| 1. |
- Barlow, Nicholas, et al.
(author)
-
Macrocyclic Peptidomimetics as Inhibitors of Insulin-Regulated Aminopeptidase (IRAP)
- 2020
-
In: RSC Medicinal chemistry. - : Royal Society of Chemistry (RSC). - 2632-8682. ; 11:2, s. 234-244
-
Journal article (peer-reviewed)abstract
- Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (∆G) and relative binding free energies (∆∆G) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.
|
|
| 2. |
- Engen, Karin, et al.
(author)
-
Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors
- 2020
-
In: ChemistryOpen. - : Wiley. - 2191-1363. ; 9:3, s. 325-337
-
Journal article (peer-reviewed)abstract
- Insulin‐regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non‐peptide IRAP inhibitors derived from a spiro‐oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)‐promoted three‐component reaction, or by a two‐step one‐pot procedure. For decoration of the oxindole ring system, rapid MW‐assisted Suzuki‐Miyaura cross‐couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S‐configuration of the spiro‐oxindole dihydroquinazolinones accounts for the inhibition of IRAP.
|
|
| 3. |
- Guo, Xiaohu, et al.
(author)
-
Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family
- 2021
-
In: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
-
Journal article (peer-reviewed)abstract
- The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.
|
|
| 4. |
- Jespers, Willem, et al.
(author)
-
Deciphering conformational selectivity in the A(2A) adenosine G protein-coupled receptor by free energy simulations
- 2021
-
In: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 17:11
-
Journal article (peer-reviewed)abstract
- Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A(2A) adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A(2A)AR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.
|
|
| 5. |
|
|
| 6. |
- Jespers, Willem
(author)
-
Free energy calculations of G protein-coupled receptor modulation : New methods and applications
- 2020
-
Doctoral thesis (other academic/artistic)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.
|
|
| 7. |
- Jespers, Willem, et al.
(author)
-
X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A(2A) Adenosine Receptor Antagonists
- 2020
-
In: Angewandte Chemie International Edition. - : WILEY-V C H VERLAG GMBH. - 1433-7851 .- 1521-3773. ; 59:38, s. 16536-16543
-
Journal article (peer-reviewed)abstract
- We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A(2A) adenosine receptor (AR). Eight A(2A)AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A(2A)AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A(2A)AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A(2A)AR, an emerging target in immunooncology.
|
|
| 8. |
- Koenekoop, Lucien, et al.
(author)
-
Principles of Cold Adaptation of Fish Lactate Dehydrogenases Revealed by Computer Simulations of the Catalytic Reaction
- 2023
-
In: Molecular biology and evolution. - : Oxford University Press. - 0737-4038 .- 1537-1719. ; 40:5
-
Journal article (peer-reviewed)abstract
- Cold-adapted enzymes from psychrophilic and psychrotolerant species are characterized by a higher catalytic activity at low temperature than their mesophilic orthologs and are also usually found to be more thermolabile. Computer simulations of the catalytic reactions have been shown to be a very powerful tool for analyzing the structural and energetic origins of these effects. Here, we examine the cold adaptation of lactate dehydrogenases from two Antarctic and sub-Antarctic fish species using this approach and compare our results with those obtained for the orthologous dogfish enzyme. Direct calculations of thermodynamic activation parameters show that the cold-adapted fish enzymes are characterized by a lower activation enthalpy and a more negative entropy term. This appears to be a universal feature of psychrophilic enzymes, and it is found to originate from a higher flexibility of certain parts of the protein surface. We also carry out free energy simulations that address the differences in thermal stability and substrate binding affinity between the two cold-adapted enzymes, which only differ by a single mutation. These calculations capture the effects previously seen in in vitro studies and provide straightforward explanations of these experimental results.
|
|
| 9. |
- Koenekoop, Lucien, et al.
(author)
-
The Activation Parameters of a Cold-Adapted Short Chain Dehydrogenase Are Insensitive to Enzyme Oligomerization
- 2022
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 61:7, s. 514-522
-
Journal article (peer-reviewed)abstract
- The structural principles of enzyme cold adaptation are of fundamental interest both for understanding protein evolution and for biotechnological applications. It has become clear in recent years that structural flexibility plays a major role in tuning enzyme activity at low temperatures, which is reflected by characteristic changes in the thermodynamic activation parameters for psychrophilic enzymes, compared to those of mesophilic and thermophilic ones. Hence, increased flexibility of the enzyme surface has been shown to lead to a lower enthalpy and a more negative entropy of activation, which leads to higher activity in the cold. This immediately raises the question of how enzyme oligomerization affects the temperature dependence of catalysis. Here, we address this issue by computer simulations of the catalytic reaction of a cold-adapted bacterial short chain dehydrogenase in different oligomeric states. Reaction free energy profiles are calculated at different temperatures for the tetrameric, dimeric, and monomeric states of the enzyme, and activation parameters are obtained from the corresponding computational Arrhenius plots. The results show that the activation free energy, enthalpy, and entropy are remarkably insensitive to the oligomeric state, leading to the conclusion that assembly of the subunit interfaces does not compromise cold adaptation, even though the mobilities of interfacial residues are indeed affected.
|
|
| 10. |
- Machado, Teresa F. G., et al.
(author)
-
Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry
- 2020
-
In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:24, s. 15019-15032
-
Journal article (peer-reviewed)abstract
- The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD(+):acetoacetate and NAD+:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.
|
|
