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Exploring non-covalent interactions between drug-like molecules and the protein acetylcholinesterase
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Anna Linusson, Umeå universitet)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
En studie av icke-kovalenta interaktioner mellan läkemedelslika molekyler och proteinet acetylkolinesteras (Swedish)
Abstract [en]

The majority of drugs are small organic molecules, so-called ligands, that influence biochemical processes by interacting with proteins. The understanding of how and why they interact and form complexes is therefore a key component for elucidating the mechanism of action of drugs. The research presented in this thesis is based on studies of acetylcholinesterase (AChE). AChE is an essential enzyme with the important function of terminating neurotransmission at cholinergic synapses. AChE is also the target of a range of biologically active molecules including drugs, pesticides, and poisons. Due to the molecular and the functional characteristics of the enzyme, it offers both challenges and possibilities for investigating protein-ligand interactions. In the thesis, complexes between AChE and drug-like ligands have been studied in detail by a combination of experimental techniques and theoretical methods. The studies provided insight into the non-covalent interactions formed between AChE and ligands, where non-classical CH∙∙∙Y hydrogen bonds (Y = O or arene) were found to be common and important. The non-classical hydrogen bonds were characterized by density functional theory calculations that revealed features that may provide unexplored possibilities in for example structure-based design. Moreover, the study of two enantiomeric inhibitors of AChE provided important insight into the structural basis of enthalpy-entropy compensation. As part of the research, available computational methods have been evaluated and new approaches have been developed. This resulted in a methodology that allowed detailed analysis of the AChE-ligand complexes. Moreover, the methodology also proved to be a useful tool in the refinement of X-ray crystallographic data. This was demonstrated by the determination of a prereaction conformation of the complex between the nerve-agent antidote HI-6 and AChE inhibited by the nerve agent sarin. The structure of the ternary complex constitutes an important contribution of relevance for the design of new and improved drugs for treatment of nerve-agent poisoning. The research presented in the thesis has contributed to the knowledge of AChE and also has implications for drug discovery and the understanding of biochemical processes in general.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 76 p.
Keyword [en]
acetylcholinesterase, drug discovery, density functional theory, hydrogen bond, nerve-agent antidote, non-covalent interaction, protein-ligand complex, structure-based design, thermodynamics, X-ray crystallography
National Category
Chemical Sciences
Research subject
läkemedelskemi
Identifiers
URN: urn:nbn:se:umu:diva-129900ISBN: 978-91-7601-644-2 (print)OAI: oai:DiVA.org:umu-129900DiVA: diva2:1063794
Public defence
2017-02-03, Stora hörsalen (KB.E3.03), KBC-huset, Umeå universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2017-01-13 Created: 2017-01-10 Last updated: 2017-01-13Bibliographically approved
List of papers
1. Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling
Open this publication in new window or tab >>Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 11, e26039Article in journal (Refereed) Published
Abstract [en]

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e. g. in Alzheimer's disease), but may also act as dangerous toxins (e. g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685.mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2011
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-52194 (URN)10.1371/journal.pone.0026039 (DOI)000298168100002 ()
Available from: 2012-02-14 Created: 2012-02-13 Last updated: 2017-01-11Bibliographically approved
2. Similar but Different: Thermodynamic and Structural Characterization of a Pair of Enantiomers Binding to Acetylcholinesterase
Open this publication in new window or tab >>Similar but Different: Thermodynamic and Structural Characterization of a Pair of Enantiomers Binding to Acetylcholinesterase
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2012 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 51, no 51, 12716-12720 p.Article in journal (Refereed) Published
Abstract [en]

Take a closer look: Unexpectedly, a pair of enantiomeric ligands proved to have similar binding affinities for acetylcholinesterase. Further studies indicated that the enantiomers exhibit different thermodynamic profiles. Analyses of the noncovalent interactions in the protein-ligand complexes revealed that these differences are partly due to nonclassical hydrogen bonds between the ligands and aromatic side chains of the protein.

Keyword
aromatic interactions, density functional calculations, molecular recognition, nonclassical hydrogen bonds, stereoselectivity
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-61594 (URN)10.1002/anie.201205113 (DOI)23161758 (PubMedID)
Available from: 2012-11-20 Created: 2012-11-20 Last updated: 2017-01-11Bibliographically approved
3. The Nature of Activated Non-classical Hydrogen Bonds: A Case Study on Acetylcholinesterase-Ligand Complexes
Open this publication in new window or tab >>The Nature of Activated Non-classical Hydrogen Bonds: A Case Study on Acetylcholinesterase-Ligand Complexes
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2016 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 22, no 8, 2672-2681 p.Article in journal (Refereed) Published
Abstract [en]

Molecular recognition events in biological systems are driven by non-covalent interactions between interacting species. Here, we have studied hydrogen bonds of the CHY type involving electron-deficient CH donors using dispersion-corrected density functional theory (DFT) calculations applied to acetylcholinesterase-ligand complexes. The strengths of CHY interactions activated by a proximal cation were considerably strong; comparable to or greater than those of classical hydrogen bonds. Significant differences in the energetic components compared to classical hydrogen bonds and non-activated CHY interactions were observed. Comparison between DFT and molecular mechanics calculations showed that common force fields could not reproduce the interaction energy values of the studied hydrogen bonds. The presented results highlight the importance of considering CHY interactions when analysing protein-ligand complexes, call for a review of current force fields, and opens up possibilities for the development of improved design tools for drug discovery.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2016
Keyword
acetylcholinesterase, density functional calculations, drug design, hydrogen bonds, quantum chemistry
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-118248 (URN)10.1002/chem.201503973 (DOI)000370193000017 ()26751405 (PubMedID)
Available from: 2016-03-16 Created: 2016-03-14 Last updated: 2017-01-11Bibliographically approved
4. Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6
Open this publication in new window or tab >>Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6
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2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 20, 5514-5519 p.Article in journal (Refereed) Published
Abstract [en]

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.

Keyword
acetylcholinesterase, density functional theory, crystallography, nerve agent, reactivation
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-121442 (URN)10.1073/pnas.1523362113 (DOI)000375977600028 ()27140636 (PubMedID)
Available from: 2016-06-23 Created: 2016-06-02 Last updated: 2017-01-11Bibliographically approved

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