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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
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 55
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1560
Keywords [en]
molecular dynamics simulations, binding free energy, molecular docking, cyclooxygenase, non-steroidal anti-inflammatory drugs, free energy perturbation, potentials of mean force
National Category
Pharmaceutical Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-328478ISBN: 978-91-513-0073-3 (print)OAI: oai:DiVA.org:uu-328478DiVA, id: diva2:1135669
Public defence
2017-11-02, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2017-10-10 Created: 2017-08-23 Last updated: 2017-10-17
List of papers
1. Toward an Optimal Docking and Free Energy Calculation Scheme in Ligand Design with Application to COX-1 Inhibitors
Open this publication in new window or tab >>Toward an Optimal Docking and Free Energy Calculation Scheme in Ligand Design with Application to COX-1 Inhibitors
2014 (English)In: JOURNAL OF CHEMICAL INFORMATION AND MODELING, ISSN 1549-9596, Vol. 54, no 5, p. 1488-1499Article in journal (Refereed) Published
Abstract [en]

Cyclooxygenase-1 (COX-1) is one of the main targets of most pain-relieving pharmaceuticals. Although the enzyme is well characterized, it is known to be a difficult target for automated molecular docking and scoring. We collected from the literature a structurally diverse set of 45 nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors (coxibs) with a wide range of binding affinities for COX-1. The binding of this data set to a homology model of human COX-1 was analyzed with different combinations of molecular docking algorithms, scoring functions, and the linear interaction energy (LIE) method for estimating binding affinities. It is found that the computational protocols for estimation of binding affinities are extremely sensitive to the initial orientations of the ligands in the binding pocket. To overcome this limitation, we propose a systematic exploration of docking poses using the LIE calculations as a postscoring function. This scheme yields predictions in excellent agreement with experiment, with a mean unsigned error of 0.9 kcal/mol for binding free energies and structures of high quality. A significant improvement of the results is also seen when averaging over experimental data from several independent measurements.

National Category
Bioinformatics and Systems Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-225268 (URN)10.1021/ci500151f (DOI)000336637400019 ()
Available from: 2014-05-29 Created: 2014-05-29 Last updated: 2017-08-23Bibliographically approved
2. Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase-1
Open this publication in new window or tab >>Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase-1
2015 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 49, p. 7283-7291Article in journal (Refereed) Published
Abstract [en]

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, inflammation, and some types of cancers. Their mechanism of action is the inhibition of isoforms 1 and 2 of the enzyme cyclooxygenase (COX-1 and COX-2, respectively). However, both nonselective and selective NSAIDs may have side effects that include gastric intestinal bleeding, peptic ulcer formation, kidney problems, and occurrences of myocardial infarction. The search for selective high-affinity COX inhibitors resulted in a number of compounds characterized by a slow, tight-binding inhibition that occurs in a two-step manner. It has been suggested that the final, only very slowly reversible, tight-binding event is the result of conformational changes in the enzyme. However, the nature of these conformational changes has remained elusive. Here we explore the structural determinants of the tight-binding phenomenon in COX-1 with molecular dynamics and free energy simulations. The calculations reveal how different classes of inhibitors affect the equilibrium between two conformational substates of the enzyme in distinctly different ways. The class of tight-binding inhibitors is found to exclusively stabilize an otherwise unfavorable enzyme conformation and bind significantly stronger to this state than to that normally observed in crystal structures. By also computing free energies of binding to the two enzyme conformations for 16 different NSAIDs, we identify an induced-fit mechanism and the key structural features associated with high-affinity tight binding. These results may facilitate the rational development of new COX inhibitors with improved selectivity profiles.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-269328 (URN)10.1021/acs.biochem.5b01024 (DOI)000366871800013 ()
Funder
Swedish Research Council
Available from: 2015-12-15 Created: 2015-12-15 Last updated: 2017-12-01Bibliographically approved
3. Probing the Time Dependency of Cyclooxygenase-1 Inhibitors by Computer Simulations
Open this publication in new window or tab >>Probing the Time Dependency of Cyclooxygenase-1 Inhibitors by Computer Simulations
2017 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 13, p. 1911-1920Article in journal (Refereed) Published
Abstract [en]

Time-dependent inhibition: of the cyclooxygenases (COX) by a range of nonsteroidal-anti-inflammatory drugs has been described since the first experimental assays of COX were performed. Slow tight-binding inhibitors of COX-1 bind in a two-step mechanism in which the EI -> EI* transition is slow and practically irreversible. Since then, various properties of the inhibitors have :been proposed to cause or affect the time dependency. Conformational changes :1-7, in the enzyme have also been proposed to cause the time, dependency, but no particular structural feature has been identified. Here, we investigated a series of inhibitors of COX 1 that are either time-independent or time-dependent using a combination of molecular dynamics simulations, binding free energy calculations, and potential of mean force calculations. We find that the time-dependent inhibitors stabilize a conformational change in the enzyme mainly identified by the rotation of a leucine Side chain adjacent to the binding pocket. The induced conformation has been previously Shown to be essential for the high binding affinities of tight-binding inhibitors in COX-1. The results of this work show that the structural features of the enzyme involved in both time-dependent and tight binding inhibition are identical and further;identify a structural mechanism responsible for the transition between the two enzyme inhibitor complexes characteristic of slow tight-binding COX-1 inhibitors.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-321184 (URN)10.1021/acs.biochem.6b01006 (DOI)000398646000012 ()28304156 (PubMedID)
Funder
Swedish Research CouncileSSENCE - An eScience CollaborationSwedish National Infrastructure for Computing (SNIC)
Available from: 2017-05-02 Created: 2017-05-02 Last updated: 2017-08-23
4. Molecular mechanisms in the selectivity of non-steroidal anti-inflammatory drugs
Open this publication in new window or tab >>Molecular mechanisms in the selectivity of non-steroidal anti-inflammatory drugs
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) 1 and 2 with varying degrees of selectivity. A group of COX-2 selective inhibitors —coxibs— bind in a time-dependent manner through a three-step mechanism, utilizing a side-pocket in the binding site. Coxibs have been extensively probed to identify the structural features regulating the slow tight-binding mechanism responsible for COX-2 selectivity. In this study, we further probe a structurally and kinetically diverse data set of COX inhibitors by molecular dynamics and free energy simulations. We found that the features regulating the high affinities associated with time-dependency in COX depend on the inhibitor kinetics. In particular, most time-dependent inhibitors share a common structural mechanism, consisting in an induced-fit rotation of the side-chain of Leu531 in the main binding pocket. The high affinities of two-step slow tight-binding inhibitors and some slow reversible inhibitors can be thus explained by the increased space in the main binding pocket after this rotation. Coxibs that belong to a separate class of slow tight-binding inhibitors benefit more from the displacement of the neighboring side-chain of Arg513, exclusive of the COX-2 side-pocket. This displacement further stabilizes the aforementioned rotation of Leu531, and can explain the selectivity of coxibs for COX-2.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-328477 (URN)
Available from: 2017-08-23 Created: 2017-08-23 Last updated: 2017-08-23
5. Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density
Open this publication in new window or tab >>Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density
Show others...
2016 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 89, no 4, p. 413-424Article in journal (Refereed) Published
Abstract [en]

Angiotensin IV (Ang IV) and related peptide analogues, as well as non-peptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocylic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N-terminal of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09 and of Ang IV in either the extended or γ-turn conformation at the C-terminal to human IRAP were predicted by docking and molecular dynamics (MD) simulations. The binding free energies calculated with the linear interaction energy (LIE) method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-275166 (URN)10.1124/mol.115.102533 (DOI)000370935700003 ()26769413 (PubMedID)
Funder
Swedish Research Council
Available from: 2016-01-31 Created: 2016-01-31 Last updated: 2018-03-20
6. Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures
Open this publication in new window or tab >>Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures
Show others...
2016 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 10, p. 1383-1392Article in journal (Refereed) Published
Abstract [en]

The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of TRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10 500 drug like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors:A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.

Keywords
Insulin-regulated aminopeptidase, aryl sulfonamides, molecular dynamics, ligand interaction energy simulations, dendritic spines, hippocampal neurons
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-307715 (URN)10.1021/acschemneuro.6b00146 (DOI)000385994000011 ()
Funder
Swedish Research Council
Available from: 2016-11-22 Created: 2016-11-21 Last updated: 2018-01-13Bibliographically approved

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