| 1. |
- Andér, Martin, 1979-
(författare)
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Computational Analysis of Molecular Recognition Involving the Ribosome and a Voltage Gated K+ Channel
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel. The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center. Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified. The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex. A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted. Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.
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| 2. |
- Jespers, Willem
(författare)
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Free energy calculations of G protein-coupled receptor modulation : New methods and applications
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- G protein-coupled receptors (GPCRs) are membrane proteins that transduce the signals of extracellular ligands, such as hormones, neurotransmitters and metabolites, through an intracellular response via G proteins. They are abundant in human physiology and approximately 34% of the marketed drugs target a GPCR. Upon activation, the receptor undergoes conformational changes to accommodate the binding of the G protein. Our insights in the structural determinants of ligand binding and receptor activation have increased tremendously over the past decade. This has largely been a cause of the growing amount of experimentally determined structures, which provide crucial insights in ligand binding mechanisms. However, in a typical drug design project it is unlikely that such structures can be generated for all ligands. In those cases, computationally derived models of the protein-ligand complex can be generated. Rigorous free energy calculations such as the free energy perturbation (FEP) method, can subsequently provide the missing link between those structures and experimental ligand binding data, and provide further insights in the binding mechanism.In this thesis, two workflows are presented to calculate free energies of binding for ligands to wildtype (QligFEP) and mutant (QresFEP) receptors. Both methods were tested on a set of solvation free energies of sidechain mimics. QligFEP was furthermore applied on three protein-ligand binding datasets, including pair comparisons between topologically unrelated molecules (scaffold hopping). QresFEP was used to calculate protein-ligand binding affinities to mutants of the neuropeptide Y1, and to predicte the effect of receptor modifications on the thermal stability of T4 lysozyme.The remainder of this work focussed on the application of these protocols in the design, synthesis and molecular pharmacology of ligands for the family of adenosine receptor (ARs). These receptors, involved in many physiological processes such as promotion of sleep (caffeine is a well-known inhibitor), have recently been pursued as drug targets in immuno-oncology. QligFEP was used in the design of novel series of antagonists for the A3AR and A2BAR. QresFEP was used to study ligand binding to the A1AR and in a multidisciplinary approach to characterize binding to the orphan receptor GPR139. Both approaches were combined to design a series of A2AAR antagonist, and to propose a binding mode later confirmed by new crystal structures. Finally, a new application of FEP is introduced based on conformational equilibria between the active and inactive A2AAR, to elucidate the regulation mechanism of receptor activation by ligands and receptor mutations.
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| 3. |
- Lind, Christoffer
(författare)
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Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ribosome is a macromolecular machine that produces proteins in all kingdoms of life. The proteins, in turn, control the biochemical processes within the cell. It is thus of extreme importance that the machine that makes the proteins works with high precision. By using three dimensional structures of the ribosome and homology modelling, we have applied molecular dynamics simulations and free-energy calculations to study the codon specificity of protein synthesis in initiation and termination on an atomistic level. In addition, we have examined the binding of small molecules to riboswitches, which can change the expression of an mRNA.The relative affinities on the ribosome between the eukaryotic initiator tRNA to the AUG start codon and six near-cognate codons were determined. The free-energy calculations show that the initiator tRNA has a strong preference for the start codon, but requires assistance from initiation factors 1 and 1A to uphold discrimination against near-cognate codons.When instead a stop codon (UAA, UGA or UAG) is positioned in the ribosomal A-site, a release factor binds and terminates protein synthesis by hydrolyzing the nascent peptide chain. However, vertebrate mitochondria have been thought to have four stop codons, namely AGA and AGG in addition to the standard UAA and UAG codons. Furthermore, two release factors have been identified, mtRF1 and mtRF1a. Free-energy calculations were used to determine if any of these two factors could bind to the two non-standard stop codons, and thereby terminate protein synthesis. Our calculations showed that the mtRF’s have similar stop codon specificity as bacterial RF1 and that it is highly unlikely that the mtRF’s are responsible for terminating at the AGA and AGG stop codons.The eukaryotic release factor 1, eRF1, on the other hand, can read all three stop codons singlehandedly. We show that eRF1 exerts a high discrimination against near-cognate codons, while having little preference for the different cognate stop codons. We also found an energetic mechanism for avoiding misreading of the UGG codon and could identify a conserved cluster of hydrophobic amino acids which prevents excessive solvent molecules to enter the codon binding site.The linear interaction energy method was used to examine binding of small molecules to the purine riboswitch and the FEP method was employed to explicitly calculate the LIE b-parameters. We show that the purine riboswitches have a remarkably high degree of electrostatic preorganization for their cognate ligands which is fundamental for discriminating against different purine analogs.
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| 4. |
- Shamsudin Khan, Yasmin
(författare)
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Non-Steroidal Anti-Inflammatory Drugs in Cyclooxygenases 1 and 2 : Binding modes and mechanisms from computational methods and free energy calculations
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used classes of drugs. They target the cyclooxygenases (COX) 1 and 2 to reduce the physiological responses of pain, fever, and inflammation. Due to their role in inducing angiogenesis, COX proteins have also been identified as targets in cancer therapies.In this thesis, I describe computational protocols of molecular docking, molecular dynamics simulations and free energy calculations. These methods were used in this thesis to determine structure-activity relationships of a diverse set of NSAIDs in binding to their target proteins COX-1 and 2. Binding affinities were calculated and used to predict the binding modes. Based on combinations of molecular dynamics simulations and free energy calculations, binding mechanisms of sub-classes of NSAIDs were also proposed. Two stable conformations of COX were probed to understand how they affect inhibitor affinities. Finally, a brief discussion on selectivity towards either COX isoform is discussed. These results will be useful in future de novo design and testing of third-generation NSAIDs.
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| 5. |
- van der Ent, Florian
(författare)
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Computational Studies of the Temperature Dependence of Enzyme Catalysis
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are known to adapt to low temperatures by lowering the enthalpy of activation of the key reaction steps. A lesser known property of some psychrophilic (cold-active) enzymes is an anomalous temperature optimum. For most enzymes the temperature optimum is determined by their denaturation temperature, but anomalous optima can occur well below the denaturation temperatures and there must thus be an alternative mechanism for their inactivation. A proposed mechanism examined in this thesis posits that there is a small conformational change or local unfolding which renders the enzyme inactive above its temperature optimum.In paper I this hypothesized mechanism of inactivation was used to guide the design of variants of a bacterial α-amylase from Pseudoalteromonas haloplanktis. This enzyme exhibits an anomalous optimum and it has been suggested that the breaking of a specific interaction is responsible for its inactivity at high temperatures. Using computational methods several designs were evaluated. When experimentally tested the best performing design raised the temperature optimum by about 6 °C. This finding demonstrates the validity of the assumed mechanism and the utility of computational modeling in enzyme design.In Paper II and III two closely related bacterial lipases were investigated, namely Lipase A from Bacillus subtilis and a psychrophilic homologue found in Bacillus pumulus. By combining computational methods and experiments, it was found that a single mutation site is responsible for the difference in activity and the mechanism of its effect on the catalysis was determined. The lipase found in Bacillus pumulus also exhibits an anomalous temperature optimum. Our preliminary findings regarding the mechanism of this optima are also presented.In the last publication, paper IV, computation of heat capacities of biomolecular systems from molecular dynamics simulations is investigated. A non-zero heat capacity of activation has been suggested to be responsible for curved Arrhenius plots in some enzymes. To further investigate these ideas, clear and robust methods for the evaluation of heat capacity must be employed. Our results show that a method relying on mean potential energies calculated at various temperatures converges quicker then alternative methods and is less sensitive to the choice of thermostat and other simulation parameters.
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| 6. |
- Vasile, Silvana, 1985-
(författare)
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Computational prediction of ligand binding in peptide G-protein coupled receptors
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- G-protein coupled receptors (GPCRs) are a superfamily of membrane receptors involved in a wide variety of biological processes, and their malfunction is associated with many diseases. Consequently, GPCRs are targeted by one-third of the drugs on the market, and constitute the focus of active public and private research in the search of more effective drugs. The GPCR families that are activated by endogenous peptides are particularly challenging for the drug design process, which in this case contemplates peptides, peptidomimetics and small molecules, as selective activators (agonists) or blockers (antagonists) of the particular receptor subtype of interest. This process benefits of a detailed understanding of how known ligands bind to the receptors. Homology modelling, molecular dynamics (MD) and free energy perturbation (FEP) are computational methods used to predict binding modes and binding affinities. In this thesis, these techniques are applied (and even further developed) in combination with novel experimental data provided by our collaborators, in order to elucidate the molecular determinants of endogenous peptide ligands, analogues and mimetics to two families of peptide-binding receptors: the neuropeptide Y (NPY) and the Angiotensin II receptors.The NPY signaling system is responsible for the regulation of food intake and its malfunction is connected to obesity, a risk factor for diseases such as diabetes and cancer. In this thesis, we focused on the elucidation of the binding mode of endogenous peptide ligands and studied the structural effect of receptor mutants, with the aim of helping in future drug design on the Y2 receptor subtype, as well as understanding the effect of receptor polymorphisms on the Y4 subtype. We further used this system to refine and test our computational protocol for the prediction of binding free energies, by characterizing the binding mode of a peptidomimetic antagonist to the Y1 receptor.The AT2 receptor is another interesting drug target, as its activation by the Angiotensin II peptide elicits responses that counterbalance the hypertensive effects caused by activation of the AT1 receptor by the same ligand. Moreover, AT2 is upregulated in events of tissue damage. We characterized the chemical evolution of peptide and peptidomimetic agonists at this receptor, with the aim to identify a set of pharmacophoric points and key interactions with AT2. The outcome of this study allowed the establishment of a clear explanation of structure-activity relationships, and will be the starting point for further ligand-design efforts at this receptor.
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| 7. |
- Almlöf, Martin, 1977-
(författare)
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Computational Methods for Calculation of Ligand-Receptor Binding Affinities Involving Protein and Nucleic Acid Complexes
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ability to accurately predict binding free energies from computer simulations is an invaluable resource in understanding biochemical processes and drug action. Several methods based on microscopic molecular dynamics simulations exist, and in this thesis the validation, application, and development of the linear interaction energy (LIE) method is presented.For a test case of several hydrophobic ligands binding to P450cam it is found that the LIE parameters do not change when simulations are performed with three different force fields. The nonpolar contribution to binding of these ligands is best reproduced with a constant offset and a previously determined scaling of the van der Waals interactions.A new methodology for prediction of binding free energies of protein-protein complexes is investigated and found to give excellent agreement with experimental results. In order to reproduce the nonpolar contribution to binding, a different scaling of the van der Waals interactions is neccesary (compared to small ligand binding) and found to be, in part, due to an electrostatic preorganization effect not present when binding small ligands.A new treatment of the electrostatic contribution to binding is also proposed. In this new scheme, the chemical makeup of the ligand determines the scaling of the electrostatic ligand interaction energies. These scaling factors are calibrated using the electrostatic contribution to hydration free energies and proposed to be applicable to ligand binding.The issue of codon-anticodon recognition on the ribosome is adressed using LIE. The calculated binding free energies are in excellent agreement with experimental results, and further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with a ribosome loaded with the Phe UUU codon. The simulations also support the previously suggested roles of A1492, A1493, and G530 in the codon-anticodon recognition process.
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| 8. |
- Carlsson, Jens, 1979-
(författare)
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Challenges in Computational Biochemistry: Solvation and Ligand Binding
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.The thermodynamic integration technique is used to calculate pKa values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated pKa values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine pKa shifts in wild-type and mutant epoxide hydrolase. The calculated pKa values support a model that can explain some of the pH dependent properties of this enzyme. Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.
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| 9. |
- Keränen, Henrik, 1983-
(författare)
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Advances in Ligand Binding Predictions using Molecular Dynamics Simulations
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biochemical processes all involve associations and dissociations of chemical entities. Understanding these is of substantial importance for many modern pharmaceutical applications. In this thesis, longstanding problems with regard to ligand binding are treated with computational methods, applied to proteins of key pharmaceutical importance. Homology modeling, docking, molecular dynamics simulations and free-energy calculations are used here for quantitative characterization of ligand binding to proteins. By combining computational tools, valuable contributions have been made for pharmaceutically relevant areas: a neglected tropical disease, an ion channel anti-drug-target, and GPCR drug-targets.We report three compounds inhibiting cruzain, the main cysteine protease of the protozoa causing Chagas’ disease. The compounds were found through an extensive virtual screening study and validated with experimental enzymatic assays. The compounds inhibit the enzyme in the μM-range and are therefore valuable in further lead optimization studies.A high-resolution crystal structure of the BRICHOS domain is reported, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry studies. This work revealed a plausible mechanism for how the chaperone activity of the domain may operate.Rationalization of structure-activity relationships for a set of analogous blockers of the hERG potassium channel is given. A homology model of the ion channel was used for docking compounds and molecular dynamics simulations together with the linear interaction energy method employed for calculating the binding free-energies.The three-dimensional coordinates of two GPCRs, 5HT1B and 5HT2B, were derived from homology modeling and evaluated in the GPCR Dock 2013 assessment. Our models were in good correlation with the experimental structures and all of them placed among the top quarter of all models assessed. Finally, a computational method, based on molecular dynamics free-energy calculations, for performing alanine scanning was validated with the A2A adenosine receptor bound to either agonist or antagonist. The calculated binding free-energies were found to be in good agreement with experimental data and the method was subsequently extended to non-alanine mutations. With extensive experimental mutation data, this scheme is a valuable tool for quantitative understanding of ligand binding and can ultimately be used for structure-based drug design.
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| 10. |
- Nervall, Martin, 1976-
(författare)
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Binding Free Energy Calculations on Ligand-Receptor Complexes Applied to Malarial Protease Inhibitors
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Malaria is a widespread disease caused by parasites of the genus Plasmodium. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, P. falciparum, accounts for ~90% of the fatal cases and has developed resistance to chloroquine. The resistant strains are a major problem and calls for novel drugs.In this thesis, the process of computational inhibitor design is illustrated through the development of P. falciparum aspartic protease inhibitors. These proteases, called plasmepsins, are part of the hemoglobin degradation chain. The hemoglobin is degraded during the intraerythrocytic cycle and serves as the major food source. By inhibiting plasmepsins the parasites can be killed by starvation.Novel inhibitors with very high affinity were found by using a combination of computational and synthetic chemistry. These inhibitors were selective and did not display any activity on human cathepsin D. The linear interaction energy (LIE) method was utilized in combination with molecular dynamics (MD) simulations to estimate free energies of binding. The MD simulations were also used to characterize the enzyme–inhibitor interactions and explain the binding on a molecular level.The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six ab initio quantum chemical charge derivation schemes were evaluated. It was found that the fast semiempirical charge models are equally useful in free energy calculations with the LIE method as the rigorous ab initio charge models.
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