New Indole Diketopiperazine Alkaloids from Soft Coral-Associated Epiphytic Fungus Aspergillus versicolor CGF 9-1-2.

Two new indole diketopiperazine alkaloids (IDAs), (+)19-epi-sclerotiamide (1) and (-)19-epi-sclerotiamide (2), along with 13 known analogs (3-15), were isolated from a soft coral-associated epiphytic fungus Aspergillus versicolor CGF 9-1-2. The structures of two new compounds were established based on the combination of HR-ESI-MS, 1D and 2D NMR spectroscopy, optical rotation measurements and quantum chemical 13 C-NMR, the absolute configurations were determined by experimental and electronic circular dichroism (ECD) calculations. The results of molecular docking showed that all the compounds had a good binding with TDP1, TDP2, TOP1, TOP2, Ache, NLRP3, EGFR, EGFR L858R, EGFR T790M and EGFR T790/L858. Biological evaluation of compounds 3, 6, 8, 11 showed that 3 exerted a strong inhibitory effect on TDP2 with a rate of 81.72 %.

2,5-Diketopiperazines: A Review of Source, Synthesis, Bioactivity, Structure, and MS Fragmentation.

BACKGROUND: 2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, are the simplest peptide derivatives in nature that are formed by the condensation of two amino acids. They are an important category of bioactive substances with various structures. OBJECTIVE: This review focuses on the natural sources, synthetic processes, biological properties and MS fragmentation regularity of simple DKPs, in order to provide a reference for exploring future scientific and therapeutic potentials of these compounds. METHODS: Pertinent information was collected and organized from several electronic scientific databases (e.g., Web of Science, China Knowledge Resource Integrated, ScienceDirect, PubMed, Wanfang Data and Google Scholar), PhD and MS dissertations. There are 107 articles published from the early 20th century to 2021 that were reviewed in this work. RESULTS: DKPs have been obtained from a broad range of natural resources, including fungi, bacteria, plants, and animals, and have been synthesized by chemical and biological methods. DKPs have various pharmacological activities, including anticancer, antibacterial, antithrombotic, neuron protective, analgesic, and other activities. Mass spectrometry is the most common method for the structural analysis of DKPs. DKPs can be quickly screened and identified by MS according to the mass spectrum fragmentation pattern. CONCLUSION: As a category of relatively unexplored compounds, DKPs have been demonstrated to have various bioactivities, especially with antitumor and antibacterial activities. However, the existing research on DKPs is still in the early stage, and their application in drug development needs to be further studied.

Development of l-Dopa-containing diketopiperazines as blood-brain barrier shuttle.

In our overall goal to develop anti-Parkinson drugs, we designed novel diketopiperazines (DKP1-6) aiming to both reach the blood-brain barrier and counteract the oxidative stress related to Parkinson's Disease (PD). The anti-Parkinson properties of DKP 1-6 were evaluated using neurotoxin-treated PC12 cells, as in vitro model of PD, while their cytotoxicity and genotoxicity potentials were investigated in newborn rat cerebral cortex (RCC) and primary human whole blood (PHWB) cell cultures. The response against free radicals was evaluated by the total antioxidant capacity (TAC) assay. Comet assay was used to detect DNA damage while the content of 8-hydroxyl-2'-deoxyguanosine (8-OH-dG) was determined as a marker of oxidative DNA damage. PAMPA-BBB and Caco-2 assays were employed to evaluate the capability of DKP1-6 to cross the membranes. Stability studies were conducted in simulated gastric and intestinal fluids and human plasma. Results showed that DKP5-6 attenuate the MPP + -induced cell death on a nanomolar scale, but a remarkable effect was observed for DKP6 on Nrf2 activation that leads to the expression of genes involved in oxidative stress response thus increasing glutathione biosynthesis and ROS buffering. DKP5-6 resulted in no toxicity for RCC neurons and PHWB cells exposed to 10-500 nM concentrations during 24 h as determined by MTT and LDH assays and TAC levels were not altered in both cultured cell types. No significant difference in the induction of DNA damage was observed for DKP5-6. Both DKPs resulted stable in simulated gastric fluids (t1/2 > 22h). In simulated intestinal fluids, DKP5 underwent immediate hydrolysis while DKP6 showed a half-life higher than 3 h. In human plasma, DKP6 resulted quite stable. DKP6 displayed both high BBB and Caco-2 permeability confirming that the DKP scaffold represents a useful tool to improve the crossing of drugs through the biological membranes.

Secondary Metabolites from Marine-Derived Fungi and Actinobacteria as Potential Sources of Novel Colorectal Cancer Drugs.

Colorectal cancer is one of the most common cancers diagnosed in the world. Chemotheraphy is one of the most common methods used for the pharmacological treatment of this cancer patients. Nevertheless, the adverse effect of chemotherapy is not optimized for improving the quality of life of people who are older, who are the most vulnerable subpopulation. This review presents recent updates regarding secondary metabolites derived from marine fungi and actinobacteria as novel alternatives for cytotoxic agents against colorectal cancer cell lines HCT116, HT29, HCT15, RKO, Caco-2, and SW480. The observed marine-derived fungi were from the species Aspergillus sp., Penicillium sp., Neosartorya sp., Dichotomomyces sp., Paradendryphiella sp., and Westerdykella sp. Additionally, Streptomyces sp. and Nocardiopsis sp. are actinobacteria discussed in this study. Seventy one compounds reviewed in this study were grouped on the basis of their chemical structures. Indole alkaloids and diketopiperazines made up most compounds with higher potencies when compared with other groups. The potency of indole alkaloids and diketopiperazines was most probably due to halogen-based functional groups and sulfide groups, respectively.

Ko143 Reverses MDR in Glioblastoma via Deactivating P-Glycoprotein, Sensitizing a Resistant Phenotype to TMZ Treatment.

BACKGROUND/AIM: Over-expression of both P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) has been associated with multidrug-resistance in glioblastoma (GBM). Though previously studied broad-spectrum inhibitors of drug efflux pumps have failed to progress in clinical studies due to in vivo toxicity, research into clinically viable targeted inhibitors is needed. This study evaluated the effects of Ko143, a non-toxic analog of fumitremorgin C, on temozolomide (TMZ) efficacy in resistant glioblastoma stem cells. MATERIALS AND METHODS: We used ATP-Glo assay to determine cell viabilities and flow cytometry to perform cell cycle analysis. Comparative gene expression was analysed through RT-qPCR. RESULTS: TMZ IC50 decreased 41.07% (p<0.01) in the resistant phenotype when delivered in combination with Ko143. Additionally, the TMZ-resistant phenotype (GBM146) displayed 44-fold greater P-gp expression than the TMZ-sensitive phenotype (GBM9) (p<0.01), yet a 0.6-fold lower BCRP expression. Ko143 potentiates TMZ efficacy and likely inhibits P-glycoprotein more potently than previously indicated. CONCLUSION: Further development of non-toxic, targeted inhibitors of drug efflux pumps for use in combinatorial chemotherapy may improve glioblastoma patient prognosis.

Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging.

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

Virus-Mimicking Polymer Nanoparticles Targeting CD169+ Macrophages as Long-Acting Nanocarriers for Combination Antiretrovirals.

Monosialodihexosylganglioside (GM3)-presenting lipid-coated polymer nanoparticles (NPs) that recapitulate the sequestration of human immunodeficiency virus-1 (HIV-1) particles in CD169+ virus-containing compartments (VCCs) of macrophages were developed as carriers for delivery and sustained release of a combination of two antiretrovirals (ARVs), rilpivirine (RPV) and cabotegravir (CAB). RPV and CAB were co-loaded into GM3-presenting lipid-coated polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) NPs without loss in potency of the drugs. GM3-presenting PLA NPs demonstrated the most favorable release properties and achieved inhibition of HIV-1 infection of primary human macrophages for up to 35 days. Intracellular localization of GM3-presenting PLA NPs in VCCs correlated with retention of intracellular ARV concentrations and sustained inhibition of HIV-1 infection. This work elucidates the design criteria of lipid-coated polymer NPs to utilize CD169+ macrophages as cellular drug depots for eradicating the viral reservoir sites or to achieve long-acting prophylaxis against HIV-1 infection.

Synthesis and Antiviral Activities of Neoechinulin B and Its Derivatives.

We have previously reported that neoechinulin B (1a), a prenylated indole diketopiperazine alkaloid, shows antiviral activities against hepatitis C virus (HCV) via the inactivation of the liver X receptors (LXRs) and the resultant disruption of double-membrane vesicles. In this study, a two-step synthesis of the diketopiperazine scaffold of 1a was achieved by the base-induced coupling of 1,4-diacetyl-3-{[(tert-butyldimethylsilyl)oxy]methyl}piperazine-2,5-dione with aldehydes, followed by the treatment of the resultant coupling products with tetra-n-butylammonium fluoride. Compound 1a and its 16 derivatives 1b-q were prepared using this method. Furthermore, variecolorin H, a related alkaloid, was obtained by the acid treatment of 1a in MeOH. The antiviral evaluation of 1a and its derivatives revealed that 1a, 1c, 1d, 1h, 1j, 1l, and 1o exhibited both anti-HCV and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activities. The results of this study indicate that the exomethylene moiety on the diketopiperazine ring is important for the antiviral activities. The antiviral compounds can inhibit the production of HCV and SARS-CoV-2 by inactivating LXRs.

Expanding the known structure space for RNA binding: a test of 2,5-diketopiperazine.

As the importance of RNA as a therapeutic target has become increasingly recognized, the need for new chemotypes able to bind RNA has grown in significance. We hypothesized that diketopiperazines (DKPs), common substructures in natural products and protein-targeting therapeutic agents, could serve as effective scaffolds for targeting RNA. To confirm this hypothesis, we designed and synthesized two analogs, one incorporating a DKP and one not, of compounds previously demonstrated to bind an RNA critical to the life cycle of HIV-1 with high affinity and specificity. Prior to compound synthesis, calculations employing density functional methods and molecular mechanics conformational searches were used to confirm that the DKP could present functionality in a similar (albeit not identical) orientation to the non DKP-containing compound. We found that both the DKP-containing and parent compound had similar affinities to the target RNA as measured by surface plasmon resonance (SPR). Both compounds were found to have modest but equal anti-HIV activity. These results establish the feasibility of using DKPs to target RNA.

Diketopiperazine derivative from marine actinomycetes Nocardiopsis sp. SCA30 with antimicrobial activity against MRSA.

Actinobacteria isolated from marine sources are a potential source of novel natural products. In this study, we report isolation, biological activity and characterization of secondary metabolites from strain Nocardiopsis sp. SCA30, isolated from marine sediments of Havelock Islands, Andaman and Nicobar, India. The ethyl acetate extracts of the isolate on screening for biological activity demonstrated antibacterial potency and antiproliferative activity. The extracts showed anticancer activity in a panel of cell lines, including HCT 15, HT 29, MCF 7 and MDA-MB 468, at concentrations ranging from 62.5 to 1000 µg/ml. A dose-dependent reduction in cell viability was observed in all the tested cell lines. The extract at 15 µg/ml and 30 µg/ml inhibited growth of methicillin-resistant Staphylococcus aureus ATCC NR-46071 and NR-46171 with MIC's of 15.62 and 7.81 µg/ml, respectively. LC-MS and NMR studies revealed that the antibacterial and anticancer compound isolated from Nocardiopsis sp. SCA30 is 1-acetyl-4-4(hydroxyphenyl)piperazine.

Neuroprotective Effect of Cyclo-(L-Pro-L-Phe) Isolated from the Jellyfish-Derived Fungus Aspergillus flavus.

Peroxisome proliferator-activated receptor (PPAR) expression has been implicated in pathological states such as cancer, inflammation, diabetes, and neurodegeneration. We isolated natural PPAR agonists-eight 2,5-diketopiperazines-from the jellyfish-derived fungus Aspergillus flavus. Cyclo-(L-Pro-L-Phe) was the most potent PPAR-γ activator among the eight 2,5-DKPs identified. Cyclo-(L-Pro-L-Phe) activated PPAR-γ in Ac2F rat liver cells and SH-SY5Y human neuroblastoma cells. The neuroprotective effect of this partial PPAR-γ agonist was examined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase release, and the Hoechst 33342 staining assay in SH-SY5Y cells. Our findings revealed that cyclo-(L-Pro-L-Phe) reduced hydrogen peroxide-induced apoptosis as well as the generation of reactive oxygen species. Rhodamine 123 staining and western blotting revealed that cyclo-(L-Pro-L-Phe) prevented the loss of mitochondrial membrane potential and inhibited the activation of mitochondria-related apoptotic proteins, such as caspase 3 and poly (ADP-ribose) polymerase. Moreover, cyclo-(L-Pro-L-Phe) inhibited the activation and translocation of nuclear factor-kappa B. Thus, the partial PPAR-γ agonist cyclo-(L-Pro-L-Phe) demonstrated potential neuroprotective activity against oxidative stress-induced neurodegeneration in SH-SY5Y cells.

Molecular Networking-Guided Isolation of New Etzionin-Type Diketopiperazine Hydroxamates from the Persian Gulf Sponge Cliona celata.

The Persian Gulf is a unique and biologically diverse marine environment dominated by invertebrates. In continuation of our research interest in the chemistry and biological activity of marine sponges from the Persian Gulf, we selected the excavating sponge Cliona celata for detailed metabolome analyses, in vitro bioactivity screening, and chemical isolation studies. A UPLC-MS/MS (MS2) molecular-networking-based dereplication strategy allowed annotation and structural prediction of various diketopiperazines (DKPs) and etzionin-type diketopiperazine hydroxamates (DKPHs) in the crude sponge extract. The molecular-networking-guided isolation approach applied to the crude extract afforded the DKPH etzionin (1) and its two new derivatives, clioetzionin A (2) and clioetzionin B (3). Another new modified DKP (4) was identified by MS/MS analyses but could not be isolated in sufficient quantities to confirm its structure. The chemical characterization of the purified DKPHs 1-3 was performed by a combination of 1D and 2D NMR spectroscopy, HRMS, HRMS/MS, and [α]D analyses. Compounds 1 and 2 exhibited broad antibacterial, antifungal, and anticancer activities, with IC50 values ranging from 19.6 to 159.1 µM. This is the first study investigating the chemical constituents of a C. celata specimen from the Persian Gulf. It is also the first report of full spectroscopic data of etzionin based on extensive spectroscopic analyses.

Structure of the Human Cholesterol Transporter ABCG1.

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.

Bioactive Polyketide and Diketopiperazine Derivatives from the Mangrove-Sediment-Derived Fungus Aspergillus sp. SCSIO41407.

Ten polyketide derivatives (1-10), including a new natural product named (E)-2,4-dihydroxy-3-methyl-6-(2-oxopent-3-en-1-yl) benzaldehyde (1), and five known diketopiperazines (11-15), were isolated from the mangrove-sediment-derived fungus Aspergillus sp. SCSIO41407. The structures of 1-15 were determined via NMR and MS spectroscopic analysis. In a variety of bioactivity screening, 3 showed weak cytotoxicity against the A549 cell line, and 2 exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 3, 5, and 6 showed inhibition against acetylcholinesterase (AChE) with IC50 values of 23.9, 39.9, and 18.6 µM. Compounds 11, 12, and 14 exhibited obvious inhibitory activities of lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) with IC50 values of 19.2, 20.9, and 8.7 µM, and they also suppressed RANKL-induced osteoclast differentiation in bone marrow macrophages cells (BMMCs), with the concentration of 5 µM. In silico molecular docking with AChE and NF-κB p65 protein were also performed to understand the inhibitory activities, and 1, 11-14 showed obvious protein/ligand-binding effects to the NF-κB p65 protein.

Anionic diketopiperazine induces osteogenic differentiation and supports osteogenesis in a 3D cryogel microenvironment.

Bioactive molecules that enhance or induce osteogenic potential of bone precursor cells have shown vital roles in bone tissue engineering. Herein, we report the design and synthesis of a novel diketopiperazine (DT) that induces osteoblastic differentiation of pre-osteoblasts and bone-marrow-derived stem cells in vitro and enhances the osteogenic potential of cryogel matrix. Such functional diketopiperazines can serve as potential scaffolds for bone healing and regeneration.

Prodrug of ICRF-193 provides promising protective effects against chronic anthracycline cardiotoxicity in a rabbit model in vivo.

The anthracycline (ANT) anticancer drugs such as doxorubicin or daunorubicin (DAU) can cause serious myocardial injury and chronic cardiac dysfunction in cancer survivors. A bisdioxopiperazine agent dexrazoxane (DEX) has been developed as a cardioprotective drug to prevent these adverse events, but it is uncertain whether it is the best representative of the class. The present study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667 (meso-(butane-2,3-diylbis(2,6-dioxopiperazine-4,1-diyl))bis(methylene)-bis(2-aminoacetate) hydrochloride), a water-soluble prodrug of ICRF-193 (meso-4,4'-(butan-2,3-diyl)bis(piperazine-2,6-dione)), as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, intravenously, weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, intravenously) was administered before each DAU dose. The treatment with GK-667 was well tolerated and provided full protection against DAU-induced mortality and left ventricular (LV) dysfunction (determined by echocardiography and LV catheterization). Markers of cardiac damage/dysfunction revealed minor cardiac damage in the group co-treated with GK-667 in the lower dose, whereas almost full protection was achieved with the higher dose. This was associated with similar prevention of DAU-induced dysregulation of redox and calcium homeostasis proteins. GK-667 dose-dependently prevented tumor suppressor p53 (p53)-mediated DNA damage response in the LV myocardium not only in the chronic experiment but also after single DAU administration. These effects appear essential for cardioprotection, presumably because of the topoisomerase IIß (TOP2B) inhibition provided by its active metabolite ICRF-193. In addition, GK-667 administration did not alter the plasma pharmacokinetics of DAU and its main metabolite daunorubicinol (DAUol) in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.

Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP).

The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA's known impact on neurodevelopment.

The inhibition of ABCB1/MDR1 or ABCG2/BCRP enables doxorubicin to eliminate liver cancer stem cells.

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

ABCG2 Is Overexpressed on Red Blood Cells in Ph-Negative Myeloproliferative Neoplasms and Potentiates Ruxolitinib-Induced Apoptosis.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

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/EBPß at the same time, thereby induced a total increase in C/EBPß levels because of AML1-ETO negative regulation to C/EBPß 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/EBPß was induced. 17-AAG did not affect the differentiation of HL-60 cells due to decreased AML1-ETO and C/EBPß 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.