Discovery of a New STAT3 Inhibitor Acting on the Linker Domain.

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that contributes to tumor cell growth and survival and is often constitutively active in several types of cancers, which makes it an attractive target for cancer therapy. We identified 5,5'-(pentane-1,5'-diyl)bis(2-methyl-1,4-benzoquinone) (BPMB) as a new STAT3 inhibitor. BPMB inhibited the transcriptional activities of STAT3, despite its inability to reduce the phosphorylation and nuclear translocation of STAT3. BPMB selectively inhibited the proliferation of human breast cancer cell lines with constitutively activated STAT3. Furthermore, a gel retardation pattern was obtained by immunoblotting only when those STAT3-activated cell lines were treated with BPMB. The shifted bands could be immunoblotted with anti-STAT3 antibody but not with anti-STAT1/STAT5 antibody, and were stable under reducing conditions. The purified recombinant STAT3 protein treated with BPMB afforded a similar band shift pattern. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of the component comprising the main shifted band suggested that the complex is a STAT3 homodimer crosslinked by BPMB through a Michael addition with Cys550 in the linker domain. Alanine replacement at this position resulted in reduction of the STAT3 dimer formation in the gel retardation assay. Thus, our results suggest that BPMB inhibits the proliferation of STAT3-activated cell lines, presumably through acylation of the linker domain and subsequent induction of the inactive STAT3 complexes.

DLiP-PPI library: An integrated chemical database of small-to-medium-sized molecules targeting protein-protein interactions.

Protein-protein interactions (PPIs) are recognized as important targets in drug discovery. The characteristics of molecules that inhibit PPIs differ from those of small-molecule compounds. We developed a novel chemical library database system (DLiP) to design PPI inhibitors. A total of 32,647 PPI-related compounds are registered in the DLiP. It contains 15,214 newly synthesized compounds, with molecular weight ranging from 450 to 650, and 17,433 active and inactive compounds registered by extracting and integrating known compound data related to 105 PPI targets from public databases and published literature. Our analysis revealed that the compounds in this database contain unique chemical structures and have physicochemical properties suitable for binding to the protein-protein interface. In addition, advanced functions have been integrated with the web interface, which allows users to search for potential PPI inhibitor compounds based on types of protein-protein interfaces, filter results by drug-likeness indicators important for PPI targeting such as rule-of-4, and display known active and inactive compounds for each PPI target. The DLiP aids the search for new candidate molecules for PPI drug discovery and is available online (https://skb-insilico.com/dlip).

Virtual screening and further development of novel ALK inhibitors.

Anaplastic lymphoma kinase (ALK) has been in the spotlight in recent years as a promising new target for therapy of non-small-cell lung cancer (NSCLC). Since the identification of the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene in some NSCLC patients was reported in 2007, various research groups have been seeking ALK inhibitors. Above all, crizotinib (PF-02341066) has been under clinical trial, and its therapeutic efficacy of inhibiting ALK in NSCLC has been reported. Among anticancer drugs, drug resistance appears frequently necessitating various kinds of inhibitors. We identified novel ALK inhibitors by virtual screening from the public chemical library collected by the Chemical Biology Research Initiative (CBRI) at the University of Tokyo, and inhibitors that are more potent were developed.

Evaluation of docking calculations on X-ray structures using CONSENSUS-DOCK.

We are participating in the challenge of identifying active compounds for target proteins using structure-based virtual screening (SBVS). We use an in-house customized docking program, CONSENSUS-DOCK, which is a customized version of the DOCK4 program in which three scoring functions (DOCK4, FlexX and PMF) and consensus scoring have been implemented. This paper compares the docking calculation results obtained using CONSENSUS-DOCK and DOCK4, and demonstrates that CONSENSUS-DOCK produces better results than DOCK4 for major X-ray structures obtained from the Protein Data Bank (PDB).

[In-silico approaches for fragment-based drug design].

An important step to promote fragment-based drug design (FBDD) is to find high-quality fragment molecules. Therefore the design of the fragment library is the most crucial stage. In our fragment library, the main considerations are ligand efficiency (LE), diversity, and solubility with drug-like properties. We especially considered LE to raise hit probability in screening. We estimated LE of the fragment molecule based on known LE values of the active compounds. We also developed a docking program suitable for screening fragments rather than drug compounds. Furthermore, we explored fragment-linking program, which links together fragments that bind to adjacent sites on a target protein so as to promote FBDD in silico.

Peroxisome proliferator-activated receptor gamma plays a critical role in inhibition of cardiac hypertrophy in vitro and in vivo.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are transcription factors of the nuclear receptor superfamily. It has been reported that the thiazolidinediones, which are antidiabetic agents and high-affinity ligands for PPARgamma, regulate growth of vascular cells. In the present study, we examined the role of PPARgamma in angiotensin II (Ang II)-induced hypertrophy of neonatal rat cardiac myocytes and in pressure overload-induced cardiac hypertrophy of mice. METHODS AND RESULTS: Treatment of cultured cardiac myocytes with PPARgamma ligands such as troglitazone, pioglitazone, and rosiglitazone inhibited Ang II-induced upregulation of skeletal alpha-actin and atrial natriuretic peptide genes and an increase in cell surface area. Treatment of mice with a PPARgamma ligand, pioglitazone, inhibited pressure overload-induced increases in the heart weight-to-body weight ratio, wall thickness, and myocyte diameter in wild-type mice and an increase in the heart weight-to-body weight ratio in heterozygous PPARgamma-deficient mice. In contrast, pressure overload-induced increases in the heart weight-to-body weight ratio and wall thickness were more prominent in heterozygous PPARgamma-deficient mice than in wild-type mice. CONCLUSIONS: These results suggest that the PPARgamma-dependent pathway is critically involved in the inhibition of cardiac hypertrophy.

Long-term endothelin a receptor blockade inhibits electrical remodeling in cardiomyopathic hamsters.

BACKGROUND: The endothelin (ET) system is activated in failing hearts. Congestive heart failure frequently is associated with ventricular arrhythmias, which may result from electrical remodeling such as changes of ionic current density and heterogeneous action potential prolongation. We examined the effects of long-term ET(A) receptor blockade on the electrophysiological properties of ventricular cells, the surface ECG, and the survival in BIO 14.6 cardiomyopathic hamsters. METHODS AND RESULTS: Membrane currents and action potentials were recorded from left ventricular cells isolated from normal F1beta hamsters and cardiomyopathic BIO 14.6 hamsters untreated and chronically treated with TA-0201, an ET(A) receptor antagonist. In ventricular cells of untreated BIO 14.6 hamsters, the action potential duration was prolonged and the densities of the L-type Ca2+ current (I(Ca,L)), the transient outward current (I(to)), the delayed rectifier K+ current (I(K)), and the inward rectifier K+ current (I(K1)) were decreased compared with those of F1beta hamsters. Long-term treatment with the ET(A) receptor antagonist significantly attenuated action potential duration prolongation and reduction of I(to), I(K), and I(Ca,L) in BIO 14.6 ventricular cells. Long-term ET(A) receptor blockade prevented the QT prolongation and ventricular arrhythmias and improved the survival rate in the cardiomyopathic hamsters. CONCLUSIONS: Long-term treatment with an ET(A) antagonist inhibits electrical remodeling such as downregulation of K+ and Ca2+ currents, action potential prolongation, and the increased QT interval and thereby suppresses ventricular arrhythmias in cardiomyopathic hearts. ET(A) receptor blockade may provide a new strategy for the prevention of ventricular arrhythmias associated with heart failure.

Antitumor activity of a novel small molecule STAT3 inhibitor against a human lymphoma cell line with high STAT3 activation.

Signal transducer and activator of transcription (STAT)3, a member of a family of DNA-binding molecules mediating numerous physiological and oncogenic signaling pathways, is a novel target in cancer cells which show high phosphorylation of STAT3. Recently, we identified a novel small-molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. We investigated the mechanisms responsible for the antitumor activity in vitro and in vivo through numerous biochemical and biological assays. Specifically, the effects of STX-0119 on target genes (c-myc, cyclin D1, survivin) and apoptosis induction were analyzed in tumors treated with STX-0119 in vivo. STX-0119 showed strong growth-inhibitory activity against a broad range of hematological cancer cell lines, particularly lymphomas. STX-0119 suppressed the growth of SCC3 cells, a human lymphoma cell line with highly activated STAT3, through apoptosis and down-regulation of STAT3 targets such as c-myc, cyclin D1, survivin and Bcl-xL. Notably, Tyr-705-phosphorylated STAT3 up-regulation was not significantly suppressed by STX-0119, as opposed to other STAT3 inhibitors. STX-0119 demonstrated potent antitumor effects in vivo in SCC3-bearing nude mice by way of the down-regulation of STAT3 target genes and induction of apoptosis in the tumors. Thus, STX-0119 may be a new type of STAT3 inhibitor exhibiting strong antitumor activity.

Identification of a New Series of STAT3 Inhibitors by Virtual Screening.

The signal transducer and activator of transcription 3 (STAT3) is considered to be an attractive therapeutic target for oncology drug development. We identified a N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarboxamide derivative, STX-0119, as a novel STAT3 dimerization inhibitor by a virtual screen using a customized version of the DOCK4 program with the crystal structure of STAT3. In addition, we used in vitro cell-based assays such as the luciferase reporter gene assay and the fluorescence resonance energy transfer-based STAT3 dimerization assay. STX-0119 selectively abrogated the DNA binding activity of STAT3 and suppressed the expression of STAT3-regulated oncoproteins such as c-myc and survivin in cancer cells. In contrast, a truncated inactive analogue, STX-0872, did not exhibit those activities. Oral administration of STX-0119 effectively abrogated the growth of human lymphoma cells in a SCC-3 subcutaneous xenograft model without visible toxicity. Structure-activity relationships of STX-0119 derivatives were investigated using the docking model of the STAT3-SH2 domain/STX-0119.

Relationship between aortic calcification and stroke in a mass screening program using a mobile helical computed tomography unit.

BACKGROUND: It is reported that there is a significant relationship between the calcification of the aortic arch detected by chest X-ray examination and stroke. However, the relationship between stroke and aortic calcification (AoC) detected during a mass screening using a mobile helical computed tomography (CT) unit remains unknown. METHODS AND RESULTS: The study population consisted of 2,618 subjects (1,345 men, and 1,273 women; mean age, 52.9+/-13.8 years) who participated in a mass CT screening for lung cancer and tuberculosis. In the present study, 28 subjects (18 men, and 10 women; mean age, 65.9+/-13.5 years) had a past history of cerebral infarction. There were no subjects with a past history of intracerebral or subarachnoid hemorrhage. The frequency of AoC was significantly higher in the infarction group who were older than 50 years of age. In logistic regression analysis, the AoC was a stronger contributor of infarction than sex, age, and risk factors. Furthermore, the odds ratio of AoC for subjects with a past history of infarction increased as the number of calcified segments increased, and these values were 1.82 (95% confidence interval (CI) 1.06-3.15) in men, and 2.53 (95% CI 1.12-5.75) in women. CONCLUSIONS: These results suggest that detection of AoC during mass chest screening using a mobile helical CT unit is an effective way to evaluate the risk of cerebral infarction.

Aortic calcification detected in a mass chest screening program using a mobile helical computed tomography unit. Relationship to risk factors and coronary artery disease.

BACKGROUND: There is a significant relationship between calcification of the aortic arch (Arch) detected by chest X-ray examination and coronary artery disease (CAD), but the relationship between risk factors, CAD and aortic calcification detected during a mass screening program using a mobile helical computed tomography (CT) unit remains unknown. METHODS AND RESULTS: In total 2,623 subjects (1,347 men, and 1,276 women; mean age, 52.9+/-13.8) underwent an examination for lung cancer and tuberculosis using a mobile helical CT unit. The frequency of calcification was 19.6% in the Arch, 2.7% in the ascending aorta, and 10.1% in the descending aorta, values that were positively associated with age in both genders. Hypertension and smoking were significantly related to calcification of the thoracic aorta. There was a significant relationship between CAD and aortic calcification. The odds ratio of aortic calcification for patients with CAD increased as the number of calcified segments increased. CONCLUSIONS: These results suggest that detection of calcification in the thoracic aorta during a mass chest screening using a mobile helical CT unit can be used to evaluate the risk of CAD.

Improvement of left ventricular dysfunction during exercise by walking in patients with successful percutaneous coronary intervention for acute myocardial infarction.

A growing body of evidence suggests that walking reduces the incidence of coronary events, so the present study investigated whether walking influences left ventricular function in 30 patients with acute myocardial infarction (AMI) who had undergone successful percutaneous coronary intervention (PCI). The patients were randomly assigned to either a 3-month exercise training program of walking (group W, n=15) or a control group (group C, n=15). At both the beginning and end of the study, patients underwent exercise stress echocardiography to determine left ventricular ejection fraction (LVEF) at rest and during exercise. At baseline, there was no difference in LVEF at rest or during exercise between the two groups. After 3 months, LVEF during exercise was significantly improved compared with at rest in group W (61+/-3% during exercise vs 57+/-5% at rest, p<0.01), whereas no difference was observed between the LVEF at rest and that during exercise in group C (54+/-5% at rest vs 52+/-7% during exercise, NS). Walking may be beneficial for improving left ventricular function during exercise in patients with AMI.

Antibody binding to fas ligand attenuates inflammatory cell infiltration and cytokine secretion, leading to reduction of myocardial infarct areas and reperfusion injury.

Fas ligand (FasL) induces apoptotic cell death when bound to Fas antigen. The engagement of FasL has anti-inflammatory effects through the prevention of cell proliferation and cytokine secretion. However, the role of FasL in myocardial ischemia/reperfusion (MI/R) injury is unclear. We examined the expression of FasL mRNA in the myocardium of MI/R rats by ligating the left coronary artery for 30 minutes and allowing reperfusion to occur for 0, 1, 3, and 24 hours. The expression of FasL mRNA was enhanced 1 hour after reperfusion, and enhanced levels were consistently seen after 24 hours of reperfusion. FasL immunostaining was observed on neutrophils, macrophages, T cells, and vascular endothelial cells. We then assessed the potential role of FasL in the cell proliferation and cytokine production seen in MI/R injury after 24 hours of reperfusion. Rats were divided into three groups; Group A, without treatment; Group B, treated with nonspecific rabbit IgG; and Group C, treated with anti-FasL antibody. Anti-FasL antibody or rabbit IgG were administered intravenously before coronary artery occlusion. In Group C, interleukin-1beta and interleukin-2 mRNA levels were decreased, and neutrophil and T cell accumulation was attenuated. The infarct area determined by triphenyltetrazolium chloride staining was significantly smaller in Group C (18 +/- 4%) than in Group A (34 +/- 2%) or Group B (33 +/- 4%) (p< 0.0001). However, there was no significant difference in the prevalence of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling-positive cardiomyocytes among the three groups. These findings suggest that the cardioprotective effect of anti-FasL antibody is due to its anti-inflammatory action, rather than antiapoptotic action. The Fas/FasL system may be involved in the development of MI/R injury.

Measurement of aortic diameters and detection of asymptomatic aortic aneurysms in a mass screening program using a mobile helical computed tomography unit.

In a mass chest computed tomography (CT) screening using a mobile helical CT unit, we measured the aortic diameter at three segments to confirm standard values and also attempted to detect any asymptomatic aortic aneurysms. The population screened in the present study consisted of 6971 subjects (3847 men and 3124 women, mean age 60.3 +/- 12.1 years). They underwent a plain chest CT to screen for lung cancer and tuberculosis. The diameters of the ascending and descending aorta were measured at the level of the pulmonary artery bifurcation. The abdominal aorta was measured at the level of the celiac bifurcation, and the mean values for each measurement were calculated. In addition, we attempted to determine whether any correlation exists among aortic diameter, age, and indices of body size and investigated the frequency of asymptomatic aortic aneurysms among the general population. The mean aortic diameter of all three segments was significantly larger in men than in women and increased with age. It also correlated significantly with indices of body size (P < 0.01). A total of 11 subjects (0.16%) had asymptomatic aortic aneurysms (3 ascending, 4 descending, and 4 thoracoabdominal). The aortic diameter in each individual case was greater than the mean aortic diameter +3 standard deviations in each age group. Chest CT screening for lung cancer can thus detect asymptomatic aneurysms through simultaneous aortic measurement.

Induction of apoptosis and ubiquitin hydrolase gene expression by human serum factors in the early phase of acute myocardial infarction.

The physiologic events leading to apoptosis in myocardial infarction and the molecules involved in the death process have not been clarified unequivocally. We developed a method to search for serum factors that induce apoptosis of human cells, using serum obtained from patients within 1 day of the onset of acute myocardial infarction (AMI). Serum factors were found to have the ability to increase the caspase-3 activity levels in human RSa cells, which are susceptible to apoptosis inducers. The factors obtained from AMI patients by elution at about 0.5 mol/L KCl from a dye-ligand column were named AMI-SFs (serum factors from AMI). Electrophoretic analysis showed DNA fragmentation in AMI-SF-treated RSa cells, but not in RSa cells treated with fractions from AMI patients 1 week after clinical onset of illness. AMI-SF-induced DNA fragmentation was also demonstrated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling analysis, whereas a suppression of fragmentation was seen in RSa cells treated with AMI-SFs in combination with a caspase-3 inhibitor. The increase in caspase-3 activity was not inhibited by neutralizing antibodies to tumor necrosis factor-alpha, interleukin-6, human interferon-beta, or interferon-gamma. Polymerase chain reaction-based messenger RNA differential display and Northern blotting revealed an increase in the messenger RNA expression level of human ubiquitin hydrolase in AMI-SF-treated RSa cells. Antisense oligonucleotides for ubiquitin hydrolase inhibited the increase in caspase-3 activity. These findings suggested that serum from AMI patients in the acute phase contains factors that induce apoptosis, possibly by inducing the expression of the ubiquitin hydrolase gene, at least in the human cells tested.

Comparison between nicorandil and magnesium as an adjunct cardioprotective agent to percutaneous coronary intervention in acute anterior myocardial infarction.

BACKGROUND: It has been reported that both nicorandil and magnesium have a cardioprotective effect in experimental ischemia - reperfusion models. In the present study, the cardioprotective effects of nicorandil and magnesium as an adjunct to reperfusion therapy in patients with acute myocardial infarction (AMI) were compared. METHODS AND RESULTS: Forty consecutive patients with AMI caused by occlusion of anterior descending coronary artery were randomized into 3 groups: (1) Group N: nicorandil was given as 4 mg iv and 4 mg ic before reperfusion, followed by continuous infusion at 4 mg/h for 24 h; (2) Group M: magnesium was administered at 10 mmol iv before reperfusion, followed by continuous infusion at 0.4 mmol/h for 24 h; and (3) Group C: neither nicorandil nor magnesium was given. Left ventriculography was performed immediately after reperfusion and 3 months later. There was no significant change in regional wall motion (RWM) in either Group C or M, whereas that of group N improved significantly. The change in RWM in Group N was significantly greater than in Group C (Group N: 0.92+/-0.92, Group M: 0.44+/-0.80, Group C: -0.01+/-0.65, p<0.05). CONCLUSIONS: The early administration of nicorandil as an adjunct to reperfusion is useful for cardioprotection in AMI, but magnesium is not.