PU-H71

PU-H71 is a potent and selective purine scaffold inhibitor of Hsp90 (Heat shock protein 90) [1].

Hsp90 is a molecular chaperone, it takes participate in a variety of cellular processes through promoting the folding, assembly and transport of its client proteins. Hsp90 is found to overexpress in many kinds of cancer cells. It serves as a protector of the oncoproteins which are also the client of Hsp90. Therefore, Hsp90 is thought to be an attractive target of the cancer therapy. The Hsp90 inhibitors found so far can be categorized into several classes according to the mechanisms. Among these, the inhibitors which bind to Hsp90 within its N-terminal ATP binding site are most explored. PU-H71 is one of this sort of inhibitors [2, 3 and 4].

As an inhibitor of Hsp90, PU-H71 is found to induce the degradation of Her2 through blocking the interaction of Hsp90 and Her2. In SKBr3 breast cancer cells, PU-H71 induced Her2 degradation with EC50 value of 50 nM. In MDA-MB-468 breast cancer cells, PU-H71 showed antimitotic efficacy with IC50 value of 70 nM. PU-H71 affected the growth of a serious of cancer cells with IC50 values of 50 nM, 58 nM and 100 to 300 nM in SKBr3, MDA-MB-468 and human myeloma cells (such as NCI-H929, U266 and MM-IR). Besides that, PU-H71 is found to cause cell cycle arrest through reducing the levels of CDK1 and Chk1 which are essential for the G2-M progression [1, 2 and 3].

PU-H71 administration significantly reduced the tumor size in mice injected with A673 tumor cells. In mice bearing MDA-MB-468 tumor xenografts, PU-H71 administration at dose of 75 mg/kg, 3 week remarkably inhibited tumor growth and proliferation by 96% and 60%, respectively. Moreover, PU-H71 is found to down-regulate some Hsp90-regulated malignancy driving proteins when treated in the animal, such as Raf-1, HER3, EGFR and PARP [5, 6].

References:
[1].  He H, Zatorska D, Kim J, et al. Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90. Journal of medicinal chemistry, 2006, 49(1): 381-390.
[2].  Usmani S Z, Bona R D, Chiosis G, et al. The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1. J Hematol Oncol, 2010, 3(1): 40.
[3].  Caldas-Lopes E, Cerchietti L, Ahn J H, et al. Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models. Proceedings of the National Academy of Sciences, 2009, 106(20): 8368-8373.
[4].  Liu M, Wang J, Wu X, et al. HPLC method development, validation and impurity characterization for an antitumor Hsp90 inhibitor—PU-H71 (NSC 750424). Journal of pharmaceutical and biomedical analysis, 2014, 89: 34-41.
[5].  Ambati S R, Lopes E C, Kosugi K, et al. Pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor, alone and in combination with bortezomib in Ewing sarcoma. Molecular oncology, 2014, 8(2): 323-336.
[6].  Gallerne C, Prola A, Lemaire C. Hsp90 inhibition by PU-H71 induces apoptosis through endoplasmic reticulum stress and mitochondrial pathway in cancer cells and overcomes the resistance conferred by Bcl-2. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2013, 1833(6): 1356-1366.

PU-H71, a novel Hsp90 inhibitor, as a potential cancer-specific sensitizer to carbon-ion beam therapy.[Pubmed:27242340]

J Radiat Res. 2016 Sep;57(5):572-575.

PU-H71, a heat shock protein 90 (Hsp90) inhibitor, has yielded therapeutic efficacy in many preclinical models and is currently in clinical trials. Carbon-ion radiotherapy (CIRT) has provided successful tumor control; however, there is still room for improvement, particularly in terms of tumor-specific radiosensitization. The Hsp90 inhibitor PU-H71 has been shown to sensitize tumor cells to X-ray radiation. A murine osteosarcoma cell line (LM8) and a normal human fibroblast cell line (AG01522) were treated with PU-H71 before X-ray, 14- or 50-keV/microm carbon-ion beam (C-ion) irradiation. Cell survival and protein expression were evaluated with colony formation and western blot, respectively. Treatment with PU-H71 alone was shown to be non-toxic to both cell lines; however, PU-H71 was shown to significantly sensitize LM8 cells to not only X-ray, but also to C-ion irradiation, while only a minimal sensitizing effect was observed in AG01522 cells. PU-H71 treatment was found to suppress the protein expression levels of Rad51 and Ku70, which are associated with the homologous recombination pathway and the non-homologous end-joining pathway of double-strand break repair. The findings reported here suggest that PU-H71 could be a promising radiosensitizer for CIRT.

HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment.[Pubmed:28114285]

Oncogene. 2017 Jun 15;36(24):3441-3449.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells in the hematopoietic system and lymphoid tissues. Although inhibitors targeting the B-cell receptor (BCR) pathway have been successful in the treatment of the disease, the underlying mechanisms leading to BCR over-activity in CLL are not fully understood. In this study, we found that HSP90, a highly conserved molecular chaperone, is overexpressed in CLL compared with resting B cells. HSP90 overexpression is accompanied by the overexpression of several BCR kinases including LYN, spleen tyrosine kinase, Bruton tyrosine kinase and AKT. Chemical and immune-precipitation demonstrated that these BCR constituents are present in a multi-client chaperone complex with HSP90. Inhibition of HSP90 with PU-H71 destabilized the BCR kinases and caused apoptosis of CLL cells through the mitochondrial apoptotic pathway. Further, PU-H71 induced apoptosis in the presence of stromal co-culture or cytoprotective survival signals. Finally, genetic knockdown of HSP90 and its client AKT, but not BTK, reduced CLL viability. Overall, our study suggests that the chaperone function of HSP90 contributes to the over-activity of the BCR signaling in CLL and inhibition of HSP90 has the potential to achieve a multi-targeting effect. Thus, HSP90 inhibition may be explored to prevent or overcome drug resistance to single targeting agents.

Radiosynthesis of the iodine-124 labeled Hsp90 inhibitor PU-H71.[Pubmed:26806023]

J Labelled Comp Radiopharm. 2016 Mar;59(3):129-32.

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone protein whose function is critical for maintaining several key proteins involved in survival and proliferation of cancer cells. PU-H71 (1), is a potent purine-scaffold based ATP pocket binding Hsp90 inhibitor which has been shown to have potent activity in a broad range of in vivo cancer models and is currently in Phase I clinical trials in patients with advanced solid malignancies, lymphomas, and myeloproliferative neoplasms. In this report, we describe the radiosynthesis of [(124)I]-PU-H71(5); this was synthesized from the corresponding Boc-protected stannane precursor 3 by iododestannylation with [(124)I]-NaI using chloramine-T as an oxidant for 2 min, followed by Boc deprotection with 6 N HCl at 50 degrees C for 30 min to yield the final compound. The final product 5 was purified using HPLC and was isolated with an overall yield of 55 +/- 6% (n = 6, isolated) from 3, and >98% purity and an average specific activity of 980 mCi/micromol. Our report sets the stage for the introduction of [(124)I]-PU-H71 as a potential non-invasive probe for understanding biodistribution and pharmacokinetics of PU-H71 in living subjects using positron emission tomography imaging.

The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.[Pubmed:27666928]

Radiother Oncol. 2016 Oct;121(1):162-168.

BACKGROUND AND PURPOSE: PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. MATERIALS AND METHODS: The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by gamma-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. RESULTS: PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of gamma-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. CONCLUSION: Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy.

Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models.[Pubmed:19416831]

Proc Natl Acad Sci U S A. 2009 May 19;106(20):8368-73.

Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G(2)-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-kappaB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.

Molecular imaging of the efficacy of heat shock protein 90 inhibitors in living subjects.[Pubmed:18172314]

Cancer Res. 2008 Jan 1;68(1):216-26.

Heat shock protein 90 alpha (Hsp90 alpha)/p23 and Hsp90 beta/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90 alpha/p23 and Hsp90 beta/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purine-scaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90 alpha/p23 and Hsp90 beta/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90 alpha/p23 and Hsp90 beta/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoform-selective Hsp90 inhibitors.

Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90.[Pubmed:16392823]

J Med Chem. 2006 Jan 12;49(1):381-90.

Hsp90 is a chaperone protein that allows cancer cells to tolerate the many components of dysregulated pathways. Its inactivation may result in targeting multiple molecular alterations and, thus, in reverting the transformed phenotype. The PU-class, a purine-scaffold Hsp90 inhibitor series, has been reported to be potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of this class was synthesized and evaluated as inhibitors of the chaperone. The structure-activity relationship and selectivity for tumor Hsp90 of compounds within the series is presented. The study identifies water soluble derivatives (>5 mM in PBS pH 7.4) of nanomolar potency (IC(50) approximately 50 nM) in cellular and animal models of cancer. Binding affinities of these compounds for Hsp90 correlate well with their biological activities. When administered in vivo to mice bearing MDA-MB-468 human breast cancer xenografted tumors, these agents result in pharmacologically relevant concentrations and, accordingly, in modulation of Hsp90-client proteins in tumors.