510 research outputs found

    Conformational transition of FGFR kinase activation revealed by site-­specific unnatural amino acid reporter and single molecule FRET

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    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo

    Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

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    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo

    Heteroreceptor complexes and their allosteric receptor-receptor interactions in the central nervous system. Focus on examples from Dopamine D2 and Serotonin 5-HT1a receptors

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    GPCR interacting proteins (specially β- arrestin) and their receptor-protein interactions are also covered but their interactions with the allosteric receptor-receptor interactions in heteroreceptor complexes remain to be elucidated. The physiological and pathological relevance of the allosteric receptor-receptor interactions in heteroreceptor complexes is emphasized and novel strategies for treatment of mental and neurological disease are introduced based on this new biological principle of integration. This work gives further experimental evidences which strongly support the current view that allosteric receptor–receptor interactions in heteroreceptor complexes appear to represent a new principle in biology making possible integration of signals already at the level of the plasma membrane. These heteroreceptor complexes and their dynamics may be part of the molecular basis of learning and memory. The receptor protomers and their allosteric receptor-receptor interactions can be disturbed in neurological and mental disorders, and in diseases of peripheral tissues like the endocrine, cardiovascular and immune systems. The dopamine (DA) neuron system most relevant for schizophrenia and Parkinson s diseases is the meso-limbic-cortical DA system inter alia densely innervating subcortical limbic regions as well as the dorsal striatum. The field of dopamine D2Rs changed significantly with the discovery of many types of D2R heteroreceptor complexes in the ventral and dorsal striatum. The results indicate that the D2R is a hub receptor (www.gpcr-hetnet.com) which interacts not only with many other GPCRs including DA isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and DA transporters. Disturbances in several of these D2R heteroreceptor complexes may contribute to the development of schizophrenia and Parkinson s diseases through changes in the balance of diverse D2R homo- and heteroreceptor complexes mediating the DA signal, especially to the ventral striato-pallidal GABA pathway. In schizophrenia, this will have consequences for the control of this pathway of the glutamate drive to the prefrontal cortex via the mediodorsal thalamic nucleus which can contribute to psychotic processes. Allosteric receptor-receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the GPCR protomers. In A2A-D2R heteroreceptor complexes allosteric A2A-D2R receptor-receptor interaction brings about a biased modulation of the D2R protomer signalling (Chapter 1). A conformational state of the D2R is induced which moves away from Gi/o signaling and instead favours b-arrestin2 mediated signalling which may be the main mechanism for its atypical antipsychotic properties especially linked to the limbic A2AR-D2R heteroreceptor complexes. Furthermore, D2R-NTS1R heterocomplexes also exist in the ventral and dorsal striatum (Chapter 2) and likely also in midbrain DA nerve cells as D2R-NTS1R autoreceptor complexes where neurotensin produces antipsychotic and propsychotic actions, respectively. D2R protomer appeared to bias the specificity of the NT orthosteric binding site towards neuromedin N vs neurotensin in the heteroreceptor complex. There is a new awareness that Receptor tyrosine kinases (RTK) and transmitter activated GPCR possess the capacity for transactivation not only via GPCR induced release of neurotrophic factors, but also during signal initiation and propagation, using shared signaling pathways or using themselves as signaling platforms via direct allosteric receptor–receptor interactions. RTK are a family of transmembrane- spanning receptors that mediate the signaling from ligands such as growth factors, like the platelet-derived growth factor (PDGF), epidermal growth factor (EGF), the brain derived neurotrophic factor (BDNF), and the fibroblast growth factor (FGF). This hypothesis on direct GPCR-RTK receptor-receptor interactions in heteroreceptor complexes was introduced by Fuxe et al 1983. They also proposed the existence of 5- HT1A-FGFR1 heteroreceptor complexes having a role in depression. The hypothesis was introduced that the neurotrophic system FGF-2/FGFR1 may be a good candidate to mediate antidepressant induced improvement in 5-HT neuronal communication and neurotrophism with regeneration of connections lost during depression. RTK transactivation in response to antidepressant drug treatment was postulated to take place via a new allosteric receptor–receptor between distinct serotonin receptor subtypes and FGFR1 in heteroreceptor complexes. The discovery of brain FGFR1-5-HT1A heteroreceptor complexes and their enhancement of neuroplasticity offers an integration of the serotonin and the neurotrophic factor hypotheses of depression at the molecular level. These heteroreceptor complexes were found in the hippocampus and midbrain raphe 5-HT nerve cells, enriched in 5-HT1A autoreceptors. Based on the triplet puzzle theory several sets of triplet homologies were identified that may be part of the receptor interface. Combined FGF-2 and 5-HT1A agonist treatment increased the formation of these heterocomplexes and the facilitatory allosteric receptor-receptor interactions within them leading to an enhancement of FGFR1 signaling (Chapter 3). This integrative phenomenon is reciprocal and RTK signaling can be placed downstream of GPCRs. Formation of such heterocomplexes involving two major classes of membrane receptors can be involved in regulating all aspects of receptor protomer function including recognition, signaling, trafficking, desensitization, and downregulation (Chapter 3). These events were associated with development of rapid antidepressant effects. These heteroreceptor complexes are a novel target for antidepressant drugs. These examples, based on solid experimental evidences, serve to illustrate that allosteric receptor-receptor interactions in GPCR heteroreceptor complexes play a significant role in receptor diversity and bias of the participating GPCR protomers.G-protein coupled receptors (GPCR)-mediated signalling is a more complicated process than described previously since every GPCR and GPCR heteromer requires a set of G protein interacting proteins (GIP) which interacts with the receptor in an orchestrated spatio-temporal fashion. Therefore, there is a high interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time, and specially, their integration in GPCR heterocomplexes of the Central Nervous System (CNS). Also, pathological protein-protein interactions in homocomplexes and heterocomplexes of Aβ, Tau, and α-Syn are at the heart of the development of conformational protein disorders. Along this work, experimental evidences are given to illustrate that GPCR interactions have relevance for neurological and mental diseases and are targets for drug development. GPCR containing heteromers and higher order heteromers through allosteric receptor- receptor interactions have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. They have become exciting new targets for neurotherapeutics in e.g. Parkinson’s disease, schizophrenia, drug addiction, anxiety and depression opening up a new field in neuropsychopharmacology. Along this work, the allosteric receptor-receptor interactions over the interfaces in A2AR-D2R, D2R-NTS1R, D2R-Sigma1R and 5-HT1A-FGFR1 heteroreceptor complexes will be explored and their biochemical, pharmacological and functional integrative implications in the CNS described. Methodologies for studies on receptor- receptor interactions are discussed including the use of FRET and BRET-based techniques in the analysis of G protein coupled receptor (GPCR) dimerization in living cells. In situ proximity ligation assay is performed to establish the existence of native heteroreceptor complexes in the CNS

    Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

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    Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan–protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan–protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan–protein–protein interactions, as well as a molecular-level understanding of those complexes

    The noni anthraquinone damnacanthal is a multi-kinase inhibitor with potent anti-angiogenic effects

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    Este es el manuscrito que fue aceptado y que finalmente se publicó en Cancer Letters con el DOI: 10.1016/j.canlet.2016.10.037The natural bioactive compound damnacanthal inhibits several tyrosine kinases. Herein, we show that -in fact- damancanthal is a multi kinase inhibitor. A docking and molecular dynamics simulation approach allows getting further insight on the inhibitory effect of damnacanthal on three different kinases: vascular endothelial growth factor receptor-2, c-Met and focal adhesion kinase. Several of the kinases targeted and inhibited by damnacanthal are involved in angiogenesis. Ex vivo and in vivo experiments clearly demonstrate that, indeed, damnacanthal is a very potent inhibitor of angiogenesis. A number of in vitro assays contribute to determine the specific effects of damnacanthal on each of the steps of the angiogenic process, including inhibition of tubulogenesis, endothelial cell proliferation, survival, migration and production of extracellular matrix remodeling enzyme. Taken altogether, these results suggest that damancanthal could have potential interest for the treatment of cancer and other angiogenesisdependent diseases.Supported by grants BIO2014-56092-R (MINECO and FEDER), P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). JAGV had the financial support of Vicerrectorado de Investigación y Transferencia (University of Málaga, Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

    Design of hFGF1 Variant(s) with Increased Stability and Enhanced Bioactivity

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    Fibroblast growth factors (FGFs) are involved in various cellular processes such as cell growth,proliferation, differentiation, migration, angiogenesis, wound healing and embryonic development. Human acidic fibroblast growth factor (hFGF1) binds non-selectively to all the four FGF-receptors and is therefore considered as a powerful mitogen with broadest specificity. However, pharmacological applications of hFGF1 are restricted due to the low thermal stability of the growth factor. hFGF1 has low thermodynamic stability under physiological temperatures which leads to impairment of cellular signaling process thereby preventing its potential mitogenic properties. hFGF1 has a heparin binding pocket at the C-terminus which comprises of positively charges residues. The interaction between the positively charged amino acids lead to electrostatic repulsions, thereby rendering instability. To overcome this instability, hFGF1 binds to the glycosaminoglycan, heparin which decreases the repulsion (s) between the positively charged residues. However, binding of heparin poses a challenge for the use of hFGF1 in wound healing. Thrombin converts fibrinogen to fibrin and works as first line of defense by blocking the loss of blood. Intriguingly, thrombin also binds to heparin. Studies on wtFGF1 have demonstrated the presence of secondary thrombin cleavage site in hFGF1. Thus, thrombin is known to cleave hFGF1 at Arg 136 and render it biologically inactive. Usually, it is considered that dependency of hFGF1 to heparin increases the plausibility of thrombin-induced degradation of the growth factor. To tackle these downfalls, I have designed and constructed several point mutations in hFGF1 to improve the thermal stability and cell proliferation ability and to subside the heparin binding affinity of the growth factor. In this dissertation, I studied single, double, triple, quadruple, and penta variants of Q54P, S61L, H107S, K126N, and R136E and examined the thermal stability, bioactivity, and heparin dependency of the protein. These studies indicate that site - directed mutagenesis in hFGF1 can impact the inherent stability of the growth factor and role of heparin in hFGF1-FGFR receptor interaction and activation

    Structural and dynamic determinants for highly selective RET kinase inhibition reveal cryptic druggability.

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    The structural and dynamic determinants for highly selective RET kinase inhibition are poorly understood. Here we demonstrate by applying an integrated structural, computational and biochemical approach that the druggability landscape of the RET active site is determined by the conformational setting of the ATP-binding (P-) loop and its coordination with the αC helix. Open and intermediate P-loop structures display additional druggable vulnerabilities within the active site that were not exploited by first generation RET inhibitors. We identify a cryptic pocket adjacent to the catalytic lysine formed by K758, L760, E768 and L772, that we name the post-lysine pocket, with higher druggability potential than the adenine-binding site and with important implications in the regulation of phospho-tyrosine kinase activity. Crystal structure and simulation data show that the binding mode of highly-selective RET kinase inhibitors LOXO-292 and BLU-667 is controlled by a synchronous open P-loop and αC-in configuration that allows accessibility to the post-lysine pocket. Molecular dynamics simulation show that these inhibitors efficiently occupy the post-lysine pocket with high stability through the simulation time-scale (300 ns), with both inhibitors forming hydrophobic contacts in the pocket further stabilized by pi-cation interactions with the catalytic K758. Engineered mutants targeting the post-lysine pocket impact on inhibitor binding and sensitivity, as well as RET tyrosine kinase activity. The identification of the post-lysine pocket as a cryptic druggable vulnerability in the RET kinase and its exploitation by second generation RET inhibitors has important implications for future drug design and the development of personalized therapies for patients with RET-driven cancers.We thank the Centro Nacional de Investigaciones Oncológicas (CNIO), which is supported by the Instituto de Salud Carlos III and recognized as a “Severo Ochoa” Centre of Excellence (ref. CEX2019-000891-S, awarded by MCIN/AEI/ 10.13039/501100011033) for core funding and supporting this study. This work was further supported by projects: BFU2017-86710-R funded by MCIN/ AEI /10.13039/501100011033 and ERDF “A way of making Europe”, PID2020-117580RB-I00 funded by MCIN/ AEI /10.13039/501100011033, RYC-2016-1938 funded by MCIN/AEI /10.13039/501100011033 and ESF “Investing in your future”, and a Marie Curie WHRI-ACADEMY International grant (number 608765) to IP-M and a CNIO-Friends predoctoral Carmen Gloria Bonnet Fellowship to MAS.N

    Structure

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    An autoinhibitory network of hydrogen bonds located at the kinase hinge (referred to as the "molecular brake") regulates the activity of several receptor tyrosine kinases. The mechanism whereby mutational disengagement of the brake allosterically activates the kinase in human disease is incompletely understood. We used a combination of NMR, bioinformatics, and molecular dynamics simulation to show that mutational disruption of the molecular brake triggers localized conformational perturbations that propagate to the active site. This entails changes in interactions of an isoleucine, one of three hydrophobic residues that lock the phenylalanine of the DFG motif in an inactive conformation. Structural analysis of tyrosine kinases provides evidence that this allosteric control mechanism is shared across the tyrosine kinase family. We also show that highly activating mutations at the brake diminish the enzyme's thermostability, thereby explaining why these mutations cause milder skeletal syndromes compared with less-activating mutations in the activation loop.R01 GM117118/GM/NIGMS NIH HHS/United StatesS10 OD016343/CD/ODCDC CDC HHS/United StatesR01 DE013686/DE/NIDCR NIH HHS/United StatesR01 GM117118/GM/NIGMS NIH HHS/United StatesR35 GM127040/GM/NIGMS NIH HHS/United StatesR01 DE013686/DE/NIDCR NIH HHS/United StatesF99 CA212474/CA/NCI NIH HHS/United StatesS10 OD016343/OD/NIH HHS/United States2020-08-06T00:00:00Z31204250PMC66875256564vault:3370

    In silico Methods for Design of Kinase Inhibitors as Anticancer Drugs

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    Rational drug design implies usage of molecular modeling techniques such as pharmacophore modeling, molecular dynamics, virtual screening, and molecular docking to explain the activity of biomolecules, define molecular determinants for interaction with the drug target, and design more efficient drug candidates. Kinases play an essential role in cell function and therefore are extensively studied targets in drug design and discovery. Kinase inhibitors are clinically very important and widely used antineoplastic drugs. In this review, computational methods used in rational drug design of kinase inhibitors are discussed and compared, considering some representative case studies

    BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

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    Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABLI360T revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.This work was supported by an investigator grant to P.V. from Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funding from the Biotechnology and Biological Sciences Research Council (BB/I023291/1 and BB/H018409/1 to AP and FF). P.B. is the recipient of an AIRC - Marie Curie fellowship
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