Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells

PubMed Central

Abe, Mariko; Pelus, Louis M.; Singh, Pratibha; Hirade, Tomohiro; Onishi, Chie; Purevsuren, Jamiyan; Taketani, Takeshi; Yamaguchi, Seiji; Fukuda, Seiji

2016-01-01

Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression. PMID:27387666

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

PubMed

Kawashita, Yujo; Deb, Niloy J; Garg, Madhur K; Kabarriti, Rafi; Fan, Zuoheng; Alfieri, Alan A; Roy-Chowdhury, Jayanta; Guha, Chandan

2014-08-01

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and

Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

PubMed Central

Park, Jung Eun; Yuen, Hiu Fung; Zhou, Jian Biao; Al-aidaroos, Abdul Qader O; Guo, Ke; Valk, Peter J; Zhang, Shu Dong; Chng, Wee Joo; Hong, Cheng William; Mills, Ken; Zeng, Qi

2013-01-01

FLT3-ITD mutations are prevalent mutations in acute myeloid leukaemia (AML). PRL-3, a metastasis-associated phosphatase, is a downstream target of FLT3-ITD. This study investigates the regulation and function of PRL-3 in leukaemia cell lines and AML patients associated with FLT3-ITD mutations. PRL-3 expression is upregulated by the FLT3-STAT5 signalling pathway in leukaemia cells, leading an activation of AP-1 transcription factors via ERK and JNK pathways. PRL-3-depleted AML cells showed a significant decrease in cell growth. Clinically, high PRL-3 mRNA expression was associated with FLT3-ITD mutations in four independent AML datasets with 1158 patients. Multivariable Cox-regression analysis on our Cohort 1 with 221 patients identified PRL-3 as a novel prognostic marker independent of other clinical parameters. Kaplan–Meier analysis showed high PRL-3 mRNA expression was significantly associated with poorer survival among 491 patients with normal karyotype. Targeting PRL-3 reversed the oncogenic effects in FLT3-ITD AML models in vitro and in vivo. Herein, we suggest that PRL-3 could serve as a prognostic marker to predict poorer survival and as a promising novel therapeutic target for AML patients. PMID:23929599

Biochemical characterization and analysis of the transforming potential of the FLT3/FLK2 receptor tyrosine kinase.

PubMed

Maroc, N; Rottapel, R; Rosnet, O; Marchetto, S; Lavezzi, C; Mannoni, P; Birnbaum, D; Dubreuil, P

1993-04-01

We recently cloned an additional member of the receptor type tyrosine kinase class III. This new gene, called Flt3 by our group [Rosnet, O., Matteï, M.G., Marchetto, S. & Birnbaum, D. (1991). Genomics, 9, 380-385; Rosnet, O., Marchetto, S., deLapeyriere, O. & Birnbaum, D. (1991). Oncogene, 6, 1641-1650] and Flk2 by others [Matthews, W., Jordan, C.T., Wieg, G.W., Pardoll, D. & Lemischka, I.R. (1991). Cell, 65, 1143-1152] is strongly related to the important developmental genes Kit, Fms and Pdgfr. The murine 3.2-kb full-length cDNA, when introduced into COS-1 cells, shows the expression of two polypeptides with apparent molecular weights of 155 kDa and 132 kDa. Treatment of cells with N-linked glycosylation inhibitors results in the expression of a 110-kDa protein. We have shown that FLT3 contains an intrinsic tyrosine kinase activity. A point mutation in a highly conserved residue within the phosphoryltransferase domain inactivates the catalytic function of this receptor, whereas activation by way of a chimeric molecule between the ligand-binding domain of colony-stimulating factor type 1 (CSF-1) receptor (CSF-1R) and the kinase domain of FLT3 results, in the presence of CSF-1, in the development of the transforming activity of this receptor as shown by anchorage-independent cell growth. Finally, expression analysis of the FLT3 protein shows that, in addition to the hematopoietic system, FLT3 is strongly expressed in neural, gonadal, hepatic and placental tissues in the mouse.

Selective FLT3 inhibitor FI-700 neutralizes Mcl-1 and enhances p53-mediated apoptosis in AML cells with activating mutations of FLT3 through Mcl-1/Noxa axis.

PubMed

Kojima, K; Konopleva, M; Tsao, T; Andreeff, M; Ishida, H; Shiotsu, Y; Jin, L; Tabe, Y; Nakakuma, H

2010-01-01

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

Dnmt3a deletion cooperates with the Flt3/ITD mutation to drive leukemogenesis in a murine model

PubMed Central

Poitras, Jennifer L.; Heiser, Diane; Li, Li; Nguyen, Bao; Nagai, Kozo; Duffield, Amy S.; Gamper, Christopher; Small, Donald

2016-01-01

Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3/ITD) are among the most common mutations in Acute Myeloid Leukemia (AML). Resulting in constitutive activation of the kinase, FLT3/ITD portends a particularly poor prognosis, with reduced overall survival and increased rates of relapse. We previously generated a knock-in mouse, harboring an internal tandem duplication at the endogenous Flt3 locus, which develops a fatal myeloproliferative neoplasm (MPN), but fails to develop acute leukemia, suggesting additional mutations are necessary for transformation. To investigate the potential cooperativity of FLT3/ITD and mutant DNMT3A, we bred a conditional Dnmt3a knockout to a substrain of our Flt3/ITD knock-in mice, and found deletion of Dnmt3a significantly reduced median survival of Flt3ITD/+ mice in a dose dependent manner. As expected, pIpC treated Flt3ITD/+ mice solely developed MPN, while Flt3ITD/+;Dnmt3af/f and Flt3ITD/+;Dnmt3af/+ developed a spectrum of neoplasms, including MPN, T-ALL, and AML. Functionally, FLT3/ITD and DNMT3A deletion cooperate to expand LT-HSCs, which exhibit enhanced self-renewal in serial re-plating assays. These results illustrate that DNMT3A loss cooperates with FLT3/ITD to generate hematopoietic neoplasms, including AML. In combination with FLT3/ITD, homozygous Dnmt3a knock-out results in reduced time to disease onset, LT-HSC expansion, and a higher incidence of T-ALL compared with loss of just one allele. The co-occurrence of FLT3 and DNMT3A mutations in AML, as well as subsets of T-ALL, suggests the Flt3ITD/+;Dnmt3af/f model may serve as a valuable resource for delineating effective therapeutic strategies in two clinically relevant contexts. PMID:27636998

The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with Mesenchymal Stem Cells.

PubMed

Oubari, Farhad; Amirizade, Naser; Mohammadpour, Hemn; Nakhlestani, Mozhdeh; Zarif, Mahin Nikougoftar

2015-01-01

Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+(CD34(+)) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05). FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture.

FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions.

PubMed

Larrosa-Garcia, Maria; Baer, Maria R

2017-06-01

The receptor tyrosine kinase fms -like tyrosine kinase 3 (FLT3), involved in regulating survival, proliferation, and differentiation of hematopoietic stem/progenitor cells, is expressed on acute myeloid leukemia (AML) cells in most patients. Mutations of FLT3 resulting in constitutive signaling are common in AML, including internal tandem duplication (ITD) in the juxtamembrane domain in 25% of patients and point mutations in the tyrosine kinase domain in 5%. Patients with AML with FLT3-ITD have a high relapse rate and short relapse-free and overall survival after chemotherapy and after transplant. A number of inhibitors of FLT3 signaling have been identified and are in clinical trials, both alone and with chemotherapy, with the goal of improving clinical outcomes in patients with AML with FLT3 mutations. While inhibitor monotherapy produces clinical responses, they are usually incomplete and transient, and resistance develops rapidly. Diverse combination therapies have been suggested to potentiate the efficacy of FLT3 inhibitors and to prevent development of resistance or overcome resistance. Combinations with epigenetic therapies, proteasome inhibitors, downstream kinase inhibitors, phosphatase activators, and other drugs that alter signaling are being explored. This review summarizes the current status of translational and clinical research on FLT3 inhibitors in AML, and discusses novel combination approaches. Mol Cancer Ther; 16(6); 991-1001. ©2017 AACR . ©2017 American Association for Cancer Research.

FLT3 D835/I836 mutations are associated with poor disease-free survival and a distinct gene-expression signature among younger adults with de novo cytogenetically normal acute myeloid leukemia lacking FLT3 internal tandem duplications

PubMed Central

Ruppert, Amy S.; Radmacher, Michael D.; Mrózek, Krzysztof; Paschka, Peter; Langer, Christian; Baldus, Claudia D.; Wen, Jing; Racke, Frederick; Powell, Bayard L.; Kolitz, Jonathan E.; Larson, Richard A.; Caligiuri, Michael A.; Marcucci, Guido; Bloomfield, Clara D.

2008-01-01

The prognostic relevance of FLT3 D835/I836 mutations (FLT3-TKD) in cytogenetically normal acute myeloid leukemia (CN-AML) remains to be established. After excluding patients with FLT3 internal tandem duplications, we compared treatment outcome of 16 de novo CN-AML patients with FLT3-TKD with that of 123 patients with wild-type FLT3 (FLT3-WT), less than 60 years of age and similarly treated on Cancer and Leukemia Group B protocols. All FLT3-TKD+ patients and 85% of FLT3-WT patients achieved a complete remission (P = .13). Disease-free survival (DFS) of FLT3-TKD+ patients was worse than DFS of FLT3-WT patients (P = .01; estimated 3-year DFS rates, 31% vs 60%, respectively). In a multivariable analysis, FLT3-TKD was associated with worse DFS (P = .02) independent of NPM1 status and percentage of bone marrow blasts. To gain further biologic insights, a gene-expression signature differentiating FLT3-TKD+ from FLT3-WT patients was identified. The signature (333 probe sets) included overexpression of VNN1, C3AR1, PTPN6, and multiple other genes involved in monocarboxylate transport activity, and underexpression of genes involved in signal transduction regulation. These associations with outcome, other prognostic markers, and the elucidated expression signature enhance our understanding of FLT3-TKD–associated biology and may lead to development of novel therapies that improve clinical outcome of CN-AML patients with FLT3-TKD. PMID:17940205

Temporal Changes in FLT3-ITD Regulation of Stem Cell Self-Renewal and Leukemogenesis

DTIC Science & Technology

2015-09-01

whether FLT3-ITD depletes HSCs, expands restricted progenitors and promotes a myeloproliferative neoplasm during the adult, but not fetal stage of...goals: Aim 1: To test whether FLT3-ITD depletes HSCs, expands restricted progenitors and promotes a myeloproliferative neoplasm during the adult, but...progenitor pool and myeloproliferative neoplasms (MPN) were also evident in 14 day old FLT3-ITD mice but not fetal mice (Fig. 1F and not shown). FLT3-ITD

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

PubMed

Pillinger, Genevra; Abdul-Aziz, Amina; Zaitseva, Lyubov; Lawes, Matthew; MacEwan, David J; Bowles, Kristian M; Rushworth, Stuart A

2015-08-21

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

DOE PAGES

Zorn, Julie A.; Wang, Qi; Fujimura, Eric; ...

2015-04-02

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain ofmore » FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.« less

FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia

PubMed Central

Kim, Hyung-Gyoon; Kojima, Kyoko; Swindle, C. Scott; Cotta, Claudiu V.; Huo, Yongliang; Reddy, Vishnu

2008-01-01

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML. PMID:17967943

Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regeneration.

PubMed

Aydin, Iraz T; Tokcaer, Zeynep; Dalgic, Aydin; Konu, Ozlen; Akcali, Kamil C

2007-12-01

The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration.

Discovery of a Diaminopyrimidine FLT3 Inhibitor Active against Acute Myeloid Leukemia

PubMed Central

2017-01-01

Profiling of the kinase-binding capabilities of an aminopyrimidine analogue detected in a cellular screen of the St. Jude small-molecule collection led to the identification of a novel series of FMS-like tyrosine kinase 3 (FLT3) inhibitors. Structure–activity relationship studies led to the development of compounds exhibiting good potency against MV4-11 and MOLM13 acute myelogenous leukemia cells driven by FLT3, regardless of their FLT3 mutation status. In vitro pharmacological profiling demonstrated that compound 5e shows characteristics suitable for further preclinical development. PMID:28580438

[Renaturation with simultaneous purification of the recombinant human Flt3 ligand from inclusion bodies by high performance hydrophobic interaction chromatography].

PubMed

Jia, Jia; Wang, Lili; Gao, Dong; Geng, Xindu

2010-06-01

Flt3 ligand (FL) is a class of cytokines with the functions of promoting early hematopoiesis. It has important clinical value in promoting growth and development of hematopoietic cells and hematopoietic mobilization. In order to obtain large quantities of recombinant human FL (rhFL) by genetic engineering methods for clinic and research, in this work, rhFL was expressed in E. coli as inclusion bodies. The inclusion bodies were recovered, cleaned and solubilized in 8 mol/L urea, the solubilized rhFL was renatured by high performance hydrophobic interaction chromatography (HPHIC) with simultaneous purification, the retention feature and renaturation regularity were studied. The results showed that when the denatured protein concentration was 8.51 g/L, and the end group of stationary phase was PEG800, under the conditions of mobile phase of pH 7.0 and with the addition of 4 mol/L urea, 1.8 mmol/L glutathione (GSH) and 0.3 mmol/L oxidative glutathione (GSSG), a mass recovery of 36.9% and a purity of 94.5% were obtained after refolding with simultaneous purification. The obtained rhFL was successfully renatured with simultaneous purification in only one step of HPHIC, and it provided a foundation for the manufacturing of high quality rhFL.

Computer-guided design, synthesis, and biological evaluation of quinoxalinebisarylureas as FLT3 inhibitors.

PubMed

Göring, Stefan; Bensinger, Dennis; Naumann, Eva C; Schmidt, Boris

2015-03-01

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in ∼30 % of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Point mutations in the tyrosine kinase domain (TKD) are observed as primary mutations or are acquired as secondary mutations in FLT3 with internal tandem duplications (ITDs) after treatment with tyrosine kinase inhibitors (TKIs). Although dozens of potent inhibitors against FLT3 ITD have been reported, activating TKD point mutations, especially at residues F691 and D835, remain the leading cause for therapy resistance, highlighting the consistent need for new potent inhibitors. Herein we report the identification and characterization of novel quinoxaline-based FLT3 inhibitors. We used the pharmacophore features of diverse known inhibitors as a starting point for a new optimization algorithm for type II TKIs, starting from an in silico library pharmacophore search and induced-fit docking in the known FLT3 structure. This led to the design of a set of diverse quinoxalinebisarylureas, which were profiled in an FLT3 kinase activity assay. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy

PubMed Central

Warkentin, Alexander A; Lopez, Michael S; Lasater, Elisabeth A; Lin, Kimberly; He, Bai-Liang; Leung, Anskar YH; Smith, Catherine C; Shah, Neil P; Shokat, Kevan M

2014-01-01

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a ‘synthetic lethal toxicity’ arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity. DOI: http://dx.doi.org/10.7554/eLife.03445.001 PMID:25531068

Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma.

PubMed

Guo, M; Miller, W M; Papoutsakis, E T; Patel, S; James, C; Goolsby, C; Winter, J N

1999-01-01

Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC. Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days. The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34(+) cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.

PubMed

Kazi, Julhash U; Chougule, Rohit A; Li, Tianfeng; Su, Xianwei; Moharram, Sausan A; Rupar, Kaja; Marhäll, Alissa; Gazi, Mohiuddin; Sun, Jianmin; Zhao, Hui; Rönnstrand, Lars

2017-07-01

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

Modification of a promiscuous inhibitor shifts the inhibition from γ-secretase to FLT-3.

PubMed

Amombo, Ghislaine Marlyse Okala; Kramer, Thomas; Lo Monte, Fabio; Göring, Stefan; Fach, Matthias; Smith, Steven; Kolb, Stephanie; Schubenel, Robert; Baumann, Karlheinz; Schmidt, Boris

2012-12-15

The inhibition of FLT-3 activity is an interesting target for the treatment of acute myeloid leukemia (AML). The serendipitous identification of FLT-3 inhibitors from a CK1/γ-secretase programme provided compounds with dual inhibitory activity. We analyzed the structure-activity relationship of these inhibitors and derivatized them to arrive at compounds with reduced impact on γ-secretase activity and enhanced FLT-3 inhibition. Copyright © 2012 Elsevier Ltd. All rights reserved.

Over-expression of Flt3 induces NF-kappaB pathway and increases the expression of IL-6.

PubMed

Takahashi, Shinichiro; Harigae, Hideo; Ishii, Keiko Kumura; Inomata, Mitsue; Fujiwara, Tohru; Yokoyama, Hisayuki; Ishizawa, Kenichi; Kameoka, Junichi; Licht, Jonathan D; Sasaki, Takeshi; Kaku, Mitsuo

2005-08-01

Activating mutations or over-expression of the Flt3 is prevalent in acute myeloblastic leukemia (AML), associated with activation of Ras/MAP kinase and other signaling pathways. In this study, we addressed the role of Flt3 in the activation of nuclear factor-kappa B (NF-kappaB), which is a target molecule of these kinase pathways. In BaF3 cells stably expressing Flt3, a NF-kappaB-responsive reporter was upregulated and its target gene, IL-6, was increased by the involvement of Flt3-ERK/MAPK-NF-kappaB pathway. Furthermore, we found a modest positive correlation (r=0.35, p=0.096) between Flt3 and IL-6 mRNA expression in 24 AML specimens. These results suggest a role of Flt3 over-expression in NF-kappaB pathway.

FLT3 is implicated in cytarabine transport by human equilibrative nucleoside transporter 1 in pediatric acute leukemia.

PubMed

Català, Albert; Pastor-Anglada, Marçal; Caviedes-Cárdenas, Liska; Malatesta, Roberta; Rives, Susana; Vega-García, Nerea; Camós, Mireia; Fernández-Calotti, Paula

2016-08-02

FLT3 abnormalities are negative prognostic markers in acute leukemia. Infant leukemias are a subgroup with frequent MLL (KMT2A) rearrangements, FLT3 overexpression and high sensitivity to cytarabine, but dismal prognosis. Cytarabine is transported into cells by Human Equilibrative Nucleoside Transporter-1 (hENT1, SLC29A1), but the mechanisms that regulate hENT1 in acute leukemia have been scarcely studied.We explored the expression and functional link between FLT3 and main cytarabine transporters in 50 pediatric patients diagnosed with acute lymphoblastic leukemia and MLL rearrangement (ALL-MLL+) and other subtypes of leukemia, and in leukemia cell lines.A significant positive correlation was found between FLT3 and hENT1 expression in patients. Cytarabine uptake into cells was mediated mainly by hENT1, hENT2 and hCNT1. hENT1-mediated uptake of cytarabine was transiently abolished by the FLT3 inhibitor PKC412, and this effect was associated with decreased hENT1 mRNA and protein levels. Noticeably, the cytotoxicity of cytarabine was lower when cells were first exposed to FLT3 inhibitors (PKC412 or AC220), probably due to decreased hENT1 activity, but we observed a higher cytotoxic effect if FLT3 inhibitors were administered after cytarabine.FLT3 regulates hENT1 activity and thereby affects cytarabine cytotoxicity. The sequence of administration of cytarabine and FLT3 inhibitors is important to maintain their efficacy.

Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

PubMed Central

Lee-Sherick, Alisa B.; Zhang, Weihe; Menachof, Kelly K.; Hill, Amanda A.; Rinella, Sean; Kirkpatrick, Gregory; Page, Lauren S.; Stashko, Michael A.; Jordan, Craig T.; Wei, Qi; Liu, Jing; Zhang, Dehui; DeRyckere, Deborah; Wang, Xiaodong; Frye, Stephen; Earp, H. Shelton; Graham, Douglas K.

2015-01-01

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML. PMID:25762638

Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia

PubMed Central

Major, Heather D.; Cambell, Robert A.; Silver, Robert M.; Branch, D. Ware; Weyrich, Andrew S.

2014-01-01

Objective Soluble fms-like tyrosine kinase (sFlt-1) is an important mediator in the pathogenesis of preeclampsia. We sought to determine if platelet-monocyte aggregates (PMAs) produced sFlt-1 and if PMAs contributed to sFlt-1 production in preeclampsia. Study Design Case-control study of sFlt-1 release from PMAs using blood samples from women with preeclampsia matched by gestational age to pregnant controls. A third group of nonpregnant, reproductive-age women comprised an additional control group. Experiments were also performed using blood from non-pregnant women to elucidate if inducing PMAs could stimulate sFlt-1 production, and if so, to determine the necessary receptors and pathways. Results Women with preeclampsia had increased total Flt-1 concentrations in platelets and monocytes at baseline compared to pregnant controls (25 vs. 10 pg/ml, p=0.0003). sFlt-1 production was elicited from monocytes incubated with thrombin-activated platelets from non-pregnant women. sFlt-1 production was regulated at the transcriptional level by p38 and NF-κB dependent pathways. Conclusion Activated platelets in preeclampsia bind monocytes to generate sFlt-1. PMAs are a previously unrecognized source of sFlt-1 that may contribute to endothelial dysfunction and systemic inflammation commonly observed in preeclampsia. PMID:24440566

Characterisation and Clinical Significance of FLT3-ITD and non-ITD in Acute Myeloid Leukaemia Patients in Kelantan, Northeast Peninsular Malaysia.

PubMed

Yunus, Noraini Mat; Johan, Muhammad Farid; Ali Nagi Al-Jamal, Hamid; Husin, Azlan; Hussein, Abdul Rahim; Hassan, Rosline

2015-01-01

Mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor gene may promote proliferation via activation of multiple signaling pathways. FLT3-internal tandem duplication (FLT3-ITD) is the most common gene alteration found in patients diagnosed with acute myeloid leukaemia (AML) and has been associated with poor prognosis. We performed mutational analysis of exons 14-15 and 20 of the FLT3 gene in 54 AML patients using PCR-CSGE (conformational sensitive gel electrophoresis) followed by sequencing analysis to characterise FLT3 mutations in adult patients diagnosed with AML at Hospital USM, Kelantan, Northeast Peninsular Malaysia. FLT3 exon 14-15 mutations were identified in 7 of 54 patients (13%) whereas no mutation was found in FLT3 exon 20. Six ITDs and one non-ITD mutation were found in exon 14 of the juxtamembrane (JM) domain of FLT3. FLT3-ITD mutations were associated with a significantly higher blast percentage (p-value=0.008) and white blood cell count (p-value=0.023) but there was no significant difference in median overall survival time for FLT3-ITD+/FLT3-ITD- within 2 years (p-value=0.374). The incidence of FLT3-ITD in AML patients in this particular region of Malaysia is low compared to the Western world and has a significant association with WBC and blast percentage.

Flt3 is a target of coumestrol in protecting against UVB-induced skin photoaging.

PubMed

Park, Gaeun; Baek, Sohee; Kim, Jong-Eun; Lim, Tae-gyu; Lee, Charles C; Yang, Hee; Kang, Young-Gyu; Park, Jun Seong; Augustin, Martin; Mrosek, Michael; Lee, Chang Yong; Dong, Zigang; Huber, Robert; Lee, Ki Won

2015-12-01

While skin aging is a naturally occurring process by senescence, exposure to ultraviolet (UV) radiation accelerates wrinkle formation and sagging of skin. UV induces skin aging by degrading collagen via activating matrix metalloproteinases (MMPs). In this study, we show that coumestrol, a metabolite of the soybean isoflavone daidzein, has a preventive effect on skin photoaging in three-dimensional human skin equivalent model. Coumestrol inhibited UVB-induced MMP-1 expression and activity. Whole human kinase profiling assay identified FLT3 kinase as a novel target protein of coumestrol in UVB-induced signaling pathway in skin. Coumestrol suppresses FLT3 kinase activity, and subsequently, Ras/MEK/ERK and Akt/p70 ribosomal S6 kinase pathway. This suppresses AP-1 activity and in turn, diminishes MMP-1 gene transcription. Using X-ray crystallography, the binding of coumestrol to FLT3 was defined and implied ATP-competitive inhibition. Residues Lys644 and Phe830 showed local changes to accommodate coumestrol in the ATP-binding pocket. 4-APIA, a pharmacological inhibitor of FLT3, inhibited MMP-1 expression and induced signal transduction changes similar to coumestrol. Taken together, coumestrol inhibits UVB-induced MMP-1 expression by suppressing FLT3 kinase activity. These findings suggest that coumestrol is a novel dietary compound with potential application in preventing and improving UVB-associated skin aging. Copyright © 2015 Elsevier Inc. All rights reserved.

PRL-3, a Metastasis Associated Tyrosine Phosphatase, Is Involved in FLT3-ITD Signaling and Implicated in Anti-AML Therapy

PubMed Central

Zhou, Jianbiao; Bi, Chonglei; Chng, Wee-Joo; Cheong, Lip-Lee; Liu, Shaw-Cheng; Mahara, Sylvia; Tay, Kian-Ghee; Zeng, Qi; Li, Jie; Guo, Ke; Tan, Cheng Peow Bobby; Yu, Hanry; Albert, Daniel H.; Chen, Chien-Shing

2011-01-01

Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation. PMID:21589872

A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia.

PubMed

Shanmugam, Rajasubramaniam; Gade, Padmaja; Wilson-Weekes, Annique; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A; Baghdadi, Tareq Al; Sargent, Katie J; Cripe, Larry D; Kalvakolanu, Dhananjaya V; Boswell, H Scott

2012-01-15

Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML. ©2011 AACR.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia.

PubMed

Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2015-10-20

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia

PubMed Central

Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2015-01-01

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML. PMID:26450903

Identification of pyrrolo[2,3-d]pyrimidines as potent HCK and FLT3-ITD dual inhibitors.

PubMed

Koda, Yasuko; Kikuzato, Ko; Mikuni, Junko; Tanaka, Akiko; Yuki, Hitomi; Honma, Teruki; Tomabechi, Yuri; Kukimoto-Niino, Mutsuko; Shirouzu, Mikako; Shirai, Fumiyuki; Koyama, Hiroo

2017-11-15

A series of novel pyrrolo[2,3-d]pyrimidines were synthesized by introducing 15 different amino acids to 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Compounds with potent activities against HCK and FLT3-ITD were evaluated in viability studies with acute myeloid leukemia cell line MV4-11. Our structure activity relationship analyses lead to the identification of compound 31, which exhibited potent HCK and FLT3-ITD inhibition and activity against the MV4-11 cell line. Copyright © 2017 Elsevier Ltd. All rights reserved.

RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

PubMed

Hospital, M-A; Jacquel, A; Mazed, F; Saland, E; Larrue, C; Mondesir, J; Birsen, R; Green, A S; Lambert, M; Sujobert, P; Gautier, E-F; Salnot, V; Le Gall, M; Decroocq, J; Poulain, L; Jacque, N; Fontenay, M; Kosmider, O; Récher, C; Auberger, P; Mayeux, P; Bouscary, D; Sarry, J-E; Tamburini, J

2018-03-01

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

Midostaurin and Decitabine in Treating Older Patients With Newly Diagnosed Acute Myeloid Leukemia and FLT3 Mutation

ClinicalTrials.gov

2017-11-29

Acute Myeloid Leukemia With FLT3/ITD Mutation; Acute Myeloid Leukemia With Gene Mutations; FLT3 Tyrosine Kinase Domain Point Mutation; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Potent Activity of Ponatinib (AP24534) in Models of FLT3-Driven Acute Myeloid Leukemia and Other Hematologic Malignancies

PubMed Central

Gozgit, Joseph M.; Wong, Matthew J.; Wardwell, Scott; Tyner, Jeffrey W.; Loriaux, Marc M.; Mohemmad, Qurish K.; Narasimhan, Narayana I.; Shakespeare, William C.; Wang, Frank; Druker, Brian J.; Clackson, Tim; Rivera, Victor M.

2011-01-01

Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD+/+) but not RS4;11 (FLT3-ITD−/−) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD–driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα. PMID:21482694

Prognostic significance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy.

PubMed

Boissel, N; Cayuela, J M; Preudhomme, C; Thomas, X; Grardel, N; Fund, X; Tigaud, I; Raffoux, E; Rousselot, P; Sigaux, F; Degos, L; Castaigne, S; Fenaux, P; Dombret, H

2002-09-01

FLT3 internal tandem duplications (FLT3-ITDs) are present in nearly 25% of patients with AML and have been associated with poor response to conventional therapy and poor outcome. We retrospectively evaluated the effect of reinforced courses of chemotherapy on the prognostic value of FLT3-ITDs in 159 AML patients prospectively enrolled in the ALFA-9000 trial, which randomly compared three reinforced induction regimens (standard 3+7 including high-dose daunorubicin, double induction, and timed-sequential therapy). FLT3-ITD was present in 40/159 (25%) blast samples and associated with high WBC (P = 0.002) and cytogenetics (P < 0.001) with a higher incidence (35%) in patients with a normal karyotype. There was no difference in CR rate between FLT3-wt and FLT3-ITD patients (80% vs 78%). Relapse-free survival (RFS) was similar in both groups (5-year RFS, 33% vs 32%; P = 0.41), even after adjustment for age, sex, WBC, cytogenetics, and treatment arm. A trend to a worse survival was observed in the FLT3-ITD group (estimated 5-year OS, 23% vs 37%; P = 0.09), mainly in patients with a normal karyotype. This was associated with a dramatic outcome in relapsing FLT3-ITD patients (estimated 3-year post-relapse survival, 0% vs 27%; P = 0.04). These results suggest that the bad prognosis associated with FLT3-ITDs in AML might be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations might thus be useful to intensify induction as well as post-remission therapy in FLT3-ITD patients.

A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML.

PubMed

Knapper, Steven; Russell, Nigel; Gilkes, Amanda; Hills, Robert K; Gale, Rosemary E; Cavenagh, James D; Jones, Gail; Kjeldsen, Lars; Grunwald, Michael R; Thomas, Ian; Konig, Heiko; Levis, Mark J; Burnett, Alan K

2017-03-02

The clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3 -internal tandem duplication mutations, 23% FLT3 -tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3 -mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside

SOCS1 cooperates with FLT3-ITD in the development of myeloproliferative disease by promoting the escape from external cytokine control.

PubMed

Reddy, Pavankumar N G; Sargin, Bülent; Choudhary, Chunaram; Stein, Stefan; Grez, Manuel; Müller-Tidow, Carsten; Berdel, Wolfgang E; Serve, Hubert; Brandts, Christian H

2012-08-23

Activating mutations in the receptor tyrosine kinase FLT3 are frequently found in acute myelogenous leukemia patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control that regulates normal hematopoiesis. We have recently demonstrated that FLT3-internal tandem duplication (ITD), when localized to the biosynthetic compartment, aberrantly activates STAT5. Here, we show that one of the target genes induced by STAT5 is suppressor of cytokine signaling (SOCS)1-a surprising finding for a known tumor suppressor. Although SOCS1 expression in murine bone marrow severely impaired cytokine-induced colony growth, it failed to inhibit FLT3-ITD-supported colony growth, indicating resistance of FLT3-ITD to SOCS1. In addition, SOCS1 coexpression did not affect FLT3-ITD-mediated signaling or proliferation. Importantly, SOCS1 coexpression inhibited interferon-α and interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-mediated growth inhibitory effects. In a murine bone marrow transplantation model, the coexpression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared with FLT3-ITD alone (P < .01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting leukemogenesis. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

PubMed Central

Cesano, Alessandra; Putta, Santosh; Rosen, David B.; Cohen, Aileen C.; Gayko, Urte; Mathi, Kavita; Woronicz, John; Hawtin, Rachael E.; Cripe, Larry; Sun, Zhuoxin; Tallman, Martin S.; Paietta, Elisabeth

2013-01-01

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML

A non-canonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia (AML)

PubMed Central

Shanmugam, Rajasubramaniam; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A.; Baghdadi, Tareq Al; Sargent, Katie J.; Cripe, Larry D.; Kalvakolanu, Dhananjaya V.; Boswell, H. Scott

2014-01-01

Purpose DAPK1, a tumor suppressor, is a rate-limiting effector in an ER stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of AML with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB- and c- jun-responsive genes, was studied. RNAi knockdown studies were performed in Flt3ITD+ve cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were performed to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results AMLs characterized by normal karyotype with Flt3ITD were found to have 10-100-fold lower DAPK1 transcripts normalized to the expression of c-jun, a transcriptional activator of DAPK1, as compared to a heterogeneous cytogenetic category. Meis1, a c-jun-responsive adverse AML prognostic gene signature was also measured as control. These Flt3ITD+ve AMLs over-express relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter along with HDAC2 and HDAC6 in the Flt3ITD+ve human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NIK, de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ve AMLs had selective nuclear activation of p52NF-κB. Conclusions Flt3ITD promotes a non-canonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ve AML. PMID:22096027

Consecutive epigenetically-active agent combinations act in ID1-RUNX3-TET2 and HOXA pathways for Flt3ITD+ve AML.

PubMed

Sayar, Hamid; Liu, Yan; Gao, Rui; Zaid, Mohammad Abu; Cripe, Larry D; Weisenbach, Jill; Sargent, Katie J; Nassiri, Mehdi; Li, Lang; Konig, Heiko; Suvannasankha, Attaya; Pan, Feng; Shanmugam, Rajasubramaniam; Goswami, Chirayu; Kapur, Reuben; Xu, Mingjiang; Boswell, H Scott

2018-01-19

Co-occurrence of Flt3ITD and TET2 mutations provoke an animal model of AML by epigenetic repression of Wnt pathway antagonists, including RUNX3, and by hyperexpression of ID1, encoding Wnt agonist. These affect HOXA over-expression and treatment resistance. A comparable epigenetic phenotype was identified among adult AML patients needing novel intervention. We chose combinations of targeted agents acting on distinct effectors, at the levels of both signal transduction and chromatin remodeling, in relapsed/refractory AML's, including Flt3ITD+ve, described with a signature of repressed tumor suppressor genes, involving Wnt antagonist RUNX3 , occurring along with ID1 and HOXA over-expressions. We tracked patient response to combination of Flt3/Raf inhibitor, Sorafenib, and Vorinostat, pan-histone deacetylase inhibitor, without or with added Bortezomib, in consecutive phase I trials. A striking association of rapid objective remissions (near-complete, complete responses) was noted to accompany induced early pharmacodynamic changes within patient blasts in situ, involving these effectors, significantly linking RUNX3 /Wnt antagonist de-repression (80%) and ID1 downregulation (85%), to a response, also preceded by profound HOXA9 repression. Response occurred in context of concurrent TET2 mutation/hypomorphy and Flt3ITD+ve mutation (83% of complete responses). Addition of Bortezomib to the combination was vital to attainment of complete response in Flt3ITD+ve cases exhibiting such Wnt pathway dysregulation.

Feature genes predicting the FLT3/ITD mutation in acute myeloid leukemia

PubMed Central

LI, CHENGLONG; ZHU, BIAO; CHEN, JIAO; HUANG, XIAOBING

2016-01-01

In the present study, gene expression profiles of acute myeloid leukemia (AML) samples were analyzed to identify feature genes with the capacity to predict the mutation status of FLT3/ITD. Two machine learning models, namely the support vector machine (SVM) and random forest (RF) methods, were used for classification. Four datasets were downloaded from the European Bioinformatics Institute, two of which (containing 371 samples, including 281 FLT3/ITD mutation-negative and 90 mutation-positive samples) were randomly defined as the training group, while the other two datasets (containing 488 samples, including 350 FLT3/ITD mutation-negative and 138 mutation-positive samples) were defined as the test group. Differentially expressed genes (DEGs) were identified by significance analysis of the micro-array data by using the training samples. The classification efficiency of the SCM and RF methods was evaluated using the following parameters: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the receiver operating characteristic curve. Functional enrichment analysis was performed for the feature genes with DAVID. A total of 585 DEGs were identified in the training group, of which 580 were upregulated and five were downregulated. The classification accuracy rates of the two methods for the training group, the test group and the combined group using the 585 feature genes were >90%. For the SVM and RF methods, the rates of correct determination, specificity and PPV were >90%, while the sensitivity and NPV were >80%. The SVM method produced a slightly better classification effect than the RF method. A total of 13 biological pathways were overrepresented by the feature genes, mainly involving energy metabolism, chromatin organization and translation. The feature genes identified in the present study may be used to predict the mutation status of FLT3/ITD in patients with AML. PMID:27177049

Feature genes predicting the FLT3/ITD mutation in acute myeloid leukemia.

PubMed

Li, Chenglong; Zhu, Biao; Chen, Jiao; Huang, Xiaobing

2016-07-01

In the present study, gene expression profiles of acute myeloid leukemia (AML) samples were analyzed to identify feature genes with the capacity to predict the mutation status of FLT3/ITD. Two machine learning models, namely the support vector machine (SVM) and random forest (RF) methods, were used for classification. Four datasets were downloaded from the European Bioinformatics Institute, two of which (containing 371 samples, including 281 FLT3/ITD mutation-negative and 90 mutation‑positive samples) were randomly defined as the training group, while the other two datasets (containing 488 samples, including 350 FLT3/ITD mutation-negative and 138 mutation-positive samples) were defined as the test group. Differentially expressed genes (DEGs) were identified by significance analysis of the microarray data by using the training samples. The classification efficiency of the SCM and RF methods was evaluated using the following parameters: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the receiver operating characteristic curve. Functional enrichment analysis was performed for the feature genes with DAVID. A total of 585 DEGs were identified in the training group, of which 580 were upregulated and five were downregulated. The classification accuracy rates of the two methods for the training group, the test group and the combined group using the 585 feature genes were >90%. For the SVM and RF methods, the rates of correct determination, specificity and PPV were >90%, while the sensitivity and NPV were >80%. The SVM method produced a slightly better classification effect than the RF method. A total of 13 biological pathways were overrepresented by the feature genes, mainly involving energy metabolism, chromatin organization and translation. The feature genes identified in the present study may be used to predict the mutation status of FLT3/ITD in patients with AML.

FLT3 and JAK2 Mutations in Acute Myeloid Leukemia Promote Interchromosomal Homologous Recombination and the Potential for Copy Neutral Loss of Heterozygosity.

PubMed

Gaymes, Terry J; Mohamedali, Azim; Eiliazadeh, Anthony L; Darling, David; Mufti, Ghulam J

2017-04-01

Acquired copy neutral LOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G 0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N -acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. Cancer Res; 77(7); 1697-708. ©2017 AACR . ©2017 American Association for Cancer Research.

Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

PubMed Central

Doshi, Kshama A.; Trotta, Rossana; Natarajan, Karthika; Rassool, Feyruz V.; Tron, Adriana E.; Huszar, Dennis; Perrotti, Danilo; Baer, Maria R.

2016-01-01

Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD. PMID:27374090

Inflammatory Flt3L is essential to mobilize dendritic cells and for T cell responses during Plasmodium infection

PubMed Central

Guermonprez, Pierre; Helft, Julie; Claser, Carla; Deroubaix, Stephanie; Karanje, Henry; Gazumyan, Anna; Darrasse-Jeze, Guillaume; Telerman, Stephanie B.; Breton, Gaëlle; Schreiber, Heidi A.; Frias-Staheli, Natalia; Billerbeck, Eva; Dorner, Marcus; Rice, Charles M.; Ploss, Alexander; Klein, Florian; Swiecki, Melissa; Colonna, Marco; Kamphorst, Alice O.; Meredith, Matthew; Niec, Rachel; Takacs, Constantin; Mikhail, Fadi; Hari, Aswin; Bosque, David; Eisenreich, Tom; Merad, Miriam; Shi, Yan; Ginhoux, Florent; Rénia, Laurent; Urban, Britta C.; Nussenzweig, Michel C.

2014-01-01

Summary Innate sensing mechanisms trigger a variety of humoral and cellular events that are essential to adaptive immune responses. Here we describe an innate sensing pathway triggered by Plasmodium infection that regulates dendritic cell (DC) homeostasis and adaptive immunity via Flt3L release. Plasmodium-induced Flt3L release requires toll-like receptor activation and type I interferon production. We find that type I interferon supports the up-regulation of xanthine dehydrogenase, which metabolizes the xanthine accumulating in infected erythrocytes to uric acid. Uric acid crystals trigger mast cells to release soluble Flt3L from a pre-synthesized membrane-associated precursor. During infection Flt3L preferentially stimulates expansion of the CD8α+/CD103+ DC subset or its BDCA3+ human DC equivalent and has a significant impact on the magnitude of T cell activation, mostly in the CD8+ compartment. Our findings highlight a new mechanism that regulates DC homeostasis and T cell responses to infection. PMID:23685841

Allogeneic Stem Cell Transplantation Improves Survival in Patients with Acute Myeloid Leukemia Characterized by a High Allelic Ratio of Mutant FLT3-ITD.

PubMed

Ho, Anthony D; Schetelig, Johannes; Bochtler, Tilmann; Schaich, Markus; Schäfer-Eckart, Kerstin; Hänel, Mathias; Rösler, Wolf; Einsele, Hermann; Kaufmann, Martin; Serve, Hubert; Berdel, Wolfgang E; Stelljes, Matthias; Mayer, Jiri; Reichle, Albrecht; Baldus, Claudia D; Schmitz, Norbert; Kramer, Michael; Röllig, Christoph; Bornhäuser, Martin; Thiede, Christian; Ehninger, Gerhard

2016-03-01

Allogeneic hematopoietic cell transplantation (alloHCT) as a postremission therapy in patients with FLT3-ITD-positive intermediate-risk acute myeloid leukemia (AML) remains controversial. FLT3-ITD mutations are heterogeneous with respect to allelic ratio, location, and length of the insertion, with a high mutant-to-wild-type ratio consistently associated with inferior prognosis. We retrospectively analyzed the role of alloHCT in first remission in relationship to the allelic ratio and presence or absence of nucleophosmin 1 mutations (NPM1) in the Study Alliance Leukemia AML2003 trial. FLT3-ITD mutations were detected in 209 patients and concomitant NPM1 mutations in 148 patients. Applying a predefined cutoff ratio of .8, AML was grouped into high- and low-ratio FLT3-ITD AML (HR(FLT3-ITD) and LR(FLT3-ITD)). Sixty-one patients (29%) were transplanted in first remission. Overall survival (OS) (HR, .3; 95% CI, .16 to .7; P = .004) and event-free survival (EFS) (HR, .4; 95% CI, .16 to .9; P = .02) were significantly increased in patients with HR(FLT3-ITD) AML who received alloHCT as consolidation treatment compared with patients who received consolidation chemotherapy. Patients with LR(FLT3-ITD) AML and wild-type NPM1 who received alloHCT in first remission had increased OS (HR, .3; 95% CI, .1 to .8; P = .02) and EFS (HR, .2; 95% CI, .1 to .8; P = .02), whereas alloHCT in first remission did not have a significant impact on OS and EFS in patients with LR(FLT3-ITD) AML and concomitant NPM1 mutation. In conclusion, our results provide additional evidence that alloHCT in first remission improves EFS and OS in patients with HR(FLT3-ITD) AML and in patients with LR(FLT3-ITD) AML and wild-type NPM1. Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy.

PubMed

Pratcorona, Marta; Brunet, Salut; Nomdedéu, Josep; Ribera, Josep Maria; Tormo, Mar; Duarte, Rafael; Escoda, Lourdes; Guàrdia, Ramon; Queipo de Llano, M Paz; Salamero, Olga; Bargay, Joan; Pedro, Carmen; Martí, Josep Maria; Torrebadell, Montserrat; Díaz-Beyá, Marina; Camós, Mireia; Colomer, Dolors; Hoyos, Montserrat; Sierra, Jorge; Esteve, Jordi

2013-04-04

Risk associated to FLT3 internal tandem duplication (FLT3-ITD) in patients with acute myeloid leukemia (AML) may depend on mutational burden and its interaction with other mutations. We analyzed the effect of FLT3-ITD/FLT3 wild-type (FLT3wt) ratio depending on NPM1 mutation (NPM1mut) in 303 patients with intermediate-risk cytogenetics AML treated with intensive chemotherapy. Among NPM1mut patients, FLT3wt and low ratio (<0.5) subgroups showed similar overall survival, relapse risk, and leukemia-free survival, whereas high ratio (≥0.5) patients had a worse outcome. In NPM1wt AML, FLT3-ITD subgroups showed a comparable outcome, with higher risk of relapse and shortened overall survival than FLT3wt patients. Allogeneic stem cell transplantation in CR1 was associated with a reduced relapse risk in all molecular subgroups with the exception of NPM1mut AML with absent or low ratio FLT3-ITD. In conclusion, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio.

In vivo imaging of therapy response to a novel Pan-HER antibody mixture using FDG and FLT positron emission tomography

PubMed Central

Kristensen, Lotte K.; Dahlman, Anna; Fröhlich, Camilla; Jacobsen, Helle J.; Poulsen, Thomas T.; Lantto, Johan; Horak, Ivan D.; Kragh, Michael; Kjaer, Andreas

2015-01-01

Purpose Overexpression of the human epidermal growth factor receptor (HER) family and their ligands plays an important role in many cancers. Targeting multiple members of the HER family simultaneously may increase the therapeutic efficacy. Here, we report the ability to image the therapeutic response obtained by targeting HER family members individually or simultaneously using the novel monoclonal antibody (mAb) mixture Pan-HER. Experimental design and results Mice with subcutaneous BxPC-3 pancreatic adenocarcinomas were divided into five groups receiving vehicle or mAb mixtures directed against either EGFR (HER1), HER2, HER3 or all three receptors combined by Pan-HER. Small animal positron emission tomography/computed tomography (PET/CT) with 2′-deoxy-2′-[18F]fluoro-D-glucose (FDG) and 3′-deoxy-3′-[18F]fluorothymidine (FLT) was performed at baseline and at day 1 or 2 after initiation of therapy. Changes in tumor uptake of tracers were quantified and compared to reduction in tumor size. Imaging results were further validated by immunohistochemistry and qPCR. Mean FDG and FLT uptake in the Pan-HER treated group decreased by 19±4.3% and 24±3.1%, respectively. The early change in FDG and FLT uptake correlated with tumor growth at day 23 relative to day 0. Ex vivo molecular analyses of markers associated with the mechanisms of FDG and FLT uptake confirmed the in vivo imaging results. Conclusions Taken together, the study supports the use of FDG and FLT as imaging biomarkers of early response to Pan-HER therapy. FDG and FLT PET/CT imaging should be considered as imaging biomarkers in clinical evaluation of the Pan-HER mAb mixture. PMID:26460961

Chidamide in FLT3-ITD positive acute myeloid leukemia and the synergistic effect in combination with cytarabine.

PubMed

Li, Xia; Yan, Xiao; Guo, Wenjian; Huang, Xin; Huang, Jiansong; Yu, Mengxia; Ma, Zhixin; Xu, Yu; Huang, ShuJuan; Li, Chenying; Zhou, Yile; Jin, Jie

2017-06-01

Chidamide, a novel histone deacetylase inhibitor (HDACi), has been approved for treatment of T-cell lymphomas in multiple clinical trials. It has been demonstrated that chidamide can inhibit cell cycle, promote apoptosis and induce differentiation in leukemia cells, whereas its effect on acute myeloid leukemia (AML) patients with FLT3-ITD mutation has not been clarified. In this study, we found that chidamide specifically induced G0/G1 arrest and apoptosis in FLT3-ITD positive AML cells in a concentration and time-dependent manner. We also found chidamide had the cytotoxicity effect on FLT3-ITD positive and negative AML cells. Moreover, with respect to relapsed/refractory patients, chidamide showed the same effectiveness as that in de novo AML patients. Notably, chidamide synergistically enhanced apoptosis caused by cytarabine. Our results support chidamide alone or combine with cytarabine may be used as an alternative therapeutic choice for AML patients especially those with FLT3-ITD mutation or relapsed/refractory ones. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Development of a potent and selective FLT3 kinase inhibitor by systematic expansion of a non-selective fragment-screening hit.

PubMed

Nakano, Hirofumi; Hasegawa, Tsukasa; Imamura, Riyo; Saito, Nae; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

2016-05-01

A non-selective inhibitor (1) of FMS-like tyrosine kinase-3 (FLT3) was identified by fragment screening and systematically modified to afford a potent and selective inhibitor 26. We confirmed that 26 inhibited the growth of FLT-3-activated human acute myeloid leukemia cell line MV4-11. Our design strategy enabled rapid development of a novel type of FLT3 inhibitor from the hit fragment in the absence of target-structural information. Copyright © 2016 Elsevier Ltd. All rights reserved.

FLT3 mutation and expression did not adversely affect clinical outcome of childhood acute leukaemia: a study of 531 Southeast Asian children by the Ma-Spore study group.

PubMed

Leow, Shuangjie; Kham, Shirley Kow-Yin; Ariffin, Hany; Quah, Thuan Chong; Yeoh, Allen Eng-Juh

2011-12-01

FMS-like tyrosine kinase 3 (FLT3) is critical for normal haematopoiesis and have been reported to be expressed in the majority of acute myeloid and lymphoid malignancies. We correlated the impact of FLT3 mutations and its expression with age, WHO 2008 classification and treatment outcome in 531 childhood acute leukaemias. Of 150 acute myeloid leukaemia (AMLs), 18 (12%) harboured FLT3-ITD while nine (6%) had FLT3-TKD. FLT3-ITD and -TKD were rare in acute megakaryoblastic leukaemia (AMKL; FLT3-ITD 0/26, FLT3-TKD 1/26) and children below 3 years (n = 2/48). Acute promyelocytic leukaemia (APL) with t(15;17);PML-RARα (n = 7/18; 39%) harboured the highest frequency of FLT3 mutations, followed by myelomonocytic (n = 4/18; 22%) and AML with t(8;21);RUNX1-RUNX1T1 (n = 2/21; 9%). FLT3 expression levels were also lowest in AMKL, both in Down's and non-Down's (p = 0.002) followed by patients <3 years (p = 0.001). The rarity of FLT3 mutations and expression levels in AMKL were independent of age. Conversely, only 2% of childhood acute lymphoblastic leukaemia (ALL) harboured FLT3 mutations (ITD = 1/381; TKD = 6/381). FLT3 was highly expressed in hyperdiploid ALL (p < 0.001). Of the 121 AMLs with clinical history, there were no significant differences in 4-year event-free survival (EFS) (46% vs. 38%; p = 0.46) and overall-survival (OS) (55% vs. 43%; p = 0.30) between FLT3-wildtype and ITD+ patients. Similarly, FLT3 expression levels did not influence survival in AML in both the good risk and non-good risk subgroups. FLT3 does not appear to be involved in the pathogenesis of AMKL, both in Down's and non-Down's. Therapeutic targets using FLT3 inhibitors may not be useful in AMKL and in young children with AML. Copyright © 2011 John Wiley & Sons, Ltd.

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain

PubMed Central

Anandasabapathy, Niroshana; Victora, Gabriel D.; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L.; Nussenzweig, Michel C.; Steinman, Ralph M.

2011-01-01

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5–7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia. PMID:21788405

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain.

PubMed

Anandasabapathy, Niroshana; Victora, Gabriel D; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L; Nussenzweig, Michel C; Steinman, Ralph M; Liu, Kang

2011-08-01

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5-7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia.

Sorafenib Dose Recommendation in Acute Myeloid Leukemia Based on Exposure-FLT3 Relationship.

PubMed

Liu, Tao; Ivaturi, Vijay; Sabato, Philip; Gobburu, Jogarao V S; Greer, Jacqueline M; Wright, John J; Smith, B Douglas; Pratz, Keith W; Rudek, Michelle A

2018-04-27

Sorafenib administered at the approved dose continuously is not tolerated long-term in patients with acute myeloid leukemia (AML). The purpose of this study was to optimize the dosing regimen by characterizing the sorafenib exposure-response relationship in patients with AML. A one-compartment model with a transit absorption compartment and enterohepatic recirculation described the exposure. The relationship between sorafenib exposure and target modulation of kinase targets (FMS-like tyrosine kinase 3 (FLT3)-ITD and extracellular signal-regulated kinase (ERK)) were described by an inhibitory maximum effect (E max ) model. Sorafenib could inhibit FLT3-ITD activity by 100% with an IC 50 of 69.3 ng/mL and ERK activity by 84% with an IC 50 of 85.7 ng/mL (both adjusted for metabolite potency). Different dosing regimens utilizing 200 or 400 mg at varying frequencies were simulated based on the exposure-response relationship. Simulations demonstrate that a 200 mg twice daily (b.i.d.) dosing regimen showed similar FLT3-ITD and ERK inhibitory activity compared with 400 mg b.i.d. and is recommended in further clinical trials in patients with AML. © 2018 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Mechanisms of apoptosis induction by simultaneous inhibition of PI3K and FLT3-ITD in AML cells in the hypoxic bone marrow microenvironment

PubMed Central

Jin, Linhua; Tabe, Yoko; Lu, Hongbo; Borthakur, Gautam; Miida, Takashi; Kantarjian, Hagop; Andreeff, Michael; Konopleva, Marina

2013-01-01

We investigated the antileukemia effects and molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in AML cells grown under hypoxia in co-cultures with bone marrow stromal cells. Combined treatment with selective class I PI3K inhibitor GDC-0941 and sorafenib reversed the protective effects of bone marrow stromal cells on FLT3-mutant AML cells in hypoxia, which was associated with downregulation of Pim-1 and Mcl-1 expression levels. These findings suggest that combined inhibition of PI3K and FLT3-ITD may constitute a targeted approach to eradicating chemoresistant AML cells sequestered in hypoxic bone marrow niches. PMID:23036488

Mechanisms of apoptosis induction by simultaneous inhibition of PI3K and FLT3-ITD in AML cells in the hypoxic bone marrow microenvironment.

PubMed

Jin, Linhua; Tabe, Yoko; Lu, Hongbo; Borthakur, Gautam; Miida, Takashi; Kantarjian, Hagop; Andreeff, Michael; Konopleva, Marina

2013-02-01

We investigated the antileukemia effects and molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in AML cells grown under hypoxia in co-cultures with bone marrow stromal cells. Combined treatment with selective class I PI3K inhibitor GDC-0941 and sorafenib reversed the protective effects of bone marrow stromal cells on FLT3-mutant AML cells in hypoxia, which was associated with downregulation of Pim-1 and Mcl-1 expression levels. These findings suggest that combined inhibition of PI3K and FLT3-ITD may constitute a targeted approach to eradicating chemoresistant AML cells sequestered in hypoxic bone marrow niches. Copyright © 2012 Elsevier Ltd. All rights reserved.

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity

PubMed Central

Zhu, Wan; Shen, Fanxia; Mao, Lei; Zhan, Lei; Kang, Shuai; Sun, Zhengda; Nelson, Jeffrey; Zhang, Rui; Zou, Dingquan; McDougall, Cameron M.; Lawton, Michael T.; Vu, Thiennu H.; Wu, Zhijian; Scaria, Abraham; Colosi, Peter; Forsayeth, John; Su, Hua

2017-01-01

Background and Purpose Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor (VEGF) has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to VEGF with high affinity, we tested intravenous (IV) delivery of an adeno-associated viral vector serotype 9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. Methods Two mouse models were used. Model 1: bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, IV-injected eight weeks after. Model 2: SM22αCre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was IV-delivered into 4–5-week-old mice. Tissue samples were collected four weeks after AAV9-sFLT1 delivery. Results AAV2-sFLT02 inhibited bAVM formation and AAV9-sFLT1 reduced abnormal vessels in Model 1 (GFP vs sFLT1: 3.66 ± 1.58/200 vessels vs 1.98 ± 1.29, p<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP vs sFLT1: 100% vs 36%) and mortality [GFP vs sFLT1: 57% (12/22 mice) vs 24% (4/19 mice), p<0.05] in Model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1-treated Model 1 mice. Conclusion By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity. PMID:28325846

Phase I Trial of Maintenance Sorafenib after Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-ITD AML

PubMed Central

Chen, Yi-Bin; Li, Shuli; Lane, Andrew A.; Connolly, Christine; Del Rio, Candice; Valles, Betsy; Curtis, Morgan; Ballen, Karen; Cutler, Corey; Dey, Bimalangshu R.; El-Jawahri, Areej; Fathi, Amir T.; Ho, Vincent T.; Joyce, Amy; McAfee, Steven; Rudek, Michelle; Rajkhowa, Trivikram; Verselis, Sigitas; Antin, Joseph H.; Spitzer, Thomas R.; Levis, Mark; Soiffer, Robert

2014-01-01

The FLT3-ITD mutation is associated with a high relapse rate for patients with AML even after allogeneic hematopoietic stem cell transplantation (HSCT). Sorafenib is a tyrosine kinase inhibitor which inhibits the FLT3 tyrosine kinase and has shown encouraging activity in FLT3-ITD AML. We conducted a phase I trial of maintenance sorafenib after HSCT in patients with FLT3-ITD AML (ClinicalTrials.gov NCT01398501). Patients received a variety of conditioning regimens and graft sources. A dose escalation 3+3 cohort design was used to define the maximum tolerated dose (MTD) with an additional 10 patients treated at the MTD. Sorafenib was initiated between days 45 and 120 after HSCT continued for twelve 28-day cycles. Twenty-two patients were enrolled (status at HSCT: CR1=16, CR2=3, refractory=3). The MTD was established at 400 mg BID with one DLT observed (pericardial effusion). Two patients died of transplant-related causes, both unrelated to sorafenib. Two patients stopped sorafenib after relapse and 5 stopped due to attributable toxicities after the DLT period. Median follow-up for surviving patients is 16.7 months after HSCT (range, 8.1–35.0). There was one case of grade II acute GVHD after starting sorafenib and the 12-month cumulative incidence of chronic GVHD was 38% (90% CI, 21%–56%). For all patients, one-year progression-free survival (PFS) is 85% (90% CI, 66%–94%) and one-year overall survival (OS) is 95% (90% CI, 79%–99%) after HSCT. For patients in CR1 / CR2 prior to HSCT (n=19), one-year PFS is 95% (90% CI, 76%–99%) and one-year OS is 100% with only one patient who has relapsed. Sorafenib is safe after HSCT for FLT3-ITD AML and merits further investigation for the prevention of relapse. PMID:25239228

The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

PubMed Central

Langen, Jan-Stephan; Schoenfelder, Gilbert; Resnick, Michael A.; Inga, Alberto

2010-01-01

Background Recently, we established that a C>T single nucleotide polymorphism (SNP) in the promoter of the VEGF receptor FLT1 gene generates a ½ site p53 response element (RE-T) that results in p53 responsiveness of the promoter. The transcriptional control required an estrogen receptor (ER) ½ site response element (ERE1) 225 nt upstream to the RE-T. Methodology/Principal Findings Here we report the identification of a second ER ½ site (ERE2) located 145 bp downstream of the RE-T and establish that both EREs can impact p53-mediated transactivation of FLT1-T in a manner that is cell type and ER level dependent. Gene reporter assays and ChIP experiments conducted in the breast cancer-derived MCF7 cells revealed that the ERE2 site was sufficient for p53-mediated ERα recruitment and transactivation of the FLT1-T promoter/reporter construct. Surprisingly, unlike the case for other p53 target promoters, p53-mediated transactivation of FLT1-T constructs or expression of the endogenous FLT1 gene, as well as binding of p53 and ER at the promoter constructs, was inducible by doxorubicin but not by 5-fluorouracil. Furthermore, ER activity at FLT1-T was differentially affected by ER ligands, compared to a control TFF1/pS2 ER target promoter. The p53-related transcription factors (TFs) p73 and p63 had no effect on FLT1 transactivation. Conclusions/Significance We establish a new dimension to the p53 master regulatory network where p53-mediated transcription from a ½ site RE can be determined by ER binding at one or more cis-acting EREs in manner that is dependent on level of ER protein, the type of ER ligand and the specific p53-inducing agent. PMID:20422012

Long-term survival of sorafenib-treated FLT3-ITD-positive acute myeloid leukaemia patients relapsing after allogeneic stem cell transplantation.

PubMed

Metzelder, S K; Schroeder, T; Lübbert, M; Ditschkowski, M; Götze, K; Scholl, S; Meyer, R G; Dreger, P; Basara, N; Fey, M F; Salih, H R; Finck, A; Pabst, T; Giagounidis, A; Kobbe, G; Wollmer, E; Finke, J; Neubauer, A; Burchert, A

2017-11-01

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive acute myeloid leukaemia (AML) relapsing after allogeneic stem cell transplantation (allo-SCT) has a dismal prognosis with limited therapeutic options. FLT3-ITD kinase inhibition is a reasonable but palliative experimental treatment alternative in this situation. Information on long-term outcome is not available. We performed a long-term follow-up analysis of a previously reported cohort of 29 FLT3-ITD-positive AML patients, which were treated in relapse after allo-SCT with sorafenib monotherapy. With a median follow-up of 7.5 years, 6 of 29 patients (21%) are still alive. Excluding one patient who received a second allo-SCT, five patients (17%) achieved sustained complete remissions with sorafenib. Four of these patients are in treatment-free remission for a median of 4.4 years. Sorafenib may enable cure of a proportion of very poor risk FLT3-ITD-positive AML relapsing after allo-SCT. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular Protection using Flt3 and PI3Kα inhibitors demonstrates multiple mechanisms of oxidative glutamate toxicity

PubMed Central

Kang, Yunyi; Tiziani, Stefano; Park, Goonho; Kaul, Marcus; Paternostro, Giovanni

2014-01-01

Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases. Here we identify small molecule inhibitors of this process. We screen a kinase inhibitor library on neuronal cells and identify Flt3 and PI3Kα inhibitors as potent protectors against glutamate toxicity. Both inhibitors prevented reactive oxygen species (ROS) generation, mitochondrial hyperpolarization, and lipid peroxidation in neuronal cells, but they do so by distinct molecular mechanisms. The PI3Kα inhibitor protects cells by inducing partial restoration of depleted glutathione levels and accumulation of intracellular amino acids, whereas the Flt3 inhibitor prevents lipid peroxidation, a key mechanism of glutamate-mediated toxicity. We also demonstrate that glutamate toxicity involves a combination of ferroptosis, necrosis, and AIF-dependent apoptosis. We confirm the protective effect by using multiple inhibitors of these kinases and multiple cell types. Our results not only identify compounds that protect against glutamate-stimulated oxidative stress, but also provide new insights into the mechanisms of glutamate toxicity in neurons. PMID:24739485

Design of FLT3 Inhibitor - Gold Nanoparticle Conjugates as Potential Therapeutic Agents for the Treatment of Acute Myeloid Leukemia.

PubMed

Simon, Timea; Tomuleasa, Ciprian; Bojan, Anca; Berindan-Neagoe, Ioana; Boca, Sanda; Astilean, Simion

2015-12-01

Releasing drug molecules at the targeted location could increase the clinical outcome of a large number of anti-tumor treatments which require low systemic damage and low side effects. Nano-carriers of drugs show great potential for such task due to their capability of accumulating and releasing their payload specifically, at the tumor site. FLT3 inhibitor - gold nanoparticle conjugates were fabricated to serve as vehicles for the delivery of anti-tumor drugs. Lestaurtinib, midostaurin, sorafenib, and quizartinib were selected among the FLT3 inhibitor drugs that are currently used in clinics for the treatment of acute myeloid leukemia. The drugs were loaded onto nanoparticle surface using a conjugation strategy based on hydrophobic-hydrophobic interactions with the Pluronic co-polymer used as nanoparticle surface coating. Optical absorption characterization of the particles in solution showed that FLT3 inhibitor-incorporated gold nanoparticles were uniformly distributed and chemically stable regardless of the drug content. Drug loading study revealed a high drug content in the case of midostaurin drug which also showed increased stability. Drug release test in simulated cancer cell conditions demonstrated more than 56 % release of the entrapped drug, a result that correlates well with the superior cytotoxicity of the nano-conjugates comparatively with the free drug. This is a pioneering study regarding the efficient loading of gold nanoparticles with selected FLT3 inhibitors. In vitro cytotoxicity assessment shows that FLT3-incorporated gold nanoparticles are promising candidates as vehicles for anti-tumor drugs and demonstrate superior therapeutic effect comparatively with the bare drugs.

Comparison of 4'-[methyl-(11)C]thiothymidine ((11)C-4DST) and 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT) PET/CT in human brain glioma imaging.

PubMed

Toyota, Yasunori; Miyake, Keisuke; Kawai, Nobuyuki; Hatakeyama, Tetsuhiro; Yamamoto, Yuka; Toyohara, Jun; Nishiyama, Yoshihiro; Tamiya, Takashi

2015-01-01

3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT) has been used to evaluate tumor malignancy and cell proliferation in human brain gliomas. However, (18)F-FLT has several limitations in clinical use. Recently, (11)C-labeled thymidine analogue, 4'-[methyl-(11)C]thiothymidine ((11)C-4DST), became available as an in vivo cell proliferation positron emission tomography (PET) tracer. The present study was conducted to evaluate the usefulness of (11)C-4DST PET in the diagnosis of human brain gliomas by comparing with the images of (18)F-FLT PET. Twenty patients with primary and recurrent brain gliomas underwent (18)F-FLT and (11)C-4DST PET scans. The uptake values in the tumors were evaluated using the maximum standardized uptake value (SUVmax), the tumor-to-normal tissue uptake (T/N) ratio, and the tumor-to-blood uptake (T/B) ratio. These values were compared among different glioma grades. Correlation between the Ki-67 labeling index and the uptake values of (11)C-4DST and (18)F-FLT in the tumor was evaluated using linear regression analysis. The relationship between the individual (18)F-FLT and (11)C-4DST uptake values in the tumors was also examined. (11)C-4DST uptake was significantly higher than that of (18)F-FLT in the normal brain. The uptake values of (11)C-4DST in the tumor were similar to those of (18)F-FLT resulting in better visualization with (18)F-FLT. No significant differences in the uptake values of (18)F-FLT and (11)C-4DST were noted among different glioma grades. Linear regression analysis showed a significant correlation between the Ki-67 labeling index and the T/N ratio of (11)C-4DST (r = 0.50, P < 0.05) and (18)F-FLT (r = 0.50, P < 0.05). Significant correlations were also found between the Ki-67 labeling index and the T/B ratio of (11)C-4DST (r = 0.52, P < 0.05) and (18)F-FLT (r = 0.55, P < 0.05). A highly significant correlation was observed between the individual T/N ratio of (11)C-4DST and (18)F-FLT in the tumor (r�

A phase I/II study of sunitinib and intensive chemotherapy in patients over 60 years of age with acute myeloid leukaemia and activating FLT3 mutations.

PubMed

Fiedler, Walter; Kayser, Sabine; Kebenko, Maxim; Janning, Melanie; Krauter, Jürgen; Schittenhelm, Marcus; Götze, Katharina; Weber, Daniela; Göhring, Gudrun; Teleanu, Veronica; Thol, Felicitas; Heuser, Michael; Döhner, Konstanze; Ganser, Arnold; Döhner, Hartmut; Schlenk, Richard F

2015-06-01

Acute myeloid leukaemia (AML) with FLT3 mutation has a dismal prognosis in elderly patients. Treatment with a combination of FLT3 inhibitors and standard chemotherapy has not been extensively studied. Therefore, we instigated a phase I/II clinical trial of chemotherapy with cytosine arabinoside (Ara-C)/daunorubicin induction (7+3) followed by three cycles of intermediate-dose Ara-C consolidation in 22 AML patients with activating FLT3 mutations. Sunitinib was added at predefined dose levels and as maintenance therapy for 2 years. At dose level 1, sunitinib 25 mg daily continuously from day 1 onwards resulted in two cases with dose-limiting toxicity (DLT), prolonged haemotoxicity and hand-foot syndrome. At dose level -1, sunitinib 25 mg was restricted to days 1-7 of each chemotherapy cycle. One DLT was observed in six evaluable patients. Six additional patients were treated in an extension phase. Thirteen of 22 patients (59%; 8/14 with FLT3-internal tandem duplication and 5/8 with FLT3-tyrosine kinase domain) achieved a complete remission/complete remission with incomplete blood count recovery. For the 17 patients included at the lower dose level, median overall, relapse-free and event-free survival were 1·6, 1·0 and 0·4 years, respectively. Four out of five analysed patients with relapse during maintenance therapy lost their initial FLT3 mutation, suggesting outgrowth of FLT3 wild-type subclones. © 2015 John Wiley & Sons Ltd.

Lower frequency of NPM1 and FLT3-ITD mutations in a South African adult de novo AML cohort

PubMed Central

Marshall, R. C.; Tlagadi, A.; Bronze, M.; Kana, V.; Naidoo, S.; Wiggill, T. M.; Carmona, S. C.

2014-01-01

Introduction Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of haemopoietic progenitor cells diagnosed in individuals of any age, but with a median age of 67 years at presentation in adults. Assessment of the mutation status of Nucleophosmin protein-1 (NPM1) and FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) are essential for the diagnosis, prognosis and treatment of AML. Methods A total of 160 de novo AML cases, both cytogenetically normal and abnormal, were analyzed for the presence of NPM1 and FLT3-ITD mutations and the results assessed in conjunction with epidemiological, clinical and laboratory findings. Results NPM1 mutations were found in 7.5%, while FLT3-ITD was present in 12% of these cases. Both of these were lower than expected. The median age at diagnosis of AML was 41 years and for the FLT3-ITD only cases, median age was 33 years; these ages were younger than expected. Conclusion The lower reported frequencies and younger median age at diagnosis of AML and these specific mutations may be contributed to by a number of factors including; effects of race on age of presentation, inclusion of patients diagnosed with de novo AML only and a generally younger median age of the South African population. PMID:24666762

Does FLT3 mutation impact survival after hematopoietic stem cell transplantation for acute myeloid leukemia? A Center for International Blood and Marrow Transplant Research (CIBMTR) analysis.

PubMed

Deol, Abhinav; Sengsayadeth, Salyka; Ahn, Kwang Woo; Wang, Hai-Lin; Aljurf, Mahmoud; Antin, Joseph Harry; Battiwalla, Minoo; Bornhauser, Martin; Cahn, Jean-Yves; Camitta, Bruce; Chen, Yi-Bin; Cutler, Corey S; Gale, Robert Peter; Ganguly, Siddhartha; Hamadani, Mehdi; Inamoto, Yoshihiro; Jagasia, Madan; Kamble, Rammurti; Koreth, John; Lazarus, Hillard M; Liesveld, Jane; Litzow, Mark R; Marks, David I; Nishihori, Taiga; Olsson, Richard F; Reshef, Ran; Rowe, Jacob M; Saad, Ayman A; Sabloff, Mitchell; Schouten, Harry C; Shea, Thomas C; Soiffer, Robert J; Uy, Geoffrey L; Waller, Edmond K; Wiernik, Peter H; Wirk, Baldeep; Woolfrey, Ann E; Bunjes, Donald; Devine, Steven; de Lima, Marcos; Sandmaier, Brenda M; Weisdorf, Dan; Khoury, Hanna Jean; Saber, Wael

2016-10-01

Patients with FMS like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML) have a poor prognosis and are referred for early allogeneic hematopoietic stem cell transplantation (HCT). Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) were used to evaluate 511 adult patients with de novo AML who underwent HCT during 2008 through 2011 to determine whether FLT3 mutations had an impact on HCT outcomes. In total, 158 patients (31%) had FLT3 mutations. Univariate and multivariate analyses revealed an increased risk of relapse at 3 years in the FLT3 mutated group compared with the wild-type (WT) group (38% [95% confidence interval (CI), 30%-45%] vs 28% [95% CI, 24%-33%]; P = .04; relative risk, 1.60 [95% CI, 1.15-2.22]; P = .0048). However, FLT3 mutation status was not significantly associated with nonrelapse mortality, leukemia-free survival, or overall survival. Although more patients in the FLT3 mutated group died from relapsed primary disease compared with those in the WT group (60% vs 46%), the 3-year overall survival rate was comparable for the 2 groups (mutated group: 49%; 95% CI, 40%-57%; WT group: 55%, 95% CI, 50%-60%; P = .20). The current data indicate that FLT3 mutation status did not adversely impact overall survival after HCT, and about 50% of patients with this mutation who underwent HCT were long-term survivors. Cancer 2016;122:3005-3014. © 2016 American Cancer Society. © 2016 American Cancer Society.

Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells

PubMed Central

Weisberg, Ellen; Banerji, Lolita; Wright, Renee D.; Barrett, Rosemary; Ray, Arghya; Moreno, Daisy; Catley, Laurence; Jiang, Jingrui; Hall-Meyers, Elizabeth; Sauveur-Michel, Maira; Stone, Richard; Galinsky, Ilene; Fox, Edward; Kung, Andrew L.

2008-01-01

Mediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo. PMID:18184863

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

PubMed Central

Zhang, Xin; Nelson, Erik; Sattler, Martin; Liu, Feiyang; Nicolais, Maria; Zhang, Jianming; Mitsiades, Constantine; Smith, Robert W.; Stone, Richard; Galinsky, Ilene; Nonami, Atsushi; Griffin, James D.; Gray, Nathanael

2013-01-01

Objectives Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment. PMID:23437141

[Use of sFlt-1/PlGF ratio in preeclampsia : a monocentric retrospective analysis].

PubMed

Verbeurgt, L; Chantraine, F; De Marchin, J; Minon, J-M; Nisolle, M

2017-09-01

Soluble Fms-like tyrosine kinase 1 (sFlt-1) is an anti-angiogenic factor released in higher amounts in preeclampsia and implicated in endothelial dysfunction. sFlt-1/PlGF ratio is used in the prediction of preeclampsia. An sFlt-1/PlGF ratio inferior to 38 predicts the short-term absence of preeclampsia. A ratio ? 85 (early-onset PE) or ? 110 (late-onset of PE) could diagnose preeclampsia. In this study, sFlt-1/PlGF ratio has been measured in 183 patients. Sixty-seven preeclampsia have been diagnosed preeclamptic at delivery. The median sFlt-1/PlGF ratio was 100.3. The median ratio among women with preeclampsia (N=67) versus no preeclampsia (N=116) was 212.7 versus 35.4. In accordance with this analysis, an sFlt-1/PlGF ratio ? 38 has a sensibility of 95,5 % and a specificity of 73.3 %. The positive predictive value and the negative predictive value were 67.4 % and 96.6 %, respectively. These results suggest that sFlt-1/PlGF ratio is helpful in the diagnosis of preeclampsia.

Optimization of Imidazo[4,5-b]pyridine-Based Kinase Inhibitors: Identification of a Dual FLT3/Aurora Kinase Inhibitor as an Orally Bioavailable Preclinical Development Candidate for the Treatment of Acute Myeloid Leukemia

PubMed Central

2012-01-01

Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A Kd = 7.5 nM, Aurora-B Kd = 48 nM), FLT3 kinase (Kd = 6.2 nM), and FLT3 mutants including FLT3-ITD (Kd = 38 nM) and FLT3(D835Y) (Kd = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20–35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4–11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children. PMID:23043539

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia

PubMed Central

Nogami, Ayako; Oshikawa, Gaku; Okada, Keigo; Fukutake, Shusaku; Umezawa, Yoshihiro; Nagao, Toshikage; Kurosu, Tetsuya; Miura, Osamu

2015-01-01

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4–11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway. PMID:25826077

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.

PubMed

Nogami, Ayako; Oshikawa, Gaku; Okada, Keigo; Fukutake, Shusaku; Umezawa, Yoshihiro; Nagao, Toshikage; Kurosu, Tetsuya; Miura, Osamu

2015-04-20

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4-11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

Flt1/VEGFR1 heterozygosity causes transient embryonic edema.

PubMed

Otowa, Yasunori; Moriwaki, Kazumasa; Sano, Keigo; Shirakabe, Masanori; Yonemura, Shigenobu; Shibuya, Masabumi; Rossant, Janet; Suda, Toshio; Kakeji, Yoshihiro; Hirashima, Masanori

2016-06-02

Vascular endothelial growth factor-A is a major player in vascular development and a potent vascular permeability factor under physiological and pathological conditions by binding to a decoy receptor Flt1 and its primary receptor Flk1. In this study, we show that Flt1 heterozygous (Flt1(+/-)) mouse embryos grow up to adult without life-threatening abnormalities but exhibit a transient embryonic edema around the nuchal and back regions, which is reminiscent of increased nuchal translucency in human fetuses. Vascular permeability is enhanced and an intricate infolding of the plasma membrane and huge vesicle-like structures are seen in Flt1(+/-) capillary endothelial cells. Flk1 tyrosine phosphorylation is elevated in Flt1(+/-) embryos, but Flk1 heterozygosity does not suppress embryonic edema caused by Flt1 heterozygosity. When Flt1 mutants are crossed with Aspp1(-/-) mice which exhibit a transient embryonic edema with delayed formation and dysfunction of lymphatic vessels, only 5.7% of Flt1(+/-); Aspp1(-/-) mice survive, compared to expected ratio (25%). Our results demonstrate that Flt1 heterozygosity causes a transient embryonic edema and can be a risk factor for embryonic lethality in combination with other mutations causing non-lethal vascular phenotype.

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

NASA Astrophysics Data System (ADS)

Poulsen, Anders; William, Anthony; Blanchard, Stéphanie; Lee, Angeline; Nagaraj, Harish; Wang, Haishan; Teo, Eeling; Tan, Evelyn; Goh, Kee Chuan; Dymock, Brian

2012-04-01

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

Gelatin-coated Gold Nanoparticles as Carriers of FLT3 Inhibitors for Acute Myeloid Leukemia Treatment.

PubMed

Suarasan, Sorina; Simon, Timea; Boca, Sanda; Tomuleasa, Ciprian; Astilean, Simion

2016-06-01

This study presents the design of a gold nanoparticle (AuNPs)-drug system with improved efficiency for the treatment of acute myeloid leukemia. The system is based on four different FLT3 inhibitors, namely midostaurin, sorafenib, lestaurtinib, and quizartinib, which were independently loaded onto gelatin-coated gold nanoparticles. Detailed investigation of the physicochemical properties of the formed complexes lead to the selection of quizartinib-loaded AuNPs for the in vitro evaluation of the biological effects of the formed complex against OCI-AML3 acute myeloid leukemia cells. Viability tests by MTT demonstrated that the proposed drug complex has improved efficacy when compared with the drug alone. The obtained results constitute a premise for further in vivo investigation of such drug vehicles based on AuNPs. To the best of our knowledge, this is the first study that investigates the delivery of the above-mentioned FLT3 inhibitors via gelatin-coated gold nanoparticles. © 2016 John Wiley & Sons A/S.

Imaging proliferation in brain tumors with 18F-FLT PET: comparison with 18F-FDG.

PubMed

Chen, Wei; Cloughesy, Timothy; Kamdar, Nirav; Satyamurthy, Nagichettiar; Bergsneider, Marvin; Liau, Linda; Mischel, Paul; Czernin, Johannes; Phelps, Michael E; Silverman, Daniel H S

2005-06-01

3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a recently developed PET tracer to image tumor cell proliferation. We characterized (18)F-FLT PET of brain gliomas and compared (18)F-FLT with (18)F-FDG PET in side-by-side studies of the same patients. Twenty-five patients with newly diagnosed or previously treated glioma underwent PET with (18)F-FLT and (18)F-FDG on consecutive days. Three stable patients in long-term remission were included as negative control subjects. Tracer kinetics in normal brain and tumor were measured. Uptake of (18)F-FLT and (18)F-FDG was quantified by the standardized uptake value (SUV) and the tumor-to-normal tissue (T/N) ratio. The accuracy of (18)F-FLT and (18)F-FDG PET in evaluating newly diagnosed and recurrent gliomas was compared. More than half of the patients underwent resection after the PET study and correlations between PET uptake and the Ki-67 proliferation index were examined. Patients were monitored for a mean of 15.4 mo (range, 12-20 mo). The predictive power of PET for tumor progression and survival was analyzed using Kaplan-Meier statistics. (18)F-FLT uptake in tumors was rapid, peaking at 5-10 min after injection and remaining stable up to 75 min. Hence, a 30-min scan beginning at 5 min after injection was sufficient for imaging. (18)F-FLT visualized all high-grade (grade III or IV) tumors. Grade II tumor did not show appreciable (18)F-FLT uptake and neither did the stable lesions. The absolute uptake of (18)F-FLT was low (maximum-pixel SUV [SUV(max)], 1.33) but image contrast was better than with (18)F-FDG (T/N ratio, 3.85 vs. 1.49). (18)F-FDG PET studies were negative in 5 patients with recurrent high-grade glioma who subsequently suffered tumor progression within 1-3 mo. (18)F-FLT SUV(max) correlated more strongly with Ki-67 index (r = 0.84; P < 0.0001) than (18)F-FDG SUV(max) (r = 0.51; P = 0.07). (18)F-FLT uptake also had more significant predictive power with respect to tumor progression and survival (P = 0

Quizartinib, an FLT3 inhibitor, as monotherapy in patients with relapsed or refractory acute myeloid leukaemia: an open-label, multicentre, single-arm, phase 2 trial.

PubMed

Cortes, Jorge; Perl, Alexander E; Döhner, Hartmut; Kantarjian, Hagop; Martinelli, Giovanni; Kovacsovics, Tibor; Rousselot, Philippe; Steffen, Björn; Dombret, Hervé; Estey, Elihu; Strickland, Stephen; Altman, Jessica K; Baldus, Claudia D; Burnett, Alan; Krämer, Alwin; Russell, Nigel; Shah, Neil P; Smith, Catherine C; Wang, Eunice S; Ifrah, Norbert; Gammon, Guy; Trone, Denise; Lazzaretto, Deborah; Levis, Mark

2018-05-30

Old age and FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukaemia are associated with early relapse and poor survival. Quizartinib is an oral, highly potent, and selective next-generation FLT3 inhibitor with clinical antileukaemic activity in relapsed or refractory acute myeloid leukaemia. We aimed to assess the efficacy and safety of single-agent quizartinib in patients with relapsed or refractory acute myeloid leukaemia. We did an open-label, multicentre, single-arm, phase 2 trial at 76 hospitals and cancer centres in the USA, Europe, and Canada. We enrolled patients with morphologically documented primary acute myeloid leukaemia or acute myeloid leukaemia secondary to myelodysplastic syndromes and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 into two predefined, independent cohorts: patients who were aged 60 years or older with relapsed or refractory acute myeloid leukaemia within 1 year after first-line therapy (cohort 1), and those who were 18 years or older with relapsed or refractory disease following salvage chemotherapy or haemopoietic stem cell transplantation (cohort 2). Patients with an FLT3-ITD allelic frequency of more than 10% were considered as FLT3-ITD positive, whereas all other patients were considered as FLT3-ITD negative. Patients received quizartinib once daily as an oral solution; the initial 17 patients received 200 mg per day but the QTcF interval was prolonged for more than 60 ms above baseline in some of these patients. Subsequently, doses were amended for all patients to 135 mg per day for men and 90 mg per day for women. The co-primary endpoints were the proportion of patients who achieved a composite complete remission (defined as complete remission + complete remission with incomplete platelet recovery + complete remission with incomplete haematological recovery) and the proportion of patients who achieved a complete remission. Efficacy

FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway.

PubMed

Okada, Keigo; Nogami, Ayako; Ishida, Shinya; Akiyama, Hiroki; Chen, Cheng; Umezawa, Yoshihiro; Miura, Osamu

2018-02-06

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that activation of STAT5 confers resistance to PI3K/Akt inhibitors on the FLT3-ITD-positive AML cell line MV4-11 and 32D cells driven by FLT3-ITD (32D/ITD) but not by FLT3 mutated in the tyrosine kinase domain (32D/TKD). Here, we report the involvement of Pim kinases expressed through STAT5 activation in acquisition of this resistance. The specific pan-Pim kinase inhibitor AZD1208 as well as PIM447 in combination with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 cooperatively downregulated the mTORC1/4EBP1 pathway, formation of the eIF4E/eIF4G complex, and Mcl-1 expression leading to activation of Bak and Bax to induce caspase-dependent apoptosis synergistically in these cells. These cooperative effects were enhanced or inhibited by knock down of mTOR or expression of its activated mutant, respectively. Overexpression of Mcl-1 conferred the resistance on 32D/ITD cells to combined inhibition of the PI3K/Akt pathway and Pim kinases, while the Mcl-1-specific BH3 mimetic A-1210477 conquered the resistance of MV4-11 cells to GDC-0941. Furthermore, overexpression of Pim-1 in 32D/TKD enhanced the mTORC1/Mcl-1 pathway and partially protected it from the PI3K/Akt inhibitors or the FLT3 inhibitor gilteritinib to confer the resistance to PI3K/Akt inhibitors. Finally, AZD1208 and GDC-0941 cooperatively inhibited the mTORC1/Mcl-1 pathway and reduced viable cell numbers of primary AML cells from some FLT3-ITD positive cases. Thus, Pim kinases may protect the mTORC1/4EBP1/Mcl-1 pathway to confer the resistance to the PI3K/Akt inhibitors on FLT3-ITD cells and represent promising therapeutic targets.

Haematopoietic cell transplantation with and without sorafenib maintenance for patients with FLT3-ITD acute myeloid leukaemia in first complete remission.

PubMed

Brunner, Andrew M; Li, Shuli; Fathi, Amir T; Wadleigh, Martha; Ho, Vincent T; Collier, Kerry; Connolly, Christine; Ballen, Karen K; Cutler, Corey S; Dey, Bimalangshu R; El-Jawahri, Areej; Nikiforow, Sarah; McAfee, Steven L; Koreth, John; Deangelo, Daniel J; Alyea, Edwin P; Antin, Joseph H; Spitzer, Thomas R; Stone, Richard M; Soiffer, Robert J; Chen, Yi-Bin

2016-11-01

We performed a retrospective study analysing the effect of sorafenib, an oral fms-Like Tyrosine Kinase 3 (FLT3)/multikinase inhibitor, as post-transplant maintenance in adult patients with FLT3-internal tandem duplication (ITD) acute myeloid leukaemia (AML). We identified consecutive patients with FLT3-ITD AML diagnosed between 2008 and 2014 who received haematopoietic cell transplantation (HCT) in first complete remission (CR1). Post-HCT initiation of sorafenib (yes/no) was evaluated as a time-varying covariate in the overall survival/progression-free survival (OS/PFS) analysis and we performed a landmark analysis of controls alive without relapse at the median date of sorafenib initiation. We identified 26 sorafenib patients and 55 controls. Median follow-up was 27·2 months post-HCT for sorafenib survivors, and 38·4 months for controls (P = 0·021). The median time to initiating sorafenib was 68 days post-HCT; 43 controls were alive without relapse at this cut-off. Sorafenib patients had improved 2-year OS in the d+68 landmark analysis (81% vs. 62%, P = 0·029). Sorafenib was associated with improved 2-year PFS (82% vs. 53%, P = 0·0081) and lower 2-year cumulative incidence of relapse (8·2% vs. 37·7%, P = 0·0077). In multivariate analysis, sorafenib significantly improved OS [Hazard ratio (HR) 0·26, P = 0·021] and PFS (HR 0·25, P = 0·016). There was no difference in 2-year non-relapse mortality (9·8% vs. 9·3%, P = 0·82) or 1-year chronic graft-versus-host disease (55·5% vs. 37·2%, P = 0·28). These findings suggest potential benefit of post-HCT sorafenib in FLT3-ITD AML, and support further evaluation of post-HCT FLT3 inhibition. © 2016 John Wiley & Sons Ltd.

A Robust Error Model for iTRAQ Quantification Reveals Divergent Signaling between Oncogenic FLT3 Mutants in Acute Myeloid Leukemia*

PubMed Central

Zhang, Yi; Askenazi, Manor; Jiang, Jingrui; Luckey, C. John; Griffin, James D.; Marto, Jarrod A.

2010-01-01

The FLT3 receptor tyrosine kinase plays an important role in normal hematopoietic development and leukemogenesis. Point mutations within the activation loop and in-frame tandem duplications of the juxtamembrane domain represent the most frequent molecular abnormalities observed in acute myeloid leukemia. Interestingly these gain-of-function mutations correlate with different clinical outcomes, suggesting that signals from constitutive FLT3 mutants activate different downstream targets. In principle, mass spectrometry offers a powerful means to quantify protein phosphorylation and identify signaling events associated with constitutively active kinases or other oncogenic events. However, regulation of individual phosphorylation sites presents a challenging case for proteomics studies whereby quantification is based on individual peptides rather than an average across different peptides derived from the same protein. Here we describe a robust experimental framework and associated error model for iTRAQ-based quantification on an Orbitrap mass spectrometer that relates variance of peptide ratios to mass spectral peak height and provides for assignment of p value, q value, and confidence interval to every peptide identification, all based on routine measurements, obviating the need for detailed characterization of individual ion peaks. Moreover, we demonstrate that our model is stable over time and can be applied in a manner directly analogous to ubiquitously used external mass calibration routines. Application of our error model to quantitative proteomics data for FLT3 signaling provides evidence that phosphorylation of tyrosine phosphatase SHP1 abrogates the transformative potential, but not overall kinase activity, of FLT3-D835Y in acute myeloid leukemia. PMID:20019052

Soluble Flt-1 links microvascular disease with heart failure in CKD.

PubMed

Di Marco, Giovana S; Kentrup, Dominik; Reuter, Stefan; Mayer, Anna B; Golle, Lina; Tiemann, Klaus; Fobker, Manfred; Engelbertz, Christiane; Breithardt, Günter; Brand, Eva; Reinecke, Holger; Pavenstädt, Hermann; Brand, Marcus

2015-05-01

Chronic kidney disease (CKD) is associated with an increased risk of heart failure (HF). Elevated plasma concentrations of soluble Flt-1 (sFlt-1) have been linked to cardiovascular disease in CKD patients, but whether sFlt-1 contributes to HF in CKD is still unknown. To provide evidence that concludes a pathophysiological role of sFlt-1 in CKD-associated HF, we measured plasma sFlt-1 concentrations in 586 patients with angiographically documented coronary artery disease and renal function classified according to estimated glomerular filtration rate (eGFR). sFlt-1 concentrations correlated negatively with eGFR and were associated with signs of heart failure, based on New York Heart Association functional class and reduced left ventricular ejection fraction (LVEF), and early mortality. Additionally, rats treated with recombinant sFlt-1 showed a 15 % reduction in LVEF and a 29 % reduction in cardiac output compared with control rats. High sFlt-1 concentrations were associated with a 15 % reduction in heart capillary density (number of vessels/cardiomyocyte) and a 24 % reduction in myocardial blood volume. Electron microscopy and histological analysis revealed mitochondrial damage and interstitial fibrosis in the hearts of sFlt-1-treated, but not control rats. In 5/6-nephrectomised rats, an animal model of CKD, sFlt-1 antagonism with recombinant VEGF121 preserved heart microvasculature and significantly improved heart function. Overall, these findings suggest that a component of cardiovascular risk in CKD patients could be directly attributed to sFlt-1. Assessment of patients with CKD confirmed that sFlt-1 concentrations were inversely correlated with renal function, while studies in rats suggested that sFlt-1 may link microvascular disease with HF in CKD.

FLT-1 gene polymorphisms and protein expression profile in rheumatoid arthritis

PubMed Central

Paradowska-Gorycka, Agnieszka; Sowinska, Anna; Pawlik, Andrzej; Malinowski, Damian; Stypinska, Barbara; Haladyj, Ewa; Romanowska-Prochnicka, Katarzyna; Olesinska, Marzena

2017-01-01

Objectives Inflammation and angiogenesis are a significant element of pathogenesis in rheumatoid arthritis (RA). The FLT-1- triggering factor for production of proinflammatory cytokines-might contributes to inflammation in patients with RA. Association of the FLT-1 polymorphisms with different “angiogenic diseases” suggests that it may be a novel genetic risk factor also for RA. The aim of the study was to identify FLT-1 genetic variants and their possible association with sFLT-1 levels, susceptibility to and severity of RA. Methods The FLT-1 gene polymorphisms were genotyped for 471 RA patients and 684 healthy individuals. Correlation analysis was performed with clinical parameters, cardiovascular disease (CVD) and anti-citrullinated peptide/protein antibody (ACPA) presence. The sFLT-1 serum levels were evaluated. Results The FLT-1 gene polymorphisms showed no significant differences in the proportion of cases and controls. Furthermore, the FLT-1 rs2296188 T/C polymorphism was associated with ACPA-positive RA. Overall, rs9943922 T/C and rs2296283 G/A are in almost completed linkage disequilibrium (LD) with D’ = 0.97 and r2 = 0.83. The FLT-1 rs7324510 A allele has shown association with VAS score (p = 0.035), DAS-28 score (p = 0.013) and ExRA presence (p = 0.027). Moreover, other clinical parameters were also higher in RA patients with this allele. In addition, FLT-1 genetic variants conferred higher sFLT-1 levels in RA patients compared to controls. Conclusion FLT-1 rs7324510 C/A variant may be a new genetic risk factor for severity of RA. Examined factor highly predispose to more severe disease activity as well as higher sFLT-1 levels in RA. PMID:28323906

Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1-2 study.

PubMed

Perl, Alexander E; Altman, Jessica K; Cortes, Jorge; Smith, Catherine; Litzow, Mark; Baer, Maria R; Claxton, David; Erba, Harry P; Gill, Stan; Goldberg, Stuart; Jurcic, Joseph G; Larson, Richard A; Liu, Chaofeng; Ritchie, Ellen; Schiller, Gary; Spira, Alexander I; Strickland, Stephen A; Tibes, Raoul; Ustun, Celalettin; Wang, Eunice S; Stuart, Robert; Röllig, Christoph; Neubauer, Andreas; Martinelli, Giovanni; Bahceci, Erkut; Levis, Mark

2017-08-01

Internal tandem duplication mutations in FLT3 are common in acute myeloid leukaemia and are associated with rapid relapse and short overall survival. The clinical benefit of FLT3 inhibitors in patients with acute myeloid leukaemia has been limited by rapid generation of resistance mutations, particularly in codon Asp835 (D835). We aimed to assess the highly selective oral FLT3 inhibitor gilteritinib in patients with relapsed or refractory acute myeloid leukaemia. In this phase 1-2 trial, we enrolled patients aged 18 years or older with acute myeloid leukaemia who either were refractory to induction therapy or had relapsed after achieving remission with previous treatment. Patients were enrolled into one of seven dose-escalation or dose-expansion cohorts assigned to receive once-daily doses of oral gilteritinib (20 mg, 40 mg, 80 mg, 120 mg, 200 mg, 300 mg, or 450 mg). Cohort expansion was based on safety and tolerability, FLT3 inhibition in correlative assays, and antileukaemic activity. Although the presence of an FLT3 mutation was not an inclusion criterion, we required ten or more patients with locally confirmed FLT3 mutations (FLT3 mut+ ) to be enrolled in expansion cohorts at each dose level. On the basis of emerging findings, we further expanded the 120 mg and 200 mg dose cohorts to include FLT3 mut+ patients only. The primary endpoints were the safety, tolerability, and pharmacokinetics of gilteritinib. Safety and tolerability were assessed in the safety analysis set (all patients who received at least one dose of gilteritinib). Responses were assessed in the full analysis set (all patients who received at least one dose of study drug and who had at least one datapoint post-treatment). Pharmacokinetics were assessed in a subset of the safety analysis set for which sufficient data for concentrations of gilteritinib in plasma were available to enable derivation of one or more pharmacokinetic variables. This study is registered with ClinicalTrials.gov, number

Dynamic PET evaluation of elevated FLT level after sorafenib treatment in mice bearing human renal cell carcinoma xenograft.

PubMed

Ukon, Naoyuki; Zhao, Songji; Yu, Wenwen; Shimizu, Yoichi; Nishijima, Ken-Ichi; Kubo, Naoki; Kitagawa, Yoshimasa; Tamaki, Nagara; Higashikawa, Kei; Yasui, Hironobu; Kuge, Yuji

2016-12-01

Sorafenib, an oral multikinase inhibitor, has anti-proliferative and anti-angiogenic activities and is therapeutically effective against renal cell carcinoma (RCC). Recently, we have evaluated the tumor responses to sorafenib treatment in a RCC xenograft using [Methyl- 3 H(N)]-3'-fluoro-3'-deoxythythymidine ([ 3 H]FLT). Contrary to our expectation, the FLT level in the tumor significantly increased after the treatment. In this study, to clarify the reason for the elevated FLT level, dynamic 3'-[ 18 F]fluoro-3'-deoxythymidine ([ 18 F]FLT) positron emission tomography (PET) and kinetic studies were performed in mice bearing a RCC xenograft (A498). The A498 xenograft was established in nude mice, and the mice were assigned to the control (n = 5) and treatment (n = 5) groups. The mice in the treatment group were orally given sorafenib (20 mg/kg/day p.o.) once daily for 3 days. Twenty-four hours after the treatment, dynamic [ 18 F]FLT PET was performed by small-animal PET. Three-dimensional regions of interest (ROIs) were manually defined for the tumors. A three-compartment model fitting was carried out to estimate four rate constants using the time activity curve (TAC) in the tumor and the blood clearance rate of [ 18 F]FLT. The dynamic pattern of [ 18 F]FLT levels in the tumor significantly changed after the treatment. The rate constant of [ 18 F]FLT phosphorylation (k 3 ) was significantly higher in the treatment group (0.111 ± 0.027 [1/min]) than in the control group (0.082 ± 0.009 [1/min]). No significant changes were observed in the distribution volume, the ratio of [ 18 F]FLT forward transport (K 1 ) to reverse transport (k 2 ), between the two groups (0.556 ± 0.073 and 0.641 ± 0.052 [mL/g] in the control group). Our dynamic PET studies indicated that the increase in FLT level may be caused by the phosphorylation of FLT in the tumor after the sorafenib treatment in the mice bearing a RCC xenograft. Dynamic PET studies with kinetic

Vascular Repair After Menstruation Involves Regulation of Vascular Endothelial Growth Factor-Receptor Phosphorylation by sFLT-1

PubMed Central

Graubert, Michael D.; Asuncion Ortega, Maria; Kessel, Bruce; Mortola, Joseph F.; Iruela-Arispe, M. Luisa

2001-01-01

Regeneration of the endometrium after menstruation requires a rapid and highly organized vascular response. Potential regulators of this process include members of the vascular endothelial growth factor (VEGF) family of proteins and their receptors. Although VEGF expression has been detected in the endometrium, the relationship between VEGF production, receptor activation, and endothelial cell proliferation during the endometrial cycle is poorly understood. To better ascertain the relevance of VEGF family members during postmenstrual repair, we have evaluated ligands, receptors, and activity by receptor phosphorylation in human endometrium throughout the menstrual cycle. We found that VEGF is significantly increased at the onset of menstruation, a result of the additive effects of hypoxia, transforming growth factor-α, and interleukin-1β. Both VEGF receptors, FLT-1 and KDR, followed a similar pattern. However, functional activity of KDR, as determined by phosphorylation studies, revealed activation in the late menstrual and early proliferative phases. The degree of KDR phosphorylation was inversely correlated with the presence of sFLT-1. Endothelial cell proliferation analysis in endometrium showed a peak during the late menstrual and early proliferative phases in concert with the presence of VEGF, VEGF receptor phosphorylation, and decrease of sFLT-1. Together, these results suggest that VEGF receptor activation and the subsequent modulation of sFLT-1 in the late menstrual phase likely contributes to the onset of angiogenesis and endothelial repair in the human endometrium. PMID:11290558

[18F]-FLT Positron Emission Tomography can be used to image the response of sensitive tumors to PI3-Kinase inhibition with the novel agent GDC-0941

PubMed Central

Cawthorne, Christopher; Burrows, Natalie; Gieling, Roben G.; Morrow, Christopher J; Forster, Duncan; Gregory, Jamil; Radigois, Marc; Smigova, Alison; Babur, Muhammad; Simpson, Kathryn; Hodgkinson, Cassandra; Brown, Gavin; McMahon, Adam; Dive, Caroline; Hiscock, Duncan; Wilson, Ian; Williams, Kaye J

2013-01-01

The Phosphatidylinositide 3-kinase (PI3-K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the PET tracer 3′-Deoxy-3′-[18F]fluorothymidine ([18F]-FLT) is suitable to mark the effect of the novel PI-3K inhibitor GDC-0941 which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [18F]-FLT and at acute (18h) and chronic (186h) timepoints after twice-daily administration of GDC-0941 (50mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3-K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [18F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3-K p85 subunit Δp85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [18F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute timepoint, and correlated with pharmacodynamic markers of PI3-K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3-K signaling via expression of Δp85α significantly reduced tumor growth and [18F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action. PMID:23427298

[18F]-FLT positron emission tomography can be used to image the response of sensitive tumors to PI3-kinase inhibition with the novel agent GDC-0941.

PubMed

Cawthorne, Christopher; Burrows, Natalie; Gieling, Roben G; Morrow, Christopher J; Forster, Duncan; Gregory, Jamil; Radigois, Marc; Smigova, Alison; Babur, Muhammad; Simpson, Kathryn; Hodgkinson, Cassandra; Brown, Gavin; McMahon, Adam; Dive, Caroline; Hiscock, Duncan; Wilson, Ian; Williams, Kaye J

2013-05-01

The phosphoinositide 3-kinase (PI3K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the positron emission tomography tracer 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]-FLT) is suitable to mark the effect of the novel PI3K inhibitor GDC-0941, which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [(18)F]-FLT and at acute (18 hours) and chronic (186 hours) time points after twice-daily administration of GDC-0941 (50 mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [(18)F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3K p85 subunit p85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [(18)F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute time point and correlated with pharmacodynamic markers of PI3K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3K signaling via expression of Δp85α significantly reduced tumor growth and [(18)F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [(18)F]-FLT is a strong candidate for the noninvasive measurement of GDC-0941 action. ©2013 AACR

Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications

PubMed Central

Fan, Xiujun; Rai, Anshita; Kambham, Neeraja; Sung, Joyce F.; Singh, Nirbhai; Petitt, Matthew; Dhal, Sabita; Agrawal, Rani; Sutton, Richard E.; Druzin, Maurice L.; Gambhir, Sanjiv S.; Ambati, Balamurali K.; Cross, James C.; Nayak, Nihar R.

2014-01-01

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications. PMID:25329693

Spatiotemporal Stability of Cu-ATSM and FLT Positron Emission Tomography Distributions During Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradshaw, Tyler J.; Yip, Stephen; Jallow, Ngoneh

2014-06-01

Purpose: In dose painting, in which functional imaging is used to define biological targets for radiation therapy dose escalation, changes in spatial distributions of biological properties during treatment can compromise the quality of therapy. The goal of this study was to assess the spatiotemporal stability of 2 potential dose painting targets—hypoxia and proliferation—in canine tumors during radiation therapy. Methods and Materials: Twenty-two canine patients with sinonasal tumors (14 carcinoma and 8 sarcoma) were imaged before hypofractionated radiation therapy with copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) positron emission tomography/computed tomography (PET/CT) for hypoxia and 3′-deoxy-3′-{sup 18}F-fluorothymidine (FLT) PET/CT for proliferation. The FLT scans were repeatedmore » after 2 fractions and the Cu-ATSM scans after 3 fractions. Midtreatment PET/CT images were deformably registered to pretreatment PET/CT images. Voxel-based Spearman correlation coefficients quantified the spatial stability of Cu-ATSM and FLT uptake distributions between pretreatment and midtreatment scans. Paired t tests determined significant differences between the patients' respective Cu-ATSM and FLT correlations coefficients. Standardized uptake value measures were also compared between pretreatment and midtreatment scans by use of paired t tests. Results: Spatial distributions of Cu-ATSM and FLT uptake were stable through midtreatment for both sarcomas and carcinomas: the population mean ± standard deviation in Spearman correlation coefficient was 0.88 ± 0.07 for Cu-ATSM and 0.79 ± 0.13 for FLT. The patients' Cu-ATSM correlation coefficients were significantly higher than their respective FLT correlation coefficients (P=.001). Changes in Cu-ATSM SUV measures from pretreatment to midtreatment were histology dependent: carcinomas experienced significant decreases in Cu-ATSM uptake (P<.05), whereas sarcomas did not (P>.20). Both histologies

Expression of FLT4 in hypoxia-induced neovascular models in vitro and in vivo.

PubMed

Liu, Jiao-Lian; Xia, Xiao-Bo; Xu, Hui-Zhuo

2011-01-01

To investigate the expression of FLT4 in retina with oxygen induced retinopathy (OIR) and in brain endothelial cell lines (bEnd3) under hypoxia conditions in mice. Fifty-two one-week-old C57BL/6J mice were divided into control group and hypoxia group. The mice of hypoxia group were exposed to 75% oxygen for 5 days and then returned to the room air to induce retinal neovascularization. Mice in control group were raised in the environment of room air at the same time. The expressions of FLT4 mRNA and protein were checked with RT-PCR and Western Blot analysis at postnatal day 14, 17 and 21 ( P14, P17 and P21) respectively. 125mmol/L CoCl(2) were added to the culture medium of bEnd3 cell, proteins were extracted in 12, 24, 48 and 72 hours and FLT4 levels were examined by Western Blot analysis. The mRNA and protein level of FLT4 expressed in P14 and P17 OIR mice retina statistically up-regulated as compared with those in control group, but there was no statistical difference between OIR group and control group at P21. FLT4 levels increased significantly in 12, 24 and 48 hours hypoxia intervened bEnd3 cells, its levels in 72 hours increased mildly but showed no significance. FLT4 levels increase in OIR mice retinas and bEnd3 cells in hypoxia. It may play an important role in endothelial cells proliferation in hypoxia and retinal neovascularization in OIR mice.

Expression of FLT4 in hypoxia-induced neovascular models in vitro and in vivo

PubMed Central

Liu, Jiao-Lian; Xia, Xiao-Bo; Xu, Hui-Zhuo

2011-01-01

AIM To investigate the expression of FLT4 in retina with oxygen induced retinopathy (OIR) and in brain endothelial cell lines (bEnd3) under hypoxia conditions in mice. METHODS Fifty-two one-week-old C57BL/6J mice were divided into control group and hypoxia group. The mice of hypoxia group were exposed to 75% oxygen for 5 days and then returned to the room air to induce retinal neovascularization. Mice in control group were raised in the environment of room air at the same time. The expressions of FLT4 mRNA and protein were checked with RT-PCR and Western Blot analysis at postnatal day 14, 17 and 21 ( P14, P17 and P21) respectively. 125mmol/L CoCl2 were added to the culture medium of bEnd3 cell, proteins were extracted in 12, 24, 48 and 72 hours and FLT4 levels were examined by Western Blot analysis. RESULTS The mRNA and protein level of FLT4 expressed in P14 and P17 OIR mice retina statistically up-regulated as compared with those in control group, but there was no statistical difference between OIR group and control group at P21. FLT4 levels increased significantly in 12, 24 and 48 hours hypoxia intervened bEnd3 cells, its levels in 72 hours increased mildly but showed no significance. CONCLUSION FLT4 levels increase in OIR mice retinas and bEnd3 cells in hypoxia. It may play an important role in endothelial cells proliferation in hypoxia and retinal neovascularization in OIR mice. PMID:22553602

[Is 3'-deoxy-3'- [18F] fluorothymidine ([18F]-FLT) the next tracer for routine clinical PET after R [18F]-FDG?].

PubMed

Couturier, Olivier; Leost, Françoise; Campone, Mario; Carlier, Thomas; Chatal, Jean-François; Hustinx, Roland

2005-09-01

Positron emission tomography (PET) with [18F]-FDG is now firmly established as a clinical tool in oncology. Its applications are however limited in some indications, due to the lack of specificity of its uptake mechanism for tumors, or the low avidity of some cancer types such as prostate. Alternative tracers are thus being developed, in order to fill up this void. Proliferation as a biological target is particularly attractive in cancer imaging. From that perspective, fluorothymidine ([18F]-FLT or FLT) has generated a strong interest among the scientific community, especially since the radiosynthesis process has been improved and simplified, thus making possible to envision a routine use for the tracer. This article aims at summarizing the status of the current scientific data regarding FLT. The uptake mechanism of FLT is well known, relying on the thymidine kinase 1 (TK1) enzymatic activity, and thus on DNA synthesis. Preclinical studies have shown a clear relationship between tracer accumulation and level of tumor proliferation, even though DNA salvage pathwayss intervene in the process and may complicate the interpretation of the results. Several clinical studies suggest a good specificity for tumor, albeit with a lower sensitivity than with FDG. In all likelihood however, the future of FLT lies in the evaluation of antitumor response and possibly the pretherapeutic prognostic characterization, rather than in the diagnosis and staging of malignancies. Although the scientific data regarding this issue remain limited, initial results are encouraging. Further significant work remains to be done in order to fully assess the clinical performances of the tracer, on the one hand, and to determine its place relative to FDG and other emerging tracers, on the other hand. Until these studies are completed, FLT should be considered as a promising tracer, but remaining at an experimental stage of its development.

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puntel, Mariana; Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689; Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048

2013-05-01

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, andmore » can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation

The favorable impact of CEBPA mutations in patients with acute myeloid leukemia is only observed in the absence of associated cytogenetic abnormalities and FLT3 internal duplication.

PubMed

Renneville, Aline; Boissel, Nicolas; Gachard, Nathalie; Naguib, Dina; Bastard, Christian; de Botton, Stéphane; Nibourel, Olivier; Pautas, Cécile; Reman, Oumedaly; Thomas, Xavier; Gardin, Claude; Terré, Christine; Castaigne, Sylvie; Preudhomme, Claude; Dombret, Hervé

2009-05-21

Mutations of the CCAAT/enhancer binding protein alpha (CEBPA) gene have been associated with a favorable outcome in patients with acute myeloid leukemia (AML), but mainly in those with a normal karyotype. Here, we analyzed the impact of associated cytogenetic abnormalities or bad-prognosis fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in 53 patients with CEBPA(+) de novo AML treated in the Acute Leukemia French Association trials. We found that only those with a normal karyotype and no FLT3-ITD displayed the expected favorable outcome. In this context, relapse-free, disease-free, and overall survival were significantly longer than in corresponding patients without the CEBPA mutation (P = .035, .016, and .047, respectively). This was not observed in the context of an abnormal karyotype or associated FLT3-ITD. Furthermore, after adjustment on age, trial, and mutation type, these features were independently predictive of shorter overall survival in the subset of patients with CEBPA(+) AML (multivariate hazard ratio = 2.7; 95% confidence interval, 1.08-6.7; and 2.9; 95% confidence interval, 1.01-8.2; with P = .034 and .05, for abnormal karyotype and FLT3-ITD, respectively).

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

PubMed

Manara, E; Basso, G; Zampini, M; Buldini, B; Tregnago, C; Rondelli, R; Masetti, R; Bisio, V; Frison, M; Polato, K; Cazzaniga, G; Menna, G; Fagioli, F; Merli, P; Biondi, A; Pession, A; Locatelli, F; Pigazzi, M

2017-01-01

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

Temporal Changes in FLT3 ITD Regulation of Stem Cell Self Renewal and Leukemogenesis

DTIC Science & Technology

2016-11-01

JAM is a 661   scholar of the Child Health Research Center for Excellence in Developmental 662   Biology at Washington University (K12-HD076224...SUPPLEMENTARY NOTES 14. ABSTRACT My goal is to understand how mechanisms that regulate normal hematopoietic development can also influence the mutation...pathways) in fetal, neonatal and adult progenitors. STAT5 was activated by FLT3-ITD at all stages of development , but MAPK was activated only in post

PML-RARα kinetics and impact of FLT3-ITD mutations in newly diagnosed acute promyelocytic leukaemia treated with ATRA and ATO or ATRA and chemotherapy.

PubMed

Cicconi, L; Divona, M; Ciardi, C; Ottone, T; Ferrantini, A; Lavorgna, S; Alfonso, V; Paoloni, F; Piciocchi, A; Avvisati, G; Ferrara, F; Di Bona, E; Albano, F; Breccia, M; Cerqui, E; Sborgia, M; Kropp, M G; Santoro, A; Levis, A; Sica, S; Amadori, S; Voso, M T; Mandelli, F; Lo-Coco, F

2016-10-01

The APL0406 study showed that arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) are not inferior to standard ATRA and chemotherapy (CHT) in newly diagnosed, low-intermediaterisk acute promyelocytic leukaemia (APL). We analysed the kinetics of promyelocytic leukaemia-retinoic acid receptor-α (PML-RARα) transcripts by real-time quantitative PCR (RQ-PCR) in bone marrow samples from 184 patients and assessed the prognostic impact of fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) in 159 patients enrolled in this trial in Italy. After induction therapy, the reduction of PML-RARα transcripts was significantly greater in patients receiving ATRA-CHT as compared with those treated with ATRA-ATO (3.4 vs 2.9 logs; P=0.0182). Conversely, at the end of consolidation, a greater log reduction of PML-RARα transcripts was detected in the ATRA-ATO as compared with the ATRA-CHT group (6.3 vs 5.3 logs; P=0.0024). FLT3-ITD mutations had no significant impact on either event-free survival (EFS) or cumulative incidence of relapse in patients receiving ATRA-ATO, whereas a trend for inferior EFS was observed in FLT3-ITD-positive patients receiving ATRA-CHT. Our study shows at the molecular level that ATRA-ATO exerts at least equal and probably superior antileukaemic efficacy compared with ATRA-CHT in low-intermediaterisk APL. The data also suggest that ATRA-ATO may abrogate the negative prognostic impact of FLT3-ITD.

TCDD and a putative endogenous AhR ligand, ITE, elicit the same immediate changes in gene expression in mouse lung fibroblasts.

PubMed

Henry, Ellen C; Welle, Stephen L; Gasiewicz, Thomas A

2010-03-01

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1'H-indolo-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5muM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible.

TCDD and a Putative Endogenous AhR Ligand, ITE, Elicit the Same Immediate Changes in Gene Expression in Mouse Lung Fibroblasts

PubMed Central

Henry, Ellen C.; Welle, Stephen L.; Gasiewicz, Thomas A.

2010-01-01

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1′H-indolo-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5μM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible. PMID:19933214

{sup 18}F-FLT uptake kinetics in head and neck squamous cell carcinoma: A PET imaging study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dan, E-mail: dan.liu@oncology.ox.ac.uk; Fenwick, John D.; Chalkidou, Anastasia

2014-04-15

Purpose: To analyze the kinetics of 3{sup ′}-deoxy-3{sup ′}-[F-18]-fluorothymidine (18F-FLT) uptake by head and neck squamous cell carcinomas and involved nodes imaged using positron emission tomography (PET). Methods: Two- and three-tissue compartment models were fitted to 12 tumor time-activity-curves (TACs) obtained for 6 structures (tumors or involved nodes) imaged in ten dynamic PET studies of 1 h duration, carried out for five patients. The ability of the models to describe the data was assessed using a runs test, the Akaike information criterion (AIC) and leave-one-out cross-validation. To generate parametric maps the models were also fitted to TACs of individual voxels.more » Correlations between maps of different parameters were characterized using Pearson'sr coefficient; in particular the phosphorylation rate-constants k{sub 3-2tiss} and k{sub 5} of the two- and three-tissue models were studied alongside the flux parameters K{sub FLT-2tiss} and K{sub FLT} of these models, and standardized uptake values (SUV). A methodology based on expectation-maximization clustering and the Bayesian information criterion (“EM-BIC clustering”) was used to distil the information from noisy parametric images. Results: Fits of two-tissue models 2C3K and 2C4K and three-tissue models 3C5K and 3C6K comprising three, four, five, and six rate-constants, respectively, pass the runs test for 4, 8, 10, and 11 of 12 tumor TACs. The three-tissue models have lower AIC and cross-validation scores for nine of the 12 tumors. Overall the 3C6K model has the lowest AIC and cross-validation scores and its fitted parameter values are of the same orders of magnitude as literature estimates. Maps ofK{sub FLT} and K{sub FLT-2tiss} are strongly correlated (r = 0.85) and also correlate closely with SUV maps (r = 0.72 for K{sub FLT-2tiss}, 0.64 for K{sub FLT}). Phosphorylation rate-constant maps are moderately correlated with flux maps (r = 0.48 for k{sub 3-2tiss} vs K{sub FLT-2tiss} and r

New Radiation Dosimetry Estimates for [18F]FLT based on Voxelized Phantoms.

PubMed

Mendes, B M; Ferreira, A V; Nascimento, L T C; Ferreira, S M Z M D; Silveira, M B; Silva, J B

2018-04-25

3'-Deoxy-3-[ 18 F]fluorothymidine, or [ 18 F]FLT, is a positron emission tomography (PET) tracer used in clinical studies for noninvasive assessment of proliferation activity in several types of cancer. Although the use of this PET tracer is expanding, to date, few studies concerning its dosimetry have been published. In this work, new [ 18 F]FLT dosimetry estimates are determined for human and mice using Monte Carlo simulations. Modern voxelized male and female phantoms and [ 18 F]FLT biokinetic data, both published by the ICRP, were used for simulations of human cases. For most human organs/tissues the absorbed doses were higher than those reported in ICRP Publication 128. An effective dose of 1.70E-02 mSv/MBq to the whole body was determined, which is 13.5% higher than the ICRP reference value. These new human dosimetry estimates obtained using more realistic human phantoms represent an advance in the knowledge of [ 18 F]FLT dosimetry. In addition, mice biokinetic data were obtained experimentally. These data and a previously developed voxelized mouse phantom were used for simulations of animal cases. Concerning animal dosimetry, absorbed doses for organs/tissues ranged from 4.47 ± 0.75 to 155.74 ± 59.36 mGy/MBq. The obtained set of organ/tissue radiation doses for healthy Swiss mice is a useful tool for application in animal experiment design.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

PubMed

Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

2012-04-01

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Interstitial diffusion and the relationship between compartment modelling and multi-scale spatial-temporal modelling of (18)F-FLT tumour uptake dynamics.

PubMed

Liu, Dan; Chalkidou, Anastasia; Landau, David B; Marsden, Paul K; Fenwick, John D

2014-09-07

Tumour cell proliferation can be imaged via positron emission tomography of the radiotracer 3'-deoxy-3'-18F-fluorothymidine (18F-FLT). Conceptually, the number of proliferating cells might be expected to correlate more closely with the kinetics of 18F-FLT uptake than with uptake at a fixed time. Radiotracer uptake kinetics are standardly visualized using parametric maps of compartment model fits to time-activity-curves (TACs) of individual voxels. However the relationship between the underlying spatiotemporal accumulation of FLT and the kinetics described by compartment models has not yet been explored. In this work tumour tracer uptake is simulated using a mechanistic spatial-temporal model based on a convection-diffusion-reaction equation solved via the finite difference method. The model describes a chain of processes: the flow of FLT between the spatially heterogeneous tumour vasculature and interstitium; diffusion and convection of FLT within the interstitium; transport of FLT into cells; and intracellular phosphorylation. Using values of model parameters estimated from the biological literature, simulated FLT TACs are generated with shapes and magnitudes similar to those seen clinically. Results show that the kinetics of the spatial-temporal model can be recovered accurately by fitting a 3-tissue compartment model to FLT TACs simulated for those tumours or tumour sub-volumes that can be viewed as approximately closed, for which tracer diffusion throughout the interstitium makes only a small fractional change to the quantity of FLT they contain. For a single PET voxel of width 2.5-5 mm we show that this condition is roughly equivalent to requiring that the relative difference in tracer uptake between the voxel and its neighbours is much less than one.

18F-FLT PET/CT in the Evaluation of Pheochromocytomas and Paragangliomas: A Pilot Study.

PubMed

Blanchet, Elise M; Taieb, David; Millo, Corina; Martucci, Victoria; Chen, Clara C; Merino, Maria; Herscovitch, Peter; Pacak, Karel

2015-12-01

(18)F-FDG PET/CT has been proven to be a highly sensitive method for pheochromocytomas/paragangliomas (PHEOs/PGLs) associated with succinate dehydrogenase (SDH) mutations. This finding has been attributed to altered tumor cell metabolism resulting from these mutations and does not provide additional prognostic information to genotype. Therefore, identification of new biomarkers for aggressiveness is needed. A high Ki-67 index was proposed to be an additional prognostic factor. This pilot study aimed to evaluate 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET/CT, a PET proliferation tracer, as a potential imaging agent in a series of 12 PHEO/PGL patients with different genetic backgrounds, to compare (18)F-FLT uptake with (18)F-FDG PET/CT, and to evaluate classic factors of aggressiveness. Twelve patients (7 metastatic and 5 nonmetastatic) were prospectively evaluated with (18)F-FDG and (18)F-FLT and followed for at least 2 y after the initial imaging work-up. Uptake was assessed at a lesion level, visually and quantitatively by maximum standardized uptake values (SUVmax) for both tracers. (18)F-FLT uptake was compared with risk factors known to be linked with a poor prognosis in PGLs (SDHB-mutated status, lesion size, dopaminergic phenotype) and with (18)F-FDG uptake. In 12 patients, 77 lesions were assessed. All lesions had low (18)F-FLT uptake (median SUVmax, 2.25; range, 0.7-4.5). There was no apparent superiority of (18)F-FLT uptake in progressive lesions, and most of the lesions showed a mismatch, with high (18)F-FDG uptake (median SUVmax, 10.8; range, 1.1-79.0) contrasting with low (18)F-FLT uptake. This study suggests that PHEOs/PGLs-even those that progress-do not exhibit intense (18)F-FLT uptake. It provides the first in vivo demonstration that proliferation may not be a major determinant of (18)F-FDG uptake in these tumors. These findings provide new insight into the biologic behavior of PGL and suggest that antiproliferative agents may be suboptimal

Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.

PubMed

Proulx, Chantal; Boyer, Lucie; Hurnanen, Darin R; Lemieux, Réal

2003-04-01

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

PubMed Central

2010-01-01

Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It

Performance of FLT-PET for pulmonary lesion diagnosis compared with traditional FDG-PET: A meta-analysis.

PubMed

Wang, Zixing; Wang, Yuyan; Sui, Xin; Zhang, Wei; Shi, Ruihong; Zhang, Yingqiang; Dang, Yonghong; Qiao, Zhen; Zhang, Biao; Song, Wei; Jiang, Jingmei

2015-07-01

Widely used (18)F 2'-deoxy-2'-fluoro-d-glucose (FDG) positron emission tomography (PET) can be problematic with false positives in cancer imaging. This study aims to investigate the diagnostic accuracy of a candidate PET tracer, (18)F 2',3'-dideoxy-3'-fluoro-2-thiothymidine (FLT), in diagnosing pulmonary lesions compared with FDG. After comprehensive search and study selection, a meta-analysis was performed on data from 548 patients pooled from 17 studies for evaluating FLT accuracy, in which data from 351 patients pooled from ten double-tracer studies was used for direct comparison with FDG. Weighted sensitivity and specificity were used as main indicators of test performance. Individual data was extracted and patient subgroup analyses were performed. Overall, direct comparisons showed lower sensitivity (0.80 vs. 0.89) yet higher specificity (0.82 vs. 0.66) for FLT compared with FDG (both p<0.01). Patient subgroup analysis showed FLT was less sensitive than FDG in detecting lung cancers staged as T1 or T2, and those ≤2.0 cm in diameter (0.81 vs. 0.93, and 0.53 vs. 0.78, respectively, both p<0.05), but was comparable for cancers staged as T3 or T4, and those >2.0 cm in diameter (0.95 vs. 1.00, 0.96 vs. 0.88, both p>0.05). For benignities, FLT performed better compared with FDG in ruling out inflammation-based lesions (0.57 vs. 0.32, p<0.05), and demonstrated greater specificity regardless of lesion sizes. Although FLT cannot replace FDG in detecting small and early lung cancers, it may help to prevent patients with larger or inflammatory lesions from cancer misdiagnosis or even over-treatment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Soluble FLT-1 rules placental destiny.

PubMed

Yamashita, Michiko; Kumasawa, Keiichi; Nakamura, Hitomi; Kimura, Tadashi

2018-02-19

Placenta previa is an abnormality in which the placenta covers the internal uterine os, and it can cause serious morbidity and mortality in both mother and fetus due to catastrophic hemorrhage. Some pregnant women recover from placenta previa due to a phenomenon called "migration." However, the mechanism of "migration" of the placenta has not been elucidated. Human placentas were collected from patients with placenta previa and those with no abnormal placentation (control). A microarray analysis was performed to detect the genes up- or down-regulated only in the caudal part in the previa group. Specific mRNA expression was evaluated using real-time quantitative reverse transcription PCR (qRT-PCR). Unilateral uterine artery ablation of 8.5 dpc mice was performed to reproduce the reduction of placental blood supply, and weights of the placentas and fetuses were evaluated in 18.5 dpc. Specific mRNA expression was also evaluated in mice placentas. According to the result of the microarray analysis, we focused on soluble fms-like tyrosine kinase-1 (sFLT-1) and hypoxia-inducible factor-1 (HIF-1) alpha. The sFLT-1 expression level is locally high in the caudal part of the human placenta in patients with placenta previa. In mice experiments, the weights of the placentas and fetuses were significantly smaller in the ablation side than those in the control side, and the sFlt-1 expression level was significantly higher in the ablation side than in the control side. Our study suggests that "migration" of the placenta is derived from placental degeneration at the caudal part of the placenta, and sFlt-1 plays a role in this placental degeneration. Copyright © 2018 Elsevier Inc. All rights reserved.

Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases

PubMed Central

Campregher, Paulo Vidal; de Mattos, Vinicius Renan Pinto; Salvino, Marco Aurélio; Santos, Fabio Pires de Souza; Hamerschlak, Nelson

2017-01-01

ABSTRACT Acute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials. PMID:28746590

Single-scan dual-tracer FLT+FDG PET tumor characterization.

PubMed

Kadrmas, Dan J; Rust, Thomas C; Hoffman, John M

2013-02-07

Rapid multi-tracer PET aims to image two or more tracers in a single scan, simultaneously characterizing multiple aspects of physiology and function without the need for repeat imaging visits. Using dynamic imaging with staggered injections, constraints on the kinetic behavior of each tracer are applied to recover individual-tracer measures from the multi-tracer PET signal. The ability to rapidly and reliably image both (18)F-fluorodeoxyglucose (FDG) and (18)F-fluorothymidine (FLT) would provide complementary measures of tumor metabolism and proliferative activity, with important applications in guiding oncologic treatment decisions and assessing response. However, this tracer combination presents one of the most challenging dual-tracer signal-separation problems--both tracers have the same radioactive half-life, and the injection delay is short relative to the half-life and tracer kinetics. This work investigates techniques for single-scan dual-tracer FLT+FDG PET tumor imaging, characterizing the performance of recovering static and dynamic imaging measures for each tracer from dual-tracer datasets. Simulation studies were performed to characterize dual-tracer signal-separation performance for imaging protocols with both injection orders and injection delays of 10-60 min. Better performance was observed when FLT was administered first, and longer delays before administration of FDG provided more robust signal-separation and recovery of the single-tracer imaging measures. An injection delay of 30 min led to good recovery (R > 0.96) of static image values (e.g. SUV), K(net), and K(1) as compared to values from separate, single-tracer time-activity curves. Recovery of higher order rate parameters (k(2), k(3)) was less robust, indicating that information regarding these parameters was harder to recover in the presence of statistical noise and dual-tracer effects. Performance of the dual-tracer FLT(0 min)+FDG(32 min) technique was further evaluated using PET/CT imaging

Single-scan dual-tracer FLT+FDG PET tumor characterization

NASA Astrophysics Data System (ADS)

Kadrmas, Dan J.; Rust, Thomas C.; Hoffman, John M.

2013-02-01

Rapid multi-tracer PET aims to image two or more tracers in a single scan, simultaneously characterizing multiple aspects of physiology and function without the need for repeat imaging visits. Using dynamic imaging with staggered injections, constraints on the kinetic behavior of each tracer are applied to recover individual-tracer measures from the multi-tracer PET signal. The ability to rapidly and reliably image both 18F-fluorodeoxyglucose (FDG) and 18F-fluorothymidine (FLT) would provide complementary measures of tumor metabolism and proliferative activity, with important applications in guiding oncologic treatment decisions and assessing response. However, this tracer combination presents one of the most challenging dual-tracer signal-separation problems—both tracers have the same radioactive half-life, and the injection delay is short relative to the half-life and tracer kinetics. This work investigates techniques for single-scan dual-tracer FLT+FDG PET tumor imaging, characterizing the performance of recovering static and dynamic imaging measures for each tracer from dual-tracer datasets. Simulation studies were performed to characterize dual-tracer signal-separation performance for imaging protocols with both injection orders and injection delays of 10-60 min. Better performance was observed when FLT was administered first, and longer delays before administration of FDG provided more robust signal-separation and recovery of the single-tracer imaging measures. An injection delay of 30 min led to good recovery (R > 0.96) of static image values (e.g. SUV), Knet, and K1 as compared to values from separate, single-tracer time-activity curves. Recovery of higher order rate parameters (k2, k3) was less robust, indicating that information regarding these parameters was harder to recover in the presence of statistical noise and dual-tracer effects. Performance of the dual-tracer FLT(0 min)+FDG(32 min) technique was further evaluated using PET/CT imaging studies in

Single-scan dual-tracer FLT+FDG PET tumor characterization

PubMed Central

Kadrmas, Dan J; Rust, Thomas C; Hoffman, John M

2013-01-01

Rapid multi-tracer PET aims to image two or more tracers in a single scan, simultaneously characterizing multiple aspects of physiology and function without the need for repeat imaging visits. Using dynamic imaging with staggered injections, constraints on the kinetic behavior of each tracer are applied to recover individual-tracer measures from the multi-tracer PET signal. The ability to rapidly and reliably image both 18F-fluorodeoxyglucose (FDG) and 18F-fluorothymidine (FLT) would provide complementary measures of tumor metabolism and proliferative activity, with important applications in guiding oncologic treatment decisions and assessing response. However, this tracer combination presents one of the most challenging dual-tracer signal-separation problems—both tracers have the same radioactive half-life, and the injection delay is short relative to the half-life and tracer kinetics. This work investigates techniques for single-scan dual-tracer FLT+FDG PET tumor imaging, characterizing the performance of recovering static and dynamic imaging measures for each tracer from dual-tracer datasets. Simulation studies were performed to characterize dual-tracer signal-separation performance for imaging protocols with both injection orders and injection delays of 10–60 min. Better performance was observed when FLT was administered first, and longer delays before administration of FDG provided more robust signal-separation and recovery of the single-tracer imaging measures. An injection delay of 30 min led to good recovery (R > 0.96) of static image values (e.g. SUV), Knet, and K1 as compared to values from separate, single-tracer time-activity curves. Recovery of higher order rate parameters (k2, k3) was less robust, indicating that information regarding these parameters was harder to recover in the presence of statistical noise and dual-tracer effects. Performance of the dual-tracer FLT(0 min)+FDG(32 min) technique was further evaluated using PET/CT imaging studies

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

PubMed

Szalai, Gabor; Romero, Roberto; Chaiworapongsa, Tinnakorn; Xu, Yi; Wang, Bing; Ahn, Hyunyoung; Xu, Zhonghui; Chiang, Po Jen; Sundell, Birgitta; Wang, Rona; Jiang, Yang; Plazyo, Olesya; Olive, Mary; Tarca, Adi L; Dong, Zhong; Qureshi, Faisal; Papp, Zoltan; Hassan, Sonia S; Hernandez-Andrade, Edgar; Than, Nandor Gabor

2015-01-01

Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 μg/mg vs. 19.3 ± 5.6 μg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One

Full-Length Human Placental sFlt-1-e15a Isoform Induces Distinct Maternal Phenotypes of Preeclampsia in Mice

PubMed Central

Szalai, Gabor; Romero, Roberto; Chaiworapongsa, Tinnakorn; Xu, Yi; Wang, Bing; Ahn, Hyunyoung; Xu, Zhonghui; Chiang, Po Jen; Sundell, Birgitta; Wang, Rona; Jiang, Yang; Plazyo, Olesya; Olive, Mary; Tarca, Adi L.; Dong, Zhong; Qureshi, Faisal; Papp, Zoltan; Hassan, Sonia S.; Hernandez-Andrade, Edgar; Than, Nandor Gabor

2015-01-01

Objective Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Methods Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Results Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10-2). Placental and fetal weights did not differ between the

The reduction of serum soluble Flt-1 in patients with neovascular age-related macular degeneration.

PubMed

Uehara, Hironori; Mamalis, Christina; McFadden, Molly; Taggart, Michael; Stagg, Brian; Passi, Samuel; Earle, Phillip; Chakravarthy, Usha; Hogg, Ruth E; Ambati, Balamurali K

2015-01-01

To evaluate serum soluble Flt-1 (sFlt-1) in age-related macular degeneration (AMD) patients. Case-control study. Study involved 56 non-AMD participants, 53 early AMD patients, and 97 neovascular AMD patients from Belfast in Northern Ireland. Serum samples were collected from each patient. Serum sFlt-1 was measured by human sVEGFR1/sFlt-1 ELISA kit. The results were analyzed by Excel and SPSS. Serum sFlt-1 concentration of non-AMD, early AMD, and neovascular AMD were 90.8 ± 2.9 pg/mL (± standard error of the mean), 88.2 ± 2.6 pg/mL, and 79.9 ± 2.2 pg/mL. sFlt-1 from neovascular AMD patients was significantly decreased compared to non-AMD and early AMD patients (ANOVA, P < .01). For each 10-point increase in sFlt-1, the odds for having neovascular AMD compared with non-AMD and neovascular AMD decrease by 27.8%, odds ratio (OR) = 0.722 (95% confidence interval [CI]: 0.588-0.888, P = .002) and 27.0%, OR = 0.730 (95% CI: 0.594-0.898, P = .003), respectively. In patients over 73 years of age, serum sFlt-1 <80 pg/mL was associated with a >6-fold higher risk of neovascular AMD. Reduced serum sFlt-1 differentiates those patients with neovascular AMD from both early AMD and non-AMD participants. In those aged over 73, serum sFlt <80 pg/mL seems to indicate a particularly high risk of neovascular AMD. Our results indicate serum sFlt-1 could be a biomarker for development of neovascular AMD. Copyright © 2015 Elsevier Inc. All rights reserved.

(18)F-FDG and (18)F-FLT PET/CT imaging in the characterization of mediastinal lymph nodes.

PubMed

Rayamajhi, Sampanna Jung; Mittal, Bhagwant Rai; Maturu, Venkata Nagarjuna; Agarwal, Ritesh; Bal, Amanjit; Dey, Pranab; Shukla, Jaya; Gupta, Dheeraj

2016-04-01

There is currently no single modality for accurate characterization of enlarged mediastinal lymph nodes into benign or malignant. Recently (18)F-fluorothymidine (FLT) has been used as a proliferation marker. In this prospective study, we examined the role of (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography/computed tomography (PET/CT) and (18)F-FLT PET/CT in categorizing mediastinal lymph nodes as benign or malignant. A total of 70 consecutive patients with mediastinal lymphadenopathy detected on computed tomography (CT) or chest radiograph underwent whole body (18)F-FLT PET/CT and (18)F-FDG PET/CT (within 1 week of each other). Lymph nodal tracer uptake was determined by calculation of standardized uptake value (SUV) with both the tracers. Results of PET/CT were compared with histopathology of the lymph nodes. Histopathology results showed thirty-seven patients with sarcoidosis, seven patients with tuberculosis, nine patients with non-small cell lung cancer, five patients with Hodgkin's lymphoma and twelve patients with non-Hodgkin's lymphoma. The mean FDG SUVmax of sarcoidosis, tuberculosis, Hodgkin's and non-Hodgkin's lymphoma was 12.7, 13.4, 8.2, and 8.8, respectively, and the mean FLT SUVmax was 6.0, 5.4, 4.4, and 3.8, respectively. It was not possible to characterize mediastinal lymphadenopathy as benign or malignant solely based on FDG SUVmax values (p > 0.05) or FLT SUVmax values (p > 0.05). There was no significant difference in FDG uptake (p > 0.9) or FLT uptake (p > 0.9) between sarcoidosis and tuberculosis. In lung cancer patients, the FDG SUVmax and FLT SUVmax of those lymph nodes with tumor infiltration on biopsy was 6.7 and 3.9, respectively, and those without nodal infiltration was 6.4 and 3.7, respectively, and both the tracers were not able to characterize the nodal status as malignant or benign (p > 0.05). Though (18)F-FLT PET/CT and (18)F-FDG PET/CT reflect different aspects of biology, i.e., proliferation and metabolism

Blocking VEGF with sFlt1 improves the chondrogenic potential of mouse skeletal muscle-derived stem cells

PubMed Central

Kubo, Seiji; Cooper, Gregory M.; Matsumoto, Tomoyuki; Phillippi, Julie A.; Corsi, Karin A.; Usas, Arvydas; Li, Guangheng; Fu, Freddie H.; Huard, Johnny

2010-01-01

Objective To investigate the effect of VEGF stimulation and the effect of blocking VEGF with its antagonist, sFlt1, on chondrogenesis using skeletal muscle-derived stem cells (MDSCs). Methods The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system followed by quantitative real time PCR and histological analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested using genetically engineered MDSCs implanted into the rat osteochondral defect. In this model, MDSCs, transduced with a retroviral vector to express BMP4, were co-implanted with MDSCs transduced to express either VEGF or sFlt1 (a VEGF antagonist) to provide a gain- and loss-of VEGF function experimental design. Histological scoring was used to compare cartilage formation among the treatment groups. Results Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt1-treated) pellet cultures, but real-time PCR revealed that sFlt1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP4 and TGF-β3. In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt1 improved the MDSC-mediated repair of articular cartilage, compared to BMP4 alone. Conclusion sFlt1 gene therapy improved BMP4- and TGF-β3-induced chondrogenic gene expression of MDSCs in vitro, and improved the persistence of repaired articular cartilage by preventing vascularization and bone invasion into the repaired articular cartilage. PMID:19116905

Soluble VEGF receptor 1 (sFLT1) induces non-apoptotic death in ovarian and colorectal cancer cells

PubMed Central

Miyake, Tatsuya; Kumasawa, Keiichi; Sato, Noriko; Takiuchi, Tsuyoshi; Nakamura, Hitomi; Kimura, Tadashi

2016-01-01

Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFLT1) is an angiogenesis inhibitor that competes with angiogenic factors such as VEGF and Placental Growth Factor (PlGF). Imbalances of VEGF and sFLT1 levels can cause pathological conditions such as tumour growth or preeclampsia. We observed direct damage caused by sFLT1 in tumour cells. We exposed several kinds of cells derived from ovarian and colorectal cancers as well as HEK293T cells to sFLT1 in two ways, transfection and exogenous application. The cell morphology and an LDH assay revealed cytotoxicity. Additional experiments were performed to clarify how sFLT1 injured cells. In this study, non-apoptotic cell damage was found to be induced by sFLT1. Moreover, sFLT1 showed an anti-tumour effect in a mouse model of ovarian cancer. Our results suggest that sFLT1 has potential as a cancer therapeutic candidate. PMID:27103202

FLT1 signaling in metastasis-associated macrophages activates an inflammatory signature that promotes breast cancer metastasis

PubMed Central

Zhang, Hui; Li, Jiufeng; He, Tianfang; Yeo, Eun-Jin; Soong, Daniel Y.H.; Carragher, Neil O.; Munro, Alison; Chang, Alvin; Bresnick, Anne R.; Lang, Richard A.

2015-01-01

Although the link between inflammation and cancer initiation is well established, its role in metastatic diseases, the primary cause of cancer deaths, has been poorly explored. Our previous studies identified a population of metastasis-associated macrophages (MAMs) recruited to the lung that promote tumor cell seeding and growth. Here we show that FMS-like tyrosine kinase 1 (Flt1, also known as VEGFR1) labels a subset of macrophages in human breast cancers that are significantly enriched in metastatic sites. In mouse models of breast cancer pulmonary metastasis, MAMs uniquely express FLT1. Using several genetic models, we show that macrophage FLT1 signaling is critical for metastasis. FLT1 inhibition does not affect MAM recruitment to metastatic lesions but regulates a set of inflammatory response genes, including colony-stimulating factor 1 (CSF1), a central regulator of macrophage biology. Using a gain-of-function approach, we show that CSF1-mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity of FLT1-inhibited MAMs. Thus, CSF1 is epistatic to FLT1, establishing a link between FLT1 and inflammatory responses within breast tumor metastases. Importantly, FLT1 inhibition reduces tumor metastatic efficiency even after initial seeding, suggesting that these pathways represent therapeutic targets in metastatic disease. PMID:26261265

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation

PubMed Central

Chappell, John C.; Cluceru, Julia G.; Nesmith, Jessica E.; Mouillesseaux, Kevin P.; Bradley, Vanessa B.; Hartland, Caitlin M.; Hashambhoy-Ramsay, Yasmin L.; Walpole, Joseph; Peirce, Shayn M.; Mac Gabhann, Feilim; Bautch, Victoria L.

2016-01-01

Aims In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. Methods and results Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages—sprout initiation, extension, connection, and stability—that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1−/− mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1−/− vascular networks, with an overall outcome of reduced numbers of new conduits. Conclusions These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies. PMID:27142980

The enhanced in vivo activity of the combination of a MEK and a PI3K inhibitor correlates with [18F]-FLT PET in human colorectal cancer xenograft tumour-bearing mice.

PubMed

Haagensen, Emma J; Thomas, Huw D; Wilson, Ian; Harnor, Suzannah J; Payne, Sara L; Rennison, Tommy; Smith, Kate M; Maxwell, Ross J; Newell, David R

2013-01-01

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [(18)F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [(18)F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [(18)F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.

Metabolically active tumour volume segmentation from dynamic [(18)F]FLT PET studies in non-small cell lung cancer.

PubMed

Hoyng, Lieke L; Frings, Virginie; Hoekstra, Otto S; Kenny, Laura M; Aboagye, Eric O; Boellaard, Ronald

2015-01-01

Positron emission tomography (PET) with (18)F-3'-deoxy-3'-fluorothymidine ([(18)F]FLT) can be used to assess tumour proliferation. A kinetic-filtering (KF) classification algorithm has been suggested for segmentation of tumours in dynamic [(18)F]FLT PET data. The aim of the present study was to evaluate KF segmentation and its test-retest performance in [(18)F]FLT PET in non-small cell lung cancer (NSCLC) patients. Nine NSCLC patients underwent two 60-min dynamic [(18)F]FLT PET scans within 7 days prior to treatment. Dynamic scans were reconstructed with filtered back projection (FBP) as well as with ordered subsets expectation maximisation (OSEM). Twenty-eight lesions were identified by an experienced physician. Segmentation was performed using KF applied to the dynamic data set and a source-to-background corrected 50% threshold (A50%) was applied to the sum image of the last three frames (45- to 60-min p.i.). Furthermore, several adaptations of KF were tested. Both for KF and A50% test-retest (TRT) variability of metabolically active tumour volume and standard uptake value (SUV) were evaluated. KF performed better on OSEM- than on FBP-reconstructed PET images. The original KF implementation segmented 15 out of 28 lesions, whereas A50% segmented each lesion. Adapted KF versions, however, were able to segment 26 out of 28 lesions. In the best performing adapted versions, metabolically active tumour volume and SUV TRT variability was similar to those of A50%. KF misclassified certain tumour areas as vertebrae or liver tissue, which was shown to be related to heterogeneous [(18)F]FLT uptake areas within the tumour. For [(18)F]FLT PET studies in NSCLC patients, KF and A50% show comparable tumour volume segmentation performance. The KF method needs, however, a site-specific optimisation. The A50% is therefore a good alternative for tumour segmentation in NSCLC [(18)F]FLT PET studies in multicentre studies. Yet, it was observed that KF has the potential to subsegment

Comparative evaluation of 18F-FLT and 18F-FDG for detecting cardiac and extra-cardiac thoracic involvement in patients with newly diagnosed sarcoidosis.

PubMed

Norikane, Takashi; Yamamoto, Yuka; Maeda, Yukito; Noma, Takahisa; Dobashi, Hiroaki; Nishiyama, Yoshihiro

2017-08-29

18 F-FDG PET has been used in sarcoidosis for diagnosis and determination of the extent of the disease. However, assessing inflammatory lesions in cardiac sarcoidosis using 18 F-FDG can be challenging because it accumulates physiologically in normal myocardium. Another radiotracer, 3'-deoxy-3'- 18 F-fluorothymidine ( 18 F-FLT), has been investigated as a promising PET tracer for evaluating tumor proliferative activity. In contrast to 18 F-FDG, 18 F-FLT uptake in the normal myocardium is low. The purpose of this retrospective study was to compare the uptake of 18 F-FLT and 18 F-FDG in the evaluation of cardiac and extra-cardiac thoracic involvement in patients with newly diagnosed sarcoidosis. Data for 20 patients with newly diagnosed sarcoidosis were examined. 18 F-FLT and 18 F-FDG PET/CT studies had been performed at 1 h after each radiotracer injection. The patients had fasted for at least 18 h before 18 F-FDG PET/CT but were given no special dietary instructions regarding the period before 18 F-FLT PET/CT. Uptake of 18 F-FLT and 18 F-FDG was examined visually and semiquantitatively using maximal standardized uptake value (SUVmax). Two patients had cardiac sarcoidosis, 7 had extra-cardiac thoracic sarcoidosis, and 11 had both cardiac and extra-cardiac thoracic sarcoidosis. On visual analysis for diagnosis of cardiac sarcoidosis, 4/20 18 F-FDG scans were rated as inconclusive because the 18 F-FDG pattern was diffuse, whereas no FLT scans were rated as inconclusive. The sensitivity of 18 F-FDG PET/CT for detection of cardiac sarcoidosis was 85%; specificity, 100%; and accuracy, 90%. The corresponding values for 18 F-FLT PET/CT were 92, 100, and 95%, respectively. Using semiquantitative analysis of cardiac sarcoidosis, the mean 18 F-FDG SUVmax was significantly higher than the mean 18 F-FLT SUVmax (P < 0.005). Both 18 F-FDG and 18 F-FLT PET/CT studies detected all 24 extra-cardiac lesions. Using semiquantitative analysis of extra-cardiac sarcoidosis, the mean 18

Repeatability of quantitative 18F-FLT uptake measurements in solid tumors: an individual patient data multi-center meta-analysis.

PubMed

Kramer, G M; Liu, Y; de Langen, A J; Jansma, E P; Trigonis, I; Asselin, M-C; Jackson, A; Kenny, L; Aboagye, E O; Hoekstra, O S; Boellaard, R

2018-06-01

3'-deoxy-3'-[ 18 F]fluorothymidine ( 18 F-FLT) positron emission tomography (PET) provides a non-invasive method to assess cellular proliferation and response to antitumor therapy. Quantitative 18 F-FLT uptake metrics are being used for evaluation of proliferative response in investigational setting, however multi-center repeatability needs to be established. The aim of this study was to determine the repeatability of 18 F-FLT tumor uptake metrics by re-analyzing individual patient data from previously published reports using the same tumor segmentation method and repeatability metrics across cohorts. A systematic search in PubMed, EMBASE.com and the Cochrane Library from inception-October 2016 yielded five 18 F-FLT repeatability cohorts in solid tumors. 18 F-FLT avid lesions were delineated using a 50% isocontour adapted for local background on test and retest scans. SUV max , SUV mean , SUV peak , proliferative volume and total lesion uptake (TLU) were calculated. Repeatability was assessed using the repeatability coefficient (RC = 1.96 × SD of test-retest differences), linear regression analysis, and the intra-class correlation coefficient (ICC). The impact of different lesion selection criteria was also evaluated. Images from four cohorts containing 30 patients with 52 lesions were obtained and analyzed (ten in breast cancer, nine in head and neck squamous cell carcinoma, and 33 in non-small cell lung cancer patients). A good correlation was found between test-retest data for all 18 F-FLT uptake metrics (R 2  ≥ 0.93; ICC ≥ 0.96). Best repeatability was found for SUV peak (RC: 23.1%), without significant differences in RC between different SUV metrics. Repeatability of proliferative volume (RC: 36.0%) and TLU (RC: 36.4%) was worse than SUV. Lesion selection methods based on SUV max  ≥ 4.0 improved the repeatability of volumetric metrics (RC: 26-28%), but did not affect the repeatability of SUV metrics. In multi-center studies

The Enhanced In Vivo Activity of the Combination of a MEK and a PI3K Inhibitor Correlates with [18F]-FLT PET in Human Colorectal Cancer Xenograft Tumour-Bearing Mice

PubMed Central

Haagensen, Emma J.; Thomas, Huw D.; Wilson, Ian; Harnor, Suzannah J.; Payne, Sara L.; Rennison, Tommy; Smith, Kate M.; Maxwell, Ross J.; Newell, David R.

2013-01-01

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3′-deoxy-3′-[18F]-fluorothymidine ([18F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [18F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [18F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [18F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment. PMID:24339963

FLT-PET/CT as a Biomarker of Therapeutic Response in Pemetrexed Therapy for Non-Small Cell Lung Cancer

DTIC Science & Technology

2016-12-01

transient burst of metabolism through the salvage pathway, an effect detected as a “flare” of activity by 18F-thymidine (FLT)- PET. FLT is a reliable...the salvage pathway, an effect detected as a “flare” of activity by 18F-thymidine (FLT)-PET. FLT is a reliable biomarker of proliferation, and post...What was accomplished under these goals? 1) Major activities : During

Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

PubMed

Capmany, G; Querol, S; Cancelas, J A; García, J

1999-08-01

The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

FLT-PET/CT as a Biomarker of Therapeutic Response in Pemetrexed Therapy for Non-Small Cell Lung Cancer

DTIC Science & Technology

2015-10-01

therapy in a human patient. These images are from a 63 y/o male with NSCLC participating in our exploratory clinical trial of FLT- PET “flare”. (a... PET in a human NSCLC patient. Results: In NSCLC cells in vitro, we identified a burst in thymidine salvage pathway activity, assessed by 3H...25 Human PET imaging IRB approval was obtained through the University of Pennsylvania Institutional Review Board for use of FLT- PET /CT

Imaging Cellular Proliferation During Chemo-Radiotherapy: A Pilot Study of Serial {sup 18}F-FLT Positron Emission Tomography/Computed Tomography Imaging for Non-Small-Cell Lung Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Everitt, Sarah, E-mail: Sarah.Everitt@petermac.or; Department of Medical Imaging and Radiation Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria; Hicks, Rodney J.

2009-11-15

Purpose: To establish whether {sup 18}F-3'-deoxy-3'-fluoro-L-thymidine ({sup 18}F-FLT) can monitor changes in cellular proliferation of non-small-cell lung cancer (NSCLC) during radical chemo-radiotherapy (chemo-RT). Methods and Materials: As part of a prospective pilot study, 5 patients with locally advanced NSCLC underwent serial {sup 18}F-FLT positron emission tomography (PET)/computed tomography (CT) scans during treatment. Baseline {sup 18}F-FLT PET/CT scans were compared with routine staging {sup 18}F-FDG PET/CT scans. Two on-treatment {sup 18}F-FLT scans were performed for each patient on Days 2, 8, 15 or 29, providing a range of time points for response assessment. Results: In all 5 patients, baseline lesional uptakemore » of {sup 18}F-FLT on PET/CT corresponded to staging {sup 18}F-FDG PET/CT abnormalities. {sup 18}F-FLT uptake in tumor was observed on five of nine (55%) on-treatment scans, on Days 2, 8 and 29, but not Day 15. A 'flare' of {sup 18}F-FLT uptake in the primary tumor of one case was observed after 2 Gy of radiation (1.22 x baseline). The remaining eight on-treatment scans demonstrated a mean reduction in {sup 18}F-FLT tumor uptake of 0.58 x baseline. A marked reduction of {sup 18}F-FLT uptake in irradiated bone marrow was observed for all cases. This reduction was observed even after only 2 Gy, and all patients demonstrated a complete absence of proliferating marrow after 10 Gy. Conclusions: This proof of concept study indicates that {sup 18}F-FLT uptake can monitor the distinctive biologic responses of epithelial cancers and highly radiosensitive normal tissue changes during radical chemo-RT. Further studies of {sup 18}F-FLT PET/CT imaging during therapy may suggest that this tracer is useful in developing response-adapted RT for NSCLC.« less

FDG-PET/CT and FLT-PET/CT for differentiating between lipid-poor benign and malignant adrenal tumours.

PubMed

Nakajo, Masatoyo; Jinguji, Megumi; Fukukura, Yoshihiko; Kajiya, Yoriko; Tani, Atushi; Nakajo, Masayuki; Nakabeppu, Yoshiaki; Arimura, Hiroshi; Nishio, Yoshihiko; Nakamura, Fumihiko; Yoshiura, Takashi

2015-12-01

To compare F-18-fluorodeoxyglucose (FDG) and F-18-fluorothymidine (FLT) PET/CT examinations for differentiating between benign and malignant adrenal tumours. Thirty lipid-poor benign and 11 malignant tumours of 40 patients were included. FDG- and FLT-based indices including visual score, maximum standardized uptake value (SUVmax) and FDG adrenal lesion/liver SUVmax (A/L SUVmax) or FLT adrenal lesion/back muscle SUVmax (A/B SUVmax) ratio were compared between benign and malignant tumours using the Mann-Whitney's U or Wilcoxon signed-rank test, and their diagnostic performances were evaluated by means of the area under the curve (AUC) values derived from the receiver operating characteristic analysis. All indices were significantly higher in malignant than benign tumours on both images (p < 0.05 each). On FDG-PET/CT, the sensitivity, specificity, and accuracy were 91 %, 63 % and 71 % for visual score, 91 %, 67 % and 73 % for SUVmax, and 100 %, 70 % and 78 % for A/L SUVmax ratio, respectively. On FLT-PET/CT, they were 100 %, 97 % and 98 % for visual score, SUVmax and A/B SUVmax ratio, respectively. All FLT indices were significantly higher than those of FDG in AUC (p < 0.05 each). FLT-PET/CT may be superior to FDG-PET/CT in differentiating lipid-poor benign from malignant adrenal tumours because of higher specificity and accuracy. • All FDG indices were significantly higher in malignant than in benign tumours. • All FLT indices were significantly higher in malignant than in benign tumours. • All FLT indices were significantly higher than those of FDG in AUC.

Prognostic Value of 18F-FLT PET in Patients with Neuroendocrine Neoplasms: A Prospective Head-to-Head Comparison with 18F-FDG PET and Ki-67 in 100 Patients.

PubMed

Johnbeck, Camilla B; Knigge, Ulrich; Langer, Seppo W; Loft, Annika; Berthelsen, Anne Kiil; Federspiel, Birgitte; Binderup, Tina; Kjaer, Andreas

2016-12-01

Neuroendocrine neoplasms (NENs) constitute a heterogeneous group of tumors arising in various organs and with a large span of aggressiveness and survival rates. The Ki-67 proliferation index is presently used as the key marker of prognosis, and treatment guidelines are largely based on this index. 3'-deoxy-3'- 18 F-fluorothymidine ( 18 F-FLT) is a proliferation tracer for PET imaging valuable in the monitoring of disease progression and treatment response in various types of cancer. However, until now only data from 10 patients with NEN were available in the literature. The aim of the present study was to investigate 18 F-FLT PET as a prognostic marker for NENs in comparison with 18 F-FDG PET and Ki-67 index. One hundred patients were PET-scanned with both 18 F-FLT and 18 F-FDG within the same week, and the prognostic value of a positive scan was examined in terms of progression-free survival (PFS) and overall survival (OS). The correlation between the Ki-67 index and 18 F-FLT uptake was also investigated. Thirty-seven percent of patients had a positive 18 F-FLT PET scan, and 49% had 18 F-FDG PET-positive foci. Patients with a high 18 F-FLT uptake had a significantly shorter OS and PFS than patients with low or no 18 F-FLT uptake. No correlation was found between Ki-67 index and 18 F-FLT uptake. In a multivariate analysis 18 F-FLT, 18 F-FDG, and Ki-67 all were significant prognostic markers of PFS. For OS, only 18 F-FDG and Ki-67 remained significant. 18 F-FLT PET has prognostic value in NEN patients but when 18 F-FDG PET and Ki-67 index are also available, a multivariate model revealed that 18 F-FLT PET only adds information regarding PFS but not OS, whereas 18 F-FDG PET remains predictive of both PFS and OS. However, a clinically robust algorithm including 18 F-FLT in addition to 18 F-FDG and Ki-67 could not be found. Accordingly, the exact role, if any, of 18 F-FLT PET in NENs remains to be established. © 2016 by the Society of Nuclear Medicine and Molecular

Assessment of glucose metabolism and cellular proliferation in multiple myeloma: a first report on combined 18F-FDG and 18F-FLT PET/CT imaging.

PubMed

Sachpekidis, C; Goldschmidt, H; Kopka, K; Kopp-Schneider, A; Dimitrakopoulou-Strauss, A

2018-04-10

Despite the significant upgrading in recent years of the role of 18 F-FDG PET/CT in multiple myeloma (MM) diagnostics, there is a still unmet need for myeloma-specific radiotracers. 3'-Deoxy-3'-[ 18 F]fluorothymidine ( 18 F-FLT) is the most studied cellular proliferation PET agent, considered a potentially new myeloma functional imaging tracer. The aim of this pilot study was to evaluate 18 F-FLT PET/CT in imaging of MM patients, in the context of its combined use with 18 F-FDG PET/CT. Eight patients, four suffering from symptomatic MM and four suffering from smoldering MM (SMM), were enrolled in the study. All patients underwent 18 F-FDG PET/CT and 18 F-FLT PET/CT imaging by means of static (whole body) and dynamic PET/CT of the lower abdomen and pelvis (dPET/CT) in two consecutive days. The evaluation of PET/CT studies was based on qualitative evaluation, semi-quantitative (SUV) calculation, and quantitative analysis based on two-tissue compartment modeling. 18 F-FDG PET/CT demonstrated focal, 18 F-FDG avid, MM-indicative bone marrow lesions in five patients. In contrary, 18 F-FLT PET/CT showed focal, 18 F-FLT avid, myeloma-indicative lesions in only two patients. In total, 48 18 F-FDG avid, focal, MM-indicative lesions were detected with 18 F-FDG PET/CT, while 17 18 F-FLT avid, focal, MM-indicative lesions were detected with 18 F-FLT PET/CT. The number of myeloma-indicative lesions was significantly higher for 18 F-FDG PET/CT than for 18 F-FLT PET/CT. A common finding was a mismatch of focally increased 18 F-FDG uptake and reduced 18 F-FLT uptake (lower than the surrounding bone marrow). Moreover, 18 F-FLT PET/CT was characterized by high background activity in the bone marrow compartment, further complicating the evaluation of bone marrow lesions. Semi-quantitative evaluation revealed that both SUV mean and SUV max were significantly higher for 18 F-FLT than for 18 F-FDG in both MM lesions and reference tissue. SUV values were higher in MM lesions than in

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

PubMed

Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity.

PubMed

Palmer, Kirsten R; Kaitu'u-Lino, Tu'uhevaha J; Hastie, Roxanne; Hannan, Natalie J; Ye, Louie; Binder, Natalie; Cannon, Ping; Tuohey, Laura; Johns, Terrance G; Shub, Alexis; Tong, Stephen

2015-12-01

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a. © 2015 American Heart Association, Inc.

Characterisation of syncytiotrophoblast vesicles in normal pregnancy and pre-eclampsia: expression of Flt-1 and endoglin.

PubMed

Tannetta, Dionne S; Dragovic, Rebecca A; Gardiner, Chris; Redman, Christopher W; Sargent, Ian L

2013-01-01

The placental syncytiotrophoblast releases micro and nanovesicles (STBM), into the maternal circulation in normal pregnancy and in increased amounts in pre-eclampsia (PE), which have proinflammatory and antiangiogenic activity and are implicated in PE pathophysiology. Better characterisation of STBM is essential to understand their role in PE. STBM prepared by placental lobe dual perfusion (pSTBM) and mechanical disruption (mSTBM) were analysed by four colour flow cytometry (4CFC), nanoparticle tracking analysis (NTA) and Western blotting to determine vesicle size, purity and Flt-1 and endoglin (Eng) expression. Biological activity of STBM associated Flt-1 and endoglin was assessed by the ability of VEGF, PlGF and TGFβ to bind to mSTBM and inhibit mSTBM induced endothelial monolayer disruption. STBM content was consistently high (~87-95%) across the different preparations. However, surface antigen intensities differed, with significantly lower placental alkaline phosphatase (P<0.05) and Eng (P<0.05) expression on mSTBM, and Flt-1 (P<0.05) expression on pSTBM. For PE placenta derived preparations, pSTBM contained lower Eng positive STBM (P<0.05) and mSTBM Eng expression was increased (P<0.05). Western blotting revealed increased Flt-1/sFlt-1 (P<0.02) and decreased placental alkaline phosphatase (P = 0.0002) content of PE placenta pSTBM. Using NTA, perfused PE placentas released significantly larger MV (P<0.001). Finally, VEGF, PlGF and TGFβ bound to mSTBM at physiologically relevant concentrations and inhibited mSTBM induced endothelial disruption (P<0.05-P<0.001). This study has found differences in physical and antigenic characteristics of normal and PE placenta STBM preparations produced by placental perfusion or mechanical disruption. We have also demonstrated that large quantities of biologically active STBM associated endoglin and Flt-1/sFlt-1 could contribute to the increased circulating levels measured in PE patients and add to the perturbation of the

Distribution Atlas of Proliferating Bone Marrow in Non-Small Cell Lung Cancer Patients Measured by FLT-PET/CT Imaging, With Potential Applicability in Radiation Therapy Planning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campbell, Belinda A., E-mail: Belinda.Campbell@petermac.org; Callahan, Jason; Bressel, Mathias

Purpose: Proliferating bone marrow is exquisitely sensitive to ionizing radiation. Knowledge of its distribution could improve radiation therapy planning to minimize unnecessary marrow exposure and avoid consequential prolonged myelosuppression. [18F]-Fluoro-3-deoxy-3-L-fluorothymidine (FLT)–positron emission tomography (PET) is a novel imaging modality that provides detailed quantitative images of proliferating tissues, including bone marrow. We used FLT-PET imaging in cancer patients to produce an atlas of marrow distribution with potential clinical utility. Methods and Materials: The FLT-PET and fused CT scans of eligible patients with non-small cell lung cancer (no distant metastases, no prior cytotoxic exposure, no hematologic disorders) were reviewed. The proportions of skeletalmore » FLT activity in 10 predefined bony regions were determined and compared according to age, sex, and recent smoking status. Results: Fifty-one patients were studied: 67% male; median age 68 (range, 31-87) years; 8% never smokers; 70% no smoking in the preceding 3 months. Significant differences in marrow distribution occurred between sex and age groups. No effect was detected from smoking in the preceding 3 months. Using the mean percentages of FLT uptake per body region, we created an atlas of the distribution of functional bone marrow in 4 subgroups defined by sex and age. Conclusions: This atlas has potential utility for estimating the distribution of active marrow in adult cancer patients to guide radiation therapy planning. However, because of interindividual variation it should be used with caution when radiation therapy risks ablating large proportions of active marrow; in such cases, individual FLT-PET scans may be required.« less

First trimester PAPP-A levels correlate with sFlt-1 levels longitudinally in pregnant women with and without preeclampsia.

PubMed

Saxena, Aditi R; Seely, Ellen W; Rich-Edwards, Janet W; Wilkins-Haug, Louise E; Karumanchi, S Ananth; McElrath, Thomas F

2013-04-04

First trimester Pregnancy Associated Plasma Protein A (PAPP-A) levels, routinely measured for aneuploidy screening, may predict development of preeclampsia. This study tests the hypothesis that first trimester PAPP-A levels correlate with soluble fms-like tyrosine kinase-1 (sFlt-1) levels, an angiogenic marker associated with preeclampsia, throughout pregnancy. sFlt-1 levels were measured longitudinally in 427 women with singleton pregnancies in all three trimesters. First trimester PAPP-A and PAPP-A Multiples of Median (MOM) were measured. Student's T and Wilcoxon tests compared preeclamptic and normal pregnancies. A linear mixed model assessed the relationship between log PAPP-A and serial log sFlt-1 levels. PAPP-A and PAPP-A MOM levels were significantly lower in preeclamptic (n = 19), versus normal pregnancies (p = 0.02). Although mean third trimester sFlt-1 levels were significantly higher in preeclampsia (p = 0.002), first trimester sFlt-1 levels were lower in women who developed preeclampsia, compared with normal pregnancies (p = 0.03). PAPP-A levels correlated significantly with serial sFlt-1 levels. Importantly, low first trimester PAPP-A MOM predicted decreased odds of normal pregnancy (OR 0.2, p = 0.002). Low first trimester PAPP-A levels suggests increased future risk of preeclampsia and correlate with serial sFlt-1 levels throughout pregnancy. Furthermore, low first trimester PAPP-A status significantly predicted decreased odds of normal pregnancy.

Diagnosis of metastases from postoperative differentiated thyroid cancer: comparison between FDG and FLT PET/CT studies.

PubMed

Nakajo, Masatoyo; Nakajo, Masayuki; Jinguji, Megumi; Tani, Atsushi; Kajiya, Yoriko; Tanabe, Hiroaki; Fukukura, Yoshihiko; Nakabeppu, Yoshiaki; Koriyama, Chihaya

2013-06-01

To compare positron emission tomography (PET)/computed tomography (CT) studies performed with the glucose analog fluorine 18 ((18)F) fluorodeoxyglucose (FDG) and the cell proliferation tracer (18)F fluorothymidine (FLT) in the diagnosis of metastases from postoperative differentiated thyroid cancer. The institutional ethics review board approved this prospective study. From March 2010 to February 2012, 20 patients (mean age, 53 years; age range, 22-79 years) with postoperative differentiated thyroid cancer underwent both FDG and FLT PET/CT as a staging work-up before radioiodine therapy. In each patient, 28 anatomic areas were set and analyzed for lymph node and distant metastases. The McNemar exact or χ(2) test was used to examine differences in diagnostic indexes in the detection of lymph node and distant metastases between both tracer PET/CT studies. There were 34 lymph node metastases and/or 73 distant metastases (70 metastases in lung and one each in bone, nasopharynx, and brain) in 13 patients. At patient-based analysis, the sensitivity, specificity, and accuracy were 92% (12 of 13 patients), 86% (six of seven patients), and 90% (18 of 20 patients), respectively, for FDG PET/CT and 69% (nine of 13 patients), 29% (two of seven patients), and 55% (11 of 20 patients) for FLT PET/CT. The accuracy of FDG PET/CT was significantly better than that of FLT PET/CT (P = .023). At lesion-based analysis, the sensitivity, specificity, and accuracy for diagnosing lymph node metastases were 85% (29 of 34 lesions), 99.6% (245 of 246 lesions), and 97.9% (274 of 280 lesions), respectively, for FDG PET/CT and 50% (17 of 34 lesions), 90.7% (223 of 246 lesions), and 85.7% (240 of 280 lesions) for FLT PET/CT. The sensitivity, specificity, and accuracy for diagnosing distant metastases were 45% (33 of 73 lesions), 100% (207 of 207 lesions), and 85.7% (240 of 280 lesions), respectively, for FDG PET/CT and 6.8% (five of 73 lesions), 100% (207 of 207 lesions), and 75.7% (212 of 280

Depicting changes in tumor biology in response to cetuximab mono- or combination therapy by apoptosis and proliferation imaging using 18F-ICMT-11 and 3'-Deoxy-3'-[18F]Fluorothymidine (18F-FLT) PET.

PubMed

Heinzmann, Kathrin; Nguyen, Quang-De; Honess, Davina Jean; Smith, Donna-Michelle; Stribbling, Stephen; Brickute, Diana; Barnes, Christopher; Griffiths, John Richard; Aboagye, Eric Ofori

2018-05-24

Imaging biomarkers must demonstrate their value in monitoring treatment. Two PET tracers, the caspase-3/7-specific isatin-5-sulfonamide 18 F-ICMT-11 and 3'-Deoxy-3'-[ 18 F]Fluorothymidine ( 18 F-FLT), were employed to detect early treatment-induced changes in tumor biology and whether any changes indicate response to cetuximab, administered as mono- or combination therapy with gemcitabine. Methods: Effects of single or repeated doses of the anti-Epidermal Growth Factor Receptor (EGFR) antibody cetuximab (10mg/kg on day 1 only or day 1 and 2) and/or a single dose of gemcitabine (125mg/kg; day 2) were investigated in mice bearing cetuximab-sensitive H1975 tumors (non-small cell lung cancer) by 18 F-ICMT-11 or 18 F-FLT-PET (day 3). Imaging was also performed in mice bearing cetuximab-insensitive HCT116 tumors (colorectal cancer) after two doses of cetuximab (day 1 and 2). For imaging/histology comparison, tumors were evaluated for proliferation (Ki67; thymidine kinase 1, TK1), cell death (cleaved caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)) and target engagement (EGFR expression) by immunohistochemistry, immunofluorescence and immunoblot, respectively. Tumor and plasma were analysed for thymidine and gemcitabine metabolites by liquid chromatography-mass spectrometry. Results: Retention of both tracers was sensitive to cetuximab in H1975 tumors. 18 F-ICMT-11 uptake and ex vivo cleaved caspase-3 staining notably increased in tumors treated with repeated doses of cetuximab- (75%) and combination-treatment (46%). While one dose of cetuximab was insufficient to induce apoptosis it did affect proliferation. Significant reduction in tumor 18 F-FLT uptake (44 to 50%; P < 0.001) induced by cetuximab mono- and combination-therapy were paralleled with a clear decrease in proliferation (%Ki67 decrease: 72 to 95%; P < 0.0001) and followed by marked tumor growth delay. TK1 expression and tumor thymidine concentrations were profoundly

Application of a whole-body pharmacokinetic model for targeted radionuclide therapy to NM404 and FLT

NASA Astrophysics Data System (ADS)

Grudzinski, Joseph J.; Floberg, John M.; Mudd, Sarah R.; Jeffery, Justin J.; Peterson, Eric T.; Nomura, Alice; Burnette, Ronald R.; Tomé, Wolfgang A.; Weichert, Jamey P.; Jeraj, Robert

2012-03-01

We have previously developed a model that provides relative dosimetry estimates for targeted radionuclide therapy (TRT) agents. The whole-body and tumor pharmacokinetic (PK) parameters of this model can be noninvasively measured with molecular imaging, providing a means of comparing potential TRT agents. Parameter sensitivities and noise will affect the accuracy and precision of the estimated PK values and hence dosimetry estimates. The aim of this work is to apply a PK model for TRT to two agents with different magnitudes of clearance rates, NM404 and FLT, explore parameter sensitivity with respect to time and investigate the effect of noise on parameter precision and accuracy. Twenty-three tumor bearing mice were injected with a ‘slow-clearing’ agent, 124I-NM404 (n = 10), or a ‘fast-clearing’ agent, 18F-FLT (3‧-deoxy-3‧-fluorothymidine) (n = 13) and imaged via micro-PET/CT pseudo-dynamically or dynamically, respectively. Regions of interest were drawn within the heart and tumor to create time-concentration curves for blood pool and tumor. PK analysis was performed to estimate the mean and standard error of the central compartment efflux-to-influx ratio (k12/k21), central elimination rate constant (kel), and tumor influx-to-efflux ratio (k34/k43), as well as the mean and standard deviation of the dosimetry estimates. NM404 and FLT parameter estimation results were used to analyze model accuracy and parameter sensitivity. The accuracy of the experimental sampling schedule was compared to that of an optimal sampling schedule found using Cramer-Rao lower bounds theory. Accuracy was assessed using correlation coefficient, bias and standard error of the estimate normalized to the mean (SEE/mean). The PK parameter estimation of NM404 yielded a central clearance, kel (0.009 ± 0.003 h-1), normal body retention, k12/k21 (0.69 ± 0.16), tumor retention, k34/k43 (1.44 ± 0.46) and predicted dosimetry, Dtumor (3.47 ± 1.24 Gy). The PK parameter estimation of FLT

Disseminated Skeletal Muscle and Cardiac Metastasis from Squamous Cell Carcinoma of the Lung Detected with FDG and FLT PET/CT.

PubMed

Jain, Tarun Kumar; Rayamajhi, Sampanna Jung; Basher, Rajender Kumar; Gupta, Dheeraj; Maturu, Venkata Nagarjuna; Mittal, Bhagwant Rai

2016-09-01

Lung cancer is one of the leading cancers all over the world. Positron emission tomography (PET) using 18F fluorodeoxyglucose (18F FDG) is useful for staging of the disease and decide the appropriate management. 3'-deoxy-3'-18 F-fluorothymidine (18F FLT) is a tracer being extensively evaluated currently and is said to represent tumor proliferation. Common sites of metastases from lung cancer include adrenal glands, bone, and brain. Muscle metastasis and cardiac metastasis are uncommon findings. We report a case of squamous cell carcinoma of the lung with metastases to multiple skeletal muscles and myocardium detected with both FDG and FLT PET/computed tomography (CT).

Intracellular pathways triggered by the selective FLT-1-agonist placental growth factor in vascular smooth muscle cells exposed to hypoxia.

PubMed

Bellik, Lydia; Vinci, Maria Cristina; Filippi, Sandra; Ledda, Fabrizio; Parenti, Astrid

2005-10-01

We have previously shown that hypoxia makes vascular smooth muscle cells (VSMCs) responsive to placental growth factor (PlGF) through the induction of functional fms-like tyrosine kinase (Flt-1) receptors. The aim of this study was to investigate the molecular mechanisms involved in the PlGF effects on proliferation and contraction of VSMCs previously exposed to hypoxia (3% O2). In cultured rat VSMCs exposed to hypoxia, PlGF increased the phosphorylation of protein kinase B (Akt), p38 and STAT3; activation of STAT3 was higher than that of other kinases. In agreement with this finding, the proliferation of hypoxia-treated VSMCs in response to PlGF was significantly impaired by the p38 and the phosphatidylinositol 3-kinase inhibitors SB202190 and LY294002, respectively, and was almost completely prevented by AG490, a janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor. Since hypoxia was able to reverse the vasorelaxant effect of PlGF into a vasoconstrictor response, the mechanism of this latter effect was also investigated. Significant Flt-1 activity was measured in isolated preparations from rat aorta exposed to hypoxia. Inhibitors of mitogen-activated protein kinase kinase, Akt and STAT3 induced a modest inhibition of the vasoconstrictor response to PlGF, while the p38 inhibitor SB202190 markedly impaired the PlGF-induced contractile response. These effects were selectively mediated by Flt-1 without any involvement of foetal liver kinase-1 receptors. These data are the first evidence that different intracellular pathways activated by Flt-1 receptor in VSMCs are involved in diverse biological effects of PlGF: while mitogen activated protein kinase kinase/extracellular signal regulated kinase(1/2) and JAK/STAT play a role in VSMC proliferation, p38 is involved in VSMC contraction. These findings may highlight the role of PlGF in vascular pathology.

Rho kinase regulates the survival and transformation of cells bearing oncogenic forms of KIT, FLT3 and BCR-ABL

PubMed Central

Mali, Raghuveer Singh; Ramdas, Baskar; Ma, Peilin; Shi, Jianjian; Munugalavadla, Veerendra; Sims, Emily; Wei, Lei; Vemula, Sasidhar; Nabinger, Sarah C.; Goodwin, Charles B.; Chan, Rebecca J.; Traina, Fabiola; Visconte, Valeria; Tiu, Ramon V.; Lewis, Timothy A.; Stern, Andrew M.; Wen, Qiang; Crispino, John D.; Boswell, H. Scott; Kapur, Reuben

2011-01-01

Summary We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3 and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth and loss of actin polymerization in oncogene bearing cells leading to significantly prolonged life span of leukemic mice. In summary, we describe a pathway involving PI3K/Rho/ROCK/MLC which may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans. PMID:21907926

Monitoring of anti-cancer treatment with 18F-FDG and 18F-FLT PET: a comprehensive review of pre-clinical studies

PubMed Central

Jensen, Mette Munk; Kjaer, Andreas

2015-01-01

Functional imaging of solid tumors with positron emission tomography (PET) imaging is an evolving field with continuous development of new PET tracers and discovery of new applications for already implemented PET tracers. During treatment of cancer patients, a general challenge is to measure treatment effect early in a treatment course and by that to stratify patients into responders and non-responders. With 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) and 3’-deoxy-3’-[18F]fluorothymidine(18F-FLT) two of the cancer hallmarks, altered energy metabolism and increased cell proliferation, can be visualized and quantified non-invasively by PET. With 18F-FDG and 18F-FLT PET changes in energy metabolism and cell proliferation can thereby be determined after initiation of cancer treatment in both clinical and pre-clinical studies in order to predict, at an early time-point, treatment response. It is hypothesized that decreases in glycolysis and cell proliferation may occur in tumors that are sensitive to the applied cancer therapeutics and that tumors that are resistant to treatment will show unchanged glucose metabolism and cell proliferation. Whether 18F-FDG and/or 18F-FLT PET can be used for prediction of treatment response has been analyzed in many studies both following treatment with conventional chemotherapeutic agents but also following treatment with different targeted therapies, e.g. monoclonal antibodies and small molecules inhibitors. The results from these studies have been most variable; in some studies early changes in 18F-FDG and 18F-FLT uptake predicted later tumor regression whereas in other studies no change in tracer uptake was observed despite the treatment being effective. The present review gives an overview of pre-clinical studies that have used 18F-FDG and/or 18F-FLT PET for response monitoring of cancer therapeutics. PMID:26550536

Soluble vascular endothelial growth factor (VEGF) receptor-1 inhibits migration of human monocytic THP-1 cells in response to VEGF.

PubMed

Zhu, Cansheng; Xiong, Zhaojun; Chen, Xiaohong; Lu, Zhengqi; Zhou, Guoyu; Wang, Dunjing; Bao, Jian; Hu, Xueqiang

2011-08-01

We aimed to investigate the regulation and contribution of vascular endothelial growth factor (VEGF) and sFlt-1(1-3) to human monocytic THP-1 migration. Ad-sFlt-1/FLAG, a recombinant adenovirus carrying the human sFlt-1(1-3) (the first three extracellular domains of FLT-1, the hVEGF receptor-1) gene, was constructed. L929 cells were infected with Ad-sFlt-1/FLAG and the expression of sFlt-1 was detected by immunofluorescent assay and ELISA. Corning(®) Transwell(®) Filter Inserts containing polyethylene terephthalate (PET) membranes with pore sizes of 3 μm were used as an experimental model to simulate THP-1 migration. Five VEGF concentrations (0, 0.1, 1, 10 and 100 ng/ml), four concentrations of sFlt-1(1-3)/FLAG expression supernatants (0.1, 1, 10 and 100 ng/ml), and monocyte chemoattractant protein-1 (MCP-1, 10 ng/ml) were used to test the ability of THP-1 cells to migrate through PET membranes. The sFlt-1(1-3) gene was successfully recombined into Ad-sFlt-1/FLAG. sFlt-1(1-3) was expressed in L929 cells transfected with Ad-sFlt-1/FLAG. THP-1 cell migration increased with increasing concentrations of VEGF, while cell migration decreased with increasing concentrations of sFlt1(1-3)/FLAG. sFlt1(1-3)/FLAG had no effect on MCP-1-induced cell migration. This study demonstrated that VEGF is able to elicit a migratory response in THP-1 cells, and that sFlt-1(1-3) is an effective inhibitor of THP-1 migration towards VEGF.

TU-F-12A-02: Quantitative Characterization of Normal Bone Marrow Proliferative Activity with FLT PET/CT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weisse, N; Jeraj, R

Purpose: [F-18]FLT PET is a tool for assessing health of bone marrow by evaluating its proliferative activity. This study establishes a baseline quantitative characterization of healthy marrow proliferation to aid in diagnosis of hematological disease. Methods: 31 patients (20 male, 11 female, 41–76 years) being treated for solid cancers with no history of hematological disease, osseous metastatic disease, or radiation therapy received pre-treatment FLT PET/CT scans. Total bone marrow was isolated from whole body FLT PET images by manually removing organs and applying a standardize uptake value (SUV) threshold of 1.0. Because adult marrow is concentrated in the axial skeleton,more » quantitative total bone marrow analysis (QTBMA) was used to isolate marrow in the lumbar spine, thoracic spine, sacrum, and pelvis for analysis. SUV mean, SUV max, and SUV CV were used to quantify bone marrow proliferation. Correlations were explored between SUV and patient characteristics including age, weight, height, and BMI using the Spearman coefficient (ρ). Results: The population-averaged whole-skeleton SUV mean, SUV max, and SUV CV were 3.0±0.6, 18.4±5.7, and 0.6±0.1, respectively. Uptake values in the axial skeleton were similar to the whole-skeleton demonstrated by SUV mean in the thoracic spine (3.6±0.6), lumbar spine (3.3±0.5), sacrum (3.0±0.6), and pelvis regions (2.8±0.5). Whole-skeleton SUV max correlated with patient weight (ρ=0.47, p<0.01) and BMI (ρ=0.60, p<0.01), suggesting marrow activity is related to the body's burden. SUV measures in the thoracic spine, lumbar spine, sacrum, and pelvis were negatively correlated with age (ρ:−0.41 to −0.46, p≤0.02). These negative correlations reflect the fact that active marrow in the adult skeleton is localized in the axial skeleton and decreases with age. Conclusions: Normal bone marrow characterizations were determined using FLT PET. These results provide a baseline characterization against which

Disseminated Skeletal Muscle and Cardiac Metastasis from Squamous Cell Carcinoma of the Lung Detected with FDG and FLT PET/CT

PubMed Central

Jain, Tarun Kumar; Rayamajhi, Sampanna Jung; Basher, Rajender Kumar; Gupta, Dheeraj; Maturu, Venkata Nagarjuna; Mittal, Bhagwant Rai

2016-01-01

Lung cancer is one of the leading cancers all over the world. Positron emission tomography (PET) using 18F fluorodeoxyglucose (18F FDG) is useful for staging of the disease and decide the appropriate management. 3’-deoxy-3’-18 F-fluorothymidine (18F FLT) is a tracer being extensively evaluated currently and is said to represent tumor proliferation. Common sites of metastases from lung cancer include adrenal glands, bone, and brain. Muscle metastasis and cardiac metastasis are uncommon findings. We report a case of squamous cell carcinoma of the lung with metastases to multiple skeletal muscles and myocardium detected with both FDG and FLT PET/computed tomography (CT). PMID:27651747

Allogeneic stem cell transplantation benefits for patients ≥ 60 years with acute myeloid leukemia and FLT3 internal tandem duplication: a study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

PubMed

Poiré, Xavier; Labopin, Myriam; Polge, Emmanuelle; Passweg, Jakob; Craddock, Charles; Blaise, Didier; Cornelissen, Jan J; Volin, Liisa; Russell, Nigel H; Socié, Gérard; Michallet, Mauricette; Fegueux, Nathalie; Chevallier, Patrice; Brecht, Arne; Hunault-Berger, Mathilde; Mohty, Mohamad; Esteve, Jordi; Nagler, Arnon

2018-02-01

Intermediate-risk cytogenetic acute myeloid leukemia with an internal tandem duplication of FLT3 ( FLT3 -ITD) is associated with a high risk of relapse, and is now a standard indication for allogeneic stem cell transplantation. Nevertheless, most studies supporting this strategy have been performed in young patients. To address the benefit of allogeneic transplantation in the elderly, we made a selection from the European Society for Blood and Marrow Transplantation registry of de novo intermediate-risk cytogenetic acute myeloid leukemia harboring FLT3 -ITD in patients aged 60 or over and transplanted from a related or unrelated donor between January 2000 and December 2015. Two hundred and ninety-one patients were identified. Most patients received a reduced-intensity conditioning (82%), while donors consisted of an unrelated donor in 161 (55%) patients. Two hundred and twelve patients received their transplantation in first remission, 37 in second remission and 42 in a more advanced stage of the disease. The 2-year leukemia-free survival rate was 56% in patients in first remission, 22% in those in second remission and 10% in patients with active disease, respectively ( P <0.005). Non-relapse mortality for the entire cohort was 20%. In multivariate analysis, disease status at transplantation was the most powerful predictor of worse leukemia-free survival, graft- versus -host disease and relapse-free survival, and overall survival. In this elderly population, age was not associated with outcome. Based on the current results, allogeneic transplantation translates into a favorable outcome in fit patients ≥ 60 with FLT3 -ITD acute myeloid leukemia in first remission, similarly to current treatment recommendations for younger patients. Copyright© 2018 Ferrata Storti Foundation.

Allogeneic stem cell transplantation benefits for patients ≥ 60 years with acute myeloid leukemia and FLT3 internal tandem duplication: a study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation

PubMed Central

Poiré, Xavier; Labopin, Myriam; Polge, Emmanuelle; Passweg, Jakob; Craddock, Charles; Blaise, Didier; Cornelissen, Jan J.; Volin, Liisa; Russell, Nigel H.; Socié, Gérard; Michallet, Mauricette; Fegueux, Nathalie; Chevallier, Patrice; Brecht, Arne; Hunault-Berger, Mathilde; Mohty, Mohamad; Esteve, Jordi; Nagler, Arnon

2018-01-01

Intermediate-risk cytogenetic acute myeloid leukemia with an internal tandem duplication of FLT3 (FLT3-ITD) is associated with a high risk of relapse, and is now a standard indication for allogeneic stem cell transplantation. Nevertheless, most studies supporting this strategy have been performed in young patients. To address the benefit of allogeneic transplantation in the elderly, we made a selection from the European Society for Blood and Marrow Transplantation registry of de novo intermediate-risk cytogenetic acute myeloid leukemia harboring FLT3-ITD in patients aged 60 or over and transplanted from a related or unrelated donor between January 2000 and December 2015. Two hundred and ninety-one patients were identified. Most patients received a reduced-intensity conditioning (82%), while donors consisted of an unrelated donor in 161 (55%) patients. Two hundred and twelve patients received their transplantation in first remission, 37 in second remission and 42 in a more advanced stage of the disease. The 2-year leukemia-free survival rate was 56% in patients in first remission, 22% in those in second remission and 10% in patients with active disease, respectively (P<0.005). Non-relapse mortality for the entire cohort was 20%. In multivariate analysis, disease status at transplantation was the most powerful predictor of worse leukemia-free survival, graft-versus-host disease and relapse-free survival, and overall survival. In this elderly population, age was not associated with outcome. Based on the current results, allogeneic transplantation translates into a favorable outcome in fit patients ≥ 60 with FLT3-ITD acute myeloid leukemia in first remission, similarly to current treatment recommendations for younger patients. PMID:29242299

Uteroplacental arterio-venous difference in soluble VEGFR-1 (sFlt-1), but not in soluble endoglin concentrations in preeclampsia.

PubMed

Paasche Roland, M C; Lorentzen, B; Godang, K; Henriksen, T

2012-03-01

The aim was to determine plasma levels of sFlt-1 and sEng on the arterial and venous side of the uteroplacental circulation in preeclamptic patients and healthy controls. Preeclamptic patients had higher arterial concentrations than controls of sFlt-1 (17,450 vs. 5055 pg/ml, p = 0.003) and sEng (68.7 vs. 28.7 ng/ml, p = 0.003). In the preclampsia group sFlt-1 was higher in the uterine vein than in the radial artery (22,450 vs. 17,450 pg/ml, p = 0.005). We found no gradient for sEng. Our results support the hypothesis that excess sFlt-1 at least partly originates in placenta, while excess sEng appears to have a different origin. Copyright © 2012 Elsevier Ltd. All rights reserved.

IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication.

PubMed

Paschka, Peter; Schlenk, Richard F; Gaidzik, Verena I; Habdank, Marianne; Krönke, Jan; Bullinger, Lars; Späth, Daniela; Kayser, Sabine; Zucknick, Manuela; Götze, Katharina; Horst, Heinz-A; Germing, Ulrich; Döhner, Hartmut; Döhner, Konstanze

2010-08-01

To analyze the frequency and prognostic impact of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations in acute myeloid leukemia (AML). We studied 805 adults (age range, 16 to 60 years) with AML enrolled on German-Austrian AML Study Group (AMLSG) treatment trials AML HD98A and APL HD95 for mutations in exon 4 of IDH1 and IDH2. Patients were also studied for NPM1, FLT3, MLL, and CEBPA mutations. The median follow-up for survival was 6.3 years. IDH mutations were found in 129 patients (16.0%) -IDH1 in 61 patients (7.6%), and IDH2 in 70 patients (8.7%). Two patients had both IDH1 and IDH2 mutations. All but one IDH1 mutation caused substitutions of residue R132; IDH2 mutations caused changes of R140 (n = 48) or R172 (n = 22). IDH mutations were associated with older age (P < .001; effect conferred by IDH2 only); lower WBC (P = .04); higher platelets (P < .001); cytogenetically normal (CN) -AML (P< .001); and NPM1 mutations, in particular with the genotype of mutated NPM1 without FLT3 internal tandem duplication (ITD; P < .001). In patients with CN-AML with the latter genotype, IDH mutations adversely impacted relapse-free survival (RFS; P = .02) and overall survival (P = .03), whereas outcome was not affected in patients with CN-AML who lacked this genotype. In CN-AML, multivariable analyses revealed a significant interaction between IDH mutation and the genotype of mutated NPM1 without FLT3-ITD (ie, the adverse impact of IDH mutation [RFS]; P = .046 was restricted to this patient subset). IDH1 and IDH2 mutations are recurring genetic changes in AML. They constitute a poor prognostic factor in CN-AML with mutated NPM1 without FLT3-ITD, which allows refined risk stratification of this AML subset.

Emerging FMS-like tyrosine kinase 3 inhibitors for the treatment of acute myelogenous leukemia

PubMed Central

Prescott, Hillary; Kantarjian, Hagop; Cortes, Jorge; Ravandi, Farhad

2014-01-01

Introduction The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute leukemias. Mutations involving FLT3 are among the most common molecular abnormalities in acute myelogenous leukemia (AML). Available evidence suggests that these molecular lesions confer a shorter disease-free survival and overall survival in patients with intermediate-risk cytogenetics. Therefore, substantial interest in FLT3 as a therapeutic target has led to the development of several promising inhibitors that target this tyrosine kinase. Areas covered This review covers the molecular pathways associated with FLT3 activation in patients with AML, the biological rationale for inhibiting FLT3 and recent clinical progress with FLT3 inhibitors for the treatment of AML. Six FLT3 inhibitors undergoing clinical evaluation are discussed. A review of selected published manuscripts on the subject of FLT3 inhibition in AML and a search of the English language manuscripts in PubMed using the index words FLT3 and AML were conducted and articles of interest selected. Expert opinion Mutated forms of FLT3, specifically FLT3-internal tandem duplication, have a significant impact on the prognosis of AML patients, particularly those with a normal karyotype. Inhibiting FLT3 may lead to clinical benefit for patients with AML. Newly developed FLT3 inhibitors have shown encouraging activity as monotherapy and in combination with other therapeutic agents. PMID:21417961

Cord blood biomarkers of vascular endothelial growth (VEGF and sFlt-1) and postnatal growth: a preterm birth cohort study

PubMed Central

Voller, Stephannie Baehl; Chock, Susanne; Ernst, Linda M.; Su, Emily; Liu, Xin; Farrow, Kathryn N.; Mestan, Karen K.

2014-01-01

Background Preterm infants are at risk for postnatal growth failure (PGF). Identification of biomarkers that are associated with neonatal growth may help reduce PGF and associated long-term morbidity. Objective To investigate the associations between cord blood vascular endothelial growth factor (VEGF) and its soluble receptor (sFlt-1) with birth weight (BW) and postnatal growth in premature infants. Study Design and Methods From an ongoing birth cohort, 123 premature infants from 23 to 36 weeks gestational age (GA) were studied. Cord blood plasma VEGF and sFlt-1 were measured via enzyme-linked immunoassay. Growth parameters and nutritional information were evaluated. Multivariate logistic regression models were constructed to evaluate the associations of VEGF and sFlt-1 on PGF, defined as weight < 10th percentile at 36 weeks corrected age or discharge. Results VEGF was positively correlated, and sFlt-1 was negatively correlated with BW and BW-for-GA percentiles. Higher cord blood VEGF levels were associated with reduced risk of PGF (OR=0.7; 95% CI=0.5–0.9), while higher sFlt-1 levels appeared to increase the risk of PGF (OR=1.6; 95% CI=1.1–2.4). The above biomarker associations were attenuated after adjustment for maternal preeclampsia, fetal growth restriction and related neonatal characteristics, and when taking into account placental vascular pathologies. Longitudinal growth patterns by mean weight and length percentiles were consistently lower among infants with low VEGF/sFlt-1 ratios. Conclusions Our data support that intrauterine regulation of angiogenesis is an important mechanism of fetal and postnatal growth. Cord blood VEGF and sFlt-1 are useful in elucidating how intrauterine processes may have long-standing effects on developing premature infants. PMID:24480606

Mechanism of the photochemical ligand substitution reactions of fac-[Re(bpy)(CO)(3)(PR(3))](+) complexes and the properties of their triplet ligand-field excited states.

PubMed

Koike, Kazuhide; Okoshi, Nobuaki; Hori, Hisao; Takeuchi, Koji; Ishitani, Osamu; Tsubaki, Hideaki; Clark, Ian P; George, Michael W; Johnson, Frank P A; Turner, James J

2002-09-25

We report herein the mechanism of the photochemical ligand substitution reactions of a series of fac-[Re(X(2)bpy)(CO)(3)(PR(3))](+) complexes (1) and the properties of their triplet ligand-field ((3)LF) excited states. The reason for the photostability of the rhenium complexes [Re(X(2)bpy)(CO)(3)(py)](+) (3) and [Re(X(2)bpy)(CO)(3)Cl] (4) was also investigated. Irradiation of an acetonitrile solution of 1 selectively gave the biscarbonyl complexes cis,trans-[Re(X(2)bpy)(CO)(2)(PR(3))(CH(3)CN)](+) (2). Isotope experiments clearly showed that the CO ligand trans to the PR(3) ligand was selectively substituted. The photochemical reactions proceeded via a dissociative mechanism from the (3)LF excited state. The thermodynamical data for the (3)LF excited states of complexes 1 and the corrective nonradiative decay rate constants for the triplet metal-to-ligand charge-transfer ((3)MLCT) states were obtained from temperature-dependence data for the emission lifetimes and for the quantum yields of the photochemical reactions and the emission. Comparison of 1 with [Re(X(2)bpy)(CO)(3)(py)](+) (3) and [Re(X(2)bpy)(CO)(3)Cl] (4) indicated that the (3)LF states of some 3- and 4-type complexes are probably accessible from the (3)MLCT state even at ambient temperature, but these complexes were stable to irradiation at 365 nm. The photostability of 3 and 4, in contrast to 1, can be explained by differences in the trans effects of the PR(3), py, and Cl(-) ligands.

STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

PubMed Central

Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

1994-01-01

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

The role of HOXB2 and HOXB3 in acute myeloid leukemia.

PubMed

Lindblad, Oscar; Chougule, Rohit A; Moharram, Sausan A; Kabir, Nuzhat N; Sun, Jianmin; Kazi, Julhash U; Rönnstrand, Lars

2015-11-27

Acute myeloid leukemia (AML) is a heterogeneous aggressive disease and the most common form of adult leukemia. Mutations in the type III receptor tyrosine kinase FLT3 are found in more than 30% of AML patients. Drugs against FLT3 have been developed for the treatment of AML, but they lack specificity, show poor response and lead to the development of a resistant phenotype upon treatment. Therefore, a deeper understanding of FLT3 signaling will facilitate identification of additional pharmacological targets in FLT3-driven AML. In this report, we identify HOXB2 and HOXB3 as novel regulators of oncogenic FLT3-ITD-driven AML. We show that HOXB2 and HOXB3 expression is upregulated in a group of AML patients carrying FLT3-ITD. Overexpression of HOXB2 or HOXB3 in mouse pro-B cells resulted in decreased FLT3-ITD-dependent cell proliferation as well as colony formation and increased apoptosis. Expression of HOXB2 or HOXB3 resulted in a significant decrease in FLT3-ITD-induced AKT, ERK, p38 and STAT5 phosphorylation. Our data suggest that HOXB2 and HOXB3 act as tumor suppressors in FLT3-ITD driven AML. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of HOXB2 and HOXB3 in acute myeloid leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindblad, Oscar; Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund; Department of Hematology and Vascular Disorders, Skåne University Hospital, Lund

2015-11-27

Acute myeloid leukemia (AML) is a heterogeneous aggressive disease and the most common form of adult leukemia. Mutations in the type III receptor tyrosine kinase FLT3 are found in more than 30% of AML patients. Drugs against FLT3 have been developed for the treatment of AML, but they lack specificity, show poor response and lead to the development of a resistant phenotype upon treatment. Therefore, a deeper understanding of FLT3 signaling will facilitate identification of additional pharmacological targets in FLT3-driven AML. In this report, we identify HOXB2 and HOXB3 as novel regulators of oncogenic FLT3-ITD-driven AML. We show that HOXB2more » and HOXB3 expression is upregulated in a group of AML patients carrying FLT3-ITD. Overexpression of HOXB2 or HOXB3 in mouse pro-B cells resulted in decreased FLT3-ITD-dependent cell proliferation as well as colony formation and increased apoptosis. Expression of HOXB2 or HOXB3 resulted in a significant decrease in FLT3-ITD-induced AKT, ERK, p38 and STAT5 phosphorylation. Our data suggest that HOXB2 and HOXB3 act as tumor suppressors in FLT3-ITD driven AML.« less

Soluble Flt-1 improves the repair of ankle joint injury in rats

PubMed Central

Tian, Jing; Xie, Bing; Xiang, Liangbi; Zhao, Yong; Zhou, Dapeng

2016-01-01

The ankle injuries create great pain to a great number of patients worldwide. Past studies have focused on the development of practical treatments to relieve pain and improve recovery, but the molecular mechanisms underlying the ankle injuries, especially the local inflammation in the damaged ankle joint, have been rarely studied. Moreover, although reduction of production and secretion of pro-inflammatory cytokines may reduce the pain and promote the recovery, a practical approach is currently lacking. Here, we detected significantly higher levels of placental growth factor (PLGF) and pro-inflammatory cytokines in the joint fluid from the patients of acute ankle joint injury (AAJI). Interestingly, the levels of PLGF and pro-inflammatory cytokines in the joint fluid strongly correlated. In order to examine whether PLGF may regulate the production and secretion of pro-inflammatory cytokines in the injured joint, we used a rat carrageenan-induced ankle injury model for AAJI in humans. We injected soluble Flt-1 (sFlt-1) into the articular cavity of the injured ankle joint to block PLGF signaling and found that injection of sFlt-1 significantly improved the rat behavior in activity wheels test, which appeared to result from reduced secretion of the pro-inflammatory cytokines in the ankle joint. Thus, our study suggests that blocking PLGF signaling may be a novel therapeutic approach for treating AAJI in humans. PMID:27904694

Anti-sFlt-1 Therapy Preserves Lung Alveolar and Vascular Growth in Antenatal Models of Bronchopulmonary Dysplasia.

PubMed

Wallace, Bradley; Peisl, Amelie; Seedorf, Gregory; Nowlin, Taylor; Kim, Christina; Bosco, Jennifer; Kenniston, Jon; Keefe, Dennis; Abman, Steven H

2018-03-15

Pregnancies complicated by antenatal stress, including preeclampsia (PE) and chorioamnionitis (CA), increase the risk for bronchopulmonary dysplasia (BPD) in preterm infants, but biologic mechanisms linking prenatal factors with BPD are uncertain. Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous antagonist to VEGF (vascular endothelial growth factor), are increased in amniotic fluid and maternal blood in PE and associated with CA. Because impaired VEGF signaling has been implicated in the pathogenesis of BPD, we hypothesized that fetal exposure to sFlt-1 decreases lung growth and causes abnormal lung structure and pulmonary hypertension during infancy. To test this hypothesis, we studied the effects of anti-sFlt-1 monoclonal antibody (mAb) treatment on lung growth in two established antenatal models of BPD that mimic PE and CA induced by intraamniotic (i.a.) injections of sFlt-1 or endotoxin, respectively. In experimental PE, mAb was administered by three different approaches, including antenatal treatment by either i.a. instillation or maternal uterine artery infusion, or by postnatal intraperitoneal injections. With each strategy, mAb therapy improved infant lung structure as assessed by radial alveolar count, vessel density, right ventricular hypertrophy, and lung function. As found in the PE model, the adverse lung effects of i.a. endotoxin were also reduced by antenatal or postnatal mAb therapy. We conclude that treatment with anti-sFlt-1 mAb preserves lung structure and function and prevents right ventricular hypertrophy in two rat models of BPD of antenatal stress and speculate that early mAb therapy may provide a novel strategy for the prevention of BPD.

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3 and KIT driven Leukemogenesis

PubMed Central

Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H.; Waskow, Emily R.; Visconte, Valeria; Tiu, Ramon V.; Smith, Catherine C.; Shah, Neil; Bunting, Kevin D.; Boswell, H. Scott; Liu, Yan; Chan, Rebecca J.; Kapur, Reuben

2015-01-01

SUMMARY Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN) and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK), whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. PMID:25456130

Dopamine D3 as well as D2 receptor ligands attenuate the cue-induced cocaine-seeking in a relapse model in rats.

PubMed

Gál, Krisztina; Gyertyán, István

2006-01-04

Environmental cues associated with the previously abused drug elicit craving and relapse to drug use in humans. Several reinstatement paradigms are used in animals to examine the relapse-preventing efficacy of possible medical treatments. The purpose of the present study was to investigate the effect of D3 dopamine receptor ligands in a relapse model where animals with stable cocaine self-administration behavior were exposed to all the environmental and reinforcement-contingent discrete cues associated for the previous cocaine-intake in a single extinction session after 3-week long abstinence period. The following compounds were studied: SB-277011-A as a selective D3 antagonist, BP-897 as a D3 partial agonist/D2 antagonist and haloperidol as a preferential D2 receptor antagonist. In addition, in the same paradigm we investigated the effect of the above ligands on relapse to natural reward-seeking behavior using sucrose as natural reward. SB-277011-A (5 and 20 mg/kg), BP-897 (1 mg/kg) and haloperidol (0.2 mg/kg) significantly inhibited the secondary cues-induced cocaine-seeking behavior. None of the above drugs significantly influenced the cue-controlled sucrose-seeking behavior. These results confirm the importance of the D3 as well as the D2 dopamine receptor in modulating the cue-induced cocaine relapse and the possible usefulness of the D3 dopamine receptor ligands as potential medication in cocaine addicts.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

PubMed

Sato, K; Yamazaki, K; Shizume, K; Kanaji, Y; Obara, T; Ohsumi, K; Demura, H; Yamaguchi, S; Shibuya, M

1995-09-01

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Weaker Ligands Can Dominate an Odor Blend due to Syntopic Interactions

PubMed Central

2013-01-01

Most odors in natural environments are mixtures of several compounds. Perceptually, these can blend into a new “perfume,” or some components may dominate as elements of the mixture. In order to understand such mixture interactions, it is necessary to study the events at the olfactory periphery, down to the level of single-odorant receptor cells. Does a strong ligand present at a low concentration outweigh the effect of weak ligands present at high concentrations? We used the fruit fly receptor dOr22a and a banana-like odor mixture as a model system. We show that an intermediate ligand at an intermediate concentration alone elicits the neuron’s blend response, despite the presence of both weaker ligands at higher concentration, and of better ligands at lower concentration in the mixture. Because all of these components, when given alone, elicited significant responses, this reveals specific mixture processing already at the periphery. By measuring complete dose–response curves we show that these mixture effects can be fully explained by a model of syntopic interaction at a single-receptor binding site. Our data have important implications for how odor mixtures are processed in general, and what preprocessing occurs before the information reaches the brain. PMID:23315042

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

PubMed

Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

2018-05-01

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

sFlt-1/PlGF ratio test for pre-eclampsia: an economic assessment for the UK.

PubMed

Vatish, M; Strunz-McKendry, T; Hund, M; Allegranza, D; Wolf, C; Smare, C

2016-12-01

To assess the economic impact of introducing into clinical practice in the UK the soluble fms-like tyrosine kinase (sFlt-1) to placental growth factor (PlGF) ratio test for guiding the management of pre-eclampsia. We used an economic model estimating the incremental value of information, from a UK National Health Service payer's perspective, generated by the sFlt-1/PlGF ratio test, compared with current diagnostic procedures, in guiding the management of women with suspected pre-eclampsia. The economic model estimated costs associated with the diagnosis and management of pre-eclampsia in pregnant women between 24 + 0 and 36 + 6 weeks' gestation, managed in either a 'test' scenario in which the sFlt-1/PlGF test is used in addition to current diagnostic procedures, or a 'no-test' scenario in which clinical decisions are based on current diagnostic procedures alone. Test characteristics and resource use were derived from PROGNOSIS, a non-interventional study in women presenting with clinical suspicion of pre-eclampsia. The main outcome measure from the economic model was the cost per patient per episode of care, from first suspicion of pre-eclampsia to birth. Introduction of the sFlt-1/PlGF ratio test into clinical practice is expected to result in cost savings of £344 per patient compared with a no-test scenario. Savings are generated primarily through an improvement in diagnostic accuracy and subsequent reduction in unnecessary hospitalization. Introducing the sFlt-1/PlGF ratio test into clinical practice in the UK was shown to be cost-saving by reducing unnecessary hospitalization of women at low risk of developing pre-eclampsia. In addition, the test ensures that those women at higher risk are identified and managed appropriately. © 2016 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology. © 2016 The Authors. Ultrasound in Obstetrics & Gynecology

Role of interfacial water molecules in proline-rich ligand recognition by the Src homology 3 domain of Abl.

PubMed

Palencia, Andres; Camara-Artigas, Ana; Pisabarro, M Teresa; Martinez, Jose C; Luque, Irene

2010-01-22

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design.

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET1

PubMed Central

Honer, Michael; Ebenhan, Thomas; Allegrini, Peter R; Ametamey, Simon M; Becquet, Mike; Cannet, Catherine; Lane, Heidi A; O'Reilly, Terence M; Schubiger, Pius A; Sticker-Jantscheff, Melanie; Stumm, Michael; McSheehy, Paul MJ

2010-01-01

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[18F]fluoro-glucose (FDG) and 3′-deoxy-3′-[18F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC50 values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing. PMID:20689768

Soluble fms-Like Tyrosine Kinase 1 (sFlt1), Endoglin and Placental Growth Factor (PlGF) in Preeclampsia among High Risk Pregnancies

PubMed Central

Powers, Robert W.; Jeyabalan, Arun; Clifton, Rebecca G.; Van Dorsten, Peter; Hauth, John C.; Klebanoff, Mark A.; Lindheimer, Marshall D.; Sibai, Baha; Landon, Mark; Miodovnik, Menachem

2010-01-01

Background Differences in circulating concentrations of antiangiogenic factors sFlt1 and soluble endoglin (sEng) and the pro-angiogenic growth factor PlGF are reported to precede the onset of preeclampsia weeks to months in low-risk pregnant women. The objective of this study was to investigate whether similar changes can be detected in pregnant women at high-risk to develop the syndrome. Methods This study is a secondary analysis of the NICHD MFMU trial of aspirin to prevent preeclampsia in high-risk pregnancies. Serum samples were available from 194 women with pre-existing diabetes, 313 with chronic hypertension, 234 with multifetal gestation, and 252 with a history of preeclampsia in a previous pregnancy. Samples collected across pregnancy were analyzed in a blinded fashion for sFlt1, sEng and PlGF. Results The odds of developing preeclampsia were significantly increased among women with multiple fetuses for each 2-fold elevation in sFlt1, sEng and the ratio of angiogenic factors (e.g. OR 2.18, 95% CI 1.46-3.32), and significantly decreased for each 2-fold elevation in circulating PlGF (OR 0.50, 95% CI 0.30-0.82) between 7 and 26 weeks' gestation. Cross-sectional analysis of the angiogenic factors across gestation showed significant differences during the third trimester in women who develop preeclampsia compared with appropriate controls in all high-risk groups. However, when data were examined in relation to the gestational week when preeclampsia was diagnosed only sFlt1 was significantly higher 2 to 5 weeks before the clinical onset of preeclampsia and only in women with previous preeclampsia. Conclusions The pattern of elevated concentrations of sFlt1 and sEng, and low PlGF in high-risk pregnant subjects who develop preeclampsia is similar to that reported in low-risk pregnant women. However, differences in these factors among high-risk women who do and do not develop preeclampsia are modest, and do not appear to be clinically useful predictors in these high

Thermodynamics and docking of agonists to the β(2)-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand.

PubMed

Toll, Lawrence; Pajak, Karolina; Plazinska, Anita; Jozwiak, Krzysztof; Jimenez, Lucita; Kozocas, Joseph A; Tanga, Mary J; Bupp, James E; Wainer, Irving W

2012-06-01

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The β(2)-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K(i) values determined for fenoterol analogs model much better the cAMP activity of the β(2)-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [(3)H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β(2)-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β(2)-adrenoceptor.

Thermodynamics and Docking of Agonists to the β2-Adrenoceptor Determined Using [3H](R,R′)-4-Methoxyfenoterol as the Marker Ligand

PubMed Central

Pajak, Karolina; Plazinska, Anita; Jozwiak, Krzysztof; Jimenez, Lucita; Kozocas, Joseph A.; Tanga, Mary J.; Bupp, James E.; Wainer, Irving W.

2012-01-01

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β2-adrenergic receptor, we developed a new agonist radioligand for binding assays. [3H](R,R′)-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β2-adrenoceptor (AR) population as determined with the antagonist [3H]CGP-12177. The β2-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and Ki values determined for fenoterol analogs model much better the cAMP activity of the β2-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [3H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β2-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β2-adrenoceptor. PMID:22434858

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis.

PubMed

Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H; Waskow, Emily R; Visconte, Valeria; Tiu, Ramon V; Smith, Catherine C; Shah, Neil; Bunting, Kevin D; Boswell, H Scott; Liu, Yan; Chan, Rebecca J; Kapur, Reuben

2014-11-20

Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Impact of pre-transplantation minimal residual disease determined by multiparameter flow cytometry on the outcome of AML patients with FLT3-ITD after allogeneic stem cell transplantation.

PubMed

Zhao, Xiaosu; Wang, Zhidong; Ruan, Guorui; Liu, Yanrong; Wang, Yu; Zhang, Xiaohui; Xu, Lanping; Huang, Xiaojun; Chang, Yingjun

2018-06-01

In this study, using multiparameter flow cytometry (FCM), we investigate the impact of minimal residual disease prior to transplantation (pre-MRD) on the transplant outcomes of AML patients with fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) mutation. A total of 20 patients who received HLA-matched sibling donor transplantation (MSDT) and 63 patients who received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were enrolled. Patients were classified into four groups based on the status of pre-FCM: group 1 with positive pre-FCM before MSDT, group 2 with negative pre-FCM before MSDT, group 3 with positive pre-FCM before haplo-HSCT, and group 4 with positive pre-FCM before haplo-HSCT. The results showed that patients in group 1 had the highest cumulative incidence of relapse (2-year CIR, 75.0%), the lowest leukemia-free survival (2-year LFS, 33.3%), and the overall survival (2-year OS, 25.0%) among all four groups. The other three groups of patients had comparable CIR (2-year CIR: group 2 vs. 3 vs. 4, 12.5% vs. 31.3% vs. 22.2%, P > 0.05) and LFS (2-year LFS: group 2 vs. 3 vs. 4, 87.5% vs. 62.5% vs. 66.5%, P > 0.05). Multivariate analysis indicated that disease status (> CR) and pre-MRD were associated with a higher CIR and a lower LFS when patients were classified by pre-MRD and transplant type. Our results suggested that AML patients with FLT3-ITD were able to be separated into high-risk and low-risk relapse groups based on pre-MRD, as determined by multiparameter FCM. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT. These results require further investigation in prospective study with large numbers of cases.

Runx1 and Cbfβ regulate the development of Flt3+ dendritic cell progenitors and restrict myeloproliferative disorder

PubMed Central

Satpathy, Ansuman T.; Briseño, Carlos G.; Cai, Xiongwei; Michael, Drew G.; Chou, Chun; Hsiung, Sunnie; Bhattacharya, Deepta; Speck, Nancy A.

2014-01-01

Runx1 and Cbfβ are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbfβ is essential for the development of Flt3+ macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105+ erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbfβ restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia. PMID:24677539

Trinuclear Mn(II) complex with paramagnetic bridging 1,2,3-dithiazolyl ligands.

PubMed

Sullivan, David J; Clérac, Rodolphe; Jennings, Michael; Lough, Alan J; Preuss, Kathryn E

2012-11-18

The first metal coordination complex of a radical ligand based on the 1,2,3-dithiazolyl heterocycle is reported. 6,7-Dimethyl-1,4-dioxo-naphtho[2,3-d][1,2,3]dithiazolyl acts as a bridging ligand in the volatile trinuclear Mn(hfac)(2)-Rad-Mn(hfac)(2)-Rad-Mn(hfac)(2) complex (hfac = 1,1,1,5,5,5-hexafluoroacetylacetonato-). The Mn(II) and radical ligand spins are coupled anti-ferromagnetically (AF) resulting in an S(T) = 13/2 spin ground state.

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuyama, Yoshiko; Tokuhara, Daisuke; Division of Mucosal Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639

Highlights: Black-Right-Pointing-Pointer Nasal Ad-FL effectively up-regulates APC function by CD11c{sup +} DCs in mucosal tissues. Black-Right-Pointing-Pointer Nasal Ad-FL induces Notch ligand (L)-expressing CD11c{sup +} DCs. Black-Right-Pointing-Pointer Notch L-expressing DCs support the induction of Th1- and Th2-type cytokine responses. -- Abstract: Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c{sup +} dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c{sup +} DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FLmore » activated CD11c{sup +} DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c{sup +} DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4{sup +} T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-{gamma}, IL-2 and IL-4 producing CD4{sup +} T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c{sup +} DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.« less

Budget impact analysis of sFlt-1/PlGF ratio as prediction test in Italian women with suspected preeclampsia.

PubMed

Frusca, Tiziana; Gervasi, Maria-Teresa; Paolini, Davide; Dionisi, Matteo; Ferre, Francesca; Cetin, Irene

2017-09-01

Preeclampsia (PE) is a pregnancy disease which represents a leading cause of maternal and perinatal mortality and morbidity. Accurate prediction of PE risk could provide an increase in health benefits and better patient management. To estimate the economic impact of introducing Elecsys sFlt-1/PlGF ratio test, in addition to standard practice, for the prediction of PE in women with suspected PE in the Italian National Health Service (INHS). A decision tree model has been developed to simulate the progression of a cohort of pregnant women from the first presentation of clinical suspicion of PE in the second and third trimesters until delivery. The model provides an estimation of the financial impact of introducing sFlt-1/PlGF versus standard practice. Clinical inputs have been derived from PROGNOSIS study and from literature review, and validated by National Clinical Experts. Resources and unit costs have been obtained from Italian-specific sources. Healthcare costs associated with the management of a pregnant woman with clinical suspicion of PE equal €2384 when following standard practice versus €1714 using sFlt-1/PlGF ratio test. Introduction of sFlt-1/PlGF into hospital practice is cost-saving. Savings are generated primarily through improvement in diagnostic accuracy and reduction in unnecessary hospitalization for women before PE's onset.

Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl*

PubMed Central

Palencia, Andres; Camara-Artigas, Ana; Pisabarro, M. Teresa; Martinez, Jose C.; Luque, Irene

2010-01-01

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design. PMID:19906645

Ligand Electron Density Shape Recognition Using 3D Zernike Descriptors

NASA Astrophysics Data System (ADS)

Gunasekaran, Prasad; Grandison, Scott; Cowtan, Kevin; Mak, Lora; Lawson, David M.; Morris, Richard J.

We present a novel approach to crystallographic ligand density interpretation based on Zernike shape descriptors. Electron density for a bound ligand is expanded in an orthogonal polynomial series (3D Zernike polynomials) and the coefficients from this expansion are employed to construct rotation-invariant descriptors. These descriptors can be compared highly efficiently against large databases of descriptors computed from other molecules. In this manuscript we describe this process and show initial results from an electron density interpretation study on a dataset containing over a hundred OMIT maps. We could identify the correct ligand as the first hit in about 30 % of the cases, within the top five in a further 30 % of the cases, and giving rise to an 80 % probability of getting the correct ligand within the top ten matches. In all but a few examples, the top hit was highly similar to the correct ligand in both shape and chemistry. Further extensions and intrinsic limitations of the method are discussed.

MO-AB-BRA-06: Dynamic FLT PET for Investigating Potential Synergistic Therapeutic Targets During Anti-Angiogenic Treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scarpelli, M; Perlman, S; Liu, G

2016-06-15

Purpose: Novel treatment strategies for metastatic cancer patients involve synergistically combining treatments with the hope of improving outcomes. This study investigated changes in tumor proliferative and vascular characteristics derived from dynamic [F-18]FLT PET during antiangiogenic treatment with the goal of identifying synergistic treatment opportunities. Methods: Patients with various solid cancers underwent continuous three-week cycles of anti-angiogenic treatment with intermittent dosing (two-weeks-on/one-week-off). Patients received up to six dynamic FLT PET/CT scans (days 0, 14, and 21 of cycle 1 (C1) and cycle 3 (C3)). Tumor proliferative (Kflt, net uptake rate) and vascular parameters (K1 blood-to-tissue transfer; Vb, vascular fraction) were calculatedmore » using a two-tissue compartment four-rate parameter kinetic model. Relative changes in these parameters, from day 0 to 14 (TxResp) and day 14 to 21 (offTxResp), were calculated. Significant differences were tested using Wilcoxon signed-rank test and significant correlations were tested using Spearman correlation. Results: Thirty patients were evaluable for C1 offTxResp with median values for Kflt, K1, and Vb of +30%, +35% and +30%, respectively. The fractions of patients with positive C1 offTxResp were: 21/30 for Ki, 24/30 for K1, 21/30 for Vb, and 12/30 had positive offTxResp for all three kinetic parameters. The offTxResp in C3 was not significantly different from C1 for any of the kinetic parameters. Significant correlations were found between TxResp and offTxResp in C1 for Kflt (ρ=-0.52, p=0.014), K1 (ρ=−0.61, p=0.003) and Vb (ρ=−0.80, p<0.001). Similar correlations were found for Kflt (ρ=-1, p=0.017) and K1 (ρ=−1, p=0.017) for the five patients evaluable in C3. Conclusion: Dynamic FLT PET showed evidence of distinct vascular and proliferative increases during off treatment weeks that could potentially be targeted with synergistic therapy. Early changes in kinetic parameters were

Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.

2009-06-24

A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from micromolar to low nanomolar.

99mTc-MDM Brain SPECT for the Detection of Recurrent/Remnant Glioma-Comparison With ceMRI and 18F-FLT PET Imaging: Initial Results.

PubMed

Singh, Baljinder; Kumar, Narendra; Sharma, Sarika; Watts, Ankit; Hazari, Puja P; Rani, Nisha; Vyas, Sameer; Anish, Bhattacharya; Mishra, Anil K

2015-10-01

To evaluate the diagnostic use of an indigenously developed single vial ready to label (with Tc) kit preparation of bis-methionine-DTPA (Tc-MDM) for the detection of recurrent/residual glioma. We prospectively studied 32 patients (21 male and 11 female subjects aged 43.0±16.0 years) with clinical suspicion of postoperative recurrent/residual glioma. After radical radiotherapy (54.0-60.0 Gy) with or without concurrent temozolomide as indicated, Tc-MDM SPECT and ceMRI of the brain was performed in all the patients and F-FLT-PET imaging in 16 of 32 patients. MDM SPECT and ceMRI findings were concordant in 28 patients (15 positive and 13 negative). The findings were discordant in the remaining 5 patients, with positive ceMRI and negative MDM-SPECT in 2 patients and negative ceMRI and positive MDM-SPECT in 3 patients. Tc-MDM-SPECT, F-FLT PET, and ceMRI scan findings were positive in 9 of 16 and negative in 5 of 16 patients. In the remaining 2 of 16 patients, both F-FLT-PET and Tc-MDM-SPECT were positive, but ceMRI was negative. Sensitivity, specificity, PPV, NPV, and DA of Tc-MDM-SPECT for diagnosing recurrent/residual glioma were 88.24%, 81.25%, 83.3%, 86.7%, and 84.8%, respectively. The diagnostic accuracy of Tc-bis-methionine (MDM)-SPECT imaging was comparable with that of ceMRI and F-FLT-PET and may be useful in the management of glioma patients in the postsurgical follow-up period. This imaging technique may be of special interest in peripheral hospitals/developing countries lacking access to expensive PET/cyclotron technology. However, comparison with the existing "gold standard" PET tracers, especially with C-11-methionine-PET imaging and histopathological correlation, is warranted in a large cohort of glioma patients through multicentric studies.

Number crunching vs. number theory: computers and FLT, from Kummer to SWAC (1850-1960), and beyond

NASA Astrophysics Data System (ADS)

Corry, Leo

2008-07-01

The article discusses the computational tools (both conceptual and material) used in various attempts to deal with individual cases of FLT [Fermat's Last Theorem], as well as the changing historical contexts in which these tools were developed and used, and affected research. It also explores the changing conceptions about the role of computations within the overall disciplinary picture of number theory, how they influenced research on the theorem, and the kinds of general insights thus achieved. After an overview of Kummer's contributions and its immediate influence, the author presents work that favored intensive computations of particular cases of FLT as a legitimate, fruitful, and worth-pursuing number-theoretical endeavor, and that were part of a coherent and active, but essentially low-profile tradition within nineteenth century number theory. This work was related to table making activity that was encouraged by institutions and individuals whose motivations came mainly from applied mathematics, astronomy, and engineering, and seldom from number theory proper. A main section of the article is devoted to the fruitful collaboration between Harry S. Vandiver and Emma and Dick Lehmer. The author shows how their early work led to the hesitant introduction of electronic computers for research related with FLT. Their joint work became a milestone for computer-assisted activity in number theory at large.

Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

PubMed

Huang, Qi-Tao; Chen, Jian-Hong; Hang, Li-Lin; Liu, Shi-San; Zhong, Mei

2015-01-01

Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future. © 2015 S. Karger AG, Basel.

Preclinical evaluation of the anti-tumor effects of the natural isoflavone genistein in two xenograft mouse models monitored by [18F]FDG, [18F]FLT, and [64Cu]NODAGA-cetuximab small animal PET.

PubMed

Honndorf, Valerie S; Wiehr, Stefan; Rolle, Anna-Maria; Schmitt, Julia; Kreft, Luisa; Quintanilla-Martinez, Letitia; Kohlhofer, Ursula; Reischl, Gerald; Maurer, Andreas; Boldt, Karsten; Schwarz, Michael; Schmidt, Holger; Pichler, Bernd J

2016-05-10

The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting. A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 3'-deoxy-3'-[18F]fluorothymidine ([18F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [18F]FDG, [18F]FLT and [64Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data. Genistein reduced tumor growth significantly in both xenografts. [18F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [18F]FDG and [64Cu]NODAGA-cetuximab were not suitable for therapy monitoring. As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [18F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

PubMed Central

2012-01-01

Introduction The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Methods Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. Results MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Conclusions Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells.

PubMed

Lappano, Rosamaria; Santolla, Maria Francesca; Pupo, Marco; Sinicropi, Maria Stefania; Caruso, Anna; Rosano, Camillo; Maggiolini, Marcello

2012-01-17

The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor

Mapping Radiation Injury and Recovery in Bone Marrow Using 18F-FLT PET/CT and USPIO MRI in a Rat Model.

PubMed

Rendon, David A; Kotedia, Khushali; Afshar, Solmaz F; Punia, Jyotinder N; Sabek, Omaima M; Shirkey, Beverly A; Zawaski, Janice A; Gaber, M Waleed

2016-02-01

We present and test the use of multimodality imaging as a topological tool to map the amount of the body exposed to ionizing radiation and the location of exposure, which are important indicators of survival and recovery. To achieve our goal, PET/CT imaging with 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) was used to measure cellular proliferation in bone marrow (BM), whereas MRI using ultra-small superparamagnetic iron oxide (USPIO) particles provided noninvasive information on radiation-induced vascular damage. Animals were x-ray-irradiated at a dose of 7.5 Gy with 1 of 3 radiation schemes-whole-body irradiation, half-body shielding (HBS), or 1-leg shielding (1LS)-and imaged repeatedly. The spatial information from the CT scan was used to segment the region corresponding to BM from the PET scan using algorithms developed in-house, allowing for quantification of proliferating cells, and BM blood volume was estimated by measuring the changes in the T2 relaxation rates (ΔR2) collected from MR scans. (18)F-FLT PET/CT imaging differentiated irradiated from unirradiated BM regions. Two days after irradiation, proliferation of 1LS animals was significantly lower than sham (P = 0.0001, femurs; P < 0.0001, tibias) and returned to sham levels by day 10 (P = 0.6344, femurs; P = 0.3962, tibias). The degree of shielding affected proliferation recovery, showing an increase in the irradiated BM of the femurs, but not the tibias, of HBS animals when compared with 1LS (P = 0.0310, femurs; P = 0.5832, tibias). MRI of irradiated spines detected radiation-induced BM vascular damage, measured by the significant increase in ΔR2 2 d after whole-body irradiation (P = 0.0022) and HBS (P = 0.0003) with a decreasing trend of values, returning to levels close to baseline over 10 d. Our data were corroborated using γ-counting and histopathology. We demonstrated that (18)F-FLT PET/CT and USPIO MRI are valuable tools in mapping regional radiation exposure and the effects of radiation on

Membrane-bound LERK2 ligand can signal through three different Eph-related receptor tyrosine kinases.

PubMed Central

Brambilla, R; Schnapp, A; Casagranda, F; Labrador, J P; Bergemann, A D; Flanagan, J G; Pasquale, E B; Klein, R

1995-01-01

The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. Recently, a small family of ligands, structurally related to the B61 protein, was identified. Binding of these ligands to Eph-related receptors did not, however, elicit measurable biological signals in cultured cells. In order to study functional interactions between B61-related ligands and Eph-related receptors, we constructed chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor. Expression and activation of such chimeric receptors in NIH 3T3 cells induced transformation in focus formation assays. Membrane-bound LERK2 ligand is shown to signal through three different Eph-related receptors, namely Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling. Images PMID:7621826

18F-FDG or 3'-deoxy-3'-18F-fluorothymidine to detect transformation of follicular lymphoma.

PubMed

Wondergem, Marielle J; Rizvi, Saiyada N F; Jauw, Yvonne; Hoekstra, Otto S; Hoetjes, Nikie; van de Ven, Peter M; Boellaard, Ronald; Chamuleau, Martine E D; Cillessen, Saskia A G M; Regelink, Josien C; Zweegman, Sonja; Zijlstra, Josée M

2015-02-01

Considering the different treatment strategy for transformed follicular lymphoma (TF) as opposed to follicular lymphoma (FL), diagnosing transformation early in the disease course is important. There is evidence that (18)F-FDG has utility as a biomarker of transformation. However, quantitative thresholds may require inclusion of homogeneous non-Hodgkin lymphoma subtypes to account for differences in tracer uptake per subtype. Moreover, because proliferation is a hallmark of transformation, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) might be superior to (18)F-FDG in this setting. To define the best tracer for detection of TF, we performed a prospective a head-to-head comparison of (18)F-FDG and (18)F-FLT in patients with FL and TF. (18)F-FDG and (18)F-FLT PET scans were obtained in 17 patients with FL and 9 patients with TF. We measured the highest maximum standardized uptake value (SUVmax), defined as the lymph node with the highest uptake per patient, and SUVrange, defined as the difference between the SUVmax of the lymph node with the highest and lowest uptake per patient. To reduce partial-volume effects, only lymph nodes larger than 3 cm(3) (A50 isocontour) were analyzed. Scans were acquired 1 h after injection of 185 MBq of (18)F-FDG or (18)F-FLT. To determine the discriminative ability of SUVmax and SUVrange of both tracers for TF, receiver-operating-characteristic curve analysis was performed. The highest SUVmax was significantly higher for TF than FL for both (18)F-FDG and (18)F-FLT (P < 0.001). SUVrange was significantly higher for TF than FL for (18)F-FDG (P = 0.029) but not for (18)F-FLT (P = 0.075). The ability of (18)F-FDG to discriminate between FL and TF was superior to that of (18)F-FLT for both the highest SUVmax (P = 0.039) and the SUVrange (P = 0.012). The cutoff value for the highest SUVmax of (18)F-FDG aiming at 100% sensitivity with a maximum specificity was found to be 14.5 (corresponding specificity, 82%). For (18)F-FLT, these values were

Theoretical study of 'Mixed' ligands superhalogens: Cl-M-NO3 (M = Li, Na, K)

NASA Astrophysics Data System (ADS)

Zhao, Xinghua; Liu, Weihui; Wang, Jiesheng; Li, Chun; Yuan, Guang

2016-08-01

MCl2-, M(NO3)2-, and (Cl-M-NO3)- (M = Li, Na, K) species are systematically investigated using the density functional theory. In all the cases studied, the vertical detachment energies (VDEs) exceed the electron affinity of chlorine atom, leading to the conclusion that MCl2-, M(NO3)2- and (Cl-M-NO3)- are superhalogens. The VDEs of (Cl-M-NO3)- are between that of MCl2- and M(NO3)2-, showing that replacing one ligand with a larger electronegative ligand leads to the higher VDE. Superhalogens with suitable VDEs can be built by using different ligands.

Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model.

PubMed

Burkholder, Timothy P; Clayton, Joshua R; Rempala, Mark E; Henry, James R; Knobeloch, John M; Mendel, David; McLean, Johnathan A; Hao, Yan; Barda, David A; Considine, Eileen L; Uhlik, Mark T; Chen, Yuefeng; Ma, Liandong; Bloem, Laura J; Akunda, Jacqueline K; McCann, Denis J; Sanchez-Felix, Manuel; Clawson, David K; Lahn, Michael M; Starling, James J

2012-06-01

LY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non

Evaluation of agreement of placental growth factor (PlGF) tests and the soluble FMS-like tyrosine kinase 1 (sFlt-1)/PlGF ratio, comparison of predictive accuracy for pre-eclampsia, and relation to uterine artery Doppler and response to aspirin.

PubMed

Navaratnam, Kate; Abreu, Patricia; Clarke, Helen; Jorgensen, Andrea; Alfirevic, Ana; Alfirevic, Zarko

2017-09-11

The objective of this study is to evaluate agreement between PlGF and sFlt-1/PlGF ratio tests and compare their predictive accuracy for pre-eclampsia in high-risk women. Also, to examine for associations of abnormal PlGF or sFlt-1/PlGF ratio with abnormal uterine artery Doppler and platelet response to aspirin. Prospective cohort study, 150 pregnant women at high risk of pre-eclampsia prescribed 75 mg aspirin daily. Uterine artery Dopplers were assessed at 20 +0 -23 +6 weeks. At 33 +0 -35 +6 weeks platelet function aspirin metabolites, PlGF and the sFlt-1/PlGF ratio were measured. Measures were all pre-eclampsia and pre-eclampsia requiring delivery prior to 37 weeks. Overall percent agreement was 89.3% for PlGF tests but 74.7-78% for PlGF tests and the sFlt-1/PlGF ratio. AUCs were 0.70-0.75 for prediction of any pre-eclampsia and 0.92-0.99 for preterm pre-eclampsia. We found a significant association between abnormal PlGF or sFlt-1/PlGF ratio and abnormal uterine artery Doppler (χ 2 5.47, p = .019), but no association with platelet response to aspirin (χ 2 0.12, p = .913). There were no associations between suboptimal aspirin adherence and either abnormal angiogenic markers or uterine artery Dopplers (χ 2 0.144, 0.038, p = .704, .846, respectively). There was good agreement between PlGF tests and limited agreement between PlGF tests and the sFlt-1/PlGF ratio. All tests have heightened predictive accuracy for preterm pre-eclampsia. Abnormal PlGF or sFlt-1/PlGF ratio relates to abnormal uterine artery Doppler but not platelet response to aspirin.

A Coordination Network with Ligand-Centered Redox Activity Based on facial-[CrIII (2-mercaptophenolato)3 ]3- Metalloligands.

PubMed

Wakizaka, Masanori; Matsumoto, Takeshi; Kobayashi, Atsushi; Kato, Masako; Chang, Ho-Chol

2017-07-21

The design of redox-active metal-organic frameworks and coordination networks (CNs), which exhibit metal- and/or ligand-centered redox activity, has recently received increased attention. In this study, the redox-active metalloligand (RML) [Me 4 N] 3 fac-[Cr III (mp) 3 ] (1) (mp=2-mercaptophenolato) was synthesized and characterized by single-crystal X-ray diffraction analysis, and its reversible ligand-centered one-electron oxidation was examined by cyclic voltammetry and spectroelectrochemical measurements. Since complex 1 contains O/S coordination sites in three directions, complexation with K + ions led to the formation of the two-dimensional honeycomb sheet-structured [K 3 fac-{Cr III (mp) 3 }(H 2 O) 6 ] n (2⋅6 H 2 O), which is the first example of a redox-active CN constructed from a RML with o-disubstituted benzene ligands. Herein, we unambiguously demonstrate the ligand-centered redox activity of the RML within the CN 2⋅6 H 2 O in the solid state. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ligand binding analysis and screening by chemical denaturation shift.

PubMed

Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

2013-12-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples. Copyright © 2013 Elsevier Inc. All rights reserved.

Ligand Binding Analysis and Screening by Chemical Denaturation Shift

PubMed Central

Sch n, Arne; Brown, Richard K.; Hutchins, Burleigh M.; Freire, Ernesto

2013-01-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Towards this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Since ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities and the ligand rank order obtained at denaturation temperatures (60°C or higher) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations in which binding changes the cooperativity of the unfolding transition. In this paper we develop the basic analytical equations and provide several experimental examples. PMID:23994566

Theoretical study on mechanism of the photochemical ligand substitution of fac-[Re(I)(bpy)(CO)3(PR3)](+) complex.

PubMed

Saita, Kenichiro; Harabuchi, Yu; Taketsugu, Tetsuya; Ishitani, Osamu; Maeda, Satoshi

2016-07-14

The mechanism of the CO ligand dissociation of fac-[Re(I)(bpy)(CO)3P(OMe)3](+) has theoretically been investigated, as the dominant process of the photochemical ligand substitution (PLS) reactions of fac-[Re(I)(bpy)(CO)3PR3](+), by using the (TD-)DFT method. The PLS reactivity can be determined by the topology of the T1 potential energy surface because the photoexcited complex is able to decay into the T1 state by internal conversions (through conical intersections) and intersystem crossings (via crossing seams) with sufficiently low energy barriers. The T1 state has a character of the metal-to-ligand charge-transfer ((3)MLCT) around the Franck-Condon region, and it changes to the metal-centered ((3)MC) state as the Re-CO bond is elongated and bent. The equatorial CO ligand has a much higher energy barrier to leave than that of the axial CO, so that the axial CO ligand selectively dissociates in the PLS reaction. The single-component artificial force induced reaction (SC-AFIR) search reveals the CO dissociation pathway in photostable fac-[Re(I)(bpy)(CO)3Cl]; however, the dissociation barrier on the T1 state is substantially higher than that in fac-[Re(I)(bpy)(CO)3PR3](+) and the minimum-energy seams of crossings (MESXs) are located before and below the barrier. The MESXs have also been searched in fac-[Re(I)(bpy)(CO)3PR3](+) and no MESXs were found before and below the barrier.

A new class of transition metal pincer ligand: tantalum complexes that feature a [CCC] X3-donor array derived from a terphenyl ligand.

PubMed

Sattler, Aaron; Parkin, Gerard

2012-02-01

A new class of [CCC] X(3)-donor pincer ligand for transition metals has been constructed via cyclometalation of a 2,6-di-p-tolylphenyl ([Ar(Tol(2))]) derivative. Specifically, addition of PMe(3) to [Ar(Tol(2))]TaMe(3)Cl induces elimination of methane and formation of the pincer complex, [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)MeCl (Tol' = C(6)H(3)Me), which may also be obtained by treatment of Ta(PMe(3))(2)Me(3)Cl(2) with [Ar(Tol(2))]Li. Solutions of [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)MeCl undergo ligand redistribution with the formation of [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)Me(2)and [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)Cl(2), which may also be synthesized by the reactions of [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)MeCl with MeMgBr and ZnCl(2), respectively. Reduction of [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)Cl(2) with KC(8) in benzene gives the benzene complex [κ(3)-Ar(Tol'(2))]Ta(PMe(3))(2)(η(6)-C(6)H(6)) that is better described as a 1,4-cyclohexadienediyl derivative. Deuterium labeling employing Ta(PMe(3))(2)(CD(3))(3)Cl(2) demonstrates that the pincer ligand is created by a pair of Ar-H/Ta-Me sigma-bond metathesis transformations, rather than by a mechanism that involves α-H abstraction by a tantalum methyl ligand. © 2012 American Chemical Society

The Effect of Ligands on FePt–Fe 3O 4 Core–Shell Magnetic Nanoparticles

DOE PAGES

Kim, Dong-Hyun; Tamada, Yoshinori; Ono, Teruo; ...

2014-03-01

FePt–Fe 3O 4 core–shell nanoparticles functionalized with 3,4-dihydroxyphenylacetic acid (DOPAC) and dimercaptosuccinic acid (DMSA) ligands were synthesized and characterized. We also found that the DOPAC ligand enhances the magnetic properties of the FePt–Fe 3O 4 particles, in comparison with the DMSA ligand, which induces the oxidation of the shell layer that causes a significant reduction of the saturation magnetization. We evaluated the synthesized magnetic nanoparticles for applications in magnetic hyperthermia and magnetic resonance imaging contrast enhancement.

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

PubMed Central

Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

2015-01-01

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

LigandRNA: computational predictor of RNA–ligand interactions

PubMed Central

Philips, Anna; Milanowska, Kaja; Łach, Grzegorz; Bujnicki, Janusz M.

2013-01-01

RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA–small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA–ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a “meta-predictor” leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl. PMID:24145824

Periodic assessment of plasma sFlt-1 and PlGF concentrations and its association with placental morphometry in gestational hypertension (GH) - a prospective follow-up study.

PubMed

Jeevaratnam, Kamalan; Nadarajah, Vishna Devi; Judson, John Paul; Nalliah, Sivalingam; Abdullah, Mohd Farouk

2010-09-28

Hypertensive disorders in pregnancy contributes to about 12% of maternal deaths in Malaysia and similarly worldwide. Early detection and adequate management are preventable strategies. Biochemical markers of abnormal angiogenesis would be more specific in early detection than routine blood pressure and proteinuria measurements. The aim of this study was to estimate maternal plasma PlGF and sFlt-1 levels in pregnant women with gestational hypertension at three intervals of pregnancy and correlate these biomarker levels with placental morphometry. Venous blood samples (antepartum, intrapartum and post partum periods) were drawn to estimate for sFlt-1 and PlGF levels while placental tissue samples were examined for placental morphometry. PlGF levels were lower in gestational hypertension (GH) compared to normotensive during antepartum and intrapartum period, whereas sFlt-1 levels were elevated in GH at antepartum, intrapartum and postpartum intervals during pregnancy. An inverse relationship between these two biomarkers was observed through correlation analysis. PlGF levels were inversely correlated with total villous surface area of the placental periphery (TCsa-C) and villous capillarization (VC-C) of the placental periphery. We established periodic values of for sFlt-1 and PlGF levels for the first time in an ethnically diverse Malaysian setting. We suggest the development of GH in women is related to defective capillarization. In demonstrating periodic changes, this study suggest the possibility of developing GH and other long term health complications as a result of prolonged exposure to sFlt-1. The correlation between PlGF levels and morphometric findings also support possible capillarization defect.

Chelation of /sup 238/Pu(IV) in vivo by 3,4,3-LICAM(C): Effects of ligand methylation and pH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Durbin, P.W.; White, D.L.; Jeung, N.L.

1989-06-01

The linear tetracarboxycatecholate ligand, 3,4,3-LICAM(C) N1,N5,N10,N14-tetrakis(2,3-dihydroxy-4-carboxybenzoyl-tetraaza tet radecane, tetra sodium salt) injected within 1 h after injection of Pu(IV) citrate, removes about the same fraction of Pu from animals as CaNa3-DTPA but removes less inhaled Pu than CaNa3-DTPA and leaves a Pu residue in the renal cortex. However, the formation constant of the expected Pu-3,4,3-LICAM(C) complexes are orders of magnitude greater than that of Pu-DTPA, and 3,4,3-LICAM(C) is 100 times more efficient than CaNa3-DTPA for removing Pu from transferrin in vitro. Because the formation constants of their actinide complexes are central to in vivo actinide chelation, ligand design strategies aremore » dominated by the search for ligands with large Pu complex stabilities, and it was necessary to explain the failure of 3,4,3-LICAM(C) to achieve its thermodynamic potential in vivo. All the batches of 3,4,3-LICAM(C) prepared at Berkeley or in France (Euro-LICAM(C)) were found by high-pressure liquid chromatography to be mixtures of the pure ligand (55% in Berkeley preparations, 8.5% in Euro-LICAM(C)) and its four methylesters. A revised synthesis for 3,4,3-LICAM(C) is appended to this report. All of the incompletely hydrolyzed 3,4,3-LICAM(C) preparations and the pure ligand were tested for removal of Pu from mice (238Pu(IV) citrate intravenous, 30 mumol kg-1 of ligand at 1 h, kill at 24 h, radioanalyze tissues and separated excretal). The presence of methylesters did not significantly impair the ability of the ligands to remove Pu from mice, and it did not alter the fraction of injected Pu deposited in kidneys. Temporary elevation (reduction) of plasma and urine pH of mice by 0.5 mL of 0.1 M NaHCO3 (NH4Cl) injected before or simultaneously with pure 3,4,3-LICAM(C) somewhat improved (significantly reduced) Pu excretion but had little influence on Pu deposition in kidneys.« less

Decreased Nephrin and GLEPP-1, But Increased VEGF, Flt-1, and Nitrotyrosine, Expressions in Kidney Tissue Sections From Women With Preeclampsia

PubMed Central

Zhao, Shuang; Gu, Xin; Groome, Lynn J.; Wang, Yuping

2011-01-01

Renal injury is a common pathophysiological feature in women with preeclampsia as evidenced by increased protein leakage (proteinuria) and glomerular injury (glomerular endotheliosis). Recently, podocyturia was found in preeclampsia, suggesting podocyte shedding occurs in this pregnancy disorder. However, podocyte function in preeclampsia is poorly understood. In this study, the authors have examined podocyte-specific protein expressions for nephrin, glomerular epithelial protein 1 (GLEPP-1), and ezrin in kidney biopsy tissue sections from women with preeclampsia. Expressions for vascular endothelial growth factor (VEGF) and its receptor Flt-1 and oxidative stress marker nitrotyrosine and antioxidant CuZn-superoxide dismutase (CuZn-SOD) were also examined. Kidney tissue sections from nonhypertensive and chronic hypertensive participants were stained as controls. The findings were (1) nephrin and GLEPP-1 were mainly expressed in glomerular podocytes; (2) ezrin was expressed in both glomerular podocytes and tubular epithelial cells; (3) compared to tissue sections from nonhypertensive and chronic hypertensive participants, nephrin and GLEPP-1 expressions were much reduced in tissue sections from preeclampsia and ezrin expression was reduced in podocytes; (4) enhanced VEGF, Flt-1, and nitrotyrosine, but reduced CuZn-SOD, expressions were observed in both glomerular podocytes and endothelial cells in tissue sections from preeclampsia; and (5) the expression pattern for nephrin, GLEPP-1, ezrin, VEGF, Flt-1, and CuZn-SOD were similar between tissue sections from nonhypertensive and chronic hypertensive participants. Although the authors could not conclude from this biopsy study whether the podocyte injury is the cause or effect of the preeclampsia phenotype, the data provide compelling evidence that podocyte injury accompanied by altered angiogenesis process and increased oxidative stress occurs in kidney of patients with preeclampsia. PMID:19528353

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, Paul  D.; Aertgeerts, Kathleen; Bauer, Cary

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank archive, ~75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery/design, and the goodness-of-fit of ligand models to electron density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide Protein Data Bank/Cambridge Crystallographicmore » Data Centre/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the Protein Data Bank? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.« less

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop

DOE PAGES

Adams, Paul  D.; Aertgeerts, Kathleen; Bauer, Cary; ...

2016-04-05

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank archive, ~75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery/design, and the goodness-of-fit of ligand models to electron density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide Protein Data Bank/Cambridge Crystallographicmore » Data Centre/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the Protein Data Bank? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.« less

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop.

PubMed

Adams, Paul D; Aertgeerts, Kathleen; Bauer, Cary; Bell, Jeffrey A; Berman, Helen M; Bhat, Talapady N; Blaney, Jeff M; Bolton, Evan; Bricogne, Gerard; Brown, David; Burley, Stephen K; Case, David A; Clark, Kirk L; Darden, Tom; Emsley, Paul; Feher, Victoria A; Feng, Zukang; Groom, Colin R; Harris, Seth F; Hendle, Jorg; Holder, Thomas; Joachimiak, Andrzej; Kleywegt, Gerard J; Krojer, Tobias; Marcotrigiano, Joseph; Mark, Alan E; Markley, John L; Miller, Matthew; Minor, Wladek; Montelione, Gaetano T; Murshudov, Garib; Nakagawa, Atsushi; Nakamura, Haruki; Nicholls, Anthony; Nicklaus, Marc; Nolte, Robert T; Padyana, Anil K; Peishoff, Catherine E; Pieniazek, Susan; Read, Randy J; Shao, Chenghua; Sheriff, Steven; Smart, Oliver; Soisson, Stephen; Spurlino, John; Stouch, Terry; Svobodova, Radka; Tempel, Wolfram; Terwilliger, Thomas C; Tronrud, Dale; Velankar, Sameer; Ward, Suzanna C; Warren, Gregory L; Westbrook, John D; Williams, Pamela; Yang, Huanwang; Young, Jasmine

2016-04-05

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Outcome of the first wwPDB/CCDC/D3R Ligand Validation Workshop

PubMed Central

Adams, Paul D.; Aertgeerts, Kathleen; Bauer, Cary; Bell, Jeffrey A.; Berman, Helen M.; Bhat, Talapady N.; Blaney, Jeff; Bolton, Evan; Bricogne, Gerard; Brown, David; Burley, Stephen K.; Case, David A.; Clark, Kirk L.; Darden, Tom; Emsley, Paul; Feher, Victoria A.; Feng, Zukang; Groom, Colin R.; Harris, Seth F.; Hendle, Jorg; Holder, Thomas; Joachimiak, Andrzej; Kleywegt, Gerard J.; Krojer, Tobias; Marcotrigiano, Joseph; Mark, Alan E.; Markley, John L.; Miller, Matthew; Minor, Wladek; Montelione, Gaetano T.; Murshudov, Garib; Nakagawa, Atsushi; Nakamura, Haruki; Nicholls, Anthony; Nicklaus, Marc; Nolte, Robert T.; Padyana, Anil K.; Peishoff, Catherine E.; Pieniazek, Susan; Read, Randy J.; Shao, Chenghua; Sheriff, Steven; Smart, Oliver; Soisson, Stephen; Spurlino, John; Stouch, Terry; Svobodova, Radka; Tempel, Wolfram; Terwilliger, Thomas C.; Tronrud, Dale; Velankar, Sameer; Ward, Suzanna; Warren, Gregory L.; Westbrook, John D.; Williams, Pamela; Yang, Huanwang; Young, Jasmine

2016-01-01

Summary Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank archive, ~75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery/design, and the goodness-of-fit of ligand models to electron density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide Protein Data Bank/Cambridge Crystallographic Data Centre/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30–31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the Protein Data Bank? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated. PMID:27050687

Diffuse Large B-Cell Lymphoma: Prospective Multicenter Comparison of Early Interim FLT PET/CT versus FDG PET/CT with IHP, EORTC, Deauville, and PERCIST Criteria for Early Therapeutic Monitoring

PubMed Central

Minamimoto, Ryogo; Fayad, Luis; Advani, Ranjana; Vose, Julie; Macapinlac, Homer; Meza, Jane; Hankins, Jordan; Mottaghy, Felix; Juweid, Malik

2016-01-01

Purpose To compare the performance characteristics of interim fluorine 18 (18F) fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) (after two cycles of chemotherapy) by using the most prominent standardized interpretive criteria (including International Harmonization Project [IHP] criteria, European Organization for Research and Treatment of Cancer [EORTC] criteria, and PET Response Criteria in Solid Tumors (PERCIST) versus those of interim 18F fluorothymidine (FLT) PET/CT and simple visual interpretation. Materials and Methods This HIPAA-compliant prospective study was approved by the institutional review boards, and written informed consent was obtained. Patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) underwent both FLT and FDG PET/CT 18–24 days after two cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone or rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin. For FDG PET/CT interpretation, IHP criteria, EORTC criteria, PERCIST, Deauville criteria, standardized uptake value, total lesion glycolysis, and metabolic tumor volume were used. FLT PET/CT images were interpreted with visual assessment by two reviewers in consensus. The interim (after cycle 2) FDG and FLT PET/CT studies were then compared with the end-of-treatment FDG PET/CT studies to determine which interim examination and/or criteria best predicted the result after six cycles of chemotherapy. Results From November 2011 to May 2014, there were 60 potential patients for inclusion, of whom 46 patients (24 men [mean age, 60.9 years ± 13.7; range, 28–78 years] and 22 women [mean age, 57.2 years ± 13.4; range, 25–76 years]) fulfilled the criteria. Thirty-four patients had complete response, and 12 had residual disease at the end of treatment. FLT PET/CT had a significantly higher positive predictive value (PPV) (91%) in predicting residual disease than did any FDG PET/CT interpretation

SU-E-J-250: A Methodology for Active Bone Marrow Protection for Cervical Cancer Intensity-Modulated Radiotherapy Using 18F-FLT PET/CT Image

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, C; Yin, Y

Purpose: The purpose of this study was to compare a radiation therapy treatment planning that would spare active bone marrow and whole pelvic bone marrow using 18F FLT PET/CT image. Methods: We have developed an IMRT planning methodology to incorporate functional PET imaging using 18F FLT/CT scans. Plans were generated for two cervical cancer patients, where pelvicactive bone marrow region was incorporated as avoidance regions based on the range: SUV>2., another region was whole pelvic bone marrow. Dose objectives were set to reduce the volume of active bone marrow and whole bone marraw. The volumes of received 10 (V10) andmore » 20 (V20) Gy for active bone marrow were evaluated. Results: Active bone marrow regions identified by 18F FLT with an SUV>2 represented an average of 48.0% of the total osseous pelvis for the two cases studied. Improved dose volume histograms for identified bone marrow SUV volumes and decreases in V10(average 18%), and V20(average 14%) were achieved without clinically significant changes to PTV or OAR doses. Conclusion: Incorporation of 18F FLT/CT PET in IMRT planning provides a methodology to reduce radiation dose to active bone marrow without compromising PTV or OAR dose objectives in cervical cancer.« less

Conversion of the. mu. ketene ligand in (PPN)(Os/sub 3/(CO)/sub 10/(. mu. -I)(. mu. -CH/sub 2/CO)) into enolate, acyl, and vinyl ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bassner, S.L.; Morrison, E.D.; Geoffroy, G.L.

1986-08-20

Free ketene is a valuable organic synthetic reagent, but its utility is somewhat limited by its high reactivity and tendency to dimerize to yield diketene. The ketene ligand is obviously stabilized by metal coordination in a variety of bonding modes, but it is not yet known how coordination influences the chemistry of this important molecule. The authors have studied the reactivity of the coordinated ketene ligand of type II found in the anionic cluster compound (PPN)(Os/sub 3/(CO)/sub 10/(..mu..-I)(..mu..-CH/sub 2/CO)) (1) (PPN/sup +/ = (Ph/sub 3/P)/sub 2/N/sup +/) and herein show that this ligand is readily converted into eta-enolate ligands uponmore » reaction with simple nucleophiles and into vinyl and acetyl ligands upon reaction with electrophiles.« less

Predicting location of recurrence using FDG, FLT, and Cu-ATSM PET in canine sinonasal tumors treated with radiotherapy

NASA Astrophysics Data System (ADS)

Bradshaw, Tyler; Fu, Rau; Bowen, Stephen; Zhu, Jun; Forrest, Lisa; Jeraj, Robert

2015-07-01

Dose painting relies on the ability of functional imaging to identify resistant tumor subvolumes to be targeted for additional boosting. This work assessed the ability of FDG, FLT, and Cu-ATSM PET imaging to predict the locations of residual FDG PET in canine tumors following radiotherapy. Nineteen canines with spontaneous sinonasal tumors underwent PET/CT imaging with radiotracers FDG, FLT, and Cu-ATSM prior to hypofractionated radiotherapy. Therapy consisted of 10 fractions of 4.2 Gy to the sinonasal cavity with or without an integrated boost of 0.8 Gy to the GTV. Patients had an additional FLT PET/CT scan after fraction 2, a Cu-ATSM PET/CT scan after fraction 3, and follow-up FDG PET/CT scans after radiotherapy. Following image registration, simple and multiple linear and logistic voxel regressions were performed to assess how well pre- and mid-treatment PET imaging predicted post-treatment FDG uptake. R2 and pseudo R2 were used to assess the goodness of fits. For simple linear regression models, regression coefficients for all pre- and mid-treatment PET images were significantly positive across the population (P < 0.05). However, there was large variability among patients in goodness of fits: R2 ranged from 0.00 to 0.85, with a median of 0.12. Results for logistic regression models were similar. Multiple linear regression models resulted in better fits (median R2 = 0.31), but there was still large variability between patients in R2. The R2 from regression models for different predictor variables were highly correlated across patients (R ≈ 0.8), indicating tumors that were poorly predicted with one tracer were also poorly predicted by other tracers. In conclusion, the high inter-patient variability in goodness of fits indicates that PET was able to predict locations of residual tumor in some patients, but not others. This suggests not all patients would be good candidates for dose painting based on a single biological target.

Predicting location of recurrence using FDG, FLT, and Cu-ATSM PET in canine sinonasal tumors treated with radiotherapy.

PubMed

Bradshaw, Tyler; Fu, Rau; Bowen, Stephen; Zhu, Jun; Forrest, Lisa; Jeraj, Robert

2015-07-07

Dose painting relies on the ability of functional imaging to identify resistant tumor subvolumes to be targeted for additional boosting. This work assessed the ability of FDG, FLT, and Cu-ATSM PET imaging to predict the locations of residual FDG PET in canine tumors following radiotherapy. Nineteen canines with spontaneous sinonasal tumors underwent PET/CT imaging with radiotracers FDG, FLT, and Cu-ATSM prior to hypofractionated radiotherapy. Therapy consisted of 10 fractions of 4.2 Gy to the sinonasal cavity with or without an integrated boost of 0.8 Gy to the GTV. Patients had an additional FLT PET/CT scan after fraction 2, a Cu-ATSM PET/CT scan after fraction 3, and follow-up FDG PET/CT scans after radiotherapy. Following image registration, simple and multiple linear and logistic voxel regressions were performed to assess how well pre- and mid-treatment PET imaging predicted post-treatment FDG uptake. R(2) and pseudo R(2) were used to assess the goodness of fits. For simple linear regression models, regression coefficients for all pre- and mid-treatment PET images were significantly positive across the population (P < 0.05). However, there was large variability among patients in goodness of fits: R(2) ranged from 0.00 to 0.85, with a median of 0.12. Results for logistic regression models were similar. Multiple linear regression models resulted in better fits (median R(2) = 0.31), but there was still large variability between patients in R(2). The R(2) from regression models for different predictor variables were highly correlated across patients (R ≈ 0.8), indicating tumors that were poorly predicted with one tracer were also poorly predicted by other tracers. In conclusion, the high inter-patient variability in goodness of fits indicates that PET was able to predict locations of residual tumor in some patients, but not others. This suggests not all patients would be good candidates for dose painting based on a single biological target.

WE-H-207A-03: The Universality of the Lognormal Behavior of [F-18]FLT PET SUV Measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scarpelli, M; Eickhoff, J; Perlman, S

Purpose: Log transforming [F-18]FDG PET standardized uptake values (SUVs) has been shown to lead to normal SUV distributions, which allows utilization of powerful parametric statistical models. This study identified the optimal transformation leading to normally distributed [F-18]FLT PET SUVs from solid tumors and offers an example of how normal distributions permits analysis of non-independent/correlated measurements. Methods: Forty patients with various metastatic diseases underwent up to six FLT PET/CT scans during treatment. Tumors were identified by nuclear medicine physician and manually segmented. Average uptake was extracted for each patient giving a global SUVmean (gSUVmean) for each scan. The Shapiro-Wilk test wasmore » used to test distribution normality. One parameter Box-Cox transformations were applied to each of the six gSUVmean distributions and the optimal transformation was found by selecting the parameter that maximized the Shapiro-Wilk test statistic. The relationship between gSUVmean and a serum biomarker (VEGF) collected at imaging timepoints was determined using a linear mixed effects model (LMEM), which accounted for correlated/non-independent measurements from the same individual. Results: Untransformed gSUVmean distributions were found to be significantly non-normal (p<0.05). The optimal transformation parameter had a value of 0.3 (95%CI: −0.4 to 1.6). Given the optimal parameter was close to zero (which corresponds to log transformation), the data were subsequently log transformed. All log transformed gSUVmean distributions were normally distributed (p>0.10 for all timepoints). Log transformed data were incorporated into the LMEM. VEGF serum levels significantly correlated with gSUVmean (p<0.001), revealing log-linear relationship between SUVs and underlying biology. Conclusion: Failure to account for correlated/non-independent measurements can lead to invalid conclusions and motivated transformation to normally distributed SUVs. The

Sensitizing Tb(III) and Eu(III) emission with triarylboron functionalized 1,3-diketonato ligands.

PubMed

Smith, Larissa F; Blight, Barry A; Park, Hee-Jun; Wang, Suning

2014-08-04

Four BMes2Ar (Mes = mesityl, Ar = phenyl or duryl) functionalized 1,3-diketonato ligands have been investigated for use in selective sensitization of Tb(III) and Eu(III) emission. These ligands have the general formula of [R1C(O)CR2C(O)R3](-) (R1 = Ph, R2 = H, R3 = p-Ph-BMes2, L1; R1 = R3 = p-Ph-BMes2, R2 = H, L2; R1 = R3 = Me, R2 = p-Ph-BMes2, L3; R1 = R3 = Me, R2 = p-duryl-BMes2, L4) and belong to class I (L1 and L2) and class II (L3 and L4), respectively. In class I, the boron unit is conjugated with the phenyl linker and the diketone backbone, while in class II, the boron unit, the linker unit, and the diketone unit are nonconjugated with a mutually orthogonal arrangement. To understand the impact of the location of the BMes2Ar unit on the electronic properties of the 1,3-diketone molecules and their ability in activating lanthanide emission, the difluoroboron chelate compounds (1-BF2 to 4-BF2) of ligands L1-L4 were synthesized and examined. The class I ligands were effective in activating Eu(III) emission, while the class II ligands were effective in activating Tb(III) emission. Four Ln(III) complexes, 1Eu, 2Eu, 3Tb, and 4Tb, based on the L1-L4 ligands, respectively, were prepared and examined. The emission quantum efficiency of 1Eu and 2Eu is low (Φ(Eu) ≤ 0.01 in THF, 0.07-0.13 in the solid state), but can be greatly enhanced by the addition of fluoride ions. In contrast, the complex 4Tb has a moderate emission efficiency (Φ(Tb) = 0.14 in THF, 0.47 in the solid state) and experiences a distinct emission quenching upon the addition of fluoride. The selective sensitization of Eu(III) and Tb(III) by L1-L4 and the distinct luminescent response of their Ln(III) complexes toward fluoride ions are caused by the distinct intraligand charge transfer transitions of the two different classes of ligands involving the BMes2 unit.

Recent advances in the rationale design of GPER ligands.

PubMed

Rosano, C; Lappano, R; Santolla, M F; Ponassi, M; Donadini, A; Maggiolini, M

2012-01-01

G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining "in silico" docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.

Virtual screening using the ligand ZINC database for novel lipoxygenase-3 inhibitors.

PubMed

Monika; Kour, Janmeet; Singh, Kulwinder

2013-01-01

The leukotrienes constitute a group of arachidonic acid-derived compounds with biologic activities suggesting important roles in inflammation and immediate hypersensitivity. Epidermis-type lipoxygenase-3 (ALOXE3), a distinct subclass within the multigene family of mammalian lipoxygenases, is a novel isoenzyme involved in the metabolism of leukotrienes and plays a very important role in skin barrier functions. Lipoxygenase selective inhibitors such as azelastine and zileuton are currently used to reduce inflammatory response. Nausea, pharyngolaryngeal pain, headache, nasal burning and somnolence are the most frequently reported adverse effects of these drugs. Therefore, there is still a need to develop more potent lipoxygenase inhibitors. In this paper, we report the screening of various compounds from the ZINC database (contains over 21 million compounds) using the Molegro Virtual Docker software against the ALOXE3 protein. Screening was performed using molecular constraints tool to filter compounds with physico-chemical properties similar to the 1N8Q bound ligand protocatechuic acid. The analysis resulted in 4319 Lipinski compliant hits which are docked and scored to identify structurally novel ligands that make similar interactions to those of known ligands or may have different interactions with other parts of the binding site. Our screening approach identified four molecules ZINC84299674; ZINC76643455; ZINC84299122 & ZINC75626957 with MolDock score of -128.901, -120.22, -116.873 & - 102.116 kcal/mol, respectively. Their energy scores were better than the 1N8Q bound co-crystallized ligand protocatechuic acid (with MolDock score of -77.225 kcal/mol). All the ligands were docked within the binding pocket forming interactions with amino acid residues.

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, M.; Canoll, P.D.; Musacchio, J.M.

1991-01-01

The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

Experimental elicitation with hydroxyisohexyl-3-cyclohexene carboxaldehyde-containing deodorants.

PubMed

Jørgensen, Pia Haslund; Jensen, Charlotte Devantier; Rastogi, Suresh; Andersen, Klaus Ejner; Johansen, Jeanne Duus

2007-03-01

Hydroxyisohexyl-3-cyclohexene carboxaldehyde (HICC) known as Lyral is a frequent allergen. It is used in more than 50% of marketed deodorants. The aim of the present study was to determine elicitation thresholds for HICC under simulated conditions of deodorant use. 15 patients with previously diagnosed contact allergy to HICC were patch tested with 5 solutions of HICC-scented and HICC-unscented deodorants. Patients and 10 healthy controls performed a use test in the axillae using deodorants scented with HICC in increasing concentrations and unscented deodorants as control. The concentration of HICC was increased every second week (200, 600, and 1800 p.p.m.) until either a reaction developed or for 6 weeks. 14 patients completed the study, and all developed unilateral eczema from the HICC-containing deodorant, while controls were all negative (P= 0.004). In 9/14 patients, a positive use test developed during the first 2 weeks to the deodorant containing 200 p.p.m. HICC. Positive correlations were found between the day of positive use and patch test threshold concentration of the HICC solutions (r= 0.71, P= 0.01) as well as the patch test thresholds of the HICC-scented deodorants (r= 0.74, P= 0.007). In conclusion, HICC elicits allergic contact dermatitis in a high proportion of sensitized individuals at common usage concentrations in deodorants.

Structural basis of ligand recognition in 5-HT3 receptors

PubMed Central

Kesters, Divya; Thompson, Andrew J; Brams, Marijke; van Elk, René; Spurny, Radovan; Geitmann, Matthis; Villalgordo, Jose M; Guskov, Albert; Helena Danielson, U; Lummis, Sarah C R; Smit, August B; Ulens, Chris

2013-01-01

The 5-HT3 receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT3 receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT3 receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT3 receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT3 receptor. PMID:23196367

Interleukin-29 induces epithelial production of CXCR3A ligands and T-cell infiltration.

PubMed

Witte, Ellen; Kokolakis, Georgios; Witte, Katrin; Warszawska, Katarzyna; Friedrich, Markus; Christou, Demetrios; Kirsch, Stefan; Sterry, Wolfram; Volk, Hans-Dieter; Sabat, Robert; Wolk, Kerstin

2016-04-01

Psoriasis is considered as a model for chronic immune-mediated disorders. Th17-cells are pivotal players in those diseases. Recently, we demonstrated that Th17-cells produce interleukin (IL)-29 and that IL-29 is highly present in psoriatic lesions. Whether IL-29, with its action on epithelial cells and melanocytes, contributes to psoriasis pathogenesis, was unknown so far. Analysis of IL-29-treated human keratinocytes revealed induction of the chemokines CXCL10, CXCL11, and, to a much lesser extent, CXCL9. Unlike these CXCR3A ligands, known to attract Th1-, CD8(+), NK-, and Th1/Th17 transient cells, no influence was found on chemokines attracting other immune cell populations or on molecules modulating the CXCR3A/CXCR3A ligand interaction. CXCR3A ligand expression was also induced by IL-29 in melanocytes and in epidermis models and explanted skin. Regarding other psoriasis-relevant cytokines, interferon-γ and, less potently, tumor necrosis factor-α and IL-1β shared and strengthened IL-29's capacity. Murine IL-29 counterpart injected into mouse skin provoked local CXCL10 and CXCL11 expression, T-cell infiltration, and, in consequence, skin swelling. The elevated IL-29 expression in psoriatic lesions was associated with upregulation of CXCR3A ligands compared to non-lesional skin of these patients and to the skin of healthy donors and atopic dermatitis patients, which lack IL-29 production. Importantly, neutralization of IL-29 reduced CXCR3A ligand levels in explant cultures of psoriatic lesions. Finally, elevated blood CXCL11 levels were found in psoriasis that might be useful for monitoring lesional activity of the IL-29 axis. In summary, the Th17-cytokine IL-29 induces specific chemokines and, in consequence, provokes skin infiltration of potentially pathogenic T-cells. IL-29 selectively induces CXCR3A-binding chemokines (CXCL9, CXCL10, CXCL11) in skin cells. Murine IL-29 counterpart induces skin T-cell infiltration and inflammation in mice. CXCR3A ligands are

The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

PubMed Central

Hoestgaard-Jensen, K; O'Connor, R M; Dalby, N O; Simonsen, C; Finger, B C; Golubeva, A; Hammer, H; Bergmann, M L; Kristiansen, U; Krogsgaard-Larsen, P; Bräuner-Osborne, H; Ebert, B; Frølund, B; Cryan, J F; Jensen, A A

2013-01-01

BACKGROUND AND PURPOSE Explorations into the heterogeneous population of native GABA type A receptors (GABAARs) and the physiological functions governed by the multiple GABAAR subtypes have for decades been hampered by the lack of subtype-selective ligands. EXPERIMENTAL APPROACH The functional properties of the orthosteric GABAA receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo. KEY RESULTS Thio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABAAR subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α5β3γ2S, α4β3δ and α6β3δ and somewhat lower efficacies at the corresponding α5β2γ2S, α4β2δ and α6β2δ subtypes (maximal responses of 4–12%). In contrast, it was an extremely low efficacious agonist at the α1β3γ2S, α1β2γ2S, α2β2γ2S, α2β3γ2S, α3β2γ2S and α3β3γ2S GABAARs (maximal responses of 0–4%). In concordance with its agonism at extrasynaptic GABAARs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties. CONCLUSION AND IMPLICATIONS The diverse signalling characteristics of Thio-4-PIOL at GABAARs represent one of the few examples of a functionally subtype-selective orthosteric GABAAR ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABAARs. PMID:23957253

Sustainable metal alkynyl chemistry: 3d metals and polyaza macrocyclic ligands.

PubMed

Ren, Tong

2016-02-25

We describe the chemistry of 3d metal alkynyls based on polyaza macrocyclic ligands, an emerging area of alkynyl chemistry that has previously been dominated by 4d and 5d metals with soft ligands. The abundance of 3d metals and low cost of tetraazacyclotetradecane ligands make these compounds more affordable, sustainable alternatives to metal alkynyls based on precious metals. Taking advantage of the rich variety of starting materials available in the literature, trans-[M(cyclam)(C2R)2]X (cyclam = 1,4,8,11-tetraazacyclotetradecane) compounds have been prepared from the reactions between [M(cyclam)X2]X (M = Cr, Fe and Co; X = Cl or OTf) and LiC2R. With [Co(cyclam)Cl2](+), both the {trans-[Co(cyclam)Cl]2(μ-(C≡C)n)}(2+) and trans-[Co(cyclam)(C2R)Cl](+) compounds have been prepared by a dehydrohalogenation reaction. The latter compounds undergo the second alkynylation reaction to afford dissymmetric trans-[Co(cyclam)(C2R)(C2R')](+) compounds. Similar alkynylation chemistry with complexes of the cyclam derivatives TMC (1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) and HMC (5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane) has been demonstrated in studies of [Ni(TMC)(C2R)](+) and trans-/cis-[Cr(HMC)(C2R)2](+). Me3TACN (1,4,7-N,N',N''-trimethyl-1,4,7-triazacyclononane) is also a supporting ligand that has been observed in transition metal alkynyls. The trans-[M(cyclam)(C2D)(C2A)](+) compounds (D = donor chromophore, A = acceptor chromophore) are excellent candidates for probing photoinduced electron transfer and related photophysical and photochemical processes. 3d Metal ions are often in high-spin ground states, which make these alkynyl compounds promising building blocks for magnetic materials.

Computational fragment-based screening using RosettaLigand: the SAMPL3 challenge

NASA Astrophysics Data System (ADS)

Kumar, Ashutosh; Zhang, Kam Y. J.

2012-05-01

SAMPL3 fragment based virtual screening challenge provides a valuable opportunity for researchers to test their programs, methods and screening protocols in a blind testing environment. We participated in SAMPL3 challenge and evaluated our virtual fragment screening protocol, which involves RosettaLigand as the core component by screening a 500 fragments Maybridge library against bovine pancreatic trypsin. Our study reaffirmed that the real test for any virtual screening approach would be in a blind testing environment. The analyses presented in this paper also showed that virtual screening performance can be improved, if a set of known active compounds is available and parameters and methods that yield better enrichment are selected. Our study also highlighted that to achieve accurate orientation and conformation of ligands within a binding site, selecting an appropriate method to calculate partial charges is important. Another finding is that using multiple receptor ensembles in docking does not always yield better enrichment than individual receptors. On the basis of our results and retrospective analyses from SAMPL3 fragment screening challenge we anticipate that chances of success in a fragment screening process could be increased significantly with careful selection of receptor structures, protein flexibility, sufficient conformational sampling within binding pocket and accurate assignment of ligand and protein partial charges.

Receptor-based 3D QSAR analysis of estrogen receptor ligands - merging the accuracy of receptor-based alignments with the computational efficiency of ligand-based methods

NASA Astrophysics Data System (ADS)

Sippl, Wolfgang

2000-08-01

One of the major challenges in computational approaches to drug design is the accurate prediction of binding affinity of biomolecules. In the present study several prediction methods for a published set of estrogen receptor ligands are investigated and compared. The binding modes of 30 ligands were determined using the docking program AutoDock and were compared with available X-ray structures of estrogen receptor-ligand complexes. On the basis of the docking results an interaction energy-based model, which uses the information of the whole ligand-receptor complex, was generated. Several parameters were modified in order to analyze their influence onto the correlation between binding affinities and calculated ligand-receptor interaction energies. The highest correlation coefficient ( r 2 = 0.617, q 2 LOO = 0.570) was obtained considering protein flexibility during the interaction energy evaluation. The second prediction method uses a combination of receptor-based and 3D quantitative structure-activity relationships (3D QSAR) methods. The ligand alignment obtained from the docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection, a significant and robust model was obtained ( r 2 = 0.991, q 2 LOO = 0.921). The predictive ability of the established model was further evaluated by using a test set of six additional compounds. The comparison with the generated interaction energy-based model and with a traditional CoMFA model obtained using a ligand-based alignment ( r 2 = 0.951, q 2 LOO = 0.796) indicates that the combination of receptor-based and 3D QSAR methods is able to improve the quality of the underlying model.

Two novel mixed-ligand complexes containing organosulfonate ligands.

PubMed

Li, Mingtian; Huang, Jun; Zhou, Xuan; Fang, Hua; Ding, Liyun

2008-07-01

The structures reported herein, viz. bis(4-aminonaphthalene-1-sulfonato-kappaO)bis(4,5-diazafluoren-9-one-kappa(2)N,N')copper(II), [Cu(C(10)H(8)NO(3)S)(2)(C(11)H(6)N(2)O)(2)], (I), and poly[[[diaquacadmium(II)]-bis(mu-4-aminonaphthalene-1-sulfonato)-kappa(2)O:N;kappa(2)N:O] dihydrate], {[Cd(C(10)H(8)NO(3)S)(2)(H(2)O)(2)].2H(2)O}(n), (II), are rare examples of sulfonate-containing complexes where the anion does not fulfill a passive charge-balancing role, but takes an active part in coordination as a monodentate and/or bridging ligand. Monomeric complex (I) possesses a crystallographic inversion center at the Cu(II) atom, and the asymmetric unit contains one-half of a Cu atom, one complete 4-aminonaphthalene-1-sulfonate (ans) ligand and one 4,5-diazafluoren-9-one (DAFO) ligand. The Cu(II) atom has an elongated distorted octahedral coordination geometry formed by two O atoms from two monodentate ans ligands and by four N atoms from two DAFO molecules. Complex (II) is polymeric and its crystal structure is built up by one-dimensional chains and solvent water molecules. Here also the cation (a Cd(II) atom) lies on a crystallographic inversion center and adopts a slightly distorted octahedral geometry. Each ans anion serves as a bridging ligand linking two Cd(II) atoms into one-dimensional infinite chains along the [010] direction, with each Cd(II) center coordinated by four ans ligands via O and N atoms and by two aqua ligands. In both structures, there are significant pi-pi stacking interactions between adjacent ligands and hydrogen bonds contribute to the formation of two- and three-dimensional networks.

Stable curcumin-loaded polymeric micellar formulation for enhancing cellular uptake and cytotoxicity to FLT3 overexpressing EoL-1 leukemic cells.

PubMed

Tima, Singkome; Anuchapreeda, Songyot; Ampasavate, Chadarat; Berkland, Cory; Okonogi, Siriporn

2017-05-01

The present study aims to develop a stable polymeric micellar formulation of curcumin (CM) with improved solubility and stability, and that is suitable for clinical applications in leukemia patients. CM-loaded polymeric micelles (CM-micelles) were prepared using poloxamers. The chemical structure of the polymers influenced micellar properties. The best formulation of CM-micelles, namely CM-P407, was obtained from poloxamer 407 at drug to polymer ratio of 1:30 and rehydrated with phosphate buffer solution pH 7.4. CM-P407 exhibited the smallest size of 30.3±1.3nm and highest entrapment efficiency of 88.4±4.1%. When stored at -80°C for 60days, CM-P407 retained high protection of CM and had no significant size change. In comparison with CM solution in dimethyl sulfoxide (CM-DMSO), CM kinetic degradation in both formulations followed a pseudo-first-order reaction, but the half-life of CM in CM-P407 was approx. 200 times longer than in CM-DMSO. Regarding the activity against FLT3 overexpressing EoL-1 leukemic cells, CM-P407 showed higher cytotoxicity than CM-DMSO. Moreover, intracellular uptake to leukemic cells of CM-P407 was 2-3 times greater than that of CM-DMSO. These promising results for CM-P407 will be further investigated in rodents and in clinical studies for leukemia treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

PubMed

Le Foll, Bernard; Collo, Ginetta; Rabiner, Eugenii A; Boileau, Isabelle; Merlo Pich, Emilio; Sokoloff, Pierre

2014-01-01

The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction. © 2014 Elsevier B.V. All rights reserved.

Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

PubMed

Istyastono, Enade P; Nijmeijer, Saskia; Lim, Herman D; van de Stolpe, Andrea; Roumen, Luc; Kooistra, Albert J; Vischer, Henry F; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

2011-12-08

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

The anti-human immunodeficiency virus agent 3'-fluorothymidine induces DNA damage and apoptosis in human lymphoblastoid cells.

PubMed Central

Sundseth, R; Joyner, S S; Moore, J T; Dornsife, R E; Dev, I K

1996-01-01

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo. PMID:8834875

Electrical and Plasmonic Properties of Ligand-Free Sn(4+) -Doped In2 O3 (ITO) Nanocrystals.

PubMed

Jagadeeswararao, Metikoti; Pal, Somnath; Nag, Angshuman; Sarma, D D

2016-03-03

Sn(4+) -doped In2 O3 (ITO) is a benchmark transparent conducting oxide material. We prepared ligand-free but colloidal ITO (8 nm, 10 % Sn(4+) ) nanocrystals (NCs) by using a post-synthesis surface-modification reaction. (CH3 )3 OBF4 removes the native oleylamine ligand from NC surfaces to give ligand-free, positively charged NCs that form a colloidal dispersion in polar solvents. Both oleylamine-capped and ligand-free ITO NCs exhibit intense absorption peaks, due to localized surface plasmon resonance (LSPR) at around λ=1950 nm. Compared with oleylamine-capped NCs, the electrical resistivity of ligand-free ITO NCs is lower by an order of magnitude (≈35 mΩ cm(-1) ). Resistivity over a wide range of temperatures can be consistently described as a composite of metallic ITO grains embedded in an insulating matrix by using a simple equivalent circuit, which provides an insight into the conduction mechanism in these systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants

PubMed Central

Wong, Terianne M.; Allen, James D.; Bebin-Blackwell, Anne-Gaelle; Carter, Donald M.; Alefantis, Timothy; DiNapoli, Joshua; Kleanthous, Harold

2017-01-01

ABSTRACT Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines. IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that

Reversibly Switchable, pH-Dependent Peptide Ligand Binding via 3,5-Diiodotyrosine Substitutions.

PubMed

Ngambenjawong, Chayanon; Sylvestre, Meilyn; Gustafson, Heather H; Pineda, Julio Marco B; Pun, Suzie H

2018-04-20

Cell type-specific targeting ligands utilized in drug delivery applications typically recognize receptors that are overexpressed on the cells of interest. Nonetheless, these receptors may also be expressed, to varying extents, on off-target cells, contributing to unintended side effects. For the selectivity profile of targeting ligands in cancer therapy to be improved, stimuli-responsive masking of these ligands with acid-, redox-, or enzyme-cleavable molecules has been reported, whereby the targeting ligands are exposed in specific environments, e.g., acidic tumor hypoxia. One possible drawback of these systems lies in their one-time, permanent trigger, which enables the "demasked" ligands to bind off-target cells if released back into the systemic circulation. A promising strategy to address the aforementioned problem is to design ligands that show selective binding based on ionization state, which may be microenvironment-dependent. In this study, we report a systematic strategy to engineer low pH-selective targeting peptides using an M2 macrophage-targeting peptide (M2pep) as an example. 3,5-Diiodotyrosine mutagenesis into native tyrosine residues of M2pep confers pH-dependent binding behavior specific to acidic environment (pH 6) when the amino acid is protonated into the native tyrosine-like state. At physiological pH of 7.4, the hydroxyl group of 3,5-diiodotyrosine on the peptide is deprotonated leading to interruption of the peptide native binding property. Our engineered pH-responsive M2pep (Ac-Y-Î-Î) binds target M2 macrophages more selectively at pH 6 than at pH 7.4. In addition, 3,5-diiodotyrosine substitutions also improve serum stability of the peptide. Finally, we demonstrate pH-dependent reversibility in target binding via a postbinding peptide elution study. The strategy presented here should be applicable for engineering pH-dependent functionality of other targeting peptides with potential applications in physiology-dependent in vivo targeting

The role of proteinase 3 (PR3) and the protease-activated receptor-2 (PAR-2) pathway in dendritic cell (DC) maturation of human-DC-like monocytes and murine DC.

PubMed

Jiang, Bo; Grage-Griebenow, Evelin; Csernok, Elena; Butherus, Kristine; Ehlers, Stefan; Gross, Wolfgang L; Holle, Julia U

2010-01-01

The aim of the study was to assess PAR-2 expression on dendritic cell (DC) subsets and other immune cells of Wegener's granulomatosis (WG) patients and healthy controls (HC) and to investigate whether Proteinase 3 (PR3, a serine protease which can activate PAR2) induces maturation of human DC-like monocytes and murine Flt-3 ligand- and GM-CSF-generated DC. Human peripheral blood cells including DC subsets and Flt-3l- and GM-CSF-generated mouse DC were analysed for expression of PAR-2 and DC maturation markers by flow cytometry before and after stimulation with PR3, trypsin, PAR-2 agonist or LPS for 24 h. There was no difference of PAR-2 expression on PMNs, monocytes, lymphocytes and DC between all WG samples and HC. However, in inactive WG, expression of PAR-2 was downregulated on the cell surface of PMNs, monocytes, lymphocytes, and CD11c+DC compared to active WG and HC. PR3 and PAR2-agonists did not induce upregulation of PAR-2 or maturation markers of human DC-like monocytes in WG and HC. Likewise, murine PR3 did not induce upregulation of PAR-2 or maturation markers in murine DC. PAR-2 expression is downregulated on human peripheral blood cells including CD11c+ DC in inactive WG compared to active WG and HC, possibly reflecting a non-activated status of these cells in inactive disease. PR3 and PAR-2- agonists did not induce maturation of human ex vivo DC-like monocytes in WG and HC and of murine DC, suggesting this pathway is not singularly involved in the maturation of these cell subsets.

Using the angiogenic factors sFlt-1 and PlGF with Doppler ultrasound of the uterine artery for confirming preeclampsia.

PubMed

Bahlmann, Franz; Al Naimi, Ammar

2016-11-01

The aim of this study is to assess the value of the angiogenic factors for diagnosing preeclampsia and predicting the severity of manifestation. A secondary aim is assessing the combination of the uterine artery Doppler with the angiogenic factors for improving the diagnostic power. This is a prospective single center study in a tertiary referral hospital. This study includes 728 individual patients. Inclusion criteria were singleton pregnancies, a referral to the hospital with suspicion of preeclampsia and any one or combination of the following symptoms: headache, upper abdominal pain, edema, and hypertension. Patients with complications that would affect the course of the pregnancy, such as placenta praevia, premature preterm rupture of membranes, breech presentation, and fetal chromosomal or structural anomalies, were excluded from the study. Blood samples collection and uterine artery Doppler ultrasound were performed at time of recruitment. The differences in sFlt-1, PlGF, and their quotient among normal collective and patients with preeclampsia were analyzed. Doppler ultrasound was performed by one of four highly qualified sonographers. Wilcoxon-Mann-Whitney U test, Spearman's rank correlation, receiver operating characteristic curves, Chi-square test, and logistic regression were used in the analysis. A total of 1003 individual samples for the angiogenic factors were included in the analysis. 584 out of the recruited 728 patients had follow-up data with delivery information at the study hospital. Patients with preeclampsia show a significant increase in sFlt-1, which directly correlate with the increased severity of manifestation (Spearman's ρ 0.49). The sFlt-1 cut-off value of 5424 pg/ml confirms preeclampsia with 83.7 % sensitivity, 68.1 % specificity, and 24 % misclassification rate. Preeclampsia patients also show a significant decrease in PlGF, which negatively correlates with the increased severity of manifestation (Spearman's ρ -0.39). A Pl

Heterogeneity in Intratumor Correlations of 18F-FDG, 18F-FLT, and 61Cu-ATSM PET in Canine Sinonasal Tumors

PubMed Central

Bradshaw, Tyler J.; Bowen, Stephen R.; Jallow, Ngoneh; Forrest, Lisa J.; Jeraj, Robert

2014-01-01

Intratumor heterogeneity in biologic properties and in relationships between various phenotypes may present a challenge for biologically targeted therapies. Understanding the relationships between different phenotypes in individual tumor types could help inform treatment selection. The goal of this study was to characterize spatial correlations of glucose metabolism, proliferation, and hypoxia in 2 histologic types of tumors. Methods Twenty canine veterinary patients with spontaneously occurring sinonasal tumors (13 carcinomas and 7 sarcomas) were imaged with 18F-FDG, 18F-labeled 39-deoxy-39-fluorothymidine (18F-FLT), and 61Cu-labeled diacetyl-bis(N4-methylthiosemicarbazone) (61Cu-ATSM) PET/CT on 3 consecutive days. Precise positioning and immobilization techniques coupled with anesthesia enabled motionless scans with repeatable positioning. Standardized uptake values (SUVs) of gross sarcoma and carcinoma volumes were compared by use of Mann– Whitney U tests. Patient images were rigidly registered together, and intratumor tracer uptake distributions were compared. Voxel-based Spearman correlation coefficients were used to quantify intertracer correlations, and the correlation coefficients of sarcomas and carcinomas were compared. The relative overlap of the highest uptake volumes of the 3 tracers was quantified, and the values were compared for sarcomas and carcinomas. Results Large degrees of heterogeneity in SUV measures and phenotype correlations were observed. Carcinoma and sarcoma tumors differed significantly in SUV measures, with carcinoma tumors having significantly higher 18F-FDG maximum SUVs than sarcoma tumors (11.1 vs. 5.0; P = 0.01) as well as higher 61Cu-ATSM mean SUVs (2.6 vs. 1.2; P = 0.02). Carcinomas had significantly higher population-averaged Spearman correlation coefficients than sarcomas in comparisons of 18F-FDG and 18F-FLT (0.80 vs. 0.61; P = 0.02), 18F-FLT and 61Cu-ATSM (0.83 vs. 0.38; P < 0.0001), and 18F-FDG and 61Cu-ATSM (0.82 vs. 0

The tryptophan-derived endogenous arylhydrocarbon receptor ligand 6-formylindolo[3,2-b]carbazole (FICZ) is a nanomolar UVA-photosensitizer in epidermal keratinocytes

PubMed Central

Williams, Joshua D.; Cabello, Christopher M.; Qiao, Shuxi; Wondrak, Georg T.

2014-01-01

Endogenous UVA-chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA-cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, ER stress, and oxidative stress response gene expression observed only upon FICZ/UVA-cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (FPG)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin. PMID:25431849

Three-Dimensional Biologically Relevant Spectrum (BRS-3D): Shape Similarity Profile Based on PDB Ligands as Molecular Descriptors.

PubMed

Hu, Ben; Kuang, Zheng-Kun; Feng, Shi-Yu; Wang, Dong; He, Song-Bing; Kong, De-Xin

2016-11-17

The crystallized ligands in the Protein Data Bank (PDB) can be treated as the inverse shapes of the active sites of corresponding proteins. Therefore, the shape similarity between a molecule and PDB ligands indicated the possibility of the molecule to bind with the targets. In this paper, we proposed a shape similarity profile that can be used as a molecular descriptor for ligand-based virtual screening. First, through three-dimensional (3D) structural clustering, 300 diverse ligands were extracted from the druggable protein-ligand database, sc-PDB. Then, each of the molecules under scrutiny was flexibly superimposed onto the 300 ligands. Superimpositions were scored by shape overlap and property similarity, producing a 300 dimensional similarity array termed the "Three-Dimensional Biologically Relevant Spectrum (BRS-3D)". Finally, quantitative or discriminant models were developed with the 300 dimensional descriptor using machine learning methods (support vector machine). The effectiveness of this approach was evaluated using 42 benchmark data sets from the G protein-coupled receptor (GPCR) ligand library and the GPCR decoy database (GLL/GDD). We compared the performance of BRS-3D with other 2D and 3D state-of-the-art molecular descriptors. The results showed that models built with BRS-3D performed best for most GLL/GDD data sets. We also applied BRS-3D in histone deacetylase 1 inhibitors screening and GPCR subtype selectivity prediction. The advantages and disadvantages of this approach are discussed.

Application of 3D-QSAR in the rational design of receptor ligands and enzyme inhibitors.

PubMed

Mor, Marco; Rivara, Silvia; Lodola, Alessio; Lorenzi, Simone; Bordi, Fabrizio; Plazzi, Pier Vincenzo; Spadoni, Gilberto; Bedini, Annalida; Duranti, Andrea; Tontini, Andrea; Tarzia, Giorgio

2005-11-01

Quantitative structure-activity relationships (QSARs) are frequently employed in medicinal chemistry projects, both to rationalize structure-activity relationships (SAR) for known series of compounds and to help in the design of innovative structures endowed with desired pharmacological actions. As a difference from the so-called structure-based drug design tools, they do not require the knowledge of the biological target structure, but are based on the comparison of drug structural features, thus being defined ligand-based drug design tools. In the 3D-QSAR approach, structural descriptors are calculated from molecular models of the ligands, as interaction fields within a three-dimensional (3D) lattice of points surrounding the ligand structure. These descriptors are collected in a large X matrix, which is submitted to multivariate analysis to look for correlations with biological activity. Like for other QSARs, the reliability and usefulness of the correlation models depends on the validity of the assumptions and on the quality of the data. A careful selection of compounds and pharmacological data can improve the application of 3D-QSAR analysis in drug design. Some examples of the application of CoMFA and CoMSIA approaches to the SAR study and design of receptor or enzyme ligands is described, pointing the attention to the fields of melatonin receptor ligands and FAAH inhibitors.

LinkIT: a ludic elicitation game for eliciting risk perceptions.

PubMed

Cao, Yan; McGill, William L

2013-06-01

The mental models approach, a leading strategy to develop risk communications, involves a time- and labor-intensive interview process and a lengthy questionnaire to elicit group-level risk perceptions. We propose that a similarity ratings approach for structural knowledge elicitation can be adopted to assist the risk mental models approach. The LinkIT game, inspired by games with a purpose (GWAP) technology, is a ludic elicitation tool designed to elicit group understanding of the relations between risk factors in a more enjoyable and productive manner when compared to traditional approaches. That is, consistent with the idea of ludic elicitation, LinkIT was designed to make the elicitation process fun and enjoyable in the hopes of increasing participation and data quality in risk studies. Like the mental models approach, the group mental model obtained via the LinkIT game can hence be generated and represented in a form of influence diagrams. In order to examine the external validity of LinkIT, we conducted a study to compare its performance with respect to a more conventional questionnaire-driven approach. Data analysis results conclude that the two group mental models elicited from the two approaches are similar to an extent. Yet, LinkIT was more productive and enjoyable than the questionnaire. However, participants commented that the current game has some usability concerns. This presentation summarizes the design and evaluation of the LinkIT game and suggests areas for future work. © 2012 Society for Risk Analysis.

Synthesis, characterization and cytotoxicity of rare earth metal ion complexes of N,N‧-bis-(2-thiophenecarboxaldimine)-3,3‧-diaminobenzidene, Schiff base ligand

NASA Astrophysics Data System (ADS)

Shakir, Mohammad; Abbasi, Ambreen; Faraz, Mohammad; Sherwani, Asif

2015-12-01

Lanthanide complexes of La3+, Pr3+, Nd3+, Gd3+, Er3+ of general formula [Ln2 L(H2O)4(NO3)4](NO3)2·2H2O have been synthesized from Schiff base, N,N‧-bis-(2-thiophenecarboxaldimine)-3,3‧-diaminobenzidene. The complexes were characterized by elemental analysis, molar conductance, UV-Vis, fluorescence, FT-IR,1H NMR, mass spectroscopy, EDX, SEM and thermal analysis. FT-IR spectral data suggested that ligand coordinate with metal ions through azomethine nitrogen and uncondensed amino group. Molar conductance data revealed 1:2 electrolytic nature of complexes. From the analytical data, the stoichiometry of the complexes was found to be 1:2 (ligand:metal). Thephysico-chemical data suggested eight coordination number for Ln(III)Schiffbase complexes. SEM analysis shows morphological changes in the surfaces of complexes as compared to free ligand. Thermal decomposition profiles were consistent with proposed formulations. The anticancer activity of the complexes and theSchiffbase ligand has been studied towards human cervical cancer celllines (HeLa) and human breast cancer cell lines (MCF-7) and it was found that complexes exhibited greater activity than theSchiffbase.

Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

PubMed

Lange, Jos H M; Venhorst, Jennifer; van Dongen, Maria J P; Frankena, Jurjen; Bassissi, Firas; de Bruin, Natasja M W J; den Besten, Cathaline; de Beer, Stephanie B A; Oostenbrink, Chris; Markova, Natalia; Kruse, Chris G

2011-10-01

Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Cationic copper (I) complexes with bulky 1,4-diaza-1,3-butadiene ligands - Synthesis, solid state structure and catalysis

NASA Astrophysics Data System (ADS)

Anga, Srinivas; Kottalanka, Ravi K.; Pal, Tigmansu; Panda, Tarun K.

2013-05-01

We report the full characterization of two glyoxal-based ligands N,N bis(diphenylmethyl)-1,4-diaza-1,3-butadiene ligand (DADPh2, 1) and more bulky N,N bis(triphenylmethyl)-1,4-diaza-1,3-butadiene ligand (DADPh3, 2) by the condensation reaction of glyoxal and diphenylmethanamine and triphenyl-methanamine respectively. The copper (I) complex of composition [Cu(DADPh2)2]PF6 (3) having two neutral bidentate N,N bis(diphenyl-methyl)-1,4-diaza-1,3-butadiene ligand was prepared by the reaction of [Cu(CH3CN)4]PF6 and 1 in 1:2 ratio in dichloromethane. In a similar reaction with N,N bis(triphenylmethyl)-1,4-diaza-1,3-butadiene ligand (2) and [Cu(CH3CN)4]PF6 in dichloromethane yielded corresponding heteroleptic copper (I) complex [Cu(DADPh3)(CH3CN)2]PF6 (4). Another copper (I) complex [Cu(DADPh2)(PPh3)]PF6 (5) can also be obtained by the one pot reaction involving ligand 1, [Cu(CH3CN)4]PF6 and triphenylphosphine. Solid state structures of all the five compounds were established by single crystal X-ray diffraction analysis. The solid state structures of the copper complexes 3-5 reveal a distorted tetrahedral geometry around the copper (I) centers. The copper complexes 3-5 were tested as catalysts for the coupling reaction of o-iodophenol and phenyl acetylene and it was observed that complex 4 exhibits the highest catalytic activity.

New Insights into Structure and Luminescence of Eu III and Sm III Complexes of the 3,4,3-LI(1,2-HOPO) Ligand

DOE PAGES

Daumann, Lena J.; Tatum, David S.; Snyder, Benjamin E. R.; ...

2015-01-21

We report the preparation and new insight into photophysical properties of luminescent hydroxypyridonate complexes [M IIIL] - (M = Eu or Sm) of the versatile 3,4,3-LI(1,2-HOPO) ligand (L). We report the crystal structure of this ligand with EuIII as well as insights into the coordination behavior and geometry in solution by using magnetic circular dichroism. In addition TD-DFT calculations were used to examine the excited states of the two different chromophores present in the 3,4,3-LI(1,2-HOPO) ligand. We find that the Eu III and Sm III complexes of this ligand undergo a transformation after in situ preparation to yield complexes withmore » higher quantum yield (QY) over time. We propose that the lower QY in the in situ complexes is not only due to water quenching but could also be due to a lower degree of f-orbital overlap (in a kinetic isomer) as indicated by magnetic circular dichroism measurements.« less

Protein-ligand docking using FFT based sampling: D3R case study.

PubMed

Padhorny, Dzmitry; Hall, David R; Mirzaei, Hanieh; Mamonov, Artem B; Moghadasi, Mohammad; Alekseenko, Andrey; Beglov, Dmitri; Kozakov, Dima

2018-01-01

Fast Fourier transform (FFT) based approaches have been successful in application to modeling of relatively rigid protein-protein complexes. Recently, we have been able to adapt the FFT methodology to treatment of flexible protein-peptide interactions. Here, we report our latest attempt to expand the capabilities of the FFT approach to treatment of flexible protein-ligand interactions in application to the D3R PL-2016-1 challenge. Based on the D3R assessment, our FFT approach in conjunction with Monte Carlo minimization off-grid refinement was among the top performing methods in the challenge. The potential advantage of our method is its ability to globally sample the protein-ligand interaction landscape, which will be explored in further applications.

Significance of the concentration of chelating ligands on Fe3+-solubility, bioavailability, and uptake in rice plant.

PubMed

Hasegawa, Hiroshi; Rahman, M Mamunur; Kadohashi, Kouta; Takasugi, Yui; Tate, Yousuke; Maki, Teruya; Rahman, M Azizur

2012-09-01

Present study investigated the significance of the concentration of chelating ligand on Fe(3+)-solubility in growth medium and its influence on Fe bioavailability and uptake in rice plant. Rice seedlings were grown in modified Murashige and Skoog (MS) hydroponic growth medium with moderate (250 μM) and high (500 μM) concentrations of ethylenediaminetetraacetate (EDTA) and hydroxyiminodisuccinate (HIDS) under sterile and non-sterile conditions. Concentrations of soluble Fe in the growth medium increased with increasing ligand concentrations, and the growth of rice seedlings was higher at moderate ligand concentration than at control (without chelant) and high ligand concentration. This explains the relationship between Fe solubility and bioavailability in the growth medium, and its effect on Fe uptake in rice plant. Fe exists in the growth medium predominantly as particulate (insoluble) forms at low ligand concentration, and as soluble [Fe(OH)(2+), Fe(OH)(2)(+), Fe-L complex] and apparently soluble (colloidal) forms at moderate ligand concentration. At high ligand concentration, most of the Fe(3+) in the growth medium forms soluble Fe-L complex, however, the bioavailability of Fe from Fe-L complex decreased due to lopsided complex formation equilibrium reaction (CFER) between Fe and the ligands. Also, Fe is solubilized forming stable and soluble Fe-L complex, which is then detached as less stable, but soluble and bioavailable substance(s) after (time-dependent) biodegradation. Therefore- i) ligand concentration and stability constant of Fe-L complex (K(Fe-L)) influence Fe bioavailability and uptake in rice plant, and ii) the biodegradable ligands (e.g., HIDS) would be more effective Fe fertilizer than the environmentally persistent and less biodegradable ligands (e.g., EDTA). Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Metal-free inorganic ligands for colloidal nanocrystals: S2-, HS-, Se2-, HSe-, Te2-, HTe-, TeS3(2-), OH-, and NH2- as surface ligands.

PubMed

Nag, Angshuman; Kovalenko, Maksym V; Lee, Jong-Soo; Liu, Wenyong; Spokoyny, Boris; Talapin, Dmitri V

2011-07-13

All-inorganic colloidal nanocrystals were synthesized by replacing organic capping ligands on chemically synthesized nanocrystals with metal-free inorganic ions such as S(2-), HS(-), Se(2-), HSe(-), Te(2-), HTe(-), TeS(3)(2-), OH(-) and NH(2)(-). These simple ligands adhered to the NC surface and provided colloidal stability in polar solvents. The versatility of such ligand exchange has been demonstrated for various semiconductor and metal nanocrystals of different size and shape. We showed that the key aspects of Pearson's hard and soft acids and bases (HSAB) principle, originally developed for metal coordination compounds, can be applied to the bonding of molecular species to the nanocrystal surface. The use of small inorganic ligands instead of traditional ligands with long hydrocarbon tails facilitated the charge transport between individual nanocrystals and opened up interesting opportunities for device integration of colloidal nanostructures.

Usefulness of 3'-[F-18]fluoro-3'-deoxythymidine with positron emission tomography in predicting breast cancer response to therapy.

PubMed

Pio, Betty S; Park, Cecilia K; Pietras, Richard; Hsueh, Wei-Ann; Satyamurthy, Nagichettiar; Pegram, Mark D; Czernin, Johannes; Phelps, Michael E; Silverman, Daniel H S

2006-01-01

The usefulness of 2-deoxy-2-[F-18]fluoro-D-glucose (FDG)-positron emission tomography (PET) in monitoring breast cancer response to chemotherapy has previously been reported. Elevated uptake of FDG by treated tumors can persist however, particularly in the early period after treatment is initiated. 3'-[F-18]Fluoro-3'-deoxythymidine (FLT) has been developed as a marker for cellular proliferation and, in principle, could be a more accurate predictor of the long-term effect of chemotherapy on tumor viability. We examined side-by-side FDG and FLT imaging for monitoring and predicting tumor response to chemotherapy. Fourteen patients with newly diagnosed primary or metastatic breast cancer, who were about to commence a new pharmacologic treatment regimen, were prospectively studied. Dynamic 3-D PET imaging of uptake into a field of view centered over tumor began immediately after administration of FDG or FLT (150 MBq). After 45 minutes of dynamic acquisition, a clinically standard whole-body PET scan was acquired. Patients were scanned with both tracers on two separate days within one week of each other (1) before beginning treatment, (2) two weeks following the end of the first cycle of the new regimen, and (3) following the final cycle of that regimen, or one year after the initial PET scans, whichever came first. (Median and mean times of early scans were 5.0 and 6.6 weeks after treatment initiation; median and mean times for late scans were 26.0 and 30.6 weeks after treatment initiation.) Scan data were analyzed on both tumor-by-tumor and patient-by-patient bases, and compared to each patient's clinical course. Mean change in FLT uptake in primary and metastatic tumors after the first course of chemotherapy showed a significant correlation with late (av. interval 5.8 months) changes in CA27.29 tumor marker levels (r = 0.79, P = 0.001). When comparing changes in tracer uptake after one chemotherapy course versus late changes in tumor size as measured by CT scans, FLT

Metal-assisted in situ formation of a tridentate acetylacetone ligand for complexation of fac-Re(CO)3+ for radiopharmaceutical applications.

PubMed

Benny, Paul D; Fugate, Glenn A; Barden, Adam O; Morley, Jennifer E; Silva-Lopez, Elsa; Twamley, Brendan

2008-04-07

Reaction of [NEt4]2[ReBr3(CO)3] with 2,4-pentanedione (acac) yields a complex of the type fac-Re(acac)(OH2)(CO)3 (1) under aqueous conditions. 1 was further reacted with a monodentate ligand (pyridine) to yield a fac-Re(acac)(pyridine)(CO)3 complex (2). Complex 1 was found to react with primary amines to generate a Schiff base (imine) in aqueous solutions. When a mixed-nitrogen donor bidentate ligand, 2-(2-aminoethyl)pyridine, that has different coordination affinities for fac-Re(acac)(OH2)(CO)3 was utilized, a unique tridentate ligand was formed in situ utilizing a metal-assisted Schiff base formation to yield a complex fac-Re(CO)3(3[(2-phenylethyl)imino]-2-pentanone) (3). Tridentate ligand formation was found to occur only with the Re-coordinated acac ligand. Reactions of acac with fac-Re(CO)3Br(2-(2-aminoethyl)pyridine) (4) or a mixture of [NEt4]2[ReBr3(CO)3], acac, and 2-(2-aminoethyl)pyridine did not yield the formation of complex 3 in water.

Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial.

PubMed

Heier, Jeffrey S; Kherani, Saleema; Desai, Shilpa; Dugel, Pravin; Kaushal, Shalesh; Cheng, Seng H; Delacono, Cheryl; Purvis, Annie; Richards, Susan; Le-Halpere, Annaig; Connelly, John; Wadsworth, Samuel C; Varona, Rafael; Buggage, Ronald; Scaria, Abraham; Campochiaro, Peter A

2017-07-01

Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 10 8 vector genomes (vg); cohort 2, 2 × 10 9 vg; cohort 3, 6 × 10 9 vg; and cohort 4, 2 × 10 10 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 10 10 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 10 10 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 10 10 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to

AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms

PubMed Central

Ravindranath, Pradeep Anand; Sanner, Michel F.

2016-01-01

Motivation: The identification of ligand-binding sites from a protein structure facilitates computational drug design and optimization, and protein function assignment. We introduce AutoSite: an efficient software tool for identifying ligand-binding sites and predicting pseudo ligand corresponding to each binding site identified. Binding sites are reported as clusters of 3D points called fills in which every point is labelled as hydrophobic or as hydrogen bond donor or acceptor. From these fills AutoSite derives feature points: a set of putative positions of hydrophobic-, and hydrogen-bond forming ligand atoms. Results: We show that AutoSite identifies ligand-binding sites with higher accuracy than other leading methods, and produces fills that better matches the ligand shape and properties, than the fills obtained with a software program with similar capabilities, AutoLigand. In addition, we demonstrate that for the Astex Diverse Set, the feature points identify 79% of hydrophobic ligand atoms, and 81% and 62% of the hydrogen acceptor and donor hydrogen ligand atoms interacting with the receptor, and predict 81.2% of water molecules mediating interactions between ligand and receptor. Finally, we illustrate potential uses of the predicted feature points in the context of lead optimization in drug discovery projects. Availability and Implementation: http://adfr.scripps.edu/AutoDockFR/autosite.html Contact: sanner@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27354702

An analysis on the roles of angiogenesis-related factors including serum vitamin D, soluble endoglin (sEng), soluble fms-like tyrosine kinase 1 (sFlt1), and vascular endothelial growth factor (VEGF) in the diagnosis and severity of late-onset preeclampsia.

PubMed

Cim, Numan; Kurdoglu, Mertihan; Ege, Serhat; Yoruk, Ibrahim; Yaman, Gorkem; Yildizhan, Recep

2017-07-01

The aim of this study was to evaluate the roles of proangiogenic factors including serum vitamin D and vascular endothelial growth factor (VEGF) and anti-angiogenic factors including soluble endoglin (sEng) and soluble fms-like tyrosine kinase 1 (sFlt1) in the diagnosis and severity of late-onset preeclampsia. The study was conducted at Yuzuncu Yil University Research and Education Hospital Department of Gynecology and Obstetrics. The study included a patient group of 40 women with late-onset preeclampsia who were pregnant at ≥32 weeks of gestation according to the last menstrual period (LMP) or ultrasonographic fetal biometric measurement and a control group of 40 healthy pregnant women who presented to our clinic for routine pregnancy examination and were at the same age and gestational period with those in the patient group. The two groups were compared in terms of maternal age, gravida, parity, week of gestation, systolic/diastolic blood pressure, total protein in spot urine sample, 24-h urine protein, white blood cell (WBC), hemoglobin (Hgb), platelet count, urea, creatinine, liver function tests (AST, ALT, LDH), vitamin D 3 , 25(OH) vitamin D 3 , 1,25(OH) vitamin D 3 , sEng, sFlt1, and VEGF levels, mode of delivery, the infant APGAR score at 1 and 5 min after delivery, and infant weight at delivery. The groups were similar in terms of age, gravida, parity, week of gestation, serum vitamin D 3 , 25(OH) vitamin D 3 , 1,25(OH) 2 vitamin D 3 and VEGF levels, and infant weight at delivery (p > 0.05). Systolic/diastolic blood pressure, total protein in spot urine sample, 24-h urine protein, WBC, Hgb, serum urea, creatine, AST, ALT, and LDH were significantly higher in the preeclamptic group compared to the healthy group (p < 0.05). However, thrombocyte level and the APGAR score at 1 and 5 min after delivery were significantly lower in the preeclamptic group compared to the healthy group (p < 0.05). No significant correlation was found between

fac-Re(CO)3 complexes of 2,6-bis(4-substituted-1,2,3-triazol-1-ylmethyl)pyridine "click" ligands: synthesis, characterisation and photophysical properties.

PubMed

Anderson, Christopher B; Elliott, Anastasia B S; Lewis, James E M; McAdam, C John; Gordon, Keith C; Crowley, James D

2012-12-28

The syntheses of the 4-n-propyl and 4-phenyl substituted fac-Re(CO)(3) complexes of the tridentate "click" ligand (2,6-bis(4-substituted-1,2,3-triazol-1-ylmethyl)pyridine) are described. The complexes were obtained by refluxing methanol solutions of [Re(CO)(5)Cl], AgPF(6) and either the 4-propyl or 4-phenyl substituted ligand for 16 h. The ligands and the two rhenium(I) complexes were characterised by elemental analysis, HR-ESMS, ATR-IR, (1)H and (13)C NMR spectroscopy and the molecular structures of both complexes were confirmed by X-ray crystallography. The electronic structure of the fac-Re(CO)(3) "click" complexes was probed using UV-Vis, Raman and emission spectroscopy, cyclic voltammetry and DFT calculations. Altering the electronic nature of the ligand's substituent, from aromatic to alkyl, had little effect on the absorption/emission maxima and electrochemical properties of the complexes indicating that the 1,2,3-triazole unit may insulate the metal centre from the electronic modification at the ligands' periphery. Both Re(I) complexes were found to be weakly emitting with short excited state lifetimes. The electrochemistry of the complexes is defined by quasi-reversible Re oxidation and irreversible triazole-based ligand reduction processes.

Modeling tyrosinase activity. Effect of ligand topology on aromatic ring hydroxylation: an overview.

PubMed

De, Anindita; Mandal, Sukanta; Mukherjee, Rabindranath

2008-01-01

Synthetic modeling of tyrosinase (o-phenol ring hydroxylation) has emerged as a novel class of successful biomimetic studies. It is a well-established fact that the reaction of dioxygen with copper(I) complexes of m-xylyl-based ligands generate putative copper-oxygen intermediate species such as side-on peroxo {CuII2(mu-O2)}2+ [in some cases bis-oxo {CuIII2(mu-O)2}2+ in equilibrium with isomeric side-on peroxo], due to oxygen activation. Electrophilic attack of such species brings about monooxygenase activity by incorporating one of the oxygens to m-xylyl ring of the ligand and the other oxygen is reduced to hydroxide ion. The goal of this review is to provide a concise overview of the present day knowledge in this field of research to emphasize the important role the designed ligands play in eliciting the desired tyrosinase-like chemistry.

Immunosuppressive Myeloid Cells' Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy.

PubMed

Kamran, Neha; Kadiyala, Padma; Saxena, Meghna; Candolfi, Marianela; Li, Youping; Moreno-Ayala, Mariela A; Raja, Nicholas; Shah, Diana; Lowenstein, Pedro R; Castro, Maria G

2017-01-04

Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Tuning of magnetic frustration in S = 1 / 2 Kagomé lattices {[Cu3(CO3)2(bpe)3](CLO4)2}n and {[Cu3(CO3)2(bpy)3](CLO4)2}n through rigid and flexible ligands

NASA Astrophysics Data System (ADS)

Ajeesh, M. O.; Yogi, A.; Padmanabhan, M.; Nath, R.

2015-04-01

Single crystalline and polycrystalline samples of S = 1 / 2 Kagomé lattices { [Cu3(CO3)2(bpe)3 ](CLO4)2 } n and { [Cu3(CO3)2(bpy)3 ](CLO4)2 } n, respectively were synthesized. Their structural and magnetic properties were characterized by means of x-ray diffraction and magnetization measurements. Both compounds crystalize in a hexagonal structure (space group P-6) consisting of CuO4 Kagomé layers in the ab-plane but linked along c direction through either rigid bpy or flexible bpe ligands to form 3D frame works. Magnetic measurements reveal that both the compounds undergo ferromagnetic ordering (TC) at low temperatures and the TC and the extent of frustration could be tuned by changing the nature of the pillar ligands. {[Cu3(CO3)2(bpe)3](CLO4)2}n which is made up of flexible bpe ligands has a TC of 5.7 K and a Curie-Weiss temperature (θCW) of -39.7 K giving rise to a frustration parameter of |θCW | /TC ≃ 6.96. But the replacement of bpe by a more rigid and electronically delocalized bpy ligand leads to an enhanced TC ≃ 9.3 K and a reduced frustration parameter of |θCW | /TC ≃ 3.54.

Comparison of Positron Emission Tomography Using 2-[18F]-fluoro-2-deoxy-D-glucose and 3-deoxy-3-[18F]-fluorothymidine in Lung Cancer Imaging

PubMed Central

Wang, Fu-Li; Tan, Ye-Ying; Gu, Xiang-Min; Li, Tian-Ran; Lu, Guang-Ming; Liu, Gang; Huo, Tian-Long

2016-01-01

Background: The detection of solitary pulmonary nodules (SPNs) that may potentially develop into a malignant lesion is essential for early clinical interventions. However, grading classification based on computed tomography (CT) imaging results remains a significant challenge. The 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET)/CT imaging produces both false-positive and false-negative findings for the diagnosis of SPNs. In this study, we compared 18F-FDG and 3-deoxy-3-[18F]-fluorothymidine (18F-FLT) in lung cancer PET/CT imaging. Methods: The binding ratios of the two tracers to A549 lung cancer cells were calculated. The mouse lung cancer model was established (n = 12), and micro-PET/CT analysis using the two tracers was performed. Images using the two tracers were collected from 55 lung cancer patients with SPNs. The correlation among the cell-tracer binding ratios, standardized uptake values (SUVs), and Ki-67 proliferation marker expression were investigated. Results: The cell-tracer binding ratio for the A549 cells using the 18F-FDG was greater than the ratio using 18F-FLT (P < 0.05). The Ki-67 expression showed a significant positive correlation with the 18F-FLT binding ratio (r = 0.824, P < 0.01). The tumor-to-nontumor uptake ratio of 18F-FDG imaging in xenografts was higher than that of 18F-FLT imaging. The diagnostic sensitivity, specificity, and the accuracy of 18F-FDG for lung cancer were 89%, 67%, and 73%, respectively. Moreover, the diagnostic sensitivity, specificity, and the accuracy of 18F-FLT for lung cancer were 71%, 79%, and 76%, respectively. There was an obvious positive correlation between the lung cancer Ki-67 expression and the mean maximum SUV of 18F-FDG and 18F-FLT (r = 0.658, P < 0.05 and r = 0.724, P < 0.01, respectively). Conclusions: The 18F-FDG uptake ratio is higher than that of 18F-FLT in A549 cells at the cellular level. 18F-FLT imaging might be superior for the quantitative diagnosis of lung tumor

The evolution of cellular deficiency in GATA2 mutation

PubMed Central

Dickinson, Rachel E.; Milne, Paul; Jardine, Laura; Zandi, Sasan; Swierczek, Sabina I.; McGovern, Naomi; Cookson, Sharon; Ferozepurwalla, Zaveyna; Langridge, Alexander; Pagan, Sarah; Gennery, Andrew; Heiskanen-Kosma, Tarja; Hämäläinen, Sari; Seppänen, Mikko; Helbert, Matthew; Tholouli, Eleni; Gambineri, Eleonora; Reykdal, Sigrún; Gottfreðsson, Magnús; Thaventhiran, James E.; Morris, Emma; Hirschfield, Gideon; Richter, Alex G.; Jolles, Stephen; Bacon, Chris M.; Hambleton, Sophie; Haniffa, Muzlifah; Bryceson, Yenan; Allen, Carl; Prchal, Josef T.; Dick, John E.; Bigley, Venetia

2014-01-01

Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56bright NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8+ memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making. PMID:24345756

Synthesis and characterisation of luminescent rhenium tricarbonyl complexes with axially coordinated 1,2,3-triazole ligands.

PubMed

Uppal, Baljinder S; Booth, Rebecca K; Ali, Noreen; Lockwood, Cindy; Rice, Craig R; Elliott, Paul I P

2011-08-07

A series of 1-alkyl-4-aryl-1,2,3-triazoles (1-methyl-4-phenyl-1,2,3-triazole (1a); 1-propyl-4-phenyl-1,2,3-triazole (1b); 1-benzyl-4-phenyl-1,2,3-triazole (1c); 1-propyl-4-p-tolyl-1,2,3-triazole (1d)) have been prepared through a one-pot procedure involving in situ generation of the alkyl azide from a halide precursor followed by copper catalysed alkyne/azide cycloaddition (CuAAC) with the appropriate aryl alkyne. Cationic Re(I) complexes [Re(bpy)(CO)(3)(1a-d)]PF(6) (2a-d) were then prepared by stirring [Re(bpy)(CO)(3)Cl] with AgPF(6) in dichloromethane in the presence of ligands 1a-d. X-ray crystal structures were obtained for 2a and 2b. In the solid state, 2a adopts a highly distorted geometry, which is not seen for 2b, in which the plane of the triazole ligand tilts by 13° with respect to the Re-N bond as a result of a π-stacking interaction between the Ph substituent and one of the rings of the bpy ligand. This π-stacking interaction also results in severe twisting of the bpy ligand. Infrared spectra of 2a-d exhibit ν(CO) bands at ∼2035 and ∼1926 cm(-1) suggesting that these ligands are marginally better donors than pyridine (ν(CO) = 2037, 1932 cm(-1)). The complexes are luminescent in aerated dichloromethane at room temperature with emission maxima at 542 to 552 nm comparable to that of the pyridine analogue (549 nm) and blue shifted relative to the parent chloride complex. Long luminescent lifetimes are observed for the triazole complexes (475 to 513 ns) in aerated dichloromethane solutions at room temperature.

Dissociation of Multisubunit Protein-Ligand Complexes in the Gas Phase. Evidence for Ligand Migration

NASA Astrophysics Data System (ADS)

Zhang, Yixuan; Deng, Lu; Kitova, Elena N.; Klassen, John S.

2013-10-01

The results of collision-induced dissociation (CID) experiments performed on gaseous protonated and deprotonated ions of complexes of cholera toxin B subunit homopentamer (CTB5) with the pentasaccharide (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p (GM1)) and corresponding glycosphingolipid (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p-Cer (GM1-Cer)) ligands, and the homotetramer streptavidin (S4) with biotin (B) and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Btl), are reported. The protonated (CTB5 + 5GM1)n+ ions dissociated predominantly by the loss of a single subunit, with the concomitant migration of ligand to another subunit. The simultaneous loss of ligand and subunit was observed as a minor pathway. In contrast, the deprotonated (CTB5 + 5GM1)n- ions dissociated preferentially by the loss of deprotonated ligand; the loss of ligand-bound and ligand-free subunit were minor pathways. The presence of ceramide (Cer) promoted ligand migration and the loss of subunit. The main dissociation pathway for the protonated and deprotonated (S4 + 4B)n+/- ions, as well as for deprotonated (S4 + 4Btl)n- ions, was loss of the ligand. However, subunit loss from the (S4 + 4B)n+ ions was observed as a minor pathway. The (S4 + 4Btl)n+ ions dissociated predominantly by the loss of free and ligand-bound subunit. The charge state of the complex and the collision energy were found to have little effect on the relative contribution of the different dissociation channels. Thermally-driven ligand migration between subunits was captured in the results of molecular dynamics simulations performed on protonated (CTB5 + 5GM1)15+ ions (with a range of charge configurations) at 800 K. Notably, the migration pathway was found to be highly dependent on the charge configuration of the ion. The main conclusion of this study is that the dissociation pathways of multisubunit protein-ligand

Ring opening and carbonylation of 3,3-dimethylthietane ligands in ruthenium carbonyl cluster complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, R.D.; Belinski, J.A.; Yamamoto, J.H.

1992-10-01

When heated to 97{degrees}C, the complex Ru{sub 4}(CO){sub 12}[{mu}-SCH{sub 2}CMe{sub 2}CH{sub 2}]2 (1) was transformed into two new hexaruthenium cluster complexes, Ru{sub 6}(CO){sub 13}({mu}{sub 3}-SCH{sub 2}CMe{sub 2}CH{sub 2}){sub 4} (2) and Ru{sub 6}(CO){sub 12}({mu}-SCH{sub 2}CMe{sub 2}CH{sub 2})({mu}{sub 3}-SCH{sub 2}CMe{sub 2}CH{sub 2}){sub 3}[{mu}{sub 3}-SCH{sub 2}C(Me)(CH{sub 2})CH{sub 2}] ({mu}-H) (3), that contain four and five ring-opened 3,3-dimethylthietane (3,3-DMT) ligands, respectively. In compound 3 one of the ring-opened DMT ligands has also undergone a CH activation on one of the methyl groups. Compound 2 reacts with additional 3,3-DMT at 97{degrees}C to form 3 in 18% yield. When treated with CO at 95{degrees}C (500more » psi), compound 2 yielded 4,4-dimethylthiobutyrolactone and Ru{sub 3}(CO){sub 12}. It was also found that the complex Os{sub 3}(CO){sub 11-}(SCH{sub 2}CMe{sub 2}CH{sub 2}C{double_bond}O) (4) yields 4,4-dimethylthiobutyrolactone when treated with CO at 120{degrees}C (1200 psi). Crystal data for 2: space group P2{sub 1}/n, {alpha} = 22.652 (7) A, {beta} = 11.712 (2) A, c = 19.965 (6) A, {Beta} = 115.75 (2){degrees} Z = 4, 3665 reflections, R = 0.021. Crystal data for 3: space group P2{sub 1}/c, {alpha} = 17.332 (8) A, {Beta} = 14.668 (9) A, c = 19.823 (9) A, {Beta} = 91.27 (4){degrees}, Z = 4, 1875 reflections, R = 0.050. 13 refs., 2 figs., 13 refs.« less

Paramagnetic oxotungsten(V) complexes containing the hydrotris(3,5-dimethylpyrazol-1-yl)borate ligand.

PubMed

Sproules, Stephen; Eagle, Aston A; Taylor, Michelle K; Gable, Robert W; White, Jonathan M; Young, Charles G

2011-05-16

Sky-blue Tp*WOCl(2) has been synthesized from the high-yielding reaction of Tp*WO(2)Cl with boron trichloride in refluxing toluene. Dark-red Tp*WOI(2) was prepared via thermal decarbonylation followed by aerial oxidation of Tp*WI(CO)(3) in acetonitrile. From these precursors, an extensive series of mononuclear tungstenyl complexes, Tp*WOXY [X = Cl(-), Y = OPh(-), SPh(-); X = Y = OPh(-), 2-(n-propyl)phenolate (PP(-)), SPh(-), SePh(-); XY = toluene-3,4-dithiolate (tdt(2-)), quinoxaline-2,3-dithiolate (qdt(2-)), benzene-1,2-diselenolate (bds(2-)); Tp* = hydrotris(3,5-dimethylpyrazol-1-yl)borate], was prepared by metathesis with the respective alkali-metal salt of X(-)/XY(2-) or (NHEt(3))(2)(qdt). The complexes were characterized by microanalysis, mass spectrometry, electrochemistry, IR, electron paramagnetic resonance (EPR), and electronic absorption spectroscopies, and X-ray crystallography (for X = Y = OPh(-), PP(-), SPh(-); XY = bds(2-)). The six-coordinate, distorted-octahedral tungsten centers are coordinated by terminal oxo [W≡O = 1.689(6)-1.704(3) Å], tridentate Tp*, and monodentate or bidentate O/S/Se-donor ligands. Spin Hamiltonian parameters derived from the simulation of fluid-solution X-band EPR spectra revealed that the soft-donor S/Se ligand complexes had larger g values and smaller (183)W hyperfine coupling constants than the less covalent hard-donor O/Cl species. The former showed low-energy ligand-to-metal charge-transfer bands in the near-IR region of their electronic absorption spectra. These oxotungsten(V) complexes display lower reduction potentials than their molybdenum counterparts, underscoring the preference of tungsten for higher oxidation states. Furthermore, the protonation of the pyrazine nitrogen atoms of the qdt(2-) ligand has been examined by spectroelectrochemistry; the product of the one-electron reduction of [Tp*WO(qdtH)](+) revealed usually intense low-energy bands.

Trigonal Mn3 and Co3 Clusters Supported by Weak-Field Ligands: A Structural, Spectroscopic, Magnetic, and Computational Investigation into the Correlation of Molecular and Electronic Structure

PubMed Central

Fout, Alison R.; Xiao, Dianne J.; Zhao, Qinliang; Harris, T. David; King, Evan R.; Eames, Emily V.; Zheng, Shao-Liang; Betley, Theodore A.

2012-01-01

Transamination of divalent transition metal starting materials (M2(N(SiMe3)2)4, M = Mn, Co) with hexadentate ligand platforms RLH6 (RLH6 = MeC(CH2NPh-o-NR)3 where R = H, Ph, Mes (Mes = Mesityl)) or H,CyLH6 = 1,3,5-C6H9(NHPh-o-NH2)3 with added pyridine or tertiary phosphine co-ligands afforded trinuclear complexes of the type (RL)Mn3(py)3 and (RL)Co3(PMe2R’)3 (R’ = Me, Ph). While the sterically less encumbered ligand varieties, HL or PhL, give rise to local square-pyramidal geometries at each of the bound metal atoms, with four anilides forming an equatorial plane and an exogenous pyridine or phosphine in the apical site, the mesityl-substituted ligand (MesL) engenders local tetrahedral coordination. Both the neutral Mn3 and Co3 clusters feature S = 1/2 ground states, as determined by dc magnetometry, 1H NMR spectroscopy, and low-temperature EPR spectroscopy. Within the Mn3 clusters, the long internuclear Mn–Mn separations suggest minimal direct metal-metal orbital overlap. Accordingly, fits to variable-temperature magnetic susceptibility data reveal the presence of weak antiferromagnetic superexchange interactions through the bridging anilide ligands with exchange couplings ranging from J = −16.8 to −42 cm−1. Conversely, the short Co–Co interatomic distances suggest a significant degree of direct metal-metal orbital overlap, akin to the related Fe3 clusters. With the Co3 series, the S = 1/2 ground state can be attributed to population of a single molecular orbital manifold that arises from mixing of the metal- and o-phenylenediamide (OPDA) ligand-based frontier orbitals. Chemical oxidation of the neutral Co3 clusters affords diamagnetic cationic clusters of the type [(RL)Co3(PMe2R)3]+. DFT calculations on the neutral (S = ½) and cationic (S = 0) Co3 clusters reveal that oxidation occurs at an oribital with contributions from both the Co3 core and OPDA subunits. The predicted bond elongations within the ligand OPDA units are corroborated by the ligand

Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.

PubMed

Bachmann, Julie; Raue, Andreas; Schilling, Marcel; Böhm, Martin E; Kreutz, Clemens; Kaschek, Daniel; Busch, Hauke; Gretz, Norbert; Lehmann, Wolf D; Timmer, Jens; Klingmüller, Ursula

2011-07-19

Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.

2,3-Di(2-pyridyl)-5-phenylpyrazine: a NN-CNN-type bridging ligand for dinuclear transition-metal complexes.

PubMed

Wu, Si-Hai; Zhong, Yu-Wu; Yao, Jiannian

2013-07-01

A new bridging ligand, 2,3-di(2-pyridyl)-5-phenylpyrazine (dpppzH), has been synthesized. This ligand was designed so that it could bind two metals through a NN-CNN-type coordination mode. The reaction of dpppzH with cis-[(bpy)2RuCl2] (bpy = 2,2'-bipyridine) affords monoruthenium complex [(bpy)2Ru(dpppzH)](2+) (1(2+)) in 64 % yield, in which dpppzH behaves as a NN bidentate ligand. The asymmetric biruthenium complex [(bpy)2Ru(dpppz)Ru(Mebip)](3+) (2(3+)) was prepared from complex 1(2+) and [(Mebip)RuCl3] (Mebip = bis(N-methylbenzimidazolyl)pyridine), in which one hydrogen atom on the phenyl ring of dpppzH is lost and the bridging ligand binds to the second ruthenium atom in a CNN tridentate fashion. In addition, the RuPt heterobimetallic complex [(bpy)2Ru(dpppz)Pt(C≡CPh)](2+) (4(2+)) has been prepared from complex 1(2+), in which the bridging ligand binds to the platinum atom through a CNN binding mode. The electronic properties of these complexes have been probed by using electrochemical and spectroscopic techniques and studied by theoretical calculations. Complex 1(2+) is emissive at room temperature, with an emission λmax = 695 nm. No emission was detected for complex 2(3+) at room temperature in MeCN, whereas complex 4(2+) displayed an emission at about 750 nm. The emission properties of these complexes are compared to those of previously reported Ru and RuPt bimetallic complexes with a related ligand, 2,3-di(2-pyridyl)-5,6-diphenylpyrazine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Issues in Requirements Elicitation

DTIC Science & Technology

1992-09-01

oriented domain analysis ( FODA ) continues that the re- quirements analyst uses the products of domain analysis when implementing a new system [Kang 90, p...Peterson, A. Spencer. Feature-Oriented Domain Analysis ( FODA ) Feasibility Study. Technical Report CMU/SEI-90-TR-21, ADA235785, Software Engineering...3.3 Problems of Volatility 12 4 Current Elicitation Techniques 15 4.1 Information Gathering 16 4.2 Requirements Expression and Analysis 19 4.3

Conformational diversity of flexible ligand in metal-organic frameworks controlled by size-matching mixed ligands

NASA Astrophysics Data System (ADS)

Hua, Xiu-Ni; Qin, Lan; Yan, Xiao-Zhi; Yu, Lei; Xie, Yi-Xin; Han, Lei

2015-12-01

Hydrothermal reactions of N-auxiliary flexible exo-bidentate ligand 1,3-bis(4-pyridyl)propane (bpp) and carboxylates ligands naphthalene-2,6-dicarboxylic acid (2,6-H2ndc) or 4,4‧-(hydroxymethylene)dibenzoic acid (H2hmdb), in the presence of cadmium(II) salts have given rise to two novel metal-organic frameworks based on flexible ligands (FL-MOFs), namely, [Cd2(2,6-ndc)2(bpp)(DMF)]·2DMF (1) and [Cd3(hmdb)3(bpp)]·2DMF·2EtOH (2) (DMF=N,N-Dimethylformamide). Single-crystal X-ray diffraction analyses revealed that compound 1 exhibits a three-dimensional self-penetrating 6-connected framework based on dinuclear cluster second building unit. Compound 2 displays an infinite three-dimensional 'Lucky Clover' shape (2,10)-connected network based on the trinuclear cluster and V-shaped organic linkers. The flexible bpp ligand displays different conformations in 1 and 2, which are successfully controlled by size-matching mixed ligands during the self-assembly process.

Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.

PubMed

Diehl, Carl; Engström, Olof; Delaine, Tamara; Håkansson, Maria; Genheden, Samuel; Modig, Kristofer; Leffler, Hakon; Ryde, Ulf; Nilsson, Ulf J; Akke, Mikael

2010-10-20

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, bothmore » sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.« less

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1.

PubMed

Bower, Joseph F; Green, Thomas D; Ross, Ted M

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

Synthesis, molecular properties estimations, and dual dopamine D2 and D3 receptor activities of Benzthiazole-based ligands.

NASA Astrophysics Data System (ADS)

Schübler, Moritz; Sadek, Bassem; Kottke, Tim; Weizel, Lilia; Stark, Holger

2017-09-01

Neurleptic drugs, e.g. aripiprazole, targeting the dopamine D2s and D3 receptors (D2sR and D3R) in the central nervous system are widely used in the treatment of several psychotic and neurodegenerative diseases. Therefore, a new series of benz[d]thiazole-based ligands (1-18) was synthesized by applying the bioisosteric approach derived from the selective D3Rs ligand BP-897 and its structurally related benz[d]imidazole derivatives. Herein, introduction of the benz[d]thiazole moiety was well tolerated by D2sR and D3R binding sites leading to antagonist affinities in the low nanomolar concentration range at both receptor subtypes. Further exploration of different substitution patterns at the benz[d]thiazole heterocycle and the basic 4-phenylpiperazine resulted in the discovery of high dually acting D2sR and D3R ligands. Moreover, the methoxy substitution at 2-position of 4-phenylpiperazine resulted in significantly (22-fold) increased D2sR binding affinity as compared to the parent ligand BP-897, and improved physicochemical and drug-likeness properties of ligands 1-9. However, the latter structural modifications failed to improve the drug-able properties in ligands having un-substituted 4-phenylpiperazine analogues (10-18). Accordingly, compound 7 showed in addition to high dual affinity at the D2sR and D3R (Ki (hD2SR) = 2.8 ± 0.8 nM; Ki (hD3R) = 3.0 ± 1.6 nM), promising clogS, clogP, LE (hD2sR, hD3R), LipE (hD2sR, hD3R), and drug-likeness score values of -4.7, 4.2, (0.4, 0.4), (4.4, 4.3), and 0.7, respectively. Also, the deaminated analogue 8 (Ki (hD2SR) = 3.2 ± 0.4 nM; Ki (hD3R) = 8.5 ± 2.2 nM) revealed clogS, clogP, LE (hD2sR, hD3R), LipE (hD2sR, hD3R) and drug-likeness score values of -4.7, 4.2, (0.4, 0.4), (3.9, 3.5), and 0.4, respectively. The results observed for the newly developed benz[d]thiazole-based ligands 1-18 provide clues for the diversity in structure activity relationships (SARs) at the D2sR and D3R subtypes.

Zinc complexes of the biomimetic N,N,O ligand family of substituted 3,3-bis(1-alkylimidazol-2-yl)propionates: the formation of oxalate from pyruvate

PubMed Central

Bruijnincx, Pieter C. A.; Lutz, Martin; den Breejen, Johan P.; van Koten, Gerard

2007-01-01

The coordination chemistry of the 2-His-1-carboxylate facial triad mimics 3,3-bis(1-methylimidazol-2-yl)propionate (MIm2Pr) and 3,3-bis(1-ethyl-4-isopropylimidazol-2-yl) propionate (iPrEtIm2Pr) towards ZnCl2 was studied both in solution and in the solid state. Different coordination modes were found depending both on the stoichiometry and on the ligand that was employed. In the 2:1 ligand-to-metal complex [Zn(MIm2Pr)2], the ligand coordinates in a tridentate, tripodal N,N,O fashion similar to the 2-His-1-carboxylate facial triad. However, the 1:1 ligand-to-metal complexes [Zn(MIm2Pr)Cl(H2O)] and [Zn(iPrEtIm2Pr)Cl] were crystallographically characterized and found to be polymeric in nature. A new, bridging coordination mode of the ligands was observed in both structures comprising N,N-bidentate coordination of the ligand to one zinc atom and O-monodentate coordination to a zinc second atom. A rather unique transformation of pyruvate into oxalate was found with [Zn(MIm2Pr)Cl], which resulted in the isolation of the new, oxalato bridged zinc coordination polymer [Zn2(MIm2Pr)2(ox)]·6H2O, the structure of which was established by X-ray crystal structure determination. PMID:17828423

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

PDZ Ligand Binding-Induced Conformational Coupling of the PDZ-SH3-GK Tandems in PSD-95 Family MAGUKs.

PubMed

Zeng, Menglong; Ye, Fei; Xu, Jia; Zhang, Mingjie

2018-01-05

Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3-GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3-GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3-GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3-GK is a common feature for other MAGUKs including CASK and PALS1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Can 3'-Deoxy-3'-((18)F) Fluorothymidine Out Perform 2-Deoxy-2-((18)F) Fluoro-D-Glucose Positron Emission Tomography/Computed Tomography in the Diagnosis of Cervical Lymphadenopathy in Patients With Oral/Head and Neck Cancer?

PubMed

Schaefferkoetter, Joshua D; Carlson, Eric R; Heidel, Robert E

2015-07-01

The present study investigated the performance of cellular metabolism imaging with 2-deoxy-2-((18)F) fluoro-D-glucose (FDG) versus cellular proliferation imaging with 3'-deoxy-3'-((18)F) fluorothymidine (FLT) in the detection of cervical lymph node metastases in oral/head and neck cancer. We conducted a prospective cohort study to assess a head-to-head performance of FLT imaging and clinical FDG imaging for characterizing cervical lymph node metastases in patients with squamous cell carcinoma (SCC) of the oral/head and neck region. The primary predictor variable of the study was the presence of FDG or FLT avidity within the cervical lymph nodes. The primary outcome variable was the histologic presence of metastatic SCC in the cervical lymph nodes. The performance was reported in terms of the sensitivity, specificity, accuracy, and positive and negative predictive values. The overall accuracy for discriminating positive from negative lymph nodes was evaluated as a function of the positron emission tomography (PET) standardized uptake value (SUV). Receiver operating characteristic (ROC) analyses were performed for both tracers. Eleven patients undergoing surgical resection of SCC of the oral/head and neck region underwent preoperative FDG and FLT PET-computed tomography (CT) scans on separate days. The interpretation of the FDG PET-CT imaging resulted in sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of 43.2, 99.5, 94.4, 88.9, and 94.7%, respectively. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value for FLT PET-CT imaging was 75.7, 99.2, 97.1, 90.3, and 97.7%, respectively. The areas under the curve for the ROC curves were 0.9 and 0.84 for FDG and FLT, respectively. Poor correlation was observed between the SUV for FDG and FLT within the lymph nodes and tumors. FLT showed better overall performance for detecting lymphadenopathy on qualitative assessment within the total

The neuropharmacology of prolactin secretion elicited by 3,4-methylenedioxymethamphetamine (“ecstasy”): A concurrent microdialysis and plasma analysis study

PubMed Central

Murnane, K.S.; Kimmel, H.L.; Rice, K.C.; Howell, L.L

2012-01-01

3,4-methylenedioxymethamphetamine (MDMA) is a substituted phenethylamine that is widely abused as the street drug “ecstasy”. Racemic MDMA (S,R(+/−)-MDMA) and its stereoisomers elicit complex spectrums of psychobiological, neurochemical, and hormonal effects. In this regard, recent findings demonstrated that S,R(+/−)-MDMA and its stereoisomer R(−)-MDMA elicit increases in striatal extracellular serotonin levels and plasma levels of the hormone prolactin in rhesus monkeys. In the present mechanistic study, we evaluated the role of the serotonin transporter and the 5-HT2A receptor in S,R(+/−)-MDMA- and R(−)-MDMA-elicited prolactin secretion in rhesus monkeys through concurrent microdialysis and plasma analysis determinations and drug interaction experiments. Concurrent neurochemical and hormone determinations showed a strong positive temporal correlation between serotonin release and prolactin secretion. Consistent with their distinct mechanisms of action and previous studies showing that the serotonin transporter inhibitor fluoxetine attenuates the behavioral and neurochemical effects of S,R(+/−)-MDMA, pretreatment with fluoxetine attenuated serotonin release elicited by either S,R(+/−)-MDMA or R(−)-MDMA. As hypothesized, at a dose that had no significant effects on circulating prolactin levels when administered alone, fluoxetine also attenuated prolactin secretion elicited by S,R(+/−)-MDMA. In contrast, combined pretreatment with both fluoxetine and the selective 5-HT2A receptor antagonist M100907 was required to attenuate prolactin secretion elicited by R(−)-MDMA, suggesting that this stereoisomer of S,R(+/−)-MDMA elicits prolactin secretion through both serotonin release and direct agonism of 5-HT2A receptors. Accordingly, these findings inform our understanding of the neuropharmacology of both S,R(+/−)-MDMA and R(−)-MDMA and the regulation of prolactin secretion. PMID:22197270

Secondary ligand-directed assembly of Co(II) coordination polymers based on a pyridine carboxylate ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Ke-Li; Zhang, Yi-Ping; Cai, Yi-Ni

2014-07-01

To investigate the influence of hydrogen bonds and secondary ligands on the structures and properties of the resulting frameworks, five new Co(II) compounds have been synthesized by the reactions of Co(II) salts and 3,5-bis(pyridin-4-ylmethoxy)benzoic acid (HL) with four rationally selected dicarboxylic acid ligands. Without secondary ligand, we got one compound [CoL{sub 2}(H{sub 2}O){sub 2}]{sub n}·2nH{sub 2}O (1), which possesses a 1D chain structure. In the presence of ancillary ligands, namely, 1,3-adamantanedicarboxylic acid (H{sub 2}adbc), terephthalic acid (H{sub 2}tpa), thiophene-2,5-dicarboxylic acid (H{sub 2}tdc) and 1,4-benzenedithioacetic acid (H{sub 2}bdtc), four 3D structures [Co{sub 2}L{sub 2}(adbc)]{sub n}·nH{sub 2}O (2), [Co{sub 2}L{sub 2}(tpa)]{sub n}more » (3), [Co{sub 2}L{sub 2}(tdc)]{sub n} (4), [Co{sub 2}L{sub 2}(bdtc)(H{sub 2}O)]{sub n} (5) were obtained, respectively. It can be observed from the architectures of 1–5 that hydrogen bonds and secondary ligands both have great effects on the spatial connective fashions, resulting in the formation of various dimensional compounds. The XRPD, TGA data of title polymers and the magnetic properties for 2 and 5 have also been investigated. - Graphical abstract: The structural differences show that the ancillary ligands have great effects on the spatial connective fashions, resulting in the formation of various dimensional compounds. - Highlights: • Five new Co(II) coordination polymers have been synthesized by solvothermal reactions based on 3,5-bis(pyridin-4-ylmethoxy)benzoic acid (HL). • The long-flexible ligand (HL) is a good candidate to produce interpenetrating architectures. • The secondary dicarboxylic acid ligands play important roles in the spatial connective fashions and the formation of various dimensional compounds. • The magnetism studies show that both 2 and 5 exhibit antiferromagnetic interactions.« less

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

PubMed Central

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C.

2015-01-01

Summary Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C-terminus. Together, these studies provide new insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential new target for therapeutic modulation of Notch signal transduction in disease. PMID:25747658

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

DOE PAGES

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; ...

2015-03-05

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

CXCR3 chemokine ligands during respiratory viral infections predict lung allograft dysfunction.

PubMed

Weigt, S S; Derhovanessian, A; Liao, E; Hu, S; Gregson, A L; Kubak, B M; Saggar, R; Saggar, R; Plachevskiy, V; Fishbein, M C; Lynch, J P; Ardehali, A; Ross, D J; Wang, H-J; Elashoff, R M; Belperio, J A

2012-02-01

Community-acquired respiratory viruses (CARV) can accelerate the development of lung allograft dysfunction, but the immunologic mechanisms are poorly understood. The chemokine receptor CXCR3 and its chemokine ligands, CXCL9, CXCL10 and CXCL11 have roles in the immune response to viruses and in the pathogenesis of bronchiolitis obliterans syndrome, the predominant manifestation of chronic lung allograft rejection. We explored the impact of CARV infection on CXCR3/ligand biology and explored the use of CXCR3 chemokines as biomarkers for subsequent lung allograft dysfunction. Seventeen lung transplant recipients with CARV infection had bronchoalveolar lavage fluid (BALF) available for analysis. For comparison, we included 34 BALF specimens (2 for each CARV case) that were negative for infection and collected at a duration posttransplant similar to a CARV case. The concentration of each CXCR3 chemokine was increased during CARV infection. Among CARV infected patients, a high BALF concentration of either CXCL10 or CXCL11 was predictive of a greater decline in forced expiratory volume in 1 s, 6 months later. CXCR3 chemokine concentrations provide prognostic information and this may have important implications for the development of novel treatment strategies to modify outcomes after CARV infection. © 2011 American Society of Transplantation and the American Society of Transplant Surgeons.

Fac-Re(bpy)(CO){sub 3}(COOMe): A model metallocarboxylate complex of rhenium with a bipyridyl ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, D.H.; Sleadd, B.A.; Vij, A.

1999-05-01

The title compound fac-(2,2{prime}-bipyridyl)(carbomethoxy)rhenium tricarbonyl, crystallizes in the monoclinic system, space group P2{sub 1}/c with the following crystal data: a = 8.37551(1), b = 6.6934(1), c = 26.2098(1) {angstrom}, V({angstrom}{sup 3}) = 1,535.93(3), Z = 4, and {beta} = 90.0971(2). The metal environment is slightly distorted octahedral with a chelating bipyridyl ligand and a facial arrangement of the carbon monoxide ligands.

ASIC3 Channels Integrate Agmatine and Multiple Inflammatory Signals through the Nonproton Ligand Sensing Domain

PubMed Central

2010-01-01

Background Acid-sensing ion channels (ASICs) have long been known to sense extracellular protons and contribute to sensory perception. Peripheral ASIC3 channels represent natural sensors of acidic and inflammatory pain. We recently reported the use of a synthetic compound, 2-guanidine-4-methylquinazoline (GMQ), to identify a novel nonproton sensing domain in the ASIC3 channel, and proposed that, based on its structural similarity with GMQ, the arginine metabolite agmatine (AGM) may be an endogenous nonproton ligand for ASIC3 channels. Results Here, we present further evidence for the physiological correlation between AGM and ASIC3. Among arginine metabolites, only AGM and its analog arcaine (ARC) activated ASIC3 channels at neutral pH in a sustained manner similar to GMQ. In addition to the homomeric ASIC3 channels, AGM also activated heteromeric ASIC3 plus ASIC1b channels, extending its potential physiological relevance. Importantly, the process of activation by AGM was highly sensitive to mild acidosis, hyperosmolarity, arachidonic acid (AA), lactic acid and reduced extracellular Ca2+. AGM-induced ASIC3 channel activation was not through the chelation of extracellular Ca2+ as occurs with increased lactate, but rather through a direct interaction with the newly identified nonproton ligand sensing domain. Finally, AGM cooperated with the multiple inflammatory signals to cause pain-related behaviors in an ASIC3-dependent manner. Conclusions Nonproton ligand sensing domain might represent a novel mechanism for activation or sensitization of ASIC3 channels underlying inflammatory pain-sensing under in vivo conditions. PMID:21143836

The synthetic substance hypoxanthine 3-N-oxide elicits alarm reactions in zebrafish (Danio rerio)

PubMed Central

Parra, Kevin V.; Adrian, James C.; Gerlai, Robert

2009-01-01

Zebrafish, one of the preferred study species of geneticists, is gaining increasing popularity in behavioral neuroscience. This small and prolific species may be an excellent tool with which the biological mechanisms of vertebrate brain function and behavior are investigated. Zebrafish has been proposed as a model organism in the analysis of fear responses and human anxiety disorders. Species-specific cues signaling the presence of predators have been successfully utilized in such research. Zebrafish has been shown to respond to its natural alarm substance with species-typical fear reactions. However, the extraction of this alarm substance and ascertaining its consistent dosing has been problematic. A synthetic substance with a known chemical identity and molecular weight would allow precise dosing and experimental control. Previously, the chemical component, hypoxanthine 3-N-oxide, common to several fish alarm substances has been identified and has been shown to elicit alarm reactions in fish species belonging to the Osteriophysan superorder. In the current study we investigate the effect of hypoxanthine 3-N-oxide by exposing zebrafish to three different concentrations of this synthetic substance. Our results show that the substance efficaciously induces species-typical fear reactions increasing the number of erratic movement episodes and jumps in zebrafish. We discuss the translational relevance of our findings and conclude that hypoxanthine 3-N-oxide will have utility to elicit fear responses in the laboratory in a precisely controlled manner in zebrafish. PMID:19583985

Serotonin 5-HT3 and 5-HT4 ligands: an update of medicinal chemistry research in the last few years.

PubMed

Modica, M N; Pittalà, V; Romeo, G; Salerno, L; Siracusa, M A

2010-01-01

The biogenic amine serotonin (5-hydroxytryptamine, 5-HT) is one of the most studied neurotransmitters in the central nervous system. It acts through the activation of at least fourteen 5-HT receptor subtypes. Over the last two decades, high attention was devoted to the 5-HT(3) and 5-HT(4) receptors due to their colocalization in the gastrointestinal tract and because their ligands are useful in the treatment of intestinal serotonergic system dysfunctions. The focus of this review is to discuss the literature concerning recent advances on 5-HT(3)R and 5-HT(4)R ligands and their structure-activity relationships from a medicinal chemistry perspective. During the last few years, new and significant progresses have been made in the field of novel potent and selective ligands, mixed ligands, agonists, partial agonists, and antagonists, and a number of patents have been filed. Furthermore several ligands targeting the 5-HT(3)R and 5-HT(4)R have been proposed for novel therapeutic indications such as the treatment of various psychiatric disorders.

Analysis of macromolecules, ligands and macromolecule-ligand complexes

DOEpatents

Von Dreele, Robert B [Los Alamos, NM

2008-12-23

A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2[W][OPEN

PubMed Central

Smith, John M.; Salamango, Daniel J.; Leslie, Michelle E.; Collins, Carina A.; Heese, Antje

2014-01-01

FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in a ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogen-activated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (>2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22. PMID:24220680

ELICIT: An alternative imprecise weight elicitation technique for use in multi-criteria decision analysis for healthcare.

PubMed

Diaby, Vakaramoko; Sanogo, Vassiki; Moussa, Kouame Richard

2016-01-01

In this paper, the readers are introduced to ELICIT, an imprecise weight elicitation technique for multicriteria decision analysis for healthcare. The application of ELICIT consists of two steps: the rank ordering of evaluation criteria based on decision-makers' (DMs) preferences using the principal component analysis; and the estimation of criteria weights and their descriptive statistics using the variable interdependent analysis and the Monte Carlo method. The application of ELICIT is illustrated with a hypothetical case study involving the elicitation of weights for five criteria used to select the best device for eye surgery. The criteria were ranked from 1-5, based on a strict preference relationship established by the DMs. For each criterion, the deterministic weight was estimated as well as the standard deviation and 95% credibility interval. ELICIT is appropriate in situations where only ordinal DMs' preferences are available to elicit decision criteria weights.

A label-free approach to detect ligand binding to cell surface proteins in real time.

PubMed

Burtscher, Verena; Hotka, Matej; Li, Yang; Freissmuth, Michael; Sandtner, Walter

2018-04-26

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a long-range conformational change. The Gouy-Chapman model predicts that adsorption of charged ligands to surface proteins must produce displacement currents and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance. © 2018, Burtscher et al.

Amino acid ionic liquids as chiral ligands in ligand-exchange chiral separations.

PubMed

Liu, Qian; Wu, Kangkang; Tang, Fei; Yao, Lihua; Yang, Fei; Nie, Zhou; Yao, Shouzhuo

2009-09-28

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1-alkyl-3-methylimidazolium L-proline (L-Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers-dl-phenylalanine (dl-Phe), dl-histidine (dl-His), dl-tryptophane (dl-Trp), and dl-tyrosine (dl-Tyr)-were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R(s))=3.26-10.81 for HPLC; R(s)=1.34-4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L-Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L-Pro are consequently attached to the support surface, thus inducing an ion-exchange type of retention for the dl-enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand-exchange-based chiral separation. It also reveals the tremendous application potential of this new type of task-specific ILs.

CXCR3 ligands are associated with the continuum of diffuse alveolar damage to chronic lung allograft dysfunction.

PubMed

Shino, Michael Y; Weigt, S Samuel; Li, Ning; Palchevskiy, Vyacheslav; Derhovanessian, Ariss; Saggar, Rajan; Sayah, David M; Gregson, Aric L; Fishbein, Michael C; Ardehali, Abbas; Ross, David J; Lynch, Joseph P; Elashoff, Robert M; Belperio, John A

2013-11-01

After lung transplantation, insults to the allograft generally result in one of four histopathologic patterns of injury: (1) acute rejection, (2) lymphocytic bronchiolitis, (3) organizing pneumonia, and (4) diffuse alveolar damage (DAD). We hypothesized that DAD, the most severe form of acute lung injury, would lead to the highest risk of chronic lung allograft dysfunction (CLAD) and that a type I immune response would mediate this process. Determine whether DAD is associated with CLAD and explore the potential role of CXCR3/ligand biology. Transbronchial biopsies from all lung transplant recipients were reviewed. The association between the four injury patterns and subsequent outcomes were evaluated using proportional hazards models with time-dependent covariates. Bronchoalveolar lavage (BAL) concentrations of the CXCR3 ligands (CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC) were compared between allograft injury patterns and "healthy" biopsies using linear mixed-effects models. The effect of these chemokine alterations on CLAD risk was assessed using Cox models with serial BAL measurements as time-dependent covariates. There were 1,585 biopsies from 441 recipients with 62 episodes of DAD. An episode of DAD was associated with increased risk of CLAD (hazard ratio, 3.0; 95% confidence interval, 1.9-4.7) and death (hazard ratio, 2.3; 95% confidence interval, 1.7-3.0). There were marked elevations in BAL CXCR3 ligand concentrations during DAD. Furthermore, prolonged elevation of these chemokines in serial BAL fluid measurements predicted the development of CLAD. DAD is associated with marked increases in the risk of CLAD and death after lung transplantation. This association may be mediated in part by an aberrant type I immune response involving CXCR3/ligands.

Interactions between alkaline earth cations and oxo ligands. DFT study of the affinity of the Mg²+ cation for phosphoryl ligands.

PubMed

da Costa, Leonardo Moreira; de Mesquita Carneiro, José Walkimar; Paes, Lilian Weitzel Coelho

2011-08-01

DFT (B3LYP/6-31+G(d)) calculations of Mg(2+) affinities for a set of phosphoryl ligands were performed. Two types of ligands were studied: a set of trivalent [O = P(R)] and a set of pentavalent phosphoryl ligands [O = P(R)(3)] (R = H, F, Cl, Br, OH, OCH(3), CH(3), CN, NH(2) and NO(2)), with R either bound directly to the phosphorus atom or to the para position of a phenyl ring. The affinity of the Mg(2+) cation for the ligands was quantified by means of the enthalpy for the substitution of one water molecule in the [Mg(H(2)O)(6)](2+) complex for a ligand. The enthalpy of substitution was correlated with electronic and geometric parameters. Electron-donor groups increase the interaction between the cation and the ligand, while electron-acceptor groups decrease the interaction enthalpy.

Ligand-accelerated enantioselective methylene C(sp3)-H bond activation.

PubMed

Chen, Gang; Gong, Wei; Zhuang, Zhe; Andrä, Michal S; Chen, Yan-Qiao; Hong, Xin; Yang, Yun-Fang; Liu, Tao; Houk, K N; Yu, Jin-Quan

2016-09-02

Effective differentiation of prochiral carbon-hydrogen (C-H) bonds on a single methylene carbon via asymmetric metal insertion remains a challenge. Here, we report the discovery of chiral acetyl-protected aminoethyl quinoline ligands that enable asymmetric palladium insertion into prochiral C-H bonds on a single methylene carbon center. We apply these palladium complexes to catalytic enantioselective functionalization of β-methylene C-H bonds in aliphatic amides. Using bidentate ligands to accelerate C-H activation of otherwise unreactive monodentate substrates is crucial for outcompeting the background reaction driven by substrate-directed cyclopalladation, thereby avoiding erosion of enantioselectivity. The potential of ligand acceleration in C-H activation is also demonstrated by enantioselective β-C-H arylation of simple carboxylic acids without installing directing groups. Copyright © 2016, American Association for the Advancement of Science.

ELICIT: An alternative imprecise weight elicitation technique for use in multi-criteria decision analysis for healthcare

PubMed Central

Diaby, Vakaramoko; Sanogo, Vassiki; Moussa, Kouame Richard

2015-01-01

Objective In this paper, the readers are introduced to ELICIT, an imprecise weight elicitation technique for multicriteria decision analysis for healthcare. Methods The application of ELICIT consists of two steps: the rank ordering of evaluation criteria based on decision-makers’ (DMs) preferences using the principal component analysis; and the estimation of criteria weights and their descriptive statistics using the variable interdependent analysis and the Monte Carlo method. The application of ELICIT is illustrated with a hypothetical case study involving the elicitation of weights for five criteria used to select the best device for eye surgery. Results The criteria were ranked from 1–5, based on a strict preference relationship established by the DMs. For each criterion, the deterministic weight was estimated as well as the standard deviation and 95% credibility interval. Conclusions ELICIT is appropriate in situations where only ordinal DMs’ preferences are available to elicit decision criteria weights. PMID:26361235

Identification of pyridinoline, a collagen crosslink, as a novel intrinsic ligand for the receptor for advanced glycation end-products (RAGE).

PubMed

Murakami, Yoto; Fujino, Takayuki; Kurachi, Ryotaro; Hasegawa, Toshiki; Usui, Teruyuki; Hayase, Fumitaka; Watanabe, Hirohito

2018-05-26

Advanced glycation end-products (AGEs) elicit inflammatory responses via the receptor for AGEs (RAGE) and participate in the pathogenesis of diabetic complications. An earlier study showed that 3-hydroxypyridinium (3-HP), a common moiety of toxic AGEs such as glyceraldehyde-derived pyridinium (GLAP) and GA-pyridine, is essential for the interaction with RAGE. However, the physiological significance of 3-HP recognition by RAGE remains unclear. We hypothesized that pyridinoline (Pyr), a collagen crosslink containing the 3-HP moiety, could have agonist activity with RAGE. To test this hypothesis, we purified Pyr from bovine achilles tendons and examined its cytotoxicity to rat neuronal PC12 cells. Pyr elicited toxicity to PC12 cells in a concentration-dependent manner, and this effect was attenuated in the presence of either the anti-RAGE antibody or the soluble form of RAGE. Moreover, surface plasmon resonance-based analysis showed specific binding of Pyr to RAGE. These data indicate that Pyr is an intrinsic ligand for RAGE. AGEs: advanced glycation end-products; RAGE: receptor for advanced glycation end-products; DAMPs: damage-associated molecular patterns; PRR: pattern recognition receptor; TLR: toll-like receptor; GLAP: glyceraldehyde-derived pyridinium; 3-HP: 3-hydroxypyridinium; Pyr: pyridinoline; HFBA: heptafluorobutyric acid; GST: glutathione S-transferase; SPR: surface plasmon resonance; ECM: extracellular matrix; EMT: epithelial to mesenchymal transition.

Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nie,Y.; Hobbs, J.; Vigues, S.

2006-01-01

Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain amore » large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.« less

Synthesis and Characterization of New Bivalent Agents as Melatonin- and Histamine H3-Ligands

PubMed Central

Pala, Daniele; Scalvini, Laura; Lodola, Alessio; Mor, Marco; Flammini, Lisa; Barocelli, Elisabetta; Lucini, Valeria; Scaglione, Francesco; Bartolucci, Silvia; Bedini, Annalida; Rivara, Silvia; Spadoni, Gilberto

2014-01-01

Melatonin is an endogenous molecule involved in many pathophysiological processes. In addition to the control of circadian rhythms, its antioxidant and neuroprotective properties have been widely described. Thus far, different bivalent compounds composed by a melatonin molecule linked to another neuroprotective agent were synthesized and tested for their ability to block neurodegenerative processes in vitro and in vivo. To identify a novel class of potential neuroprotective compounds, we prepared a series of bivalent ligands, in which a prototypic melatonergic ligand is connected to an imidazole-based H3 receptor antagonist through a flexible linker. Four imidazolyl-alkyloxy-anilinoethylamide derivatives, characterized by linkers of different length, were synthesized and their binding affinity for human MT1, MT2 and H3 receptor subtypes was evaluated. Among the tested compounds, 14c and 14d, bearing a pentyl and a hexyl linker, respectively, were able to bind to all receptor subtypes at micromolar concentrations and represent the first bivalent melatonergic/histaminergic ligands reported so far. These preliminary results, based on binding affinity evaluation, pave the way for the future development of new dual-acting compounds targeting both melatonin and histamine receptors, which could represent promising therapeutic agents for the treatment of neurodegenerative pathologies. PMID:25222552

Spectroscopic characterization of ligands on the surface of water dispersible NaGdF4:Ln3+ nanocrystals

NASA Astrophysics Data System (ADS)

Cichos, J.; Karbowiak, M.

2012-05-01

For electronic or biomedical applications it is desirable to have ligand-free water-dispersible nanocrystals (NCs). The commonly used FTIR spectroscopy often provides a direct evidence for molecules on the surface. In some cases, however, the strong bands of solvent molecules may obscure the peaks of surface bounded ligands. We show that in this regard the emission spectroscopy may be used as a more reliable probing tool. The relevant information can be obtained from emission and excitation spectra, emission decay times as well as from analysis of relative efficiency of excitation energy transfer from Gd3+ to Eu3+ ions. Using these methods we tested samples obtained by various synthetic routes and indicated that only nitrosonium tetrafluoroborate (NOBF4) removes successfully the organic ligands from the nanocrystals surface, yielding organic ligand-free NCs dispersible in aqueous solutions. The conclusions drawn from emission spectroscopy are useful for interpretation of results of FTIR, Raman and NMR studies. The detailed assignment of FTIR peaks for oleate-capped and oleate-free NCs is also provided. Finally, we point to the risk of drawing erroneous conclusions about colloidal stability of nanocrystals if refractive indexes of NCs and medium are similar.

Influence of ligand chemistry on silver nanoparticles for colorimetric detection of Cr3+ and Hg2+ ions

NASA Astrophysics Data System (ADS)

Kailasa, Suresh Kumar; Chandel, Madhurya; Mehta, Vaibhavkumar N.; Park, Tae Jung

2018-04-01

In this work, we describe the role of ligand chemistry on the surfaces of silver nanoparticles (Ag NPs) for tuning their analytical applications. The citrate and melamine (MA) molecules were used as ligands for the surface modification of Ag NPs. The addition of Cr3+ ion in citrate-Ag NPs (Cit-Ag NPs) and of Hg2+ ion in melamine-Ag NPs (MA-Ag NPs) cause Ag NPs aggregation, and are accompanied by a color change and a red-shift. The resulting distinctly visual readouts are favorable for colorimetric detection of Cr3+ and Hg2+ ions. Under optimal conditions, the linear ranges are observed in the concentration ranges of 1.0-50.0 and of 10.0-100.0 μM, and with detection limit of 0.52 and 1.80 μM for Cr3+ and Hg2+ ions. The simultaneous detection of Cr3+ and Hg2+ ion is driven by the changing the ligand chemistry on the surfaces of Ag NPs that allows to tune their specific interactions with target analytes. Finally, the functionalized Ag NPs were successfully applied to detect Cr3+ and Hg2+ ions in water samples with satisfactory recoveries.

An unexpected semi-hydrogenation of a ligand in the complexation of 2,7-bispyridinyl-1,8-naphthyridine with Ru3(CO)12.

PubMed

Liao, Bei-Sih; Liu, Yi-Hung; Peng, Shie-Ming; Reddy, K Rajender; Liu, Shin-Hung; Chou, Pi-Tai; Liu, Shiuh-Tzung

2014-03-07

Thermal reaction of 2,7-bis(2-pyridinyl)-l,8-naphthyridine () with Ru3(CO)12 in the presence of moisture resulted in the formation of a formate-bridged diruthenium complex [(-H3)Ru2(μ-HCOO)(CO)4] (), in which the ligand was partially hydrogenated. Complex was fully characterized by spectroscopic analyses and X-ray single crystal determination. Regarding the partially reduced ligand in , it occurs through a water-gas shift type reduction. The bridging formate ligand can be substituted by other carboxylate ligands. Physical and chemical properties of the newly prepared complexes were investigated.

Ligand-biased ensemble receptor docking (LigBEnD): a hybrid ligand/receptor structure-based approach

NASA Astrophysics Data System (ADS)

Lam, Polo C.-H.; Abagyan, Ruben; Totrov, Maxim

2018-01-01

Ligand docking to flexible protein molecules can be efficiently carried out through ensemble docking to multiple protein conformations, either from experimental X-ray structures or from in silico simulations. The success of ensemble docking often requires the careful selection of complementary protein conformations, through docking and scoring of known co-crystallized ligands. False positives, in which a ligand in a wrong pose achieves a better docking score than that of native pose, arise as additional protein conformations are added. In the current study, we developed a new ligand-biased ensemble receptor docking method and composite scoring function which combine the use of ligand-based atomic property field (APF) method with receptor structure-based docking. This method helps us to correctly dock 30 out of 36 ligands presented by the D3R docking challenge. For the six mis-docked ligands, the cognate receptor structures prove to be too different from the 40 available experimental Pocketome conformations used for docking and could be identified only by receptor sampling beyond experimentally explored conformational subspace.

Synthesis, characterization, and ligand exchange reactivity of a series of first row divalent metal 3-hydroxyflavonolate complexes.

PubMed

Grubel, Katarzyna; Rudzka, Katarzyna; Arif, Atta M; Klotz, Katie L; Halfen, Jason A; Berreau, Lisa M

2010-01-04

A series of divalent metal flavonolate complexes of the general formula [(6-Ph(2)TPA)M(3-Hfl)]X (1-5-X; X = OTf(-) or ClO(4)(-); 6-Ph(2)TPA = N,N-bis((6-phenyl-2-pyridyl)methyl)-N-((2-pyridyl)methyl)amine; M = Mn(II), Co(II), Ni(II), Cu(II), Zn(II); 3-Hfl = 3-hydroxyflavonolate) were prepared and characterized by X-ray crystallography, elemental analysis, FTIR, UV-vis, (1)H NMR or EPR, and cyclic voltammetry. All of the complexes have a bidentate coordinated flavonolate ligand. The difference in M-O distances (Delta(M-O)) involving this ligand varies through the series, with the asymmetry of flavonolate coordination increasing in the order Mn(II) approximately Ni(II) < Cu(II) < Zn(II) < Co(II). The hypsochromic shift of the absorption band I (pi-->pi*) of the coordinated flavonolate ligand in 1-5-OTf (relative to that in free anion) increases in the order Ni(II) < Mn(II) < Cu(II) < Zn(II), Co(II). Previously reported 3-Hfl complexes of divalent metals fit well with this ordering. (1)H NMR studies indicate that the 3-Hfl complexes of Co(II), Ni(II), and Zn(II) exhibit a pseudo-octahedral geometry in solution. EPR studies suggest that the Mn(II) complex 1-OTf may form binuclear structures in solution. The mononuclear Cu(II) complex 4-OTf has a distorted square pyramidal geometry. The oxidation potential of the flavonolate ligand depends on the metal ion present and/or the solution structure of the complex, with the Mn(II) complex 1-OTf exhibiting the lowest potential, followed by the pseudo-octahedral Ni(II) and Zn(II) 3-Hfl complexes, and the distorted square pyramidal Cu(II) complex 4-OTf. The Mn(II) complex [(6-Ph(2)TPA)Mn(3-Hfl)]OTf (1-OTf) is unique in the series in undergoing ligand exchange reactions in the presence of M(ClO(4))(2).6H(2)O (M = Co, Ni, Zn) in CD(3)CN to produce [(6-Ph(2)TPA)M(CD(3)CN)(n)](X)(2), [Mn(3-Hfl)(2).0.5H(2)O], and MnX(2) (X = OTf(-) or ClO(4)(-)). Under similar conditions, the 3-Hfl complexes of Co(II), Ni(II), and Cu(II) undergo

Synthesis of the water soluble ligands dmPTA and dmoPTA and the complex [RuClCp(HdmoPTA)(PPh(3))](OSO(2)CF(3)) (dmPTA = N,N'-Dimethyl-1,3,5-triaza-7-phosphaadamantane, dmoPTA = 3,7-Dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane, HdmoPTA = 3,7-H-3,7-Dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane).

PubMed

Mena-Cruz, Adrian; Lorenzo-Luis, Pablo; Romerosa, Antonio; Saoud, Mustapha; Serrano-Ruiz, Manuel

2007-07-23

The new water-soluble ligand dmPTA(OSO(2)CF(3))(2) (1) (dmPTA = N,N'-dimethyl-1,3,5-triaza-7-phosphaadamantane) has been synthesized by reaction of PTA with MeOSO(2)CF(3) in acetone (PTA = 1,3,5-triaza-7-phosphatricycle[3.3.1.1(3,7)]decane). The reaction of 1 with KOH gave rise to the new water-soluble ligand dmoPTA (3) (dmoPTA = 3,7-dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) by elimination of the -CH(2)- group located between both NCH(3) units. Compound dmPTA(BF(4))(2) (2) and complex [RuClCp(HdmoPTA)(PPh(3))](OSO(2)CF(3)) (4) have also been synthesized, while compounds HdmoPTA(BF(4)) (3a) and [RuClCp(dmPTA)(PPh(3))](OSO(2)CF(3)) (5) were characterized but not isolated. The new ligands and the complex have been fully characterized by NMR, IR, elemental analysis, and X-ray crystal structure determination (ligand 1 and complex 4). The synthetic processes for 3 and 4 were studied.

A new analysis of charge transfer and polarization for ligand-metal bonding - Model studies of Al4CO and Al4NH3

NASA Technical Reports Server (NTRS)

Bagus, P. S.; Hermann, K.; Bauschlicher, C. W., Jr.

1984-01-01

The nature of the bonding of CO and NH3 ligands to Al is analyzed, and the intra-unit charge polarization and inter-unit donation for the interaction of ligands with metals are studied. The consequences of metal-to-ligand and ligand-to-metal charge transfer are separately considered by performing a constrained space orbital variation (CSOV) with the electrons of the metal member of the complex in the field of frozen ligand. The electrons of the metal atoms are then frozen in the relaxed distribution given by the CSOV SCF wave function and the ligand electrons are allowed to relax. Quantitative measures of the importance of inter-unit charge transfers and intra-unit polarization are obtained using results of SCF studies of Al4CO and Al4NH3 clusters chosen to simulate the adsorption of the ligands at an on-top side of the Al(111) surface. The electrostatic attraction of the effective dipole moments of the metal and ligand units makes an important contribution to the bond.

Ligand specificity of MobR, a transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Mariko; Hiromoto, Takeshi; Hosokawa, Keiichi

2007-10-19

MobR from Comamonas testosteroni KH122-3s is a member of the MarR family of transcriptional regulators and functions as a repressor for the mobA gene that encodes a 3-hydroxybenzoate 4-hydroxylase. 3-Hydroxybenzoate binds to MobR as a ligand, resulting in an efficient induction of mobA. Various 3-hydroxybenzoate analogues were examined for their inducibilities using the mobA::lacZ transcriptional fusion system. {beta}-Galactosidase was induced by the addition of 2,3-dihydroxybenzoate or 3,5-dihydroxybenzoate besides 3-hydroxybenzoate, suggesting that the hydroxyl group at position 3 is critical in addition to the carboxyl group on the aromatic ring. A gel mobility-shift assay also showed that MobR was released frommore » the target DNA in the presence of these compounds. Circular dichroism studies demonstrated that MobR adopted two conformational states corresponding to the 3-hydroxybenzoate-bound and unbound forms. Other ligands also induced the structural change as well; however, the tertiary structures of converted forms were different from those by 3-hydroxybenzoate.« less

PDB-Ligand: a ligand database based on PDB for the automated and customized classification of ligand-binding structures.

PubMed

Shin, Jae-Min; Cho, Doo-Ho

2005-01-01

PDB-Ligand (http://www.idrtech.com/PDB-Ligand/) is a three-dimensional structure database of small molecular ligands that are bound to larger biomolecules deposited in the Protein Data Bank (PDB). It is also a database tool that allows one to browse, classify, superimpose and visualize these structures. As of May 2004, there are about 4870 types of small molecular ligands, experimentally determined as a complex with protein or DNA in the PDB. The proteins that a given ligand binds are often homologous and present the same binding structure to the ligand. However, there are also many instances wherein a given ligand binds to two or more unrelated proteins, or to the same or homologous protein in different binding environments. PDB-Ligand serves as an interactive structural analysis and clustering tool for all the ligand-binding structures in the PDB. PDB-Ligand also provides an easier way to obtain a number of different structure alignments of many related ligand-binding structures based on a simple and flexible ligand clustering method. PDB-Ligand will be a good resource for both a better interpretation of ligand-binding structures and the development of better scoring functions to be used in many drug discovery applications.

B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

PubMed

Candolfi, Marianela; Curtin, James F; Yagiz, Kader; Assi, Hikmat; Wibowo, Mia K; Alzadeh, Gabrielle E; Foulad, David; Muhammad, A K M G; Salehi, Sofia; Keech, Naomi; Puntel, Mariana; Liu, Chunyan; Sanderson, Nicholas R; Kroeger, Kurt M; Dunn, Robert; Martins, Gislaine; Lowenstein, Pedro R; Castro, Maria G

2011-10-01

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.

Quartz crystal microbalance (QCM) with immobilized protein receptors: comparison of response to ligand binding for direct protein immobilization and protein attachment via disulfide linker.

PubMed

Baltus, Ruth E; Carmon, Kendra S; Luck, Linda A

2007-03-27

Results from an investigation of the frequency response resulting from ligand binding for a genetically engineered hormone-binding domain of the alpha-estrogen receptor immobilized to a piezoelectric quartz crystal are reported. Two different approaches were used to attach a genetically altered receptor to the gold electrode on the quartz surface: (1) the mutant receptor containing a single solvent-exposed cysteine was directly attached to the crystal via a sulfur to gold covalent bond, forming a self-assembled protein monolayer, and (2) the N-terminal histidine-tagged end was utilized to attach the receptor via a 3,3-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid] linker complexed with nickel. Previous studies have shown that these engineered constructs bind 17beta-estradiol and are fully functional. Exposure of the receptor directly attached to the piezoelectric crystal to the known ligand 17beta-estradiol resulted in a measurable frequency response, consistent with a change in conformation of the receptor with ligand binding. However, no response was observed when the receptor immobilized via the linker was exposed to the same ligand. The presence of the linker between the quartz surface and the protein receptor does not allow the crystal to sense the conformational change in the receptor that occurs with ligand binding. These results illustrate that the immobilization strategy used to bind the receptor to the sensor platform is key to eliciting an appropriate response from this biosensor. This study has important implications for the development of QCM-based sensors using protein receptors.

sc-PDB: a 3D-database of ligandable binding sites—10 years on

PubMed Central

Desaphy, Jérémy; Bret, Guillaume; Rognan, Didier; Kellenberger, Esther

2015-01-01

The sc-PDB database (available at http://bioinfo-pharma.u-strasbg.fr/scPDB/) is a comprehensive and up-to-date selection of ligandable binding sites of the Protein Data Bank. Sites are defined from complexes between a protein and a pharmacological ligand. The database provides the all-atom description of the protein, its ligand, their binding site and their binding mode. Currently, the sc-PDB archive registers 9283 binding sites from 3678 unique proteins and 5608 unique ligands. The sc-PDB database was publicly launched in 2004 with the aim of providing structure files suitable for computational approaches to drug design, such as docking. During the last 10 years we have improved and standardized the processes for (i) identifying binding sites, (ii) correcting structures, (iii) annotating protein function and ligand properties and (iv) characterizing their binding mode. This paper presents the latest enhancements in the database, specifically pertaining to the representation of molecular interaction and to the similarity between ligand/protein binding patterns. The new website puts emphasis in pictorial analysis of data. PMID:25300483

A higher maternal choline intake among third-trimester pregnant women lowers placental and circulating concentrations of the antiangiogenic factor fms-like tyrosine kinase-1 (sFLT1).

PubMed