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Hochdorffer K.,Tumor Biology Center | Abu Ajaj K.,Tumor Biology Center | Abu Ajaj K.,Leibniz Institute for Molecular Pharmacology | Schafer-Obodozie C.,ProQinase GmbH | Kratz F.,Tumor Biology Center
Journal of Medicinal Chemistry | Year: 2012

Bone metastases are a frequent cause of morbidity in cancer patients. The present palliative therapeutic options are chemotherapy, hormone therapy, and the administration of bisphosphonates. The affinity between bisphosphonates and the apatite structure of bone metastases is strong. Thus, we designed two low-molecular-weight and water-soluble prodrugs which incorporate a bisphosphonate group as a bone targeting ligand, doxorubicin as the anticancer agent, and either an acid-sensitive bond (1) or a cathepsin B cleavable bond (3) for ensuring an effective release of doxorubicin at the site of action. Cleavage studies of both prodrugs showed a fast release of doxorubicin but sufficient stability over several hours in human plasma. Effective binding of prodrug 1 and 3 was demonstrated with hydroxyapatite and with native bone. In orientating toxicity studies in nude mice, the MTD of 1 was 3-fold higher compared to conventional doxorubicin, whereas 3 showed essentially the same MTD as doxorubicin. © 2012 American Chemical Society.


Lee Y.,Gwangju Institute of Science and Technology | Graeser R.,ProQinase GmbH | Kratz F.,Tumor Biology Center | Geckeler K.E.,Gwangju Institute of Science and Technology
Advanced Functional Materials | Year: 2011

Novel paclitaxel-loaded polymer nanoparticles were developed for circumventing multidrug resistance (MDR) of malignant cancerous diseases, which is an unsolved clinical problem in cancer chemotherapy. In many cases, MDR is due to the intrinsic or acquired expression of an efflux pump, the P-170 glycoprotein (P-gp). By encapsulating paclitaxel in a water-soluble and biocompatible synthetic polyampholyte using a solid-state reaction the highly water-soluble paclitaxel-loaded nanoparticles are formed. The resulting paclitaxel nanoparticles with an average diameter of 250 nm show a significant reversal of chemoresistance in the drug-resistant variants (MCF7/ADR, MT3/ADR) by a factor of 100 or more. The novel paclitaxel nanoparticles enter MDR breast cancer cells by adsorptive endocytosis bypassing the P-gp, preventing the efflux of paclitaxel and thus restoring the anti-proliferative effect of paclitaxel. Novel paclitaxel-loaded polymer nanoparticles are synthesized by supramolecular complexation in the solid state. They are highly soluble in water and taken up by wild and MDR-type breast cancer cells via adsorptive endocytosis and bypass the P-glycoprotein efflux in MDR-type cells. Their significantly enhanced cancer cell inhibition effect against MDR cells shows high potential for a new anticancer agent. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Goettert M.,University of Tübingen | Shaalan N.,German University in Cairo | Graeser R.,ProQinase GmbH | Laufer S.A.,University of Tübingen
Journal of Pharmaceutical and Biomedical Analysis | Year: 2012

The p38 mitogen activated protein kinase (MAPK) has emerged as a target for treating inflammatory diseases, like rheumatoid arthritis (RA). Expression of p38δ is induced in rheumatoid arthritis synovial fibroblasts (RASFs) by a cytokine-independent pathway substantially different from other MAPK pathways. To identify inhibitors of p38δ MAPK, we developed a direct ELISA assay based on a previously described p38α assay for monitoring the phosphorylation of ATF-2. This work presents a straightforward assay for evaluating the potency of small-molecule inhibitors. To validate the assay under optimized conditions, we used reference compounds and achieved results comparable to published data. © 2012 Elsevier B.V.


Thaher B.A.,Islamic University of Gaza | Arnsmann M.,University of Tübingen | Totzke F.,ProQinase GmbH | Ehlert J.E.,ProQinase GmbH | And 6 more authors.
Journal of Medicinal Chemistry | Year: 2012

In the course of searching for new p38α MAP kinase inhibitors, we found that the regioisomeric switch from 3-(4-fluorophenyl)-4-(pyridin-4-yl)-1- (aryl)-1H-pyrazol-5-amine to 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(aryl)-1H- pyrazol-5-amine led to an almost complete loss of p38α inhibition, but they showed activity against important cancer kinases. Among the tested derivatives, 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(2,4,6-trichlorophenyl)-1H- pyrazol-5-amine (6a) exhibited the best activity, with IC50 in the nanomolar range against Src, B-Raf wt, B-Raf V600E, EGFRs, and VEGFR-2, making it a good lead for novel anticancer programs. © 2011 American Chemical Society.


Goettert M.,University of Tübingen | Luik S.,University of Tübingen | Graeser R.,ProQinase GmbH | Laufer S.A.,University of Tübingen
Journal of Pharmaceutical and Biomedical Analysis | Year: 2011

The c-jun N-terminal kinase 3 (JNK3) is a promising drug target for the treatment of neurological disorders. Here we report a direct ELISA including the optimization of a nonradioactive immunosorbent JNK3 activity assay to determine inhibitory potency of small-molecule inhibitors. Based on our previous JNK3 assay and our recently optimized p38α mitogen activated protein kinase (MAPK) protocol for monitoring the phosphorylation of activating-transcription factor 2 (ATF-2), we present a rapid and straightforward alternative to conventional radioactive and indirect ELISA kinase assays. To validate the assay with the optimized assay conditions we used reference compounds and achieved well comparable IC 50 results to published data. The use of a linked monoclonal antibody increased the specificity and the sensitivity of the assay, reducing the required antibody concentration by approximately 100-fold. The novel protocol is an accurate, easy-to-handle and robust screening assay for JNK3 and the assay performance was reduced from 7.5 to 3h. © 2011 Elsevier B.V.


Goettert M.,University of Tübingen | Graeser R.,ProQinase GmbH | Laufer S.A.,University of Tübingen
Analytical Biochemistry | Year: 2010

Here we describe an optimization of a nonradioactive immunosorbent p38α mitogen-activated protein kinase (MAPK) activity assay to determine inhibitory potency of small molecule inhibitors. The assay omits the secondary antibody and, therefore, is shorter, more accurate, and easier to handle (total assay time of 3 h). This direct assay uses a new monoclonal anti-phospho-ATF-2 (Thr69/71) peroxidase-conjugated antibody, increasing specificity and eliminating problems with cross-reactivities of secondary antibodies. © 2010 Elsevier Inc.


Piersma S.R.,VU University Amsterdam | Fiedler U.,ProQinase GmbH | Span S.,VU University Amsterdam | Lingnau A.,ProQinase GmbH | And 4 more authors.
Journal of Proteome Research | Year: 2010

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed. © 2010 American Chemical Society.


Abu Ajaj K.,Tumor Biology Center | Abu Ajaj K.,Leibniz Institute for Molecular Pharmacology | Graeser R.,ProQinase GmbH | Graeser R.,Janssen Pharmaceutical | Kratz F.,Tumor Biology Center
Breast Cancer Research and Treatment | Year: 2012

The P-glycoprotein (P-gp) is a 170-kDa protein that acts as an energy dependent, transmembrane efflux pump and is encoded by the MDR1 gene. It has been shown to be responsible for multidrug resistance (MDR) in a defined subpopulation of breast cancer patients and thus represents a molecular target for circumventing MDR in this tumor indication. MDR modulators have been developed and demonstrated high selectivity for P-gp with inhibitory activities in the low nanomolar range. Although some objective responses were achieved in clinical trials, combination therapy with these MDR modulators, such as Ca 2+ antagonists caused unacceptable toxicity. Targeting P-gp inhibitors to the tumor site is a mean to increase their therapeutic index, and in this context binding of tailor-made prodrugs to circulating albumin is an established technology to reduce the toxicity and enhance the efficacy of anticancer drugs. In this study, we consequently developed an acid-sensitive albumin-binding prodrug of the P-gp inhibitor zosuquidar (LY335979) in a two-step synthesis using a maleimide hydrazone linker system established in our laboratory that first introduces acetylbenzoic acid at the HO-group of zosuquidar followed by derivatization with 6-maleimidocaproyl hydrazide to form the acid-sensitive hydrazone bond. The maleimide group enables the prodrug to bind rapidly and selectively to the cysteine-34 position of endogenous albumin after intravenous administration. HPLC analysis demonstrated rapid albumin binding of the zosuquidar prodrug as well as the quantitative release of the acetylbenzoic ester derivative of zosuquidar at pH 5.0. Subsequently, its ability to circumvent MDR was tested in two doxorubicin-resistant breast carcinoma cell lines (MCF-7/ADR and MT-3/ADR). The MDR status of these cell lines can be reversed by zosuquidar which was confirmed in a rhodamine 123 assay using fluorescence microscopy and FACS analysis. Furthermore, zosuquidar as well its acid-sensitive albumin conjugate re-sensitized cells to doxorubicin as well as to an albumin-binding prodrug of doxorubicin, i.e., the 6-maleimidocaproyl hydrazone derivative of doxorubicin, achieving IC 50 values in the same order of magnitude as the parental cell lines. Thus, a novel formulation of zosuquidar has been developed that could have the potential to improve the toxicity issues and tumor targeting properties of the original compound. © 2012 Springer Science+Business Media, LLC.


Fasol U.,Albert Ludwigs University of Freiburg | Frost A.,Albert Ludwigs University of Freiburg | Buchert M.,Albert Ludwigs University of Freiburg | Arends J.,Albert Ludwigs University of Freiburg | And 4 more authors.
Annals of Oncology | Year: 2012

Background: EndoTAG-1 (ET), a novel formulation of cationic liposomes carrying embedded paclitaxel (Taxol), shows antitumoral activity, targeting tumor endothelial cells in solid tumors. Patients with advanced metastatic cancer were evaluated investigating effects on pharmacokinetics and tumor vasculature using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and contrast-enhanced ultrasound (CEUS). Patients and methods: The pharmacokinetic (PK) profile of ET (22 mg/m 2 i.v.) was evaluated after single and repeated doses. DCE-MRI and CEUS explored hepatic metastases before, during and after the 4-week treatment cycle. Angiogenic biomarkers were assessed. Tumor response was evaluated by modified RECIST. Results: The PK profile demonstrated slight accumulation of paclitaxel after repeated doses. DCE-MRI parameters K trans and/or iAUC 60 showed a trend to decrease. Changes of blood flow-dependent parameters of DCE-MRI and CEUS were well correlated. Angiogenic biomarkers revealed no clear trend. ET was generally well tolerated; common toxic effects were fatigue and hypersensitivity reactions. Nine (9 of 18) patients had stable disease after the first treatment cycle. Four patients without disease progression continued treatment. Conclusions: This study including multiple pretreated patients with different metastatic cancer revealed individually distinctive hemodynamic alterations by DCE-MRI. The PK profiles of ET were similar as observed previously. © The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.


Wang W.-L.,Yeshiva University | Wang W.-L.,ProQinase GmbH | Shechter D.,Yeshiva University
International Journal of Developmental Biology | Year: 2016

Chromatin, primarily a complex of DNA and histone proteins, is the physiological form of the genome. Chromatin is generally repressive for transcription and other information transactions that occur on DNA. A wealth of post-translational modifications on canonical histones and histone variants encode regulatory information to recruit or repel effector proteins on chromatin, promoting and further repressing transcription and thereby form the basis of epigenetic information. During metazoan oogenesis, large quantities of histone proteins are synthesized and stored in preparation for the rapid early cell cycles of development and to elicit maternal control of chromatin assembly pathways. Oocyte and egg cell-free extracts of the frog Xenopus laevis are a compelling model system for the study of chromatin assembly and transcription, precisely because they exist in an extreme state primed for rapid chromatin assembly or for transcriptional activity. We show that chromatin assembly rates are slower in the X. laevis oocyte than in egg extracts, while conversely, only oocyte extracts transcribe template plasmids. We demonstrate that rapid chromatin assembly in egg extracts represses RNA Polymerase II dependent transcription, while pre-binding of TATA-Binding Protein (TBP) to a template plasmid promotes transcription. Our experimental evidence presented here supports a model in which chromatin assembly and transcription are in competition and that the onset of zygotic genomic activation may be in part due to stable transcriptional complex assembly. © 2016 UPV/EHU Press.

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