In silico identification of small molecule modulators for disruption of Hsp90-Cdc37 protein-protein interaction interface for cancer therapeutic application.

The protein-protein interactions (PPIs) in the biological systems are important to maintain a number of cellular processes. Several disorders including cancer may be developed due to dysfunction in the assembly of PPI networks. Hence, targeting intracellular PPIs can be considered as a crucial drug target for cancer therapy. Among the enormous and diverse group of cancer-enabling PPIs, the Hsp90-Cdc37 is prominent for cancer therapeutic development. The successful inhibition of Hsp90-Cdc37 PPI interface can be an important therapeutic option for cancer management. In the current study, a set of more than sixty thousand compounds belong to four databases were screened through a multi-steps molecular docking study in Glide against the Hsp90-Cdc37 interaction interface. The Glide-score and Prime-MM-GBSA based binding free energy of DCZ3112, standard Hsp90-Cdc37 inhibitor were found to be -6.96 and -40.46 kcal/mol, respectively. The above two parameters were used as cut-off score to reduce the chemical space from all successfully docked molecules. Furthermore, the in-silico pharmacokinetics parameters, common-feature pharmacophore analyses and the molecular binding interactions were used to wipe out the inactive molecules. Finally, four molecules were found to be important to modulate the Hsp90-Cdc37 interface. The potentiality of the final four molecules was checked through several drug-likeness characteristics. The molecular dynamics (MD) simulation study explained that all four molecules retained inside the interface of Hsp90-Cdc37. The binding free energy of each molecule obtained from the MD simulation trajectory was clearly explained the strong affection towards the Hsp90-Cdc37. Hence, the proposed molecule might be crucial for successful inhibition of the Hsp90-Cdc37 interface.Communicated by Ramaswamy H. Sarma.

Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity.

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein-protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.

Synthesis and Biological Evaluation of Celastrol Derivatives with Improved Cytotoxic Selectivity and Antitumor Activities.

Cdc37 associates kinase clients to Hsp90 and promotes the development of cancers. Celastrol, a natural friedelane triterpenoid, can disrupt the Hsp90-Cdc37 interaction to provide antitumor effects. In this study, 31 new celastrol derivatives, 2a-2d, 3a-3g, and 4a-4t, were designed and synthesized, and their Hsp90-Cdc37 disruption activities and antiproliferative activities against cancer cells were evaluated. Among these compounds, 4f, with the highest tumor cell selectivity (15.4-fold), potent Hsp90-Cdc37 disruption activity (IC50 = 1.9 µM), and antiproliferative activity against MDA-MB-231 cells (IC50 = 0.2 µM), was selected as the lead compound. Further studies demonstrated 4f has strong antitumor activities both in vitro and in vivo through disrupting the Hsp90-Cdc37 interaction and inhibiting angiogenesis. In addition, 4f exhibited less toxicity than celastrol and showed a good pharmacokinetics profile in vivo. These findings suggest that 4f may be a promising candidate for development of new cancer therapies.

Discovery of novel celastrol-triazole derivatives with Hsp90-Cdc37 disruption to induce tumor cell apoptosis.

To enhance the disruption of Hsp90-Cdc37, we designed and synthesized a series (27) of CEL-triazole derivatives. Most of the target compounds showed enhanced anti-proliferative activity on four cancer cell lines (MDA-MB-231, MCF-7, HepG2 and A459). Among them, compound 6 showed the best anti-proliferation (IC50 = 0.34 ± 0.01 µM) on MDA-MB-231. Pharmacological studies had found that compound 6 showed a higher ability to disrupt Hsp90-Cdc37 interaction in cells and inhibited the expression of the key Hsp90-Cdc37 clients in a concentration-dependent manner. Further studies indicated that an enhanced covalent binding between compound 6 and thiols (cysteine) might be one of the reasons for the increased activity. Furthermore, compound 6 arrested cells in the G0/G1 phase and induced tumor cell apoptosis significantly. Overall, for cancer treatment, compound 6 was worth further exploring.

The CDC37-HSP90 chaperone complex co-translationally degrades the nascent kinase-dead mutant of HIPK2.

Homeodomain-interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto-phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin-dependent degradation. By comparing the expression of wild-type and kinase-defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co-translational degradation of kinase-defective versus wild-type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37-HSP90 chaperone complex and points to a novel role of CDC37 in the co-translational degradation of a client protein.

Discovery and Optimization of Small Molecules Targeting the Protein-Protein Interaction of Heat Shock Protein 90 (Hsp90) and Cell Division Cycle 37 as Orally Active Inhibitors for the Treatment of Colorectal Cancer.

Cell division cycle 37 (Cdc37) is known to work as a kinase-specific cochaperone, which selectively regulates the maturation of kinases through protein-protein interaction (PPI) with Hsp90. Directly disrupting the Hsp90-Cdc37 PPI is emerging as an alternative strategy to develop anticancer agents through a specific inhibition manner of kinase clients of Hsp90. Based on a first specific small-molecule inhibitor targeting Hsp90-Cdc37 PPI (DDO-5936), which was previously reported by our group, we conducted a preliminary investigation of the structure-activity relationships and pharmacodynamic evaluations to improve the potency and drug-like properties. Here, our efforts resulted in the currently best inhibitor 18h with improved binding affinity (Kd = 0.5 µM) and cellular inhibitory activity (IC50 = 1.73 µM). Both in vitro and in vivo assays revealed that 18h could efficiently block the Hsp90-Cdc37 interaction to specifically inhibit kinase clients of Hsp90. Furthermore, 18h showed ideal physiochemical properties with favorable stability, leading to an oral efficacy in vivo.

Design of Disruptors of the Hsp90-Cdc37 Interface.

The molecular chaperone Hsp90 is a ubiquitous ATPase-directed protein responsible for the activation and structural stabilization of a large clientele of proteins. As such, Hsp90 has emerged as a suitable candidate for the treatment of a diverse set of diseases, such as cancer and neurodegeneration. The inhibition of the chaperone through ATP-competitive inhibitors, however, was shown to lead to undesirable side effects. One strategy to alleviate this problem is the development of molecules that are able to disrupt specific protein-protein interactions, thus modulating the activity of Hsp90 only in the particular cellular pathway that needs to be targeted. Here, we exploit novel computational and theoretical approaches to design a set of peptides that are able to bind Hsp90 and compete for its interaction with the co-chaperone Cdc37, which is found to be responsible for the promotion of cancer cell proliferation. In spite of their capability to disrupt the Hsp90-Cdc37 interaction, no important cytotoxicity was observed in human cancer cells exposed to designed compounds. These findings imply the need for further optimization of the compounds, which may lead to new ways of interfering with the Hsp90 mechanisms that are important for tumour growth.

Small-molecule inhibitor targeting the Hsp90-Cdc37 protein-protein interaction in colorectal cancer.

Disrupting the interactions between Hsp90 and Cdc37 is emerging as an alternative and specific way to regulate the Hsp90 chaperone cycle in a manner not involving adenosine triphosphatase inhibition. Here, we identified DDO-5936 as a small-molecule inhibitor of the Hsp90-Cdc37 protein-protein interaction (PPI) in colorectal cancer. DDO-5936 disrupted the Hsp90-Cdc37 PPI both in vitro and in vivo via binding to a previously unknown site on Hsp90 involving Glu47, one of the binding determinants for the Hsp90-Cdc37 PPI, leading to selective down-regulation of Hsp90 kinase clients in HCT116 cells. In addition, inhibition of Hsp90-Cdc37 complex formation by DDO-5936 resulted in a remarkable cyclin-dependent kinase 4 decrease and consequent inhibition of cell proliferation through Cdc37-dependent cell cycle arrest. Together, our results demonstrated DDO-5936 as an identified specific small-molecule inhibitor of the Hsp90-Cdc37 PPI that could be used to comprehensively investigate alternative approaches targeting Hsp90 chaperone cycles for cancer therapy.

Discovery of 18ß-glycyrrhetinic acid conjugated aminobenzothiazole derivatives as Hsp90-Cdc37 interaction disruptors that inhibit cell migration and reverse drug resistance.

A series of 18ß-glycyrrhetinic acid (GA) conjugated aminobenzothiazole derivatives were designed, synthesized and evaluated for disruption activity of Hsp90-Cdc37 as well as the effects of in vitro cell migration. These compounds exhibited relatively good disruption activity against Hsp90-Cdc37 with IC50 values in low micromolar range. A docking study of the most active compound 11g revealed key interactions between 11g and Hsp90-Cdc37 complex in which the benzothiazole moiety and the amine chain group were important for improving activity. It is noteworthy that further antitumor activity screening revealed that some compounds exhibited better inhibitory activity than the commercial anticancer drug 5-FU and showed potent suppression activity against drug-resistant cancer cells. In particular, compound 11 g appeared to be the most potent compound against the A549 cell line, at least partly, by inhibition of the activity of Hsp90 and apoptosis induction. The treatment of A549 cells with compound 11g resulted in inhibition of in vitro cell migration through wound healing assay and S phase of cell cycle arrested. In addition, 11g-induced apoptosis was significantly facilitated in A549 cells. Thus, we conclude that GA aminobenzothiazole derivatives may be the potential Hsp90-Cdc37 disruptors with the ability to suppress cells migration and reversed drug-resistant.

The targeted inhibition of Hsp90 by a synthetic small molecule, DPide offers an effective treatment strategy against TNBCs.

Triple-negative breast cancers (TNBCs) are the most aggressive and metastatic subtype of breast cancers and exhibit poor clinical outcome due to the lack of drug target receptors such as estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (Her2). The limited effectiveness of therapeutic options and the poor prognosis of TNBC patients emphasize the urgent need for identifying new therapeutic agents. In this regard, heat shock protein 90 (Hsp90) has emerged as a promising therapeutic target for the treatment of TNBCs. Hsp90 is a molecular chaperone that regulates the folding, stability, and function of many oncogenic proteins. Hence, the inhibition of Hsp90 chaperone function leads to a simultaneous blockage of multiple signaling pathways in the proliferation and survival of cancers. In the present study, we performed the design, synthesis, and biological evaluation of Hsp90 inhibitors and found that a synthetic small molecule, DPide exerted a potent anticancer activity against TNBC cell line, MDA­MB­231 and non­small cell lung cancer (NSCLC) cell line, H1975 with GI50 values of 0.478 and 1.67 µM, respectively. Soft­agar colony formation assay also revealed that DPide suppressed the anchorage­independent growth of MDA­MB­231 cells. Western blot analysis indicated that the treatment of MDA­MB­231 cells with DPide induced the proteasomal degradation of EGFR, Her2, Met, Akt, c­Raf, and Cdk4 and the consequent cleavage of PARP, leading to apoptotic cell death. DPide also inhibited the migration and MMP9 activity of MDA­MB­231 cells, suggesting that the metastatic potential of TNBCs could be suppressed by DPide. Collectively, DPide offers an effective therapeutic option for the treatment TNBCs.

Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System.

Selective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions.

A chemical compound inhibiting the Aha1-Hsp90 chaperone complex.

The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90-co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1-Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90-Aha1-dependent client proteins in vivo We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90.

Structure-based virtual screening and optimization of modulators targeting Hsp90-Cdc37 interaction.

Identification of novel Hsp90 inhibitors to disrupt Hsp90-Cdc37 protein-protein interaction (PPI) could be an alternative strategy to achieve Hsp90 inhibition. In this paper, a series of small molecules targeting Hsp90-Cdc37 complex are addressed and characterized. The molecules' key characters are determined by utilizing a structure-based virtual screening workflow, derivatives synthesis, and biological evaluation. Structural optimization and structure-activity relationship (SAR) analysis were then carried out on the virtual hit of VS-8 with potent activity, which resulted in the discovery of compound 10 as a more potent regulator of Hsp90-Cdc37 interaction with a promising inhibitory effect (IC50 = 27 µM), a moderate binding capacity (KD = 40 µM) and a preferable antiproliferative activity against several cancer lines including MCF-7, SKBR3 and A549 cell lines (IC50 = 26 µM, 15 µM and 38 µM respectively). All the data suggest that compound 10 exhibits moderate inhibitory effect on Hsp90-Cdc37 and could be regard as a first evidence of a non-natural compound targeting Hsp90-Cdc37 PPI.

Computational Discovery of Niclosamide Ethanolamine, a Repurposed Drug Candidate That Reduces Growth of Hepatocellular Carcinoma Cells In Vitro and in Mice by Inhibiting Cell Division Cycle 37 Signaling.

BACKGROUND & AIMS: Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC). METHODS: We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated ß-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored. RESULTS: Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-ß-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90. CONCLUSIONS: In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC.

Optimization and biological evaluation of celastrol derivatives as Hsp90-Cdc37 interaction disruptors with improved druglike properties.

Heat shock protein 90 (Hsp90) as a molecular target for oncology therapeutics has attracted much attention in the last decade. The Hsp90 multichaperone complex has important roles in the growth and/or survival of cancer cells. Cdc37, as a cochaperone, associates kinase clients to Hsp90 and promotes the development of malignant tumors. Disrupting the Hsp90-Cdc37 interaction provides an alternative strategy to inhibit the function of Hsp90 for cancer therapy. Celastrol, as a natural product, can disrupt the Hsp90-Cdc37 interaction and induce degradation of kinase clients. The study conducted here attempted to elucidate the structure-activity relationship of celastrol derivatives as Hsp90-Cdc37 disruptors and to improve the druglike properties. 23 celastrol derivatives were designed, synthesized, and the biological activities and physicochemical properties were determined. The derivative CEL20 showed improved Hsp90-Cdc37 disruption activity, anti-proliferative activities as well as druglike properties. Additionally, CEL20 induced clients degradation, cell cycle arrest and apoptosis in Panc-1 cells. This study can provide reference for the discovery of novel Hsp90-Cdc37 disruptors.

FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation.

BACKGROUND: Heat shock protein 90 (Hsp90) is a promising therapeutic target and inhibition of Hsp90 will presumably result in suppression of multiple signaling pathways. FW-04-806, a bis-oxazolyl macrolide compound extracted from China-native Streptomyces FIM-04-806, was reported to be identical in structure to the polyketide Conglobatin. METHODS: We adopted the methods of chemproteomics, computational docking, immunoprecipitation, siRNA gene knock down, Quantitative Real-time PCR and xenograft models on the research of FW-04-806 antitumor mechanism, through the HER2-overexpressing breast cancer SKBR3 and HER2-underexpressing breast cancer MCF-7 cell line. RESULTS: We have verified the direct binding of FW-04-806 to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits Hsp90/cell division cycle protein 37 (Cdc37) chaperone/co-chaperone interactions, but does not affect ATP-binding capability of Hsp90, thereby leading to the degradation of multiple Hsp90 client proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibits cell proliferation, caused G2/M cell cycle arrest, induced apoptosis, and downregulated Hsp90 client proteins HER2, Akt, Raf-1 and their phosphorylated forms (p-HER2, p-Akt) in a dose and time-dependent manner. Importantly, FW-04-806 displays a better anti-tumor effect in HER2-overexpressed SKBR3 tumor xenograft model than in HER2-underexpressed MCF-7 model. The result is consistent with cell proliferation assay and in vitro apoptosis assay applied for SKBR-3 and MCF-7. Furthermore, FW-04-806 has a favorable toxicity profile. CONCLUSIONS: As a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminal of Hsp90 and inhibits Hsp90/Cdc37 interaction, resulting in the disassociation of Hsp90/Cdc37/client complexes and the degradation of Hsp90 client proteins. FW-04-806 displays promising antitumor activity against breast cancer cells both in vitro and in vivo, especially for HER2-overexpressed breast cancer cells.

Expression and purification of recombinant NRL-Hsp90α and Cdc37-CRL proteins for in vitro Hsp90/Cdc37 inhibitors screening.

Hsp90 has emerged as a promising target for cancer treatment. Hsp90 interacts with co-chaperone Cdc37 to mediate the conformational maturation of its kinase client proteins. Screening small molecule inhibitors targeting Hsp90/Cdc37 might be a promising strategy for further cancer therapeutic. In order to establish a recombinant protein system, the novel cloning and purification of full-length human Hsp90α and Cdc37 from BL21 (DE3) Escherichia coli is described here. In this work, we cloned and expressed recombinant NRL-Hsp90α and Cdc37-CRL that represent the full-length human Hsp90α and Cdc37 fused with the split Renilla luciferase (RL) protein fragments. We also expressed the full-length RL protein as a control for inhibitors screening. Moreover, we confirmed that the interaction proteins were able to complement split luciferase fragments and show the RL activity when substrate was added. In comparison, two mutations NRL-Hsp90α (Q133A) and Cdc37 (R167A)-CRL retained only 20% of the complemented RL activities. Six small molecule compounds were tested using this recombinant system. Very interestingly, Sulforaphane, Withaferin A, Celastrol and EGCG all decreased the complemented NRL-Hsp90α/Cdc37-CRL activities in the concentration-dependent manner. In addition, neither Sulforaphane nor Withaferin A showed non-specific inhibition on full length RL activity. However, Celastrol and EGCG showed different RL inhibition levels. The other two compounds LBH-589 and 17-AAG showed neither NRL-Hsp90α/Cdc37-CRL nor RL inhibition activities. These results indicate that purified NRL-Hsp90α and Cdc37-CRL appeared as pure, stable and active conformation, and can be used as an in vitro bioluminescence system for Hsp90/Cdc37 inhibitors screening.

Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach.

The innate immune system senses viral DNA that enters mammalian cells, or in aberrant situations self-DNA, and triggers type I interferon production. Here we present an integrative approach that combines quantitative proteomics, genomics and small molecule perturbations to identify genes involved in this pathway. We silenced 809 candidate genes, measured the response to dsDNA and connected resulting hits with the known signaling network. We identified ABCF1 as a critical protein that associates with dsDNA and the DNA-sensing components HMGB2 and IFI204. We also found that CDC37 regulates the stability of the signaling molecule TBK1 and that chemical inhibition of the CDC37-HSP90 interaction and several other pathway regulators potently modulates the innate immune response to DNA and retroviral infection.

Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90.

BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. RESULTS: In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. CONCLUSIONS: Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.