Hsp90 C-terminal Inhibitors Research Articles

The Investigation of Hsp90C-Terminal Inhibitors Containing Amide Bioisosteres.

Heat Shock Protein 90 (Hsp90) is responsible for the proper folding and maturation of ~400 client protein substrates, many of which are directly associated with the ten hallmarks of cancer. Hsp90 is a great target for cancer therapy including melanoma, since Hsp90 inhibition can disrupt multiple oncogenic pathways simultaneously. In this study, we report the synthesis and anti-proliferative activity manifested by a series of Hsp90 C-terminal inhibitors against mutant BRAF and wild-type BRAF melanoma cells. Furthermore, we explored structure-activity relationships (SAR) for the amide moiety of 6 (B1), a novel Hsp90C-terminal inhibitor via introduction of amide bioisosteres. Compound 6 displayed an IC50 of 1.01 μM, 0.782 μM, 0.607 μM and 1.413 μM against SKMel173, SKMel103, SKMel19 and A375 cells, respectively.

Synthesis of C3'-Foused Aryl/Penta-1,4-Dien-3-One/Amine Hybrids as HSP90C-Terminal Inhibitors.

24 C3'-focused hybrids of aryl/penta-1,4-dien-3-one/amine (APDA) were designed and synthesized. Of these hybrids, 2 n demonstrated improved antiproliferative effects on HER2-positive breast cancer cells (SKBr3 and BT474) and triple-negative breast cancer (TNBC) cells (MDA-MB-231 and MDA-MB-468) with IC50 values ranging from 7.45 to 10.75 μM, but less toxicity to normal breast cells MCF-10A than the first generation of hybrid 1. Additionally, 2 n retained its ability to inhibit HSP90C-terminus, leading to the degradation of HSP90 client proteins HER2, EGFR, pAKT, AKT, and CDK4, without inducing a heat-shock response. Notably, 2 n also demonstrated improved thermostability compared to 1 and maintained in vitro metabolic stability in simulated intestinal fluid. These findings will provide a scientific basis for developing HSP90C-terminal inhibitors in the future.

Exploration and optimisation of structure-activity relationships of new triazole-based C-terminal Hsp90 inhibitors towards in vivo anticancer potency

The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.

The HSP90 inhibitor HVH-2930 exhibits potent efficacy against trastuzumab-resistant HER2-positive breast cancer.

Rationale: Resistance to targeted therapies like trastuzumab remains a critical challenge for HER2-positive breast cancer patients. Despite the progress of several N-terminal HSP90 inhibitors in clinical trials, none have achieved approval for clinical use, primarily due to issues such as induction of the heat shock response (HSR), off-target effects, and unfavorable toxicity profiles. We sought to examine the effects of HVH-2930, a novel C-terminal HSP90 inhibitor, in overcoming trastuzumab resistance. Methods: The effect of HVH-2930 on trastuzumab-sensitive and -resistant cell lines in vitro was evaluated in terms of cell viability, expression of HSP90 client proteins, and impact on cancer stem cells. An in vivo model with trastuzumab-resistant JIMT-1 cells was used to examine the efficacy and toxicity of HVH-2930. Results: HVH-2930 was rationally designed to fit into the ATP-binding pocket interface cavity of the hHSP90 homodimer in the C-terminal domain of HSP90, stabilizing its open conformation and hindering ATP binding. HVH-2930 induces apoptosis without inducing the HSR but by specifically suppressing the HER2 signaling pathway. This occurs with the downregulation of HER2/p95HER2 and disruption of HER2 family member heterodimerization. Attenuation of cancer stem cell (CSC)-like properties was associated with the downregulation of stemness factors such as ALDH1, CD44, Nanog and Oct4. Furthermore, HVH-2930 administration inhibited angiogenesis and tumor growth in trastuzumab-resistant xenograft mice. A synergistic effect was observed when combining HVH-2930 and paclitaxel in JIMT-1 xenografts. Conclusion: Our findings highlight the potent efficacy of HVH-2930 in overcoming trastuzumab resistance in HER2-positive breast cancer. Further investigation is warranted to fully establish its therapeutic potential.

Development of Hsp90 C-terminal inhibitors with noviomimetics that manifest anti-proliferative activities.

Inhibition of the Hsp90 C-terminal domain offers a promising opportunity to treat numerous diseases/indications. Furthermore, the development of Hsp90 C-terminal inhibitors (CTIs) is advantageous over N-terminal inhibitors because it avoids the detriments associated with induction of the heat shock response (HSR). However, the lack of co-crystal structures of small molecules bound to the C-terminus have hindered their development. Therefore, structure-activity relationship (SAR) studies have been pursued to optimize such inhibitors. Noviose sugar surrogates, also known as noviomimetics have been prepared to investigate the size and nature of the C-terminal domain binding pocket. Herein, we report the synthesis and anti-proliferative activity manifested by this new series of Hsp90 C-terminal inhibitors.

Ligand-based pharmacophore modelling, structure optimisation, and biological evaluation for the identification of 2-heteroarylthio-N-arylacetamides as novel HSP90 C-terminal inhibitors

Targeting Heat shock protein 90 (HSP90) C-terminus is an important strategy to develop HSP90 inhibitors without inducing heat shock response. The development of C-terminal inhibitors, however, is hampered by a lack of understanding regarding the interaction between the HSP90 C-terminus and the present inhibitors. We collected seven classical and structurally diverse HSP90 C-terminal inhibitors and constructed a ligand-based pharmacophore model. The subsequent virtual screening and structural optimisation led to the identification of 2-heteroarylthio-N-arylacetamides as novel HSP90 C-terminal inhibitors. 9 and 27 exhibited strong antitumour activity in vitro by inhibiting proliferation and inducing apoptosis in multiple cancer cell lines. These compounds disrupted the interaction between HSP90 C-terminus and peptidylprolyl isomerase D, exerting a stronger inhibitory effect than novobiocin. 27 significantly induced the degradation of HSP90 clients without triggering heat shock response. In an in vivo study using 4T1 mice breast cancer models, 9 showed a potent antitumour effect without obvious toxicity.

HSP90 C-terminal domain inhibition promotes VDAC1 oligomerization via decreasing K274 mono-ubiquitination in Hepatocellular Carcinoma

Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.

Heat shock protein 90 C-terminal inhibitor PNSA promotes anticancer immunology of CD8+ T cells.

Penisuloxazin A (PNSA), a new compound from the fungus, is a novel C-terminal Hsp90 inhibitor reported by us before. It has been reported to possess antitumor activity and suppresses metastasis of breast cancer cells. However, the influence of PNSA on T cells is not fully understood. Here, we found that PNSA was much less toxic to lymphocytes than to tumor cells and it had no significant effect on populations of CD3+, CD4+ and CD8+ T lymphocytes. We discovered that PNSA directly enhanced the killing capacities of the CD8+ T and CD3+CD25- to CT26 cells, but not that of CD3+ cells due to the increase of Treg cells. What's more, PNSA pretreated tumor cells increase the sensitivity to CD8+ T cells mainly through the degradation of client protein of Hsp90 and declination of PD-L1 expression. Eventually, PNSA enhanced the killing ability of CD8+ and CD3+ T cells by simultaneously acting on lymphocytes and cancer cells. In vivo experiments, PNSA exhibited inhibition effects in the colon adenocarcinoma with increase of CD8 T cell infiltration in tumor tissues. All these results indicate that the novel Hsp90 C-terminal inhibitor-PNSA can promote lytic T cell immunological function to improve anticancer effect of PNSA, which provides a better foundation for anticancer drug development of PNSA in future.

A silicon-containing aryl/penta-1,4-dien-3-one/amine hybrid exhibits antiproliferative effects on breast cancer cells by targeting the HSP90 C-terminus without inducing heat-shock response.

A pharmacophore-hybridized strategy based on previously reported HSP90 C-terminal inhibitors was utilized to prepare 32 aryl/penta-1,4-dien-3-one/amine hybrids. Among them, a silicon-containing compound 1z exhibited remarkable broad-spectrum antiproliferative effects on various human breast cancer cell lines. Through fluorescence polarization and AlphaScreen-based assays, we demonstrated that 1z specifically inhibited the HSP90 C-terminus without affecting HSP90 N-terminus. Furthermore, 1z effectively inhibited the HSP90 C-terminus without inducing heat-shock response (HSR), leading to the degradation of its client proteins HER2, pAKT, AKT, and CDK4, causing G1 arrest of MCF-7 and SKBr3 cells, and ultimately contributing to apoptosis of these cells through caspase-3, caspase-8, and caspase-9 activation. Additionally, the penta-1,4-dien-3-one linker in the hybrid, a large bulky lipophilic substitution in the aryl fragment at the 3'-site, and the presence of N-methylpiperazine as the amine fragment were identified as crucial factors that significantly contributed to the observed antiproliferative activity through structure-activity relationship (SAR) analysis. Lastly, we found that 1z exhibited superior thermostability compared to vibsanin B derivatives and good in vitro metabolic stability in simulated intestinal fluid, representing one of the few reported silicon-containing HSP90 C-terminal inhibitors.

Strategy of Virtual Screening based Discovery of HSP90 C-terminal Inhibitors and Network Pharmacological Analysis.

HSP90 has been considered an important anticancer target for several decades, but traditional HSP90 N-terminal inhibitors often suffered from organ toxicity and/or drug resistance. The development of HSP90 C-terminal inhibitors represents a reliable alternative strategy. In view of rare examples of structure-based identification of HSP90 C-terminal inhibitors, we report a virtual screening based strategy for the discovery of HSP90 C-terminal inhibitors as anticancer agents from natural products. 13 chemical ingredients from licorice were identified as possible HSP90 inhibitors and 3 of them have been reported as anticancer agents. The binding modes towards HSP90 C-terminus were predicted by molecular docking and refined by molecular dynamics simulation. Further network pharmacological analysis predicted overall possible targets involved in the pathways in cancer and revealed that 8 molecules possibly interact with HSP90. A structure based virtual screening strategy was established for the discovery of HSP90 Cterminal inhibitors.

Synthesis and evaluation of 3′- and 4′-substituted cyclohexyl noviomimetics that modulate mitochondrial respiration

KU-32 (2) and KU-596 (3), are first and second generation cytoprotective novologues that are derivatives of novobiocin (1), a heat shock protein 90 (Hsp90) C-terminal inhibitor. Although 2 and 3 improve mitochondrial bioenergetics and have demonstrated considerable cytoprotective activity, they contain a synthetically demanding noviose sugar. This issue was initially addressed by creating noviomimetics, such as KU-1202 (4), which replaced the noviose sugar with ether-linked cyclohexyl derivatives that retained some cytoprotective potential due to their ability to increase mitochondrial bioenergetics. Based on structure–activity relationship (SAR) studies of KU-1202 (4), the current study investigated 3′- and 4′-substituted cyclohexyl scaffolds as noviomimetics and determined their efficacy at increasing mitochondrial bioenergetic as a marker for cytoprotective potential.

Abstract 1009: A novel C-terminal HSP90 inhibitor NCT-58 targets cancer stem-like properties and suppresses migratory ability in triple-negative breast cancer cells

Abstract Background: Although several N-terminal HSP90 inhibitors have been investigated in clinical trials, none have yet been approved for clinical use due to issues including induction of the heat shock response (HSR), off-target effects and toxicity. A major issue for N-terminal inhibitors is induction of the heat shock response (HSR) which triggers cell survival. In the present study, we investigated the effects of NCT-58, a rationally-designed novel HSP90 inhibitor that instead targets the C-terminal domain, and evaluated its capacity to induce apoptosis and target cancer stem-like properties in triple-negative breast cancer. Materials and Methods: The effect of NCT-58 on TNBC cell lines in vitro was evaluated with cell viability, apoptosis, heat shock response and cell migration. Cancer stem-like properties were examined by Aldefluor positivity, CD44+/CD24- stem-like population and mammosphere formation assays. Results: NCT-58 induces apoptosis in TNBC cells without triggering the heat shock response (HSR) due to its targeting of the C-terminal region. This is accompanied by potent and simultaneous degradation of AKT, MEK and STAT3. Importantly, NCT-58 kills not only the rapidly proliferating tumor cells and but also effectively eradicates the breast cancer stem-like population (BCSCs). The latter phenomena are accompanied by reductions in the activity of stem/progenitor marker ALDH1 and the CD44+/CD24- stem-like population and as well as impairment of mammosphere formation. Furthermore, NCT-58 markedly impairs cell migratory ability, coinciding with collapse of HSP90 client cytoskeletal proteins including vimentin and F-actin in TNBC cells in vitro. It is noteworthy that NCT-58 exhibits more cytotoxic to tumor cells but minimally cytotoxicity to non-malignant cells. Conclusion: These findings suggest that NCT-58 may represent an effective therapeutic approach for the simultaneous targeting of HSP90 and its client oncoproteins for the treatment of molecularly heterogeneous TNBC. Citation Format: Soeun Park, Jung Min Park, Minsu Park, Dongmi Ko, Seongjae Kim, Juyeon Seo, Kee Dal Nam, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. A novel C-terminal HSP90 inhibitor NCT-58 targets cancer stem-like properties and suppresses migratory ability in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1009.

A novel HSP90 inhibitor SL-145 suppresses metastatic triple-negative breast cancer without triggering the heat shock response

Despite recent advances, there remains a significant unmet need for the development of new targeted therapies for triple-negative breast cancer (TNBC). Although the heat shock protein HSP90 is a promising target, previous inhibitors have had issues during development including undesirable induction of the heat shock response (HSR) and off-target effects leading to toxicity. SL-145 is a novel, rationally-designed C-terminal HSP90 inhibitor that induces apoptosis in TNBC cells via the suppression of oncogenic AKT, MEK/ERK, and JAK2/STAT3 signaling and does not trigger the HSR, in contrast to other inhibitors. In an orthotopic allograft model incorporating breast cancer stem cell-enriched TNBC tumors, SL-145 potently suppressed tumor growth, angiogenesis, and metastases concomitant with dysregulation of the JAK2/STAT3 signaling pathway. Our findings highlight the potential of SL-145 in suppressing metastatic TNBC independent of the HSR.

Synthesis and Biological Activity of 3-(Heteroaryl)quinolin-2(1H)-ones Bis-Heterocycles as Potential Inhibitors of the Protein Folding Machinery Hsp90.

In the context of our SAR study concerning 6BrCaQ analogues as C-terminal Hsp90 inhibitors, we designed and synthesized a novel series of 3-(heteroaryl)quinolin-2(1H), of types 3, 4, and 5, as a novel class of analogues. A Pd-catalyzed Liebeskind–Srogl cross-coupling was developed as a convenient approach for easy access to complex purine architectures. This series of analogues showed a promising biological effect against MDA-MB231 and PC-3 cancer cell lines. This study led to the identification of the best compounds, 3b (IC50 = 28 µM) and 4e, which induce a significant decrease of CDK-1 client protein and stabilize the levels of Hsp90 and Hsp70 without triggering the HSR response.

The C-terminal HSP90 inhibitor NCT-58 kills trastuzumab-resistant breast cancer stem-like cells

N-terminal HSP90 inhibitors in development have had issues arising from heat shock response (HSR) induction and off-target effects. We sought to investigate the capacity of NCT-58, a rationally-synthesized C-terminal HSP90 inhibitor, to kill trastuzumab-resistant HER2-positive breast cancer stem-like cells. NCT-58 does not induce the HSR due to its targeting of the C-terminal region and elicits anti-tumor activity via the simultaneous downregulation of HER family members as well as inhibition of Akt phosphorylation. NCT-58 kills the rapidly proliferating bulk tumor cells as well as the breast cancer stem-like population, coinciding with significant reductions in stem/progenitor markers and pluripotent transcription factors. NCT-58 treatment suppressed growth and angiogenesis in a trastuzumab-resistant xenograft model, concomitant with downregulation of ICD-HER2 and HSF-1/HSP70/HSP90. These findings warrant further investigation of NCT-58 to address trastuzumab resistance in heterogeneous HER2-positive cancers.

Virtual screening based identification of miltefosine and octenidine as inhibitors of heat shock protein 90

The molecular chaperone HSP90 facilitates the maturation of newly synthesized and unfolded proteins. The client proteins of HSP90 are involved in many processes of cancer occurrence and development, and therefore, HSP90 is considered as a promising target for the development of anticancer drugs. In contrast to N-terminal inhibitor, C-terminal inhibitor does not induce the pro-survival heat shock response. In order to get novel HSP90 C-terminal inhibitors and more evidences that HSP90 inhibitors could be applied in the therapy of cancer, we conducted a virtual screening toward HSP90 C-terminus from FDA-approved drugs. In this study, miltefosine and octenidine were identified as new HSP90 inhibitors. Miltefosine and octenidine exhibited strong and broad-spectrum anticancer activity and inhibited the proliferation of cancer cell by promoting apoptosis. Western blotting analysis revealed that miltefosine and octenidine significantly down-regulated the expression levels of HSP90 client proteins including p-AKT, CDK6, and ERK, and did not induce overexpression of heat shock proteins including HSP70 and HSP90 in MCF-7 cells. These results were in accordance with the characteristics of HSP90 C-terminal inhibitor. In conclusion, miltefosine and octenidine could disrupt the molecular chaperone function of HSP90, and thus, their strong and broad-spectrum anticancer activity is at least in part attributed to the inhibition activity against HSP90.

A novel C-terminal Hsp90 inhibitor KU758 synergizes efficacy in combination with BRAF or MEK inhibitors and targets drug-resistant pathways in BRAF-mutant melanomas.

Melanoma remains the most aggressive and fatal form of skin cancer, despite several FDA-approved targeted chemotherapies and immunotherapies for use in advanced disease. Of the 100 350 new patients diagnosed with melanoma in 2020 in the US, more than half will develop metastatic disease leading to a 5-year survival rate <30%, with a majority of these developing drug-resistance within the first year of treatment. These statistics underscore the critical need in the field to develop more durable therapeutics as well as those that can overcome chemotherapy-induced drug resistance from currently approved agents. Fortunately, several of the drug-resistance pathways in melanoma, including the proteins in those pathways, rely in part on Hsp90 chaperone function. This presents a unique and novel opportunity to simultaneously target multiple proteins and drug-resistant pathways in this disease via molecular chaperone inhibition. Taken together, we hypothesize that our novel C-terminal Hsp90 inhibitor, KU758, in combination with the current standard of care targeted therapies (e.g. vemurafenib and cobimetinib) can both synergize melanoma treatment efficacy in BRAF-mutant tumors, as well as target and overcome several major resistance pathways in this disease. Using in vitro proliferation and protein-based Western Blot analyses, our novel inhibitor, KU758, potently inhibited melanoma cell proliferation (without induction of the heat shock response) in vitro and synergized with both BRAF and MEK inhibitors in inhibition of cell migration and protein expression from resistance pathways. Overall, our work provides early support for further translation of C-terminal Hsp90 inhibitor and mitogen-activated protein kinase pathway inhibitor combinations as a novel therapeutic strategy for BRAF-mutant melanomas.

HDN-1 induces cell differentiation toward apoptosis in promyelocytic leukemia cells depending on its selective effect on client proteins of Hsp90

Heat Shock Protein 90 (Hsp90) is frequently upregulated in many cancers, and its inhibition simultaneously blocks multiple signaling pathways, resulting in cell differentiation or apoptosis. However, the complexity of Hsp90 in differentiation and its relation with apoptosis have remained unsettled. In this study, we demonstrated that HDN-1, a C-terminal inhibitor of Hsp90, induced the differentiation of HL-60 cells toward apoptosis. HDN-1 induced the differentiation of cells containing mutant AML1-ETO into mature granulocytes, which was related to its selective effect on client proteins of Hsp90. HDN-1 destabilized AML1-ETO and preserved C/EBPβ at the same time, thereby induced a total increase in C/EBPβ levels because of AML1-ETO negative regulation to C/EBPβ expression. Neither HDN-1 nor 17-AAG (an N-terminal inhibitor of Hsp90) led to the differentiation of NB4 cells because mutant PML-RARα was not affected as a client protein of Hsp90; thus, no additional expression of C/EBPβ was induced. 17-AAG did not affect the differentiation of HL-60 cells due to decreased AML1-ETO and C/EBPβ levels. These results indicate that HDN-1 drives cell differentiation toward apoptosis depending on its selective influence on client proteins of Hsp90, establishing the relationship between differentiation and apoptosis and uncovering the mechanism of HDN-1 in promyelocytic leukemia cell differentiation. Moreover, HDN-1 is very promising for the development of anticancer agents with the induction of differentiation.

Using NMR to identify binding regions for N and C-terminal Hsp90 inhibitors using Hsp90 domains.

We present the first NMR study of the interaction between heat shock protein 90 (Hsp90) and amino (N)-terminal inhibitors 17-AAG, and AUY922, and carboxy (C)-terminal modulators SM253, and LB51. We show that the two ATP mimics, 17-AAG and AUY922, bind deeply within the ATP binding pocket of the N-terminal domain, consistent with the crystal structures. In contrast, SM253, a C-terminal Hsp90 modulator, binds to the linker region between the N and middle domains. We also show that C-terminal inhibitor LB51 binds to the C-terminus with a more significant spectroscopic change than previously reported using NMR binding studies of C-terminal inhibitors novobiocin and silybin. These data provide key insights into how the allosteric inhibitor SM253 controls the C-terminal co-chaperones and confirms the binding domain of LB51.

SUV39H1 is a New Client Protein of Hsp90 Degradated by Chaetocin as a Novel C-Terminal Inhibitor of Hsp90.

Hsp90 is often overexpressed with activated form in cancer cells, and many key cellular proteins are dependent upon the Hsp90 machinery (these proteins are called “client protein”). Nowadays, more client proteins and more inhibitors of Hsp90 are being discovered. Chaetocin has been identified as an inhibitor of histone methyl transferase SUV39H1. Herein, we find that Chaetocin is an inhibitor of Hsp90 which binds to the C-terminal of Hsp90α. Chaetocin inhibited a variety of Hsp90 client proteins including AMl1-ETO and BCL-ABL, the mutant fusion-protein in the K562 and HL-60 cells. SUV39H1 mediates epigenetic events in the pathophysiology of hematopoietic disorders. We found that inhibition of Hsp90 by Chaetocin and 17-AAG had ability to induce degradation of SUV39H1 through proteasome pathway. In addition, SUV39H1 interacted with Hsp90 through co-chaperone HOP. These results suggest that SUV39H1 belongs to a client protein of Hsp90. Moreover, Chaetocin was able to induce cell differentiation in the two cells in the concentration range of Hsp90 inhibition. Altogether, our results demonstrate that SUV39H1 is a new client protein of Hsp90 degradated by Chaetocin as a novel C-terminal inhibitor of Hsp90. The study establishes a new relationship of Chaetocin and SUV39H1, and paves an avenue for exploring a new strategy to target SUV39H1 by inhibition of Hsp90 in leukemia.