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1.
Int J Cancer ; 145(5): 1346-1357, 2019 09 01.
Article in English | MEDLINE | ID: mdl-30807645

ABSTRACT

Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan-FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR-addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR-amplified cell lines suggests that the anti-proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line- and patient-derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756).


Subject(s)
Breast Neoplasms/drug therapy , Neoplasms/drug therapy , Piperazines/pharmacology , Pyrroles/pharmacology , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Thiophenes/pharmacology , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Drug Screening Assays, Antitumor , Female , Human Umbilical Vein Endothelial Cells , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms/metabolism , Phosphorylation/drug effects , Random Allocation , Rats , Xenograft Model Antitumor Assays
2.
Proc Natl Acad Sci U S A ; 116(7): 2551-2560, 2019 02 12.
Article in English | MEDLINE | ID: mdl-30683722

ABSTRACT

Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C-SOS1, SOS1, and SOS2. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS-SOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations.


Subject(s)
Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , SOS1 Protein/antagonists & inhibitors , Cell Line , Crystallography, X-Ray , Drug Discovery , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Humans , Protein Binding , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , SOS1 Protein/chemistry , SOS1 Protein/metabolism , Signal Transduction
3.
J Med Chem ; 62(2): 928-940, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30563338

ABSTRACT

The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein, we report the identification of a highly potent and selective ERK5 inhibitor BAY-885 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction, and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis, and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology.


Subject(s)
Mitogen-Activated Protein Kinase 7/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Pyrimidines/chemistry , Apoptosis/drug effects , Binding Sites , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Evaluation, Preclinical , Half-Life , Humans , Mitogen-Activated Protein Kinase 7/metabolism , Molecular Docking Simulation , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Pyridines/metabolism , Pyridines/pharmacology , Pyrimidines/metabolism , Pyrimidines/pharmacology , Signal Transduction/drug effects , Structure-Activity Relationship , Transcription, Genetic/drug effects
4.
J Nucl Med ; 53(11): 1794-801, 2012 Nov.
Article in English | MEDLINE | ID: mdl-23008501

ABSTRACT

UNLABELLED: Imaging of amyloid-ß (Aß) plaques by PET is more and more integrated into concepts for Alzheimer disease (AD) diagnosis and drug development. The objective of this study was to find novel chemical entities that can be transformed into (18)F-labeled Aß tracers with favorable brain washout kinetics and low background signal. METHODS: High-throughput screening of a large chemical library was used to identify new ligands for fibrillar aggregates of Aß(1-42) peptide. Thirty-two fluorinated derivatives were synthesized and tested for their affinity toward AD brain homogenate. Twelve ligands have been radiolabeled with (18)F. The pharmacokinetic properties of the radioligands were investigated in mouse and monkey biodistribution studies. Binding characteristics were determined by autoradiography of AD brain sections in vitro and using amyloid precursor protein transgenic mice in vivo. RESULTS: The systematic search for Aß imaging agents revealed several fluorinated derivatives with nanomolar affinity for Aß. The fluoropyridyl derivative BAY 1008472 showed a high initial brain uptake (6.45 percentage injected dose per gram at 2 min) and rapid brain washout (ratio of percentage of injected dose per gram of tissue at 2 and 30 min after injection, 9.2) in mice. PET studies of healthy rhesus monkeys confirmed the high initial brain uptake of BAY 1008472 (2.52 standardized uptake value at peak) and a fast elimination of total radioactivity from gray and white matter areas (ratio of standardized uptake value at peak uptake and 60 min 11.0). In autoradiographic analysis, BAY 1008472 selectively detected Aß deposits in human AD brain sections with high contrast and did not bind to τ- or α-synuclein pathologies. Finally, ex vivo autoradiography of brain sections from amyloid precursor protein-transgenic mice confirmed that BAY 1008472 is indeed suitable for the in vivo detection of Aß plaques. CONCLUSION: A new chemical class of Aß tracers has been identified by high-throughput screening. The fluoropyridyl derivative BAY 1008472 shows a favorable preclinical profile including low background binding in gray and white matter. These properties might qualify this new tracer, in particular, to detect subtle amounts or changes of Aß burden in presymptomatic AD and during therapy.


Subject(s)
Amyloid beta-Peptides/metabolism , Fluorine Radioisotopes , Positron-Emission Tomography/methods , Animals , Female , Frontal Lobe/diagnostic imaging , Frontal Lobe/metabolism , Half-Life , Humans , Macaca mulatta , Male , Mice , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacokinetics , Radioactive Tracers
5.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 3): 156-66, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21358046

ABSTRACT

A crystallographic fragment screen was carried out to identify starting points for the development of inhibitors of protein kinase Pim-1, a potential target for tumour therapy. All fragment hits identified via soaking in this study turned out to bind to the unusually hydrophobic pocket at the hinge region. The most potent fragments, two cinnamic acid derivatives (with a best IC(50) of 130 µM), additionally form a well defined hydrogen bond. The balance between hydrophobic and polar interactions makes these molecules good starting points for further optimization. Pim-2 inhibitors from a recently reported high-throughput screening campaign also feature a cinnamic acid moiety. Two of these Pim-2 inhibitors were synthesized, their potencies against Pim-1 were determined and their cocrystal structures were elucidated in order to determine to what degree the binding modes identified by fragment screening are conserved in optimized inhibitors. The structures show that the cinnamic acid moieties indeed adopt the same binding mode. Fragment screening thus correctly identified binding modes which are maintained when fragments are grown into larger and higher affinity inhibitors. The high-throughput screening-derived compound (E)-3-{3-[6-(4-aminocyclohexylamino)-pyrazin-2-yl]phenyl}acrylic acid (compound 1) is the most potent inhibitor of the cinnamic acid series for which the three-dimensional binding mode is known (IC(50) = 17 nM, K(d) = 28 nM). The structure reveals the molecular basis for the large gain in potency between the initial fragment hit and this optimized inhibitor.


Subject(s)
Cinnamates/chemistry , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-pim-1/chemistry , Cinnamates/metabolism , Cinnamates/pharmacology , Crystallography, X-Ray , Inhibitory Concentration 50 , Ligands , Models, Molecular , Protein Binding , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/metabolism , Structure-Activity Relationship , Thermodynamics
6.
Assay Drug Dev Technol ; 6(1): 39-53, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18315499

ABSTRACT

Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay.


Subject(s)
Drug Evaluation, Preclinical/methods , GTP-Binding Protein alpha Subunits, Gq-G11/drug effects , Animals , Automation , CHO Cells , Calibration , Cell Adhesion , Cells, Cultured , Cricetinae , Cricetulus , GTP-Binding Protein alpha Subunits, Gq-G11/antagonists & inhibitors , Gene Library , Indicators and Reagents , Kinetics , Linear Models , Robotics
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