Verba K.A.
      • Wang R.Y.
      • Arakawa A.
      • Liu Y.
      • Shirouzu M.
      • Yokoyama S.
      • Agard D.A.
      Atomic structure of Hsp90–Cdc37–Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.
      ), isoelectric-focusing capillary electrophoresis (
      • Ouimet C.M.
      • Dawod M.
      • Grinias J.
      • Assimon V.A.
      • Lodge J.
      • Mapp A.K.
      • Gestwicki J.E.
      • Kennedy R.T.
      Protein cross-linking capillary electrophoresis at increased throughput for a range of protein–protein interactions.
      ), and others, are accelerating the rate of probe discovery and optimization. At the same time, increasingly sophisticated chemical libraries, such as macrocycles (
      • Villar E.A.
      • Beglov D.
      • Chennamadhavuni S.
      • Porco Jr, J.A.
      • Kozakov D.
      • Vajda S.
      • Whitty A.
      How proteins bind macrocycles.
      ) and natural product–inspired libraries (
      • Dougherty P.G.
      • Qian Z.
      • Pei D.
      Macrocycles as protein–protein interaction inhibitors.
      ), which tend to be enriched in PPI inhibitors, are being built. It seems likely that these advances will combine to produce additional chemical probes for a wider range of chaperones and their PPIs.

      Acknowledgments

      We thank our many colleagues in the proteostasis and chemical biology fields, whose combined wisdom we tried to reflect here. We also apologize to those groups whose work we were not able to include, and we thank Dan Schwarz for helpful discussions.

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