ABSTRACT
Reversible protein kinase inhibitors that bind in the ATP cleft can be classified as typeâ I or typeâ II binders. Of these, typeâ I inhibitors address the active form, whereas typeâ II inhibitors typically lock the kinase in an inactive form. At the molecular level, the conformation of the flexible activation loop holding the key DFG motif controls access to the ATP site, thereby determining an active or inactive kinase state. Accordingly, typeâ I and typeâ II kinase inhibitors bind to so-called DFG-in or DFG-out conformations, respectively. Based on our former study on highly selective platelet-derived growth factor receptorâ ß (PDGFRß) pyrazin-2-one typeâ I inhibitors, we expanded this scaffold toward the deep pocket, yielding the highly potent and effective typeâ II inhibitor 5 (4-[(4-methylpiperazin-1-yl)methyl]-N-[3-[[6-oxo-5-(3,4,5-trimethoxyphenyl)-1H-pyrazin-3-yl]methyl]phenyl]benzamide). Inâ vitro characterization, including selectivity panel data from activity-based assays (300 kinases) and affinity-based assays (97 kinases) of these PDGFRß typeâ I (1; 5-(4-hydroxy-3-methoxy-phenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazin-2-one) and II (5) inhibitors showing the same pyrazin-2-one chemotype are compared. Implications are discussed regarding the data for selectivity and efficacy of typeâ I and typeâ II ligands.
Subject(s)
Drug Design , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Binding Sites , Blotting, Western , Imatinib Mesylate/chemistry , Imatinib Mesylate/pharmacology , Molecular Structure , Protein Binding/drug effects , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazines/chemistryABSTRACT
Dabrafenib (Tafinlar) was approved in 2013 by the FDA as a selective single agent treatment for patients with BRAFV600E mutation-positive advanced melanoma. One year later, a combination of dabrafenib and trametinib was used for treatment of BRAFV600E/K mutant metastatic melanoma. In the present study, we report on hitherto not described photosensitivity of dabrafenib both in organic and aqueous media. The half-lives for dabrafenib degradation were determined. Moreover, we revealed photoinduced chemical conversion of dabrafenib to its planar fluorescent derivative dabrafenib_photo 2. This novel compound could be isolated and biologically characterized in vitro. Both enzymatic and cellular assays proved that 2 is still a potent BRAFV600E inhibitor. The intracellular formation of 2 from dabrafenib upon ultraviolet irradiation is shown. The herein presented findings should be taken in account when handling dabrafenib both in preclinical research and in clinical applications.
ABSTRACT
In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.
Subject(s)
Prodrugs , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Cell Line , Cell Proliferation/drug effects , Drug Design , Molecular Structure , Photochemical Processes , Prodrugs/radiation effects , Ultraviolet Rays , Vascular Endothelial Growth Factor Receptor-2/administration & dosageABSTRACT
In this study, we report on novel photoactivatable caged prodrugs of vemurafenib. This kinase inhibitor was the first approved drug for the personalized treatment of BRAF-mutated melanoma and showed impressive results in clinical studies. However, the occurrence of severe side effects and drug resistance illustrates the urgent need for innovative therapeutic approaches. To conquer these limitations, we implemented photoremovable protecting groups into vemurafenib. In general, this caging concept provides spatial and temporal control over the activation of molecules triggered by ultraviolet light. Thus, higher inhibitor concentrations in tumor tissues might be reached with less systemic effects. Our study describes the first development of caged vemurafenib prodrugs useful as pharmacological tools. We investigated their photochemical characteristics and photoactivation. In vitro evaluation proved the intended loss-of-function and the light-dependent recovery of efficacy in kinase and cellular assays. The reported vemurafenib photo prodrugs represent a powerful biological tool for novel pharmacological approaches in cancer research.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Melanoma/drug therapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Prodrugs/chemistry , Prodrugs/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Humans , Melanoma/genetics , Melanoma/pathology , Models, Molecular , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , VemurafenibABSTRACT
Imatinib is the first protein kinase inhibitor approved for clinical use and is a seminal drug for the concept of targeted therapy. Herein we report on the design, synthesis, photokinetic properties, and in vitro enzymatic evaluation of a photoactivatable caged prodrug of imatinib. This approach allows spatial and temporal control over the activation of imatinib triggered by ultraviolet light. The successful application of the photoactivation concept to this significant kinase inhibitor provides further evidence for the caging technique as a feasible approach in the kinase field. The presented photoactivatable imatinib prodrug will be highly useful as a pharmacological tool to study the impact of imatinib toward biological systems in greater detail.
Subject(s)
Drug Design , Imatinib Mesylate/chemistry , Prodrugs/chemistry , Protein Kinase Inhibitors/chemical synthesis , Binding Sites , Catalytic Domain , Imatinib Mesylate/chemical synthesis , Molecular Docking Simulation , Prodrugs/chemical synthesis , Protein Kinase Inhibitors/chemistry , Receptor, Platelet-Derived Growth Factor beta/chemistry , Receptor, Platelet-Derived Growth Factor beta/metabolism , Ultraviolet RaysABSTRACT
In this study we report on the hit optimization of substituted 3,5-diaryl-pyrazin-2(1H)-ones toward potent and effective platelet-derived growth factor receptor (PDGF-R) ß-inhibitors. Originally, the 3,5-diaryl-pyrazin-2-one core was derived from the marine sponge alkaloid family of hamacanthins. In our first series compound 2 was discovered as a promising hit showing strong activity against PDGF-Rß in the kinase assay (IC50 = 0.5 µM). Furthermore, 2 was shown to be selective for PDGF-Rß in a panel of 24 therapeutically relevant protein kinases. Molecular modeling studies on a PDGF-Rß homology model using prediction of water thermodynamics suggested an optimization strategy for the 3,5-diaryl-pyrazin-2-ones as DFG-in binders by using a phenolic OH function to replace a structural water molecule in the ATP binding site. Indeed, we identified compound 38 as a highly potent inhibitor with an IC50 value of 0.02 µM in a PDGF-Rß enzymatic assay also showing activity against PDGF-R dependent cancer cells.
Subject(s)
Protein Kinase Inhibitors/chemistry , Receptor, Platelet-Derived Growth Factor beta/chemistry , Small Molecule Libraries/chemistry , Thermodynamics , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Binding Sites , Binding, Competitive , Humans , Models, Chemical , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Pyrazines/chemistry , Pyrazines/metabolism , Pyrazines/pharmacology , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/metabolism , Signal Transduction/drug effects , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Water/chemistryABSTRACT
In this study, we report on pyrazin-2(1H)-ones as lead for the development of potent adenosine triphosphate (ATP) competitive protein kinase inhibitors with implications as anti-cancer drugs. Initially, we identified the pyrazin-2(1H)-one scaffold from hamacanthins (deep sea marine sponge alkaloids) by Molecular Modeling studies as core binding motif in the ATP pocket of receptor tyrosine kinases (RTK), which are validated drug targets for the treatment of various neoplastic diseases. Structure-based design studies on a human RTK member PDGFR (platelet-derived growth factor receptor) suggested a straight forward lead optimization strategy. Accordingly, we focused on a Medicinal Chemistry project to develop pyrazin-2(1H)-ones as optimized PDGFR binders. In order to reveal Structure-Activity-Relationships (SAR), we established a flexible synthetic route via microwave mediated ring closure to asymmetric 3,5-substituted pyrazin-2(1H)-ones and produced a set of novel compounds. Herein, we identified highly potent PDGFR binders with IC50 values in an enzymatic assay below µM range, and possessing significant activity against PDGFR dependent cancer cells. Thus, marine hamacanthin-derived pyrazin-2(1H)-ones showing interesting properties as lead for their further development towards potent PDGFR-inhibitors.