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Treatment-induced neuroendocrine prostate cancer (t-NEPC) often arises from adenocarcinoma via lineage plasticity in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the specific regulators and targets involved in the transition to NEPC are not well understood. Plexin D1 (PLXND1) is a cellular receptor of the semaphorin (SEMA) family that plays important roles in modulating the cytoskeleton and cell adhesion. Here, we found that PLXND1 was highly expressed and positively correlated with neuroendocrine markers in patients with NEPC. High PLXND1 expression was associated with poorer prognosis in prostate cancer patients. Additionally, PLXND1 was upregulated and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells. Knockdown or knockout of PLXND1 inhibited neural lineage pathways, thereby suppressing NEPC cell proliferation, patient derived xenograft (PDX) tumor organoid viability, and xenograft tumor growth. Mechanistically, the heat shock protein 70 (HSP70) regulated PLXND1 protein stability through degradation, and inhibition of HSP70 decreased PLXND1 expression and NEPC organoid growth. In summary, our findings indicate that PLXND1 could serve as a promising therapeutic target and molecular marker for NEPC.

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

The androgen receptor is a key regulator of prostate cancer and the principal target of current prostate cancer therapies collectively termed androgen deprivation therapies. Insensitivity to these drugs is a hallmark of progression to a terminal disease state termed castration-resistant prostate cancer. Therefore, novel therapeutic options that slow progression of castration-resistant prostate cancer and combine effectively with existing agents are in urgent need. We show that JG-98, an allosteric inhibitor of HSP70, re-sensitizes castration-resistant prostate cancer to androgen deprivation drugs by targeting mitochondrial HSP70 (HSPA9) to suppress aerobic respiration. Rather than impacting androgen receptor stability as previously described, JG-98's primary effect is inhibition of mitochondrial translation, leading to disruption of electron transport chain activity. Although functionally distinct from HSPA9 inhibition, direct inhibition of the electron transport chain with a complex I or II inhibitor creates a similar physiological state capable of re-sensitizing castration-resistant prostate cancer to androgen deprivation therapies. These data identify a significant role for HspA9 in mitochondrial ribosome function and highlight an actionable metabolic vulnerability of castration-resistant prostate cancer.

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic.SignificanceTranslatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.

The potential of small molecules to localize within subcellular compartments is rarely explored. To probe this question, we measured the localization of Hsp70 inhibitors using fluorescence microscopy. We found that even closely related analogs had dramatically different distributions, with some residing predominantly in the mitochondria and others in the ER. CRISPRi screens supported this idea, showing that different compounds had distinct chemogenetic interactions with Hsp70s of the ER (HSPA5/BiP) and mitochondria (HSPA9/mortalin) and their co-chaperones. Moreover, localization seemed to determine function, even for molecules with conserved binding sites. Compounds with distinct partitioning have distinct anti-proliferative activity in breast cancer cells compared with anti-viral activity in cellular models of Dengue virus replication, likely because different sets of Hsp70s are required in these processes. These findings highlight the contributions of subcellular partitioning and chemogenetic interactions to small molecule activity, features that are rarely explored during medicinal chemistry campaigns.

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70's binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein-protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both "canonical" interactions, which are universally conserved, and "non-canonical" (or "secondary") contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70's secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis.

Castration-resistant prostate cancer (CRPC) is associated with an increased reliance on heat shock protein 70 (HSP70), but it is not clear what other protein homeostasis (proteostasis) factors might be involved. To address this question, we performed functional and synthetic lethal screens in four prostate cancer cell lines. These screens confirmed key roles for HSP70, HSP90, and their co-chaperones, but also suggested that the mitochondrial chaperone, HSP60/HSPD1, is selectively required in CRPC cell lines. Knockdown of HSP60 does not impact the stability of androgen receptor (AR) or its variants; rather, it is associated with loss of mitochondrial spare respiratory capacity, partly owing to increased proton leakage. Finally, transcriptional data revealed a correlation between HSP60 levels and poor survival of prostate cancer patients. These findings suggest that re-wiring of the proteostasis network is associated with CRPC, creating selective vulnerabilities that might be targeted to treat the disease.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

The formation of membraneless organelles can be a proteotoxic stress control mechanism that locally condenses a set of components capable of mediating protein degradation decisions. The breadth of mechanisms by which cells respond to stressors and form specific functional types of membraneless organelles, is incompletely understood. We found that Bcl2-associated athanogene 2 (BAG2) marks a distinct phase-separated membraneless organelle, triggered by several forms of stress, particularly hyper-osmotic stress. Distinct from well-known condensates such as stress granules and processing bodies, BAG2-containing granules lack RNA, lack ubiquitin and promote client degradation in a ubiquitin-independent manner via the 20S proteasome. These organelles protect the viability of cells from stress and can traffic to the client protein, in the case of Tau protein, on the microtubule. Components of these ubiquitin-independent degradation organelles include the chaperone HSP-70 and the 20S proteasome activated by members of the PA28 (PMSE) family. BAG2 condensates did not co-localize with LAMP-1 or p62/SQSTM1. When the proteasome is inhibited, BAG2 condensates and the autophagy markers traffic to an aggresome-like structure.

Activation of microglia is a prominent pathological feature in tauopathies, including Alzheimer's disease. How microglia activation contributes to tau toxicity remains largely unknown. Here we show that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, activated by tau, drives microglial-mediated tau propagation and toxicity. Constitutive activation of microglial NF-κB exacerbated, while inactivation diminished, tau seeding and spreading in young PS19 mice. Inhibition of NF-κB activation enhanced the retention while reduced the release of internalized pathogenic tau fibrils from primary microglia and rescued microglial autophagy deficits. Inhibition of microglial NF-κB in aged PS19 mice rescued tau-mediated learning and memory deficits, restored overall transcriptomic changes while increasing neuronal tau inclusions. Single cell RNA-seq revealed that tau-associated disease states in microglia were diminished by NF-κB inactivation and further transformed by constitutive NF-κB activation. Our study establishes a role for microglial NF-κB signaling in mediating tau spreading and toxicity in tauopathy.

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A. A cryo-EM structure of PA26E102A-h20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC.

Post-translational modifications (PTMs) direct the assembly of protein complexes. In this context, proteolysis is a unique PTM because it is irreversible; the hydrolysis of the peptide backbone generates separate fragments bearing a new N and C terminus. Proteolysis can "re-wire" protein-protein interactions (PPIs) via the recruitment of end-binding proteins to new termini. In this review, we focus on the role of proteolysis in specifically creating complexes by recruiting E3 ubiquitin ligases to new N and C termini. These complexes potentiate proteolytic signaling by "erasing" proteolytic modifications. This activity tunes the duration and magnitude of protease signaling events. Recent work has shown that the stepwise process of proteolysis, end-binding by E3 ubiquitin ligases, and fragment turnover is associated with both the nascent N terminus (i.e., N-degron pathways) and the nascent C terminus (i.e., the C-degron pathways). Here, we discuss how these pathways might harmonize protease signaling with protein homeostasis (i.e., proteostasis).

The major classes of molecular chaperones have highly variable sequences, sizes, and shapes, yet they all bind to unfolded proteins, limit their aggregation, and assist in their folding. Despite the central importance of this process to protein homeostasis, it has not been clear exactly how chaperones guide this process or whether the diverse families of chaperones use similar mechanisms. For the first time, recent advances in NMR spectroscopy have enabled detailed studies of how unfolded, "client" proteins interact with both ATP-dependent and ATP-independent classes of chaperones. Here, we review examples from four distinct chaperones, Spy, Trigger Factor, DnaK, and HscA-HscB, highlighting the similarities and differences between their mechanisms. One striking similarity is that the chaperones all bind weakly to their clients, such that the chaperone-client interactions are readily outcompeted by stronger, intra- and intermolecular contacts in the folded state. Thus, the relatively weak affinity of these interactions seems to provide directionality to the folding process. However, there are also key differences, especially in the details of how the chaperones release clients and how ATP cycling impacts that process. For example, Spy releases clients in a largely folded state, while clients seem to be unfolded upon release from Trigger Factor or DnaK. Together, these studies are beginning to uncover the similarities and differences in how chaperones use weak interactions to guide protein folding.

The protein homeostasis (proteostasis) network is composed of multiple pathways that work together to balance protein folding, stability, and turnover. Cancer cells are particularly reliant on this network; however, it is hypothesized that inhibition of one node might lead to compensation. To better understand these connections, we dosed 22Rv1 prostate cancer cells with inhibitors of four proteostasis targets (Hsp70, Hsp90, proteasome, and p97), either alone or in binary combinations, and measured the effects on cell growth. The results reveal a series of additive, synergistic, and antagonistic relationships, including strong synergy between inhibitors of p97 and the proteasome and striking antagonism between inhibitors of Hsp90 and the proteasome. Based on RNA-seq, these relationships are associated, in part, with activation of stress pathways. Together, these results suggest that cocktails of proteostasis inhibitors might be a powerful way of treating some cancers, although antagonism that blunts the efficacy of both molecules is also possible.

The Hsp90 chaperone promotes folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during folding of kinases, nuclear receptors, and tau. Here we determined the cryoelectron microscopy (cryo-EM) structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å, which, together with mutagenesis and crosslinking analyses, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent to Hsp90 client binding sites, whereas a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 is positioned to act on specific client residues presented during Hsp90-catalyzed remodeling.

The molecular chaperone, Heat Shock Protein 70 (Hsp70), is an emerging drug target for neurodegenerative diseases, because of its ability to promote degradation of microtubule-associated protein tau (MAPT/tau). Recently, we reported YM-08 as a brain penetrant, allosteric Hsp70 inhibitor, which reduces tau levels. However, the benzothiazole moiety of YM-08 is vulnerable to metabolism by CYP3A4, limiting its further application as a chemical probe. In this manuscript, we designed and synthesized seventeen YM-08 derivatives by systematically introducing halogen atoms to the benzothiazole ring and shifting the position of the heteroatom in a distal pyridine. In microsome assays, we found that compound JG-23 has 12-fold better metabolic stability and it retained the ability to reduce tau levels in two cell-based models. These chemical probes of Hsp70 are expected to be useful tools for studying tau homeostasis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression.

Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically "undruggable" targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes.

Prion protein (PrP) adopts either a helical conformation (PrPC) or an alternative, beta sheet-rich, misfolded conformation (PrPSc). The PrPSc form has the ability to "infect" PrPC and force it into the misfolded state. Accumulation of PrPSc is associated with a number of lethal neurodegenerative disorders, including Creutzfeldt-Jacob disease (CJD). Knockout of PrPC protects cells and animals from PrPSc infection; thus, there is interest in identifying factors that regulate PrPC stability, with the therapeutic goal of reducing PrPC levels and limiting infection by PrPSc. Here, we assembled a short-hairpin RNA (shRNA) library composed of 25+ shRNA sequences for each of 133 protein homeostasis (aka proteostasis) factors, such as molecular chaperones and co-chaperones. This Proteostasis shRNA Library was used to identify regulators of PrPC stability in HEK293 Hu129M cells. Strikingly, the screen identified a number of Hsp70 family members and their co-chaperones as putative targets. Indeed, a chemical pan-inhibitor of Hsp70s reduced PrPC levels and limited conversion to PrPSc in N2a cells. These results implicate specific proteostasis sub-networks, especially the Hsp70 system, as potential new targets for the treatment of CJD. More broadly, the Proteostasis shRNA Library might be a useful tool for asking which proteostasis factors are important for a given protein.

Plasmodium falciparum is a unicellular protozoan parasite and causative agent of a severe form of malaria in humans, accounting for very high worldwide fatality rates. At the molecular level, survival of the parasite within the human host is mediated by P. falciparum heat shock proteins (PfHsps) that provide protection during febrile episodes. The ATP-dependent chaperone activity of Hsp70 relies on the co-chaperone J domain protein (JDP), with which it forms a chaperone-co-chaperone complex. The exported P. falciparum JDP (PfJDP), PFA0660w, has been shown to stimulate the ATPase activity of the exported chaperone, PfHsp70-x. Furthermore, PFA0660w has been shown to associate with another exported PfJDP, PFE0055c, and PfHsp70-x in J-dots, highly mobile structures found in the infected erythrocyte cytosol. Therefore, the present study aims to conduct a structural and functional characterization of the full-length exported PfJDP, PFE0055c. Recombinant PFE0055c was successfully expressed and purified and found to stimulate the basal ATPase activity of PfHsp70-x to a greater extent than PFA0660w but, like PFA0660w, did not significantly stimulate the basal ATPase activity of human Hsp70. Small-molecule inhibition assays were conducted to determine the effect of known inhibitors of JDPs (chalcone, C86) and Hsp70 (benzothiazole rhodacyanines, JG231 and JG98) on the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. In this study, JG231 and JG98 were found to inhibit both the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. C86 only inhibited the PFE0055c-stimulated ATPase activity of PfHsp70-x, consistent with PFE0055c binding to PfHsp70-x through its J domain. This research has provided further insight into the molecular basis of the interaction between these exported plasmodial chaperones, which could inform future antimalarial drug discovery studies.

Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism.

Protein-protein interactions (PPIs) involving the extreme C-terminus serve important scaffolding and regulatory functions. Here, we leveraged NanoBiT technology to build a luminescent complementation assay for use in studying this subcategory of PPI. As a model system, we fused one component of NanoBiT to the disordered C-terminus of heat shock protein (Hsp70) and the other to its binding partner, the tetratricopeptide repeat (TPR) domain of CHIP/STUB1. We found that HEK293 cells that stably express these chimeras under a doxycycline promoter produced a robust luminescence signal. This signal was sensitive to mutations and it was further tuned by the expression of competitive C-termini. Using this system, we identified a promising, membrane permeable inhibitor of the Hsp70-CHIP interaction. More broadly, we anticipate that NanoBiT is well-suited for studying PPIs that involve C-termini.

BackgroundThe trans-neuronal propagation of tau has been implicated in the progression of tau-mediated neurodegeneration. There is critical knowledge gap in understanding how tau is released and transmitted, and how that is dysregulated in diseases. Previously, we reported that lysine acetyltransferase p300/CBP acetylates tau and regulates its degradation and toxicity. However, whether p300/CBP is involved in regulation of tau secretion and propagation is unknown.MethodWe investigated the relationship between p300/CBP activity, the autophagy-lysosomal pathway (ALP) and tau secretion in mouse models of tauopathy and in cultured rodent and human neurons. Through a high-through-put compound screen, we identified a new p300 inhibitor that promotes autophagic flux and reduces tau secretion. Using fibril-induced tau spreading models in vitro and in vivo, we examined how p300/CBP regulates tau propagation.ResultsIncreased p300/CBP activity was associated with aberrant accumulation of ALP markers in a tau transgenic mouse model. p300/CBP hyperactivation blocked autophagic flux and increased tau secretion in neurons. Conversely, inhibiting p300/CBP promoted autophagic flux, reduced tau secretion, and reduced tau propagation in fibril-induced tau spreading models in vitro and in vivo.ConclusionsWe report that p300/CBP, a lysine acetyltransferase aberrantly activated in tauopathies, causes impairment in ALP, leading to excess tau secretion. This effect, together with increased intracellular tau accumulation, contributes to enhanced spreading of tau. Our findings suggest that inhibition of p300/CBP as a novel approach to correct ALP dysfunction and block disease progression in tauopathy.

The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its permeability. Moreover, this compound was processed by both esterase and phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in cells. Thus, Hint1 appears to recognize a broader set of substrates than previously appreciated. It seems possible that other potent, but impermeable, phosphate-containing inhibitors might likewise benefit from this approach.

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.

The complexity of glucocorticoid receptor (GR) signaling cannot be measured with direct tissue analysis in living subjects, which has stifled our understanding of GR's role in human physiology or disease and impeded the development of selective GR modulators. Herein, we report 18F-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]quinoline (18F-YJH08), a radioligand that enables noninvasive measurements of tissue autonomous GR expression levels in vivo with positron emission tomography (PET). YJH08 potently binds GR (Ki ∼ 0.4 nM) with ∼100-fold selectivity compared to nuclear hormone receptors in the same subfamily. 18F-YJH08 was prepared via Cu(OTf)2(py)4-mediated radiofluorination of an arylboronic acid pinacol ester with ∼12% decay corrected radiochemical yield from the starting 18F-fluoride ion. We applied treatment with the tissue-wide GR agonist dexamethasone and adrenalectomy and generated an adipocyte specific GR knockout mouse to show that 18F-YJH08 specifically binds GR in normal mouse tissues, including those for which aberrant GR expression is thought to drive severe diseases (e.g., brain, adipose tissue, kidneys). Remarkably, 18F-YJH08 PET also revealed that JG231, a potent and bioavailable HSP70 inhibitor, selectively degrades GR only in the adipose tissue of mice, a finding that foreshadows how GR targeted PET might be integrated into drug discovery to screen for selective GR modulation at the tissue level, beyond the historical screening that was performed at the transcriptional level. In summary, 18F-YJH08 enables a quantitative assessment of GR expression levels in real time among multiple tissues simultaneously, and this technology is a first step toward unraveling the daunting complexity of GR signaling and rationally engineering tissue specific therapeutic modulators in vivo.

The mitochondrial HSP70 chaperone mortalin (HSPA9/GRP75) is often upregulated and mislocalized in MEK/ERK-deregulated tumors. Here, we show that mortalin depletion can selectively induce death of immortalized normal fibroblasts IMR90E1A when combined with K-RasG12V expression, but not with wild-type K-Ras expression, and that K-RasG12V-driven MEK/ERK activity is necessary for this lethality. This cell death was attenuated by knockdown or inhibition of adenine nucleotide translocase (ANT), cyclophilin D (CypD), or mitochondrial Ca2+ uniporter (MCU), which implicates a mitochondria-originated death mechanism. Indeed, mortalin depletion increased mitochondrial membrane permeability and induced cell death in KRAS-mutated human pancreatic ductal adenocarcinoma (PDAC) and colon cancer lines, which were attenuated by knockdown or inhibition of ANT, CypD, or MCU, and occurred independently of TP53 and p21CIP1. Intriguingly, JG-98, an advanced MKT-077 derivative, phenocopied the lethal effects of mortalin depletion in K-RasG12V-expressing IMR90E1A and KRAS-mutated tumor cell lines in vitro. Moreover, JG-231, a JG-98 analog with improved microsomal stability effectively suppressed the xenograft of MIA PaCa-2, a K-RasG12C-expressing human PDAC line, in athymic nude mice. These data demonstrate that oncogenic KRAS activity sensitizes cells to the effects of mortalin depletion, suggesting that mortalin has potential as a selective therapeutic target for KRAS-mutated tumors.

BackgroundPlatelets play a pivotal role in hemostasis, wound healing, and inflammation, and are thus implicated in a variety of diseases, including cancer. Platelet function is associated with release of granule content, cellular shape change, and upregulation of receptors that promote establishment of a thrombus and maintenance of hemostasis.ObjectivesThe role of heat shock proteins (Hsps) in modulating platelet function has been studied for a number of years, but comparative roles of individual Hsps have not been thoroughly examined.MethodsWe utilized a panel of specific inhibitors of Hsp40, Hsp70, Hsp90, and Grp94 (the endoplasmic reticulum homolog of Hsp90) to assess their impact on several aspects of platelet function.ResultsInhibition of each of the aforementioned Hsps reduced alpha granule release. In contrast, there was some selectivity in impacts on dense granule release. Thromboxane synthesis was impaired after exposure to inhibitors of Hsp40, Hsp90, and Grp94, but not after inhibition of Hsp70. Both expression of active glycoprotein IIb/IIIa (GPIIb/IIIa) and fibrinogen-induced platelet shape change were diminished by our inhibitors. In contrast, aggregation was selectively abrogated after inhibition of Hsp40 or Hsp90. Lastly, activated platelet-cancer cell interactions were reduced by inhibition of both Hsp70 and Grp94.ConclusionsThese data suggest the importance of Hsp networks in regulating platelet activity.

Mortalin [also known as heat shock protein family A (HSP70) member 9 (HSPA9) or glucose-regulated protein 75 (GRP75)] is a mitochondrial molecular chaperone that is often up-regulated and mislocalized in tumors with abnormal activation of the kinases MEK and ERK. Here, we found that mortalin depletion was selectively lethal to tumor and immortalized normal cells expressing the mutant kinase B-RafV600E or the chimeric protein ΔRaf-1:ER and that MEK-ERK-sensitive regulation of the peptide-binding domain in mortalin was critical to cell survival or death. Proteomics screening identified adenine nucleotide translocase 3 (ANT3) as a previously unknown mortalin substrate and cell survival/death effector. Mechanistically, increased MEK-ERK signaling activity and mortalin function converged opposingly on the regulation of mitochondrial permeability. Specifically, whereas MEK-ERK activity increased mitochondrial permeability by promoting the interaction between ANT3 and the peptidyl-prolyl isomerase cyclophilin D (CypD), mortalin decreased mitochondrial permeability by inhibiting this interaction. Hence, mortalin depletion increased mitochondrial permeability in MEK-ERK-deregulated cells to an extent that triggered cell death. HSP70 inhibitor derivatives that effectively inhibited mortalin suppressed the proliferation of B-RafV600E tumor cells in culture and in vivo, including their B-Raf inhibitor-resistant progenies. These findings suggest that targeting mortalin has potential as a selective therapeutic strategy in B-Raf-mutant or MEK-ERK-driven tumors.

The heat shock protein 70 (Hsp70) family of molecular chaperones are highly expressed in tumors. Inhibitors containing a pyridinium-modified benzothiazole, such as JG-98, bind to a conserved, allosteric site in Hsp70, showing promising anti-proliferative activity in cancer cells. When bound to Hsp70, the charged pyridinium makes favorable contacts; however, this moiety also increases the inhibitor's fluorescence, giving rise to undesirable interference in biochemical and cell-based assays. Here, we explore whether the pyridinium can be replaced with a neutral pyridine. We report that pyridine-modified benzothiazoles, such as compound 17h (JG2-38), have reduced fluorescence, yet retain promising anti-proliferative activity (EC50 values ~0.1 to 0.07 µM) in breast and prostate cancer cell lines. These chemical probes are expected to be useful in exploring the roles of Hsp70s in tumorigenesis and cell survival.

Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase. Nuclear magnetic resonance analysis reveals that SEW84 binds to the C-terminal domain of Aha1 to weaken its asymmetric binding to Hsp90. Consistent with this observation, SEW84 blocks Aha1-dependent Hsp90 chaperoning activities, including the in vitro and in vivo refolding of firefly luciferase, and the transcriptional activity of the androgen receptor in cell-based models of prostate cancer and promotes the clearance of phosphorylated tau in cellular and tissue models of neurodegenerative tauopathy. We propose that SEW84 provides a novel lead scaffold for developing therapeutic approaches to treat proteostatic disease.

Understanding the selectivity of a small molecule for its target(s) in cells is an important goal in chemical biology and drug discovery. One powerful way to address this question is with dominant negative (DN) mutants, in which an active site residue in the putative target is mutated. While powerful, this approach is less straightforward for allosteric sites. Here, we introduce tryptophan scanning mutagenesis as an expansion of this idea. As a test case, we focused on the challenging drug target, heat shock cognate protein 70 (Hsc70), and its allosteric inhibitor JG-98. Structure-based modelling predicted that mutating Y149W in human Hsc70 or Y145W in the bacterial ortholog DnaK would place an indole side chain into the allosteric pocket normally occupied by the compound. Indeed, we found that the tryptophan mutants acted as if they were engaged with JG-98. We then used DnaK Y145W to suggest that this protein may be an anti-bacterial target. Indeed, we found that DnaK inhibitors have minimum inhibitory concentration (MIC) values

Genetic diversity provides a rich repository for understanding the role of proteostasis in the management of the protein fold in human biology. Failure in proteostasis can trigger multiple disease states, affecting both human health and lifespan. Niemann-Pick C1 (NPC1) disease is a rare genetic disorder triggered by mutations in NPC1, a multi-spanning transmembrane protein that is trafficked through the exocytic pathway to late endosomes (LE) and lysosomes (Ly) (LE/Ly) to globally manage cholesterol homeostasis. Defects triggered by >300 NPC1 variants found in the human population inhibit export of NPC1 protein from the endoplasmic reticulum (ER) and/or function in downstream LE/Ly, leading to cholesterol accumulation and onset of neurodegeneration in childhood. We now show that the allosteric inhibitor JG98, that targets the cytosolic Hsp70 chaperone/co-chaperone complex, can significantly improve the trafficking and post-ER protein level of diverse NPC1 variants. Using a new approach to model genetic diversity in human disease, referred to as variation spatial profiling, we show quantitatively how JG98 alters the Hsp70 chaperone/co-chaperone system to adjust the spatial covariance (SCV) tolerance and set-points on an amino acid residue-by-residue basis in NPC1 to differentially regulate variant trafficking, stability, and cholesterol homeostasis, results consistent with the role of BCL2-associated athanogene family co-chaperones in managing the folding status of NPC1 variants. We propose that targeting the cytosolic Hsp70 system by allosteric regulation of its chaperone/co-chaperone based client relationships can be used to adjust the SCV tolerance of proteostasis buffering capacity to provide an approach to mitigate systemic and neurological disease in the NPC1 population.

ABSTRACT The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.

Dysregulation of neuronal excitability underlies the pathogenesis of tauopathies, including frontotemporal dementia (FTD) with tau inclusions. A majority of FTD-causing tau mutations are located in the microtubule-binding domain, but how these mutations alter neuronal excitability is largely unknown. Here, using CRISPR/Cas9-based gene editing in human pluripotent stem cell (iPSC)-derived neurons and isogenic controls, we show that the FTD-causing V337M tau mutation impairs activity-dependent plasticity of the cytoskeleton in the axon initial segment (AIS). Extracellular recordings by multi-electrode arrays (MEAs) revealed that the V337M tau mutation in human neurons leads to an abnormal increase in neuronal activity in response to chronic depolarization. Stochastic optical reconstruction microscopy of human neurons with this mutation showed that AIS plasticity is impaired by the abnormal accumulation of end-binding protein 3 (EB3) in the AIS submembrane region. These findings expand our understanding of how FTD-causing tau mutations dysregulate components of the neuronal cytoskeleton, leading to network dysfunction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.