Computational approaches to study mechanisms of regulation and inhibition of enzymes involved in phospho-transfer reactions

Protein kinases are the enzymes in the cell that catalyze phosphorylation reactions. They are essential for almost all cellular processes and many of them are considered promising pharmaceutical targets since they are involved in a large number of tumorigenic functions such as proliferation, immune evasion, anti-apoptosis, metastasis and angiogenesis. The progress in high-resolution structure determination techniques has contributed enormously to a better understanding of the structural basis of kinase regulation and the associated structural plasticity. However, because of the high sequence and structural conservation across the kinome, new efforts are required that combine a variety of methodologies, which in particular exploit the differential dynamical behaviour of kinases. In the following doctoral thesis different computational methodologies are employed to study three topics related to phosphorylation: 1.Understanding the reaction mechanism of phosphorylation and dephosphorylation: -Using PKA and GSK3β as model kinases we perform molecular dynamics simulations and carry out hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on the evolution of the Michaelis complexes formed between these kinases and their bona fide substrates towards the respective phosphorylated products and characterize each step of the phosphorylation reactions in atomic detail paying particular attention to the roles and fates of the catalytic metal ions . -We analyse the dephosphorylation reaction catalyzed by the SHIP2 inositol phosphatase. Models of the two substrates, PI(4,5)P3 and IP4, in complex with SHIP2 phosphatase are built to understand the reaction mechanism in atomic detail . In addition, Principal Component Analysis and molecular dynamics simulations are used to study the allosteric role of the C2 domain and to propose and test different mutants with a view to confirming or rejecting our hypothesis. 2.Analysis of conformational changes involved in the activation of two prototypical kinases: -Free energy calculations using umbrella sampling and metadynamics are applied to validate the energetic profiles of the opening and closing of the activation loop in non-receptor Abelson tyrosine kinase (Abl) codificated in the protooncogene ABL1 and to characterize the differences between the phosphorylated and the unphosphorylated forms of this pharmacologically important enzyme. -Molecular dynamics simulations and normal mode analysis are performed on focal adhesion kinase (FAK), another non-receptor tyrosine kinase involved in cancer, in the presence or absence of ATP/Mg2+ in order to understand the allosteric effect of ATP on the conformational and dynamic properties of the enzyme. 3.Computational search for specific protein kinase inhibitors: -We perform extensive molecular dynamics simulations of the apo enzymes to identify transient and potentially targetable allosteric pockets. -We calculate molecular interaction fields and putative hotspots on the active site and regulatory domains of these kinases to characterize the potential ligand-binding sites. -We make use of a variety of docking tools to identify new potential hits present in chemical libraries and/or fragment databases (large-scale virtual screening)

Computational approaches to study mechanisms of regulation and inhibition of enzymes involved in phospho-transfer reactions

Protein kinases are the enzymes in the cell that catalyze phosphorylation reactions. They are essential for almost all cellular processes and many of them are considered promising pharmaceutical targets since they are involved in a large number of tumorigenic functions such as proliferation, immune evasion, anti-apoptosis, metastasis and angiogenesis. The progress in high-resolution structure determination techniques has contributed enormously to a better understanding of the structural basis of kinase regulation and the associated structural plasticity. However, because of the high sequence and structural conservation across the kinome, new efforts are required that combine a variety of methodologies, which in particular exploit the differential dynamical behaviour of kinases. In the following doctoral thesis different computational methodologies are employed to study three topics related to phosphorylation: 1.Understanding the reaction mechanism of phosphorylation and dephosphorylation: -Using PKA and GSK3β as model kinases we perform molecular dynamics simulations and carry out hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on the evolution of the Michaelis complexes formed between these kinases and their bona fide substrates towards the respective phosphorylated products and characterize each step of the phosphorylation reactions in atomic detail paying particular attention to the roles and fates of the catalytic metal ions . -We analyse the dephosphorylation reaction catalyzed by the SHIP2 inositol phosphatase. Models of the two substrates, PI(4,5)P3 and IP4, in complex with SHIP2 phosphatase are built to understand the reaction mechanism in atomic detail . In addition, Principal Component Analysis and molecular dynamics simulations are used to study the allosteric role of the C2 domain and to propose and test different mutants with a view to confirming or rejecting our hypothesis. 2.Analysis of conformational changes involved in the activation of two prototypical kinases: -Free energy calculations using umbrella sampling and metadynamics are applied to validate the energetic profiles of the opening and closing of the activation loop in non-receptor Abelson tyrosine kinase (Abl) codificated in the protooncogene ABL1 and to characterize the differences between the phosphorylated and the unphosphorylated forms of this pharmacologically important enzyme. -Molecular dynamics simulations and normal mode analysis are performed on focal adhesion kinase (FAK), another non-receptor tyrosine kinase involved in cancer, in the presence or absence of ATP/Mg2+ in order to understand the allosteric effect of ATP on the conformational and dynamic properties of the enzyme. 3.Computational search for specific protein kinase inhibitors: -We perform extensive molecular dynamics simulations of the apo enzymes to identify transient and potentially targetable allosteric pockets. -We calculate molecular interaction fields and putative hotspots on the active site and regulatory domains of these kinases to characterize the potential ligand-binding sites. -We make use of a variety of docking tools to identify new potential hits present in chemical libraries and/or fragment databases (large-scale virtual screening)

Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques

Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.We thank Jose´ Terro´ n Bautista for help with MD analysis. We thank the ESRF and ALBA for provid- ing the synchrotron-radiation facilities and the staff for their assistance in the data collection. We are grateful to the Barcelona Supercomputing Centre and National Supercomputing Centre (BSC-CNS) for allocating computer time to run the reported simulations. The work was supported by the Span- ish Ministry of Economy, Industry and Competitiveness (MEIC) Grants BFU2010-15923 (DL) and MEIC Project Retos BFU2016-77665-R co-funded by the European Regional Development Fund (ERDF) (DL), the Comunidad Auto´ noma de Madrid Grant S2010/BMD-2457 (DL), and by the National Cancer Research Centre. DL is also a recipient of awards from the Volkswagen Foundation (Az: 86 416–1) and Worldwide Cancer Research (15-1177).S

Stepwise Simulation of 3,5-Dihydro-5-methylidene‑4<i>H-</i>imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

A 3,5-dihydro-5-methylidene-4<i>H-</i>imidazol-4-one (MIO) electrophilic moiety is post-translationally and autocatalytically generated in homotetrameric histidine ammonia-lyase (HAL) and other enzymes containing the tripeptide Ala-Ser-Gly in a suitably positioned loop. The backbone cyclization step is identical to that taking place during fluorophore formation in green fluorescent protein from the tripeptide Ser-Tyr-Gly, but dehydration, rather than dehydrogenation by molecular oxygen, is the reaction that gives rise to the mature MIO ring system. To gain additional insight into this unique process and shed light on some still unresolved issues, we have made use of extensive molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations implementing the self-consistent charge density functional tight-binding method on a fully solvated tetramer of <i>Pseudomonas putida</i> HAL. Our results strongly support the idea that mechanical compression of the reacting loop by neighboring protein residues in the precursor state is absolutely required to prevent formation of inhibitory main-chain hydrogen bonds and to enforce proper alignment of donor and acceptor orbitals for bond creation. The consideration of the protein environment in our computations shows that water molecules, which have been mostly neglected in previous theoretical work, play a highly relevant role in the reaction mechanism and, more importantly, that backbone cyclization resulting from the nucleophilic attack of the Gly amide lone pair on the π* orbital of the Ala carbonyl precedes side-chain dehydration of the central serine

Stepwise Simulation of 3,5-Dihydro-5-methylidene‑4<i>H-</i>imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

A 3,5-dihydro-5-methylidene-4<i>H-</i>imidazol-4-one (MIO) electrophilic moiety is post-translationally and autocatalytically generated in homotetrameric histidine ammonia-lyase (HAL) and other enzymes containing the tripeptide Ala-Ser-Gly in a suitably positioned loop. The backbone cyclization step is identical to that taking place during fluorophore formation in green fluorescent protein from the tripeptide Ser-Tyr-Gly, but dehydration, rather than dehydrogenation by molecular oxygen, is the reaction that gives rise to the mature MIO ring system. To gain additional insight into this unique process and shed light on some still unresolved issues, we have made use of extensive molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations implementing the self-consistent charge density functional tight-binding method on a fully solvated tetramer of <i>Pseudomonas putida</i> HAL. Our results strongly support the idea that mechanical compression of the reacting loop by neighboring protein residues in the precursor state is absolutely required to prevent formation of inhibitory main-chain hydrogen bonds and to enforce proper alignment of donor and acceptor orbitals for bond creation. The consideration of the protein environment in our computations shows that water molecules, which have been mostly neglected in previous theoretical work, play a highly relevant role in the reaction mechanism and, more importantly, that backbone cyclization resulting from the nucleophilic attack of the Gly amide lone pair on the π* orbital of the Ala carbonyl precedes side-chain dehydration of the central serine

Design, Synthesis, and Evaluation of Novel Imidazo[1,2‑<i>a</i>][1,3,5]triazines and Their Derivatives as Focal Adhesion Kinase Inhibitors with Antitumor Activity

A series of triazinic inhibitors of focal adhesion kinase (FAK) have been recently shown to exert antiangiogenic activity against HUVEC cells and anticancer efficacy against several cancer cell lines. We report herein that we further explored the heterocyclic core of these inhibitors by a fused imidazole ring with the triazine to provide imidazo­[1,2-<i>a</i>]­[1,3,5]­triazines. Importantly, these new compounds displayed 10^–7–10^–8 M IC₅₀ values, and the best inhibitor showed IC₅₀ value of 50 nM against FAK enzymatic activity. Several inhibitors potently inhibited the proliferation of a panel of cancer cell lines expressing high levels of FAK. Apoptosis analysis in U87-MG and HCT-116 cell lines suggested that these compounds delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further investigation demonstrated that these compounds strongly inhibited cell-matrix adhesion, migration, and invasion of U87-MG cells