Hsp90 Binding Research Articles

Computational Modeling of the Hsp90 Interactions with Cochaperones and Small-Molecule Inhibitors.

Allosteric interactions of the molecular chaperone Hsp90 with a diverse array of cochaperones and client proteins, such as protein kinases and transcription factors, allow for efficient molecular communication in signal transduction networks. Deregulation of pathways involving these proteins is commonly associated with cancer pathologies and allosteric inhibition of oncogenic clients by targeting Hsp90 provides a powerful therapeutic strategy in cancer research. We review several validated computational approaches and tools used in the studies of the Hsp90 interactions with proteins and small molecules. These methods include experimentally guided docking to predict Hs90-protein interactions, molecular and binding free energy simulations to analyze Hsp90 binding with small molecules, and structure-based network modeling to evaluate allosteric interactions and communications in the Hsp90 regulatory complexes. Through the lens of allosteric-centric view on Hsp90 function and regulation, we discuss newly emerging computational tools that link protein structure modeling with biophysical simulations and network-based systems biology approaches.

The regulation mechanisms of AhR by molecular chaperone complex.

The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR.

Pih1p-Tah1p Puts a Lid on Hexameric AAA+ ATPases Rvb1/2p

The Saccharomyces cerevisiae (Sc) R2TP complex affords an Hsp90-mediated and nucleotide-driven chaperone activity to proteins of small ribonucleoprotein particles (snoRNPs). The current lack of structural information on the ScR2TP complex, however, prevents a mechanistic understanding of this biological process. We characterized the structure of the ScR2TP complex made up of two AAA+ ATPases, Rvb1/2p, and two Hsp90 binding proteins, Tah1p and Pih1p, and its interaction with the snoRNP protein Nop58p by a combination of analytical ultracentrifugation, isothermal titration calorimetry, chemical crosslinking, hydrogen-deuterium exchange, and cryoelectron microscopy methods. We find that Pih1p-Tah1p interacts with Rvb1/2p cooperatively through the nucleotide-sensitive domain of Rvb1/2p. Nop58p further binds Pih1p-Tahp1 on top of thedome-shaped R2TP. Consequently, nucleotide binding releases Pih1p-Tah1p from Rvb1/2p, which offers a mechanism for nucleotide-driven binding and release of snoRNP intermediates.

Abstract 3446: Melatonin suppresses hepatocellular carcinoma progression via lncRNA-CPS1-IT-mediated HIF-1α inactivation

Abstract Background: Melatonin, the main pineal hormone that relays light/dark cycle information to the circadian system, was recently reported to have intrinsic antitumor activity in various cancers. However, the detailed regulatory mechanisms associated with antitumor activity of melatonin are not well understood yet. Moreover, limited studies have addressed the role of melatonin in hepatocellular carcinoma (HCC), the major life-threatening malignancy for both sexes in Taiwan. Hence, in this study, we investigated the antitumor activity of melatonin on HCC and explored its regulatory mechanism. Methods: Human hepatoma cell lines HepG2 and Huh7 were treated with melatonin followed by functional assays to dissect antitumor effect of melatonin in HCC. The whole transcriptome sequence analysis was performed to explore the long non-coding RNAs (lncRNAs) involved in the anti-HCC activity of melatonin. Further, quantitative RT-PCR, western blot, immunohistochemistry assays, and in vivo animal model experiments were performed to elucidate the regulatory mechanism of lncRNAs in melatonin-mediated HCC suppression. Results: Melatonin significantly inhibited cell proliferation, migration, and invasion capacity of HCC cell lines, while the expression of transcription factor FOXA2 was significantly induced in cells treated with melatonin. The increase in expression of FOXA2 resulted in upregulation of lncRNA-CPS1-IT1 expression, which in turn reduced binding and activation of Hsp90 with HIF-1α. The inactivation of HIF-1α resulted in the suppression of epithelial-mesenchymal transition (EMT) progression and HCC metastasis. Furthermore, the results of the in vivo animal model experiments also validated the tumor-suppressor role of melatonin via a reduction in tumor growth. Conclusions: Taken together, our findings suggested that melatonin inhibited HCC progression by modulating lncRNA-CPS1-IT1-mediated EMT suppression and further supported melatonin as a promising therapeutic agent for the treatment of HCC. Note: This abstract was not presented at the meeting. Citation Format: Tong Hong Wang, Chi Hao Wu, Chau Ting Yeh, Kung Hao Liang, Chuen Hsueh, Chi Yuan Chen. Melatonin suppresses hepatocellular carcinoma progression via lncRNA-CPS1-IT-mediated HIF-1α inactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3446. doi:10.1158/1538-7445.AM2017-3446

Interactions and Dynamics Within CHIP/Hsp70 Complexes

Interactions between the E3 ubiquitin ligase CHIP (C‐terminus of Hsp70 interacting protein) and the chaperone Hsp70 represent the physical intersection of two competing protein quality control efforts: refolding, and degradation. Interaction with CHIP allows for the formation of a productive encounter complex that facilitates ubiquitination of Hsp70‐bound substrates, thereby targeting those proteins for proteasomal degradation. Central to the CHIP/Hsp70 interaction are changes in structure and dynamics that occur upon binding. Although recruitment of Hsp70 to CHIP via interaction with the CHIP tetratricopeptide repeat (TPR) domain is well established, alterations in structure and dynamics of CHIP and Hsp70 upon binding are not well understood. Using NMR we describe residue‐level changes in dynamics to the CHIP‐TPR domain that occur upon binding Hsp70. We also utilize small angle X‐ray scattering (SAXS) and electron paramagnetic resonance (EPR) to identify motions with the CHIP/Hsp70 complex. Independent SAXS and EPR experiments both point to a mobile Hsp70 substrate binding domain that is only partially constrained through interactions with the CHIP‐TPR. Building upon static models of the full‐length CHIP/Hsp70 complex, afforded by our recent crystal structure of the CHIP‐TPR/Hsp70‐lid‐tail complex, we have assembled a dynamic model of the CHIP‐Hsp70 complex. This dynamic model sheds light on the function of the CHIP/Hsp70 chaperoned ubiquitination complex and how CHIP harnesses dynamics of Hsp70 to overcome significant distance separations identified in earlier static models of the CHIP/Hsp70 complex.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding.

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp-substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp-substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.

HSP90 Shapes the Consequences of Human Genetic Variation

HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases.

Luteolin selectively kills STAT3 highly activated gastric cancer cells through enhancing the binding of STAT3 to SHP-1

The antitumor effect of luteolin, a plant flavonoid, in gastric cancer (GC) cells has not been fully understood. Here we show that luteolin selectively kills STAT3 overactivated GC cells that are often drug resistant. The treatment of luteolin in these GC cells significantly inhibited STAT3 phosphorylation and reduced the expression of STAT3 targeting gene Mcl-1, Survivin and Bcl-xl. Silencing of SHP-1, a protein tyrosine phosphatase, abolished the inhibitory effect of luteolin on STAT3 and cell apoptosis, suggesting that SHP-1 is crucial in luteolin-mediated cellular function. Moreover, this luteolin effect of STAT3 dephosphorylation by SHP-1 involved in HSP-90, which protected STAT3 phosphorylation by forming HSP-90/STAT3 complex. Thus, luteolin inhibited STAT3 activation through disrupting the binding of HSP-90 to STAT3, which promoted its interaction to SHP-1, resulted in the dephosphorylation of STAT3. The GC cell xenograft mouse model confirmed the effectiveness of luteolin induced inhibition of tumor growth in vivo.

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70

Background/Aims: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. Methods: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. Results: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. Conclusion: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

Research Article: Simian virus 40 large T antigen-mediated transactivation of the cyclin A promoter is independent of p53 and TBP binding functions

Abstract Simian virus 40 is a small DNA tumor virus capable of transforming cells in culture and causing tumors in animal models. The viral protein large T antigen specifically binds the tumor suppressors p53 and hypophosphorylated retinoblastoma protein (pRb) to prevent G1 arrest. T antigen also transactivates the cyclin-A promoter. This activity is independent of pRb binding, yet dependent upon the J-domain Hsp70-binding region. T antigen also binds transcription factors, including TATA box binding protein (TBP). Thus, we assessed whether cyclin A promoter activation relied on this activity and/or the ability to complex with p53. Using a luciferase reporter assay, we show that T antigen mutant proteins defective in TBP and p53 binding are able to transactivate the cyclin A promoter to wild type levels. These data indicate that T antigen mediated transactivation of the cyclin A promoter uses a p53 and TBP-binding-independent mechanism, yet relies on hsp70 binding to the N-terminal, J-domain. This suggest...

Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation.

Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways.

Bipartite Role of Heat Shock Protein 90 (Hsp90) Keeps CRAF Kinase Poised for Activation

CRAF kinase maintains cell viability, growth, and proliferation by participating in the MAPK pathway. Unlike BRAF, CRAF requires continuous chaperoning by Hsp90 to retain MAPK signaling. However, the reason behind the continuous association of Hsp90 with CRAF is still elusive. In this study, we have identified the bipartite role of Hsp90 in chaperoning CRAF kinase. Hsp90 facilitates Ser-621 phosphorylation of CRAF and prevents the kinase from degradation. Co-chaperone Cdc37 assists in this phosphorylation event. However, after folding, the stability of the kinase becomes insensitive to Hsp90 inhibition, although the physical association between Hsp90 and CRAF remains intact. We observed that overexpression of Hsp90 stimulates MAPK signaling by activating CRAF. The interaction between Hsp90 and CRAF is substantially increased under an elevated level of cellular Hsp90 and in the presence of either active Ras (RasV12) or EGF. Surprisingly, enhanced binding of Hsp90 to CRAF occurs prior to the Ras-CRAF association and facilitates actin recruitment to CRAF for efficient Ras-CRAF interaction, which is independent of the ATPase activity of Hsp90. However, monomeric CRAF (CRAFR401H) shows abrogated interaction with both Hsp90 and actin, thereby affecting Hsp90-dependent CRAF activation. This finding suggests that stringent assemblage of Hsp90 keeps CRAF kinase equipped for participating in the MAPK pathway. Thus, the role of Hsp90 in CRAF maturation and activation acts as a limiting factor to maintain the function of a strong client like CRAF kinase.

Design, synthesis and pharmacological evaluation of 4,5-diarylisoxazols bearing amino acid residues within the 3-amido motif as potent heat shock protein 90 (Hsp90) inhibitors

A structure-based medicinal chemistry optimization was conducted on the clinical Hsp90 inhibitor diarylisoxazole 3. Several series of new compounds were designed and synthesized by incorporating diversified amino acid derivatives with various lengths to the 3-amido motif of the isoxazole scaffold. Compound 14j was identified to have high Hsp90 binding potency (14 nM) and antiproliferative activity against H3122 human lung cancer and BT-474 breast cancer cells. Treatment of 14j with H3122 cell led to degradation of client protein ALK, reduction of downstream phosphorylation of AKT and ERK, and up-regulation of Hsp70. Molecular docking suggested that the terminal valine moiety and the ethyleneglycol linker in compound 14j formed additional apolar and polar interaction network with a number of amino acid residues.

Bacterial and Yeast AAA + Disaggregases ClpB and Hsp104 Operate through Conserved Mechanism Involving Cooperation with Hsp70

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are members of the Hsp100 family of ring-forming hexameric AAA+ chaperones that promote the solubilization of aggregated proteins and the propagation of prions. ClpB and Hsp104 cooperate with cognate Hsp70 chaperones for substrate targeting and activation of ATPase and substrate threading, achieved by transient Hsp70 binding to the repressing ClpB/Hsp104 M-domain. Fundamental differences in ATPase regulation and disaggregation mechanisms have been reported; however, these differences are raising doubts regarding the working principle of this AAA+ chaperone. In particular, unique functional plasticity was suggested to specifically enable Hsp104 to circumvent Hsp70 requirement for derepression in protein disaggregation and prion propagation. We show here that both ClpB and Hsp104 cooperation with Hsp70 is crucial for efficient protein disaggregation and, in contrast to earlier claims, cannot be circumvented by activating M-domain mutations. Activation of ClpB and Hsp104 requires two signals, relief of M-domain repression and substrate binding, leading to increased ATPase subunit coupling. These data demonstrate that ClpB and Hsp104 operate by the same basic mechanism, underscore a dominant function of Hsp70 in regulating ClpB/Hsp104 activity, and explain a plethora of in vivo studies showing a crucial function of Hsp70 in proteostasis and prion propagation.

Discovery of new heat shock protein 90 inhibitors using virtual co-crystallized pharmacophore generation

The pharmacophoric features of the virtual cocrystallized protein of 178 Hsp90 proteins were obtained from the protein data bank and explored to generate 1260 pharmacophores evaluated using the decoy list composed of 1022 compounds. Accordingly, 51 pharmacophores were selected with high receiver operating characteristic (ROC) value for further processing. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of pharmacophoric models and 2D physicochemical descriptors capable of accessing a self-consistent quantitative structure-activity relationship (QSAR) of optimal predictive potential (R672 = 0.819, F = 43.0, R2LOO = 0.782, R2PRESS against 16 external test inhibitors equal 0.735). Two orthogonal pharmacophores emerged in the QSAR equation suggesting the existence of at least two binding modes accessible to ligands within the Hsp90 binding pocket. The fifth generated pharmacophoric model from Hsp90 protein 2XJX (2XJX_2_05), and the forth generated cocrystallized pharmacophoric model from Hsp90 protein 4LWF (4LWF_2_04) with area under the curve AUC–ROC values 0.812 and 0.876, respectively were selected to be used as a searching tool sequentially of the National Cancer Institute (NCI) database. The captured hits were mapped based on successful hypotheses and the best predicted hits were selected. Twenty-four hits showed Hsp90 inhibition, 15 hits were measured with low micromolar IC50 ranged from 5.0 μM to 77.1 μM

Thermodynamic Analysis of the Geldanamycin-Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach.

Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 μM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ.

C-terminal domain of p42 Ebp1 is essential for down regulation of p85 subunit of PI3K, inhibiting tumor growth.

Potential tumor suppressor p42, ErbB3-binding protein 1 (EBP1) inhibits phosphoinositide 3-kinase (PI3K) activity reducing the p85 regulatory subunit. In this study, we demonstrated that overexpression of p42 promoted not only a reduction of wild type of p85 subunit but also oncogenic mutant forms of p85 which were identified in human cancers. Moreover, we identified the small fragment of C-terminal domain of p42 is sufficient to exhibit tumor suppressing activity of p42-WT, revealing that this small fragment (280–394) of p42 is required for the binding of both HSP70 and CHIP for a degradation of p85. Furthermore, we showed the small fragment of p42 markedly inhibited the tumor growth in mouse xenograft models of brain and breast cancer, resembling tumor suppressing activity of p42. Through identification of the smallest fragment of p42 that is responsible for its tumor suppressor activity, our findings represent a novel approach for targeted therapy of cancers that overexpress PI3K.

Structural and functional basis of protein phosphatase 5 substrate specificity

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.

Abstract 106: G Protein-coupled Receptor Kinase 2 Associates With Mitochondrial Proteins and Negatively Alters Mitochondrial Respiration After Myocardial Infarction

During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). Consequently, inhibition of GRK2 has been shown to rescue several animal models of HF, as demonstrated by cardiac expression of the βARKct, a peptide inhibitor of GRK2. There is new evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, during conditions that increase oxidative stress, GRK2 has been shown to localize to the mitochondria, where it is a pro-death kinase. βARKct has also been shown to inhibit the mitochondrial translocation of GRK2 due to inhibition of Hsp90 binding, a chaperone found to bind and localize GRK2 to mitochondria. Our objective is to identify the functions and targets of GRK2 in the mitochondria and its role in the development of HF. We hypothesize that GRK2 has mitochondrial targets that impair mitochondrial function in vivo, contributing to the pathogenesis of HF. Our results show that ADP-stimulated and maximal respiration are significantly decreased in mitochondria isolated from mouse hearts 2 weeks post-myocardial infarction (MI). Cardiac-specific expression of the βARKct rescued the reduction in mitochondrial respiration post-MI, in the presence of pyruvate or palmitoylcarnitine. To identify the mitochondrial targets of GRK2, we performed interactome and phospho-interactome analyses on GRK2 immunoprecipitates from neonatal rat cardiomyocytes subjected to oxidative stress. These results revealed that GRK2 interacts with several mitochondrial proteins, including those involved in metabolism, oxidative stress, calcium handling and cell death. Thus we propose that during ischemia, an increase in mitochondrial GRK2 results in its association with proteins that compromise respiration and contribute to myocardial damage and HF. This demonstrates that understanding both the canonical and non-canonical roles of GRK2 in HF is essential for more targeted intervention for therapy.

C15-methoxyphenylated 18-deoxy-herbimycin A analogues, their in vitro anticancer activity and heat shock protein 90 binding affinity

Benzoquinone ansamycins are important leads for the discovery of novel inhibitors of heat shock protein 90 (Hsp90), a promising target of cancer chemotherapeutics. Intrinsic hepatotoxicity caused by the benzoquinone moiety appeared to be a serious limitation to the development of these compounds. To solve this problem by rational structure optimization, a short series of C18-deoxy analogues of herbimycin A were designed based on putative interactions between the compound and the protein. Chemical synthesis of the target molecules were attempted by following the established synthetic route to the natural product, but resulted in the isolation of four serendipitous C15 phenylated final products. In vitro antiproliferative activity and Hsp90 binding affinity of the compounds were determined, suggesting the C18-oxygen of herbimycin A is removable and bulky lipophilic groups can be accommodated at C15 without loss of activity.