Discovery of a novel chemotype of tyrosine kinase inhibitors by fragment-based docking and molecular dynamics.
ABSTRACT: We have discovered a novel chemical class of inhibitors of the EphB4 tyrosine kinase by fragment-based high-throughput docking followed by explicit solvent molecular dynamics simulations for assessment of the binding mode. The synthesis of a single derivative (compound 7) of the hit identified in silico has resulted in an improvement of the inhibitory potency in an enzymatic assay from 8.4 ?M to 160 nM and a ligand efficiency of 0.39 kcal/mol per non-hydrogen atom. Such remarkable improvement in affinity is due to an additional hydroxyl group involved in two favorable (buried) hydrogen bonds as predicted by molecular dynamics and validated by the crystal structure of the complex with EphA3 solved at 1.7 Å resolution.
Project description:We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I1/2 (DFG in) and type II (DFG out). The three structures with type I1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases.
Project description:The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.
Project description:Many protein-protein interactions are mediated by a short linear motif. Usually, amino acid sequences of those motifs are known or can be predicted. It is much harder to experimentally characterize or predict their structure in the bound form. In this work, we test a possibility of using flexible docking of a short linear motif to predict the interaction interface of the EphB4-EphrinB2 complex (a system extensively studied for its significance in tumor progression).In the modeling, we only use knowledge about the motif sequence and experimental structures of EphB4-EphrinB2 complex partners. The proposed protocol enables efficient modeling of significant conformational changes in the short linear motif fragment during molecular docking simulation. For the docking simulations, we use the CABS-dock method for docking fully flexible peptides to flexible protein receptors (available as a server at http://biocomp.chem.uw.edu.pl/CABSdock/ ). Based on the docking result, the protein-protein complex is reconstructed and refined.Using this novel protocol, we obtained an accurate EphB4-EphrinB2 interaction model.The results show that the CABS-dock method may be useful as the primary docking tool in specific protein-protein docking cases similar to EphB4-EphrinB2 complex-that is, where a short linear motif fragment can be identified.
Project description:Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.
Project description:Specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis. Carriers do not exhibit increased cardiovascular disease risk despite reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of L75P and L174S patients presents a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.
Project description:Mammalian type B (mitochondrial) b(5) cytochromes exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-strand ?-sheet, which can be attributed to fully buried residue 21 in strand ?4. The greater bulk of Leu21 in hCYB5B in comparison to that of Thr21 in rCYB5B results in a substantial displacement of the first two residues in ?5, and consequent loss of two of the three hydrogen bonds between ?5 and ?4. Hydrogen bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between ?4 and ?5. When the buried water molecules were removed prior to a second 10 ns simulation, ?4 and ?5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of access of water to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic, and redox properties. The results provide new insight into the factors stabilizing the cytochrome b(5) fold.
Project description:The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using state-of-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.
Project description:Cancer genome sequencing efforts recently identified EPHA3, which encodes the EPHA3 receptor tyrosine kinase, as one of the most frequently mutated genes in lung cancer. Although receptor tyrosine kinase mutations often drive oncogenic conversion and tumorigenesis, the oncogenic potential of the EPHA3 mutations in lung cancer remains unknown.We used immunoprecipitation, western blotting, and kinase assays to determine the activity and signaling of mutant EPHA3 receptors. A mutation-associated gene signature was generated from one large dataset, mapped to another training dataset with survival information, and tested in a third independent dataset. EPHA3 expression levels were determined by quantitative reverse transcription-polymerase chain reaction in paired normal-tumor clinical specimens and by immunohistochemistry in human lung cancer tissue microarrays. We assessed tumor growth in vivo using A549 and H1299 human lung carcinoma cell xenografts in mice (n = 7-8 mice per group). Tumor cell proliferation was measured by bromodeoxyuridine incorporation and apoptosis by multiple assays. All P values are from two-sided tests.At least two cancer-associated EPHA3 somatic mutations functioned as dominant inhibitors of the normal (wild type) EPHA3 protein. An EPHA3 mutation-associated gene signature that was associated with poor patient survival was identified. Moreover, EPHA3 gene copy numbers and/or expression levels were decreased in tumors from large cohorts of patients with lung cancer (eg, the gene was deleted in 157 of 371 [42%] primary lung adenocarcinomas). Reexpression of wild-type EPHA3 in human lung cancer lines increased apoptosis by suppression of AKT activation in vitro and inhibited the growth of tumor xenografts (eg, for H1299 cells, mean tumor volume with wild-type EPHA3 = 437.4 mm(3) vs control = 774.7 mm(3), P < .001). Tumor-suppressive effects of wild-type EPHA3 could be overridden in trans by dominant negative EPHA3 somatic mutations discovered in patients with lung cancer.Cancer-associated EPHA3 mutations attenuate the tumor-suppressive effects of normal EPHA3 in lung cancer.
Project description:This study investigates the role of ephrin receptor A3 (EphA3) in the angiogenesis of Multiple Myeloma (MM) and the effects of a selective target of EphA3 by a specific monoclonal antibody on primary bone marrow endothelial cells (ECs) of MM patients.EphA3 mRNA and protein were evaluated in ECs of MM patients (MMECs), in ECs of patients with monoclonal gammopathies of undetermined significance (MGECs) and in ECs of healthy subjects (control ECs). The effects of EphA3 targeting by mRNA silencing (siRNA) or by the anti EphA3 antibody on the angiogenesis were evaluated. We found that EphA3 is highly expressed in MMECs compared to the other EC types. Loss of function of EphA3 by siRNA significantly inhibited the ability of MMECs to adhere to fibronectin, to migrate and to form tube like structures in vitro, without affecting cell proliferation or viability. In addition, gene expression profiling showed that knockdown of EphA3 down modulated some molecules that regulate adhesion, migration and invasion processes. Interestingly, EphA3 targeting by an anti EphA3 antibody reduced all the MMEC angiogenesis-related functions in vitro. In conclusion, our findings suggest that EphA3 plays an important role in MM angiogenesis.
Project description:Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo.