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)

Hit validation of ERK5 inhibitors: Expectations and challenges

Extracellular signal-regulated kinase 5 (ERK5) plays a key role in the transduction of extracellular signals to intracellular effectors. Activation of the ERK5 signalling pathway is associated with cell survival and proliferation, and thus ERK5 over-expression has implications in carcinogenesis. Inhibiting ERK5 is therefore an effective approach for anti-cancer therapy. Following a high throughput screening campaign, three chemical series (1 -3) were selected for validation. The syntheses and biological evaluation of novel benzo[d]thiazoles (1), 4-aminopyrimidine-5-carbonitriles (2) and 3 cyanopyridines (3) will be discussed

Carbohydrate-Active Enzymes

Carbohydrate-active enzymes are responsible for both biosynthesis and the breakdown of carbohydrates and glycoconjugates. They are involved in many metabolic pathways; in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose and lignin; and in the glycosylation of proteins and lipids. Carbohydrate-active enzymes are classified into glycoside hydrolases, glycosyltransferases, polysaccharide lyases, carbohydrate esterases, and enzymes with auxiliary activities (CAZy database, www.cazy.org). Glycosyltransferases synthesize a huge variety of complex carbohydrates with different degrees of polymerization, moieties and branching. On the other hand, complex carbohydrate breakdown is carried out by glycoside hydrolases, polysaccharide lyases and carbohydrate esterases. Their interesting reactions have attracted the attention of researchers across scientific fields, ranging from basic research to biotechnology. Interest in carbohydrate-active enzymes is due not only to their ability to build and degrade biopolymers—which is highly relevant in biotechnology—but also because they are involved in bacterial biofilm formation, and in glycosylation of proteins and lipids, with important health implications. This book gathers new research results and reviews to broaden our understanding of carbohydrate-active enzymes, their mutants and their reaction products at the molecular level

Modulation of the Mitogen Activated Protein Kinase Pathway Spatiotemporal Signalling Components: Influence on Pathway Activation Behaviour Using an Agent Based Model

The subtleties of how the Mitogen Activated Protein Kinase works (MAPK) biochemical signalling pathway works, its emergent oscillatory behaviour and sensitivity is explained though an analysis of a computational agent based model that takes into account the distribution of the MAPK cascade components into multiple compartment

Characterisation and structural biology of protein arginine methyltransferases

PhD ThesisPost-translational and epigenetic modifications of proteins and nucleic acids are known to play major roles in influencing cell fate. Enzymes that catalyse modifications such as phosphorylation, acetylation and methylation have been identified as promising drug targets. Protein methyltransferase 2 (PRMT2) and Coactivator-associated arginine methyltransferase 1 (CARM1) belong to the class of Type 1 PRMTs which catalyse the asymmetric dimethylation of substrate arginine residues. CARM1 has been shown to be overexpressed in different cancer types including breast and prostate cancer. PRMT2 has been identified as a potential target for oncology with reported links to androgen receptor signalling, NF-κB signalling and induction of apoptosis. However, selective chemical probes that could be used as tools for target validation and which could potentially be a starting point for drug discovery are still missing. The work presented here aims to identify selective CARM1 and PRMT2 inhibitors that target the cofactor- and substrate-binding sites. Crystal structures of mouse PRMT2 in the apo-state and in complex with Sinefungin are presented. Crystal structures of the catalytic domain of CARM1 in complex with the cofactor S-adenosyl Lhomocysteine (SAH) and different small molecule inhibitors were also determined. Surface plasmon resonance was used to characterise inhibitor binding to CARM1 and identify structure-activity relationships. To further map the CARM1 active site, ligand soaks of CARM1 with a library of small fragments called FragLites were performed. These small fragments can more readily find potential binding pockets than larger more drug-like inhibitors. A direct and label-free mass spectrometry-based assay was developed to measure CARM1 activity and its inhibition. Together these findings can be used to further develop inhibitors that target the PRMT family. These inhibitors will be useful tools to investigate the biology of PRMT2 and CARM1 and to understand their biological role in cancer

Marine Compounds and Cancer 2020

The very first marine-derived anticancer drug, Cytarabine (aka Ara-C, Cytosar-U®), was approved by the FDA in 1969 for the treatment of leukemia. At the beginning of 2021, the list of approved marine-derived anticancer drugs consists of nine substances, five of which received approval within the last two years, demonstrating the rapid evolution of the field. The current book is a collection of scientific articles related to the exponentially growing field of anticancer marine compounds. These articles cover the whole field, from agents with cancer-preventive activity, to novel and previously characterized compounds with anticancer activity, both in vitro and in vivo, as well as the latest status of compounds under clinical development

The Tumor Microenvironment of High Grade Serous Ovarian Cancer

The Special Issue on high grade serous ovarian cancer (HGSOC) and the contribution of the tumor microenviroment (TME) consists of reviews contributed by leaders in the OC field. As HGSOC metastases have a highly complex TME, there is an urgent need to better understand the TME in general, its distinct components in particular, and the role of the TME in the context of disease recurrence and development of chemoresistance. The Special Issue incorporates the current understanding of the different parts of thd TME components, including the cancer cells themselves, the cells surrounding the cancer cells or stromal cells, and the cells of the immune system, which are attracted to the site of metastases. In addition to these cells of the TME, the role of various cellular factors made by the cells of the TME are also the subject of the reviews. In addition, reviews in this Special Issue cover the complex relationships between the molecular mechanisms of HGSOC progression, including genomic, epigenomic and transcriptomic changes and changes in the immune cell landscape, as these may provide attractive new molecular targets for HGSOC therapy

Effect of rare and common single amino acid substitutions on DISC1 subcellular targeting and functional interaction with ATF4

DISC1, a strong genetic candidate for psychiatric illness, is a molecular scaffold residing in multiple subcellular compartments, where it regulates the function of interacting proteins with key roles in neurodevelopment and plasticity. Both common and rare DISC1 missense variants are associated with risk of mental illness and/or brain abnormalities in healthy carriers, but the underlying mechanisms are unclear. In this thesis, I initially examine the effect of a panel of common and rare single amino acid substitutions on DISC1 subcellular targeting, establishing that the rare mutation R37W and the common variant L607F disrupt DISC1 nuclear targeting in a dominant-negative fashion. This finding predicts that DISC1 nuclear expression is severely impaired in 37W and 607F carriers. In addition, I show that the L607F substitution results in aberrant cytoplasmic and cytoskeletal distribution of DISC1. In the nucleus, DISC1 interacts with the transcription factor ATF4, which is involved in the regulation of cellular stress responses and memory consolidation. Here I show that at basal cAMP levels, wild-type DISC1 strongly inhibits the transcriptional activity of ATF4, and this effect is ablated by 37W and 607F, most likely as a consequence of their defective nuclear targeting. 607F additionally reduces DISC1/ATF4 interaction, which likely contributes to its weakened inhibitory effect. I also demonstrate that DISC1 modulates transcriptional responses to endoplasmic reticulum stress, and that this modulatory effect is also ablated by 37W and 607F. By providing evidence that single amino acid substitutions of DISC1 associated with psychiatric illness impair its regulatory function on ATF4-dependent transcription, I highlight a potential mechanism by which these protein variants may impact on molecular pathways underlying cognition and stress responses, two processes of direct relevance to psychiatric disease