IIα Protein Research Articles

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

Molecular Docking and QSAR Study of 5-O-acylpinostrobin Derivatives as Topoisomerase IIα Inhibitors

Background: Cancer is one of the top causes of death worldwide. A wide range of illnesses known as cancer can start in almost any organ or tissue in the body when abnormal cells multiply uncontrollably. Cancer patients have higher levels of the Topo IIα protein in their cells, this protein has been proposed as a relevant target for anticancer treatment development. Objective: This study aims to predict the anticancer activity of pinostrobin and 5-O-acylpinostrobin derivatives against topoisomerase IIα by docking molecular and QSAR study. Methods: In silico analysis was performed using the structure of the topoisomerase IIα (PDB: 5GWK)) as templates. Molecular docking analysis was performed with AutoDock Vina. Result: All 5-O-acyl pinostrobin derivatives, showed lower ΔG values than the parent pinostrobin. The 5-O-acetyl pinostrobin compound showed the highest score, namely -9.14 kcal/mol. 5-O-acetyl pinostrobin is predicted as the most powerful inhibitor that can cause inhibition of topoisomerase IIα. Conclution: The results of the best QSAR equation obtained can be used as a reference for predicting the activity of the new pinostrobin derivatives to be synthesized by inserting the electronic (Etot) parameter values of the compounds into the equation.

Expanding the therapeutic arsenal against cancer: a computational investigation of hybrid xanthone derivatives as selective Topoisomerase 2α ATPase inhibitors

The DNA topoisomerase II (topo II) enzyme plays an important role in the replication, recombination, and repair of DNA. Despite their widespread applications in cancer therapy, new, selective, and potent topo II inhibitors with better pharmaceutical profiles are needed to handle drug resistance and severe adverse effects. In this respect, an array of 36 new anticancer compounds was designed based on a Xanthone core tethered to multifunctional Pyridine-amines and Imidazole scaffold via alkyl chain linkers. An integrated in silico approach was used to understand the structural basis and mechanism of inhibition of the hybrid xanthone derivatives. In this study, we established an initial virtual screening workflow based on pharmacophore mapping, docking, and cancer target association to validate the target selection process. Next, a simulation-based docking was conducted along with pharmacokinetic analysis to filter out the five best compounds (7, 10, 25, 27, and 30) having binding energies within the range of −60.45 to −40.97 kcal/mol. The screened compounds were further subjected to molecular dynamics simulation for 200 ns followed by MM-GBSA and ligand properties analysis to assess the stability and binding affinity to hTOP2α. The top-ranking hits 3,7-bis(3-(2-aminopyridin-3-ylhydroxy)propoxy)-1-hydroxy-9H-xanthen-9-one (ligand 7) and 3,8-bis(3-(2-aminopyridin-3-ylhydroxy)propoxy)-1-hydroxy-9H-xanthen-9-one (ligand 25) were found to have no toxicity, optimum pharmacokinetic and, DFT properties and stable intermolecular interactions with the active site of hTopo IIα protein. In conclusion, further in vitro and in vivo experimental validation of the identified lead molecules is warranted for the discovery of new human Topoisomerase 2 alpha inhibitors. Communicated by Ramaswamy H. Sarma

Topoisomerase IIα immunoexpression in feline mammary carcinomas: A correlation with Ki67 immunoexpression and the mitotic count

The aim of the study was to compare the immunohistochemical expression of topoisomerase IIα protein (Topo IIα) with Ki67 expression and mitotic count in feline mammary carcinomas (FMCs). Topo IIα is considered as a proliferation indicator as well as a molecular target of anthracycline chemotherapy. The studied material included 70 FMCs from female cats treated with mastectomy. Primary mouse monoclonal antibodies directed against Topo IIα and Ki67 were used in immunohistochemical reactions. The number of mitotic figures was counted at 400× magnification in a field of 2.37 mm2. Immunohistochemical reaction for Topo IIα occurred in cell nuclei. The Topo IIα index ranged from 6.12% to 54.60% and was positively correlated with the values of the Ki67 index (r = 0.7193) and the mitotic count (r = 0. 2858). This indicates the potential possibility of use of the immunohistochemical expression of Topo IIα to assess the rate of proliferation in FMCs. The wide range of expression of Topo IIα in individual tumorus found in the conducted studies allows us to hypothesize that its assessment could be used as a predictive marker in chemotherapy of FMCs with the use of anthracyclines. However, this requires confirmation in clinical trials.

Novel 2-amino-1,4-naphthoquinone hybrids: Design, synthesis, cytotoxicity evaluation and in silico studies

In the present work, a novel series of 2-amino-1,4-naphthoquinones bearing oxyphenyl moiety (5a-5m) were designed and synthesized via a two-step route and evaluated for their in vitro cytotoxic activity against three different cancer cell lines (MCF-7, HL-60 and U937) and normal human cell line (HEK-293) by MTT assay. Compounds 5b (4-nitro-benzyl-) and 5k (4-bromo-benzyl-) were identified to possess the highest cytotoxic activity against MCF-7 cancerous cells (IC50 values of 27.76 and 27.86 μM, respectively). At the same time, none of the compounds exert significant toxicity against HEK-293 normal human kidney cells. Cell cycle analysis showed that the selected derivatives increased the population of MCF-7 cells in the S phase at 25 and 50 μM concentrations. Annexin V-FITC/PI staining assay also confirmed that compounds 5b and 5k induced apoptosis in the cell death pathway. Molecular docking and molecular dynamics studies were also performed to evaluate the probable interactions between the hybrids and human ATP binding domain of topo IIα protein. Our findings may provide new insight for further development of novel naphthoquinone-containing compounds.

Inhibition of topoisomerase IIα and induction of DNA damage in cholangiocarcinoma cells by altholactone and its halogenated benzoate derivatives

Topoisomerase IIα enzyme (Topo IIα) plays a critical function in DNA replication process and is considered to be a promising target of anti-cancer drugs. In the present study, we reported that the altholactone derivatives modified by adding a halogenated benzoate group showed greater inhibitory activity on Topo IIα enzyme in cell-free system concomitant with cytotoxicity against the CCA cell lines (KKU-M055 and KKU-M213) than those of the parent altholactone. However, the cytotoxic activities of four halogenated benzoate altholactone derivatives including iodo-, fluoro-, chloro-, and bromobenzoate derivatives (compound 1, 2, 3, and 4, respectively) were of equal potency. The fluorobenzoate derivative (compound 2) was chosen for investigating the underlying mechanism in CCA cells. Compound 2 arrested CCA cell cycle at sub G1 phase and induced apoptotic cell death. It markedly inhibited Topo IIα protein expression in both KKU-M055 and KKU-M213 cells, which was accompanied by DNA double-strand breaks demonstrated by an increase in phosphorylated H2A.X protein. Interestingly, KKU-M055 cells, which express higher Topo IIα mRNA compared to KKU-M213 cells, showed greater sensitivity to the compound, indicating the selectivity of the compound to Topo IIα enzyme. By computational docking analysis, the binding affinity of altholactone (-52.5 kcal/mol) and compound 2 (-56.7 kcal/mol) were similar to that of the Topo II poison salvicine (-53.7 kcal/mol). The aromatic moiety of both altholactones embedded in a hydrophobic pocket of Topo II ATPase domain. In addition, compound 2 induced the formation of linear DNA in Topo II-mediated cleavage assay. Collectively, our results demonstrate that the addition of fluorobenzoyl group to altholactone enhances potency and selectivity to inhibit type IIα topoisomerases. Atholactone and fluorobenzoate derivative act as Topo II cleavage complexes stabilizing compounds or Topo II poisons preferentially through binding at ATPase domain of Topo IIα, leading to DNA double-strand breaks and apoptosis induction. Such activity of 3-fluorobenzoate derivative of altholactone should be further explored for the development of an anti-cancer drug for CCA.

Hsa-miR-9-3p and hsa-miR-9-5p as Post-Transcriptional Modulators of DNA Topoisomerase IIα in Human Leukemia K562 Cells with Acquired Resistance to Etoposide.

DNA topoisomerase IIα protein (TOP2α) 170 kDa (TOP2α/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2α/170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2α/170. miRNA-sequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3'-untranslated region (UTR) of TOP2α/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2α/170 3'-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2α/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2α/170 protein levels without a change in TOP2α/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNA-inhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2α/170) led to increased TOP2α/170 protein expression without a change in TOP2α/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase IIα protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.

The influence of propofol anesthesia exposure on nonaversive memory retrieval and expression of molecules involved in memory process in the dorsal hippocampus in peripubertal rats.

The effects of anesthetic drugs on postoperative cognitive function in children are not well defined and have not been experimentally addressed. The present study aimed to examine the influence of propofol anesthesia exposure on nonaversive hippocampus-dependent learning and biochemical changes involved in memory process in the dorsal hippocampus, in peripubertal rats as the rodent model of periadolescence. The intersession spatial habituation and the novel object recognition tasks were used to assess spatial and nonspatial, nonaversive hippocampus-dependent learning. The exposure to anesthesia was performed after comparably long acquisition phases in both tasks. Behavioral testing lasted for 2 consecutive days (24-hour retention period). Changes in the expression of molecules involved in memory retrieval/reconsolidation were examined in the dorsal hippocampus by Western blot and immunohistochemistry, at the time of behavioral testing. Exposure to propofol anesthesia resulted in inappropriate assessment of spatial novelty at the beginning of the test session and affected continuation of acquisition in the spatial habituation test. The treatment did not affect recognition of the novel object at the beginning of the test session but it attenuated overall preference to novelty, reflecting retrieval of a weak memory. The expression of phosphorylated extracellular signal-regulated kinase 2 (involved in memory retrieval) was decreased while the level of phosphorylated Ca2+ /calmodulin-dependent protein kinase IIα and early growth response protein 1 (involved in memory reconsolidation) was increased in the dorsal hippocampus. The level of Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog B (neuronal activity indicator) was increased in the dorsal dentate gyrus. Enhanced exploratory activity was still evident in the propofol anesthesia exposure (PAE) group 48hour after the treatment in both tasks. In peripubertal rats, propofol anesthesia exposure affects memory retrieval and acquisition of new learning in the spatial and nonspatial, nonaversive learning tasks 24hour after the treatment, along with the expression of molecules that participate in memory retrieval/reconsolidation in the dorsal hippocampus. These results may have clinical implications, favoring control of basic cognitive functions in older children after the propofol exposure.

Antinociception of the spirocyclopiperazinium salt compound LXM-15 via activating α7 nAChR and M4 mAChR and inhibiting CaMKIIα/cAMP/CREB/CGRP signalling pathway in mice

The aim of this study was to investigate the analgesic effect of the spirocyclopiperazinium salt compound LXM-15 by intragastric administration in thermal and chemical pain models and further to elucidate the possible molecular mechanisms. The results showed that LXM-15 exerted significant antinociception in hot-plate test, formalin test and acetic acid writhing test. Western blot analysis showed that LXM-15 significantly reduced the upregulation of phosphorylation of calcium/calmodulin -dependent protein kinase IIα (CaMKIIα) and cAMP response element-binding protein (CREB), and further decreased the elevation of calcitonin gene related peptide (CGRP) in the dorsal root ganglion (DRG) and spinal cord in mice. ELISA analysis showed the level of cAMP in the spinal cord was decreased by LXM-15. All effects of LXM-15 could be blocked by methyllycaconitine citrate (MLA, a selective α7 nicotinic receptor antagonist) or tropicamide (TRO, a selective M4 muscarinic receptor antagonist). This study first reported that intragastric administration of LXM-15 produced significant analgesic effect, which may be related to the activation of α7 nicotinic acetylcholine receptor and M4 muscarine acetylcholine receptor, and thereby inhibiting CaMKIIα/cAMP/CREB/CGRP signalling pathway.

Smurf2-Mediated Stabilization of DNA Topoisomerase IIα Controls Genomic Integrity

DNA topoisomerase IIα (Topo IIα) ensures genomic integrity and unaltered chromosome inheritance and serves as a major target of several anticancer drugs. Topo IIα function is well understood, but how its expression is regulated remains unclear. Here, we identify the E3 ubiquitin ligase Smurf2 as a physiologic regulator of Topo IIα levels. Smurf2 physically interacted with Topo IIα and modified its ubiquitination status to protect Topo IIα from the proteasomal degradation in dose- and catalytically dependent manners. Smurf2-depleted cells exhibited a reduced ability to resolve DNA catenanes and pathological chromatin bridges formed during mitosis, a trait of Topo IIα-deficient cells and a hallmark of chromosome instability. Introducing Topo IIα into Smurf2-depleted cells rescued this phenomenon. Smurf2 was a determinant of Topo IIα protein levels in normal and cancer cells and tissues, and its levels affected cell sensitivity to the Topo II-targeting drug etoposide. Our results identified Smurf2 as an essential regulator of Topo IIα, providing novel insights into its control and into the suggested tumor-suppressor functions of Smurf2. Cancer Res; 77(16); 4217-27. ©2017 AACR.

Abstract P5-16-04: A randomized phase II neoadjuvant study comparing docetaxel and cyclophosphamide (TC) with 5-fluorouracil, epirubicin, and cyclophosphamide followed by docetaxel (FEC-D) for hormone receptor-negative breast cancer: The Kanagawa breast oncology group (KBOG) 1101 study

Abstract Purpose: This study aimed to evaluate response to neoadjuvant chemotherapy (NAC) for patients with hormone receptor-negative (HR-negative) breast cancer (BC) to identify subtypes that require anthracycline treatment. Methods: In total, 103 patients with operable HR-negative BC were registered. They were randomely assigned to administration of 6 cycles of docetaxel (75mg/m2) and cyclophosphamide (600 mg/m2) (TC6) or 3 cycles of 5-fluorouracil (500 mg/m2), epirubicin (100mg/m2), and cyclophosphamide (500mg/m2) followed by 3 cycles of docetaxel (100mg/m2) (FEC-D). Cytokeratin (CK) 5/6 and EGFR expression were used to identify basal and non-basal triple-negative (TN) BC. The primary endpoint was pathological complete response (pCR); secondary endpoints were safety, breast-conserving surgery, disease-free survival, and overall survival. Predictive factors of pCR for each regimen were also evaluated. Results: The pCR rate was 36% for FEC-D and 25.5% for TC6, which did not differ significantly (P=0.265). When TN BC was subdivided into basal and non-basal subtypes, the pCR rate in the basal subtype was significantly lower for TC6 (13.6%) than for FEC-D (42.9%) (P=0.033), but did not significantly differ in the non-basal (TC6, 36.4%; FEC-D, 25.0%) and HER2-positive (TC6, 41.7%; FEC-D, 35.7%) cases. The relative dose intensities of epirubicin and docetaxel in FEC-D and docetaxel in TC6 were 96.3±13.0%, 93.5±14.6%, and 93.9±16.3% (mean±SD), respectively. Occurrence of grade ≥2 adverse events was significant in FEC-D-treated patients. Poor appetite (P<0.001), nausea (P<0.001), vomiting (P<0.001), dysgeusia (P=0.03), and fatigue (P=0.05) were significantly more common for FEC-D than TC6. Patients treated with FEC-D experienced significantly more febrile neutropenia and anemia (P=0.016 and 0.017, respectively). The rates of breast-conserving surgery were 68.0 and 72.3% for FEC-D and TC6, respectively (P=0.641). Patients achieved pCR had better DFS (log rank test, P = 0.287) and OS (log rank test, P = 0.069), though not significant. Patients treated with FEC-D had better DFS (log rank test, P = 0.107) and OS (log rank test, P = 0.159), though not significant. Among patients with TN BC, those treated with FEC-D had significantly better DFS (log rank test, P = 0.016) and OS (log rank test, P = 0.034) than treated with TC6. Low ALDH1 expression and high topo IIα protein expression were strongly correlated with pCR in FEC-D, with odds ratios (ORs) of 4.33 [95% CI, 1.02–18.38] and 4.08 [0.97–17.2], respectively. ALDH1 was also associated with pCR in TC, OR=3.50 [0.84–14.6]. Other factors, including age, tumor size, nodal status, tumor grade, Ki67, p53, and TOP 2A status were not associated with pCR in either regimen. Conclusions:We found that TC6 was less effective than FEC-D for treating HR-negative BC because it was insufficient for TNBC, particularly for basal BC. This suggests that anthracycline is more important than taxane for basal BC. Additionally, ALDH1 could be a marker for resistance to conventional chemotherapy. Citation Format: Narui K, Ishikawa T, Shimizu D, Tanabe M, Sasaki T, Oba MS, Morita S, Nawata S, Kida K, Mogaki M, Doi T, Tsugawa K, Ogata H, Ota T, Kosaka Y, Sengoku N, Kuranami M, Saito Y, Suzuki Y, Suto A, Arioka H, Chishima T, Ichikawa Y, Endo I, Tokuda Y. A randomized phase II neoadjuvant study comparing docetaxel and cyclophosphamide (TC) with 5-fluorouracil, epirubicin, and cyclophosphamide followed by docetaxel (FEC-D) for hormone receptor-negative breast cancer: The Kanagawa breast oncology group (KBOG) 1101 study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-16-04.

XPO1 Inhibition using Selinexor Synergizes with Chemotherapy in Acute Myeloid Leukemia by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus.

Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy. Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC-dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142-52. ©2016 AACR.

Expressions of HER2 and Topo IIα in breast cancer and its clinical significance

To detect the expressions of human epidermal growth factor receptor 2 (HER2) and Topo IIα in breast cancer, and to analyze the clinical significance of neoadjuvant chemotherapy for the anthracycline-based drugs. Methods: The HER2 and Topo IIα gene and protein expressions in cancer tissues from 189 patients with breast cancer were detected by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). And the objective response rate (ORR) and pathological complete rate (pCR) were analyzed. Results: The HER2 protein expression in 46 patients (24.3%) and Topo IIα protein expression in 55 patients (29.1%) was 3+ by IHC or they were 49 (25.9%) and 94 (49.0%) by FISH, respectively. The ORR and pCR in HER2 negative or positive patients were 47.4% and 20.3% or 32.7% and 16.3%, respectively, with significant differences (All P<0.05). The ORR and pCR in Topo IIα positive or negative patients were 69.1% and 36.0% or 28.4% and 2.2%, respectively, with significant differences (All P<0.05). Conclusion: FISH and IHC were consistent in the determination of HER2 expression whereas they were inconsistent in the determination of Topo IIα expression. The amplification of Topo IIα can effectively improve the effect of the adjuvant treatment effect of the anthracyclines.

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

BackgroundSequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. MethodsDNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. ResultsIntroduction of the N1-alkyl amino group reduced selectivity for the target sequence 5′-TACGAT-3′ on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1–2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. ConclusionsPolyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. General significancePharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Design, synthesis, cytotoxicity, HuTopoIIα inhibitory activity and molecular docking studies of pyrazole derivatives as potential anticancer agents

In an attempt to find potential anticancer agents, a series of novel ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylates 5a-i and 5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamides 6a-i were designed, synthesized and evaluated for their topoisomerase IIα inhibitory activity and in vitro cytotoxicity against a panel of cancerous cell lines (MCF-7, NCI-H460, HeLa) and a normal cell line (HEK-293T). Molecular docking studies of all the synthesized compounds into the binding site of topoisomerase IIα protein (PDB ID: 1ZXM) were performed to gain a comprehensive understanding into plausible binding modes. These compounds were also screened for in silico drug-likeliness properties on the basis of the absorption, distribution, metabolism and excretion (ADME) prediction. Among all the synthesized compounds, analogue 5d showed superior cytotoxicity with an IC50 value of 7.01±0.60μM for HeLa, 8.55±0.35μM for NCI-H460 and 14.31±0.90 for MCF-7 cancer cell lines. Further, compound 5d showed 70.82% inhibition of topoisomerase IIα at a concentration of 100μM with maximum docking score of −8.24. Results of ADME prediction revealed that most of these compounds showed in silico drug-likeliness properties within the ideal range.

Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells

BackgroundEtoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions. MethodsHere, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin. ResultsTreatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines. ConclusionInhibition of topo II function by NO/NO-derived species induces significant resistance to etoposide, without affecting doxorubicin cytotoxicity in human tumor cells. General significanceAs tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

Effect of Gastrodia elata on learning and memory abilities and synaptic transmission protein expression of hippocampus in model rats with Alzheimer's disease

Objective To explore the effect of gastrodia elata on learning and memory abilities and synaptic transmission protein(P38, Ca2+ -CaMK Ⅱα, CREB)expression of hippocampus in model rats with Alzheimer's disease(AD). Methods 24 adult Wistar rats were randomly divided into control group, test group and intervention group. A dose of Aβ1-40 was injected into the hippocampus of rats on test group and intervention group, the control rats were injected with normal saline. When the models were successful, the rats of control group and test groups were given with sodium carboxymethyl cellulose(50g/kg), the rats of intervention group were given with gastrodia elata powder(50g/kg), lasting 15 days, Morris water maze test was used for learning and memory abilities study.The expression of P38, Ca2+ -CaMK Ⅱα and CREB protein were detected by immunohistochemistry method. Results Behavioral tests showed the mean escape latencies and search time of test group were obviously increased than those of control group and intervention group, the percentage of search distance on test groups was less than that of control group and intervention group(P<0.01). Immunohistochemistry results showed P38, Ca2+ -CaMK Ⅱα and CREB positive cells and optical density in hippocampus CA1 on test group were less than those of control group and intervention group(all P<0.01) (P38: 58.92±10.82, 0.208±0.037; Ca2+ -CaMK Ⅱα: 72.38±14.67, 0.174±0.036; CREB: 53.86±5.31, 0.161±0.043 in test group; P38: 87.32±9.56, 0.371±0.046; Ca2+ -CaMK Ⅱα: 98.16±16.29, 0.283±0.051; CREB: 86.76±7.73, 0.356±0.052 in intervention group; P38: 102.54±16.73, 0.563±0.078; Ca2+ -CaMK Ⅱα: 123.46±17.65, 0.436±0.057; CREB: 125.43±9.16, 0.524±0.057 in control group). Conclusion Gastrodia elata can treat AD by increasing expression of P38, Ca2+ -CaMK Ⅱα and CREB. Key words: Alzheimer‘s disease; Gastrodia elata; P38; Ca2+ -CaMK Ⅱα; CREB

Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d=25.2±4.6 and 0.47±0.08µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total=-7.6 and -9.2kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to wild type peptide. Thus, the two peptides are considered as promising lead entities to develop therapeutic molecular agents with high efficacy and specificity to target CaMKIIα-MUPP1 interaction. Other five designed peptides (SILPSV(-COOH), SGLPNV(-COOH), SIVMSV(-COOH), SIVPNV(-COOH) and SIAMNV(-COOH)) possessed comparable affinity with the wild type, and they may be further optimized to obtain higher potency.

Thapsigargin-induced activation of Ca2+-CaMKII-ERK in brainstem contributes to substance P release and induction of emesis in the least shrew

Cytoplasmic calcium (Ca2+) mobilization has been proposed to be an important factor in the induction of emesis. The selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, is known to deplete intracellular Ca2+ stores, which consequently evokes extracellular Ca2+ entry through cell membrane-associated channels, accompanied by a prominent rise in cytosolic Ca2+. A pro-drug form of thapsigargin is currently under clinical trial as a targeted cancer chemotherapeutic. We envisioned that the intracellular effects of thapsigargin could cause emesis and planned to investigate its mechanisms of emetic action. Indeed, thapsigargin did induce vomiting in the least shrew in a dose-dependent and bell-shaped manner, with maximal efficacy (100%) at 0.5 mg/kg (i.p.). Thapsigargin (0.5 mg/kg) also caused increases in c-Fos immunoreactivity in the brainstem emetic nuclei including the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX), as well as enhancement of substance P (SP) immunoreactivity in DMNX. In addition, thapsigargin (0.5 mg/kg, i.p.) led to vomit-associated and time-dependent increases in phosphorylation of Ca2+/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) in the brainstem. We then explored the suppressive potential of diverse chemicals against thapsigargin-evoked emesis including antagonists of: i) neurokinin-1 receptors (netupitant), ii) the type 3 serotonin receptors (palonosetron), iii) store-operated Ca2+ entry (YM-58483), iv) L-type Ca2+ channels (nifedipine), and v) SER Ca2+-release channels inositol trisphosphate (IP3Rs) (2-APB)-, and ryanodine (RyRs) (dantrolene)-receptors. In addition, the antiemetic potential of inhibitors of CaMKII (KN93) and ERK1/2 (PD98059) were investigated. All tested antagonists/blockers attenuated emetic parameters to varying degrees except palonosetron, however a combination of non-effective doses of netupitant and palonosetron exhibited additive antiemetic efficacy. A low-dose combination of nifedipine and 2-APB plus dantrolene mixture completely abolished thapsigargin-evoked vomiting, CaMKII-ERK1/2 activation and SP elevation. In addition, pretreatment with KN93 or PD98059 suppressed thapsigargin-induced increases in SP and ERK1/2 activation. Intracerebroventricular injection of netupitant suppressed vomiting caused by thapsigargin which suggests that the principal site of evoked emesis is the brainstem. In sum, this is the first study to demonstrate that thapsigargin causes vomiting via the activation of the Ca2+-CaMKII-ERK1/2 cascade, which is associated with an increase in the brainstem tissue content of SP, and the evoked emesis occurs through SP-induced activation of neurokinin-1 receptors.

Multiple domains in the C-terminus of NMDA receptor GluN2B subunit contribute to neuronal death following in vitro ischemia

Global cerebral ischemia induces selective degeneration of specific subsets of neurons throughout the brain, particularly in the hippocampus and cortex. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca2+ overload and ultimately neuronal demise. N-methyl-d-aspartate receptors (NMDARs) are considered to be largely responsible for excitotoxic injury due to their high Ca2+ permeability. In the hippocampus and cortex, these receptors are most prominently composed of combinations of two GluN1 subunits and two GluN2A and/or GluN2B subunits. Due to the controversy regarding the differential role of GluN2A and GluN2B subunits in excitotoxic cell death, we investigated the role of GluN2B in the activation of pro-death signaling following an in vitro model of global ischemia, oxygen and glucose deprivation (OGD). For this purpose, we used GluN2B−/− mouse cortical cultures and observed that OGD-induced damage was reduced in these neurons, and partially prevented in wild-type rat neurons by a selective GluN2B antagonist. Notably, we found a crucial role of the C-terminal domain of the GluN2B subunit in triggering excitotoxic signaling. Indeed, expression of YFP–GluN2B C-terminus mutants for the binding sites to post-synaptic density protein 95 (PSD95), Ca2+-calmodulin kinase IIα (CaMKIIα) or clathrin adaptor protein 2 (AP2) failed to mediate neuronal death in OGD conditions. We focused on the GluN2B–CaMKIIα interaction and found a determinant role of this interaction in OGD-induced death. Inhibition or knock-down of CaMKIIα exerted a neuroprotective effect against OGD-induced death, whereas overexpression of this kinase had a detrimental effect. Importantly, in comparison with neurons overexpressing wild-type CaMKIIα, neurons overexpressing a mutant form of the kinase (CaMKII-I205K), unable to interact with GluN2B, were partially protected against OGD-induced damage. Taken together, our results identify crucial determinants in the C-terminal domain of GluN2B subunits in promoting neuronal death in ischemic conditions. These mechanisms underlie the divergent roles of the GluN2A- and GluN2B–NMDARs in determining neuronal fate in cerebral ischemia.