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Computational Studies of the Biogenic Amines of the Brain for Targeting Neurological Diseases

Final Report Summary - COMPBAND (Computational Studies of the Biogenic Amines of the Brain for Targeting Neurological Diseases)

The focus of this computational research was to improve the understanding of a link between features, metabolic pathways and signal transduction of amine neurotransmitters, and the treatment of neurological diseases, thus addressing challenges raised by the ever-increasing prevalence of brain disorders. Brain monoaminergic systems have been extensively implicated in the etiology and course of various neurodegenerative diseases, and their binding proteins such as receptors, transporters and common metabolic enzymes are the starting points for development of tools to diagnose and drugs to treat specific clusters of symptoms. Therefore, the research performed in this project concentrated on two important biological systems, namely on monoamine oxidase enzymes (MAOs) responsible for regulating amine levels in the synaptic region, and on histamine H2 receptor that is activated by the biogenic amine histamine as its primary endogenous ligand to exert various regulatory actions.

The work on this project resulted in elucidating the precise molecular mechanism of the MAO catalytic activity and convincingly demonstrated the prevailing feasibility of our newly proposed two-step hydride transfer mechanism over other alternatives, being a vital achievement for the pharmaceutical industry in terms of developing new target molecules as MAO inhibitors. By investigating MAO B catalyzed degradation of dopamine, we obtained the activation free energy corresponding to the hydride anion abstraction from the substrate to the enzyme's flavin co-factor of 16.1 kcal/mol, in excellent agreement with the experimental value of 16.5 kcal/mol, thus strongly supporting the proposed mechanism. On the other hand, our analysis of the same mechanism for the noradrenaline metabolism by the other MAO isoform, MAO A, gave the activation free energy of 20.3 kcal/mol, in reasonable agreement with the correlated experimental value of 16.5 kcal/mol. In this way, we credibly demonstrated that both A and B MAO isoforms operate by the same hydride mechanism, which is something that has been debated and denied in the literature. Additionally, we considered a few point mutations of the "aromatic cage" tyrosine Tyr444 residue in MAO A and the calculated changes in the reaction barriers are in agreement with the experimental values, thus emphasizing the functional importance of the Tyr444 residue and providing additional support to the proposed mechanism. To further strengthen our conclusions, we inspected pKa values of the matching residues in the active sites of both MAO A and MAO B, and showed that these assume identical values, suggesting that the electrostatic environment in both enzymes is alike, lending credence to the idea that both operate through the same mechanism. Finally, we considered the effects of nuclear tunnelling related to the MAO B catalyzed dopamine degradation. The calculated H/D kinetic isotope effect of 12.8 ± 0.3 is found in a very good agreement with the available experimental data for closely related biogenic amines, which gives strong support for our hydride mechanism.

Although sharing around 70% sequence identity, both MAO A and B isoforms strongly differ in substrate affinities and inhibitor sensitivities. Inhibitors that act on MAO A are used to treat depression, due to their ability to raise serotonin concentrations, whereas MAO B inhibitors decrease dopamine degradation and improve motor control in patients with Parkinson disease. Despite this functional importance, the factors affecting MAO selectivity are poorly understood. To address this, we used a combination of MD simulations, MM–PBSA binding free energy evaluations, and QM cluster calculations to tackle the unexpected, yet challenging MAO B selectivity for N-methylhistamine (NMH) over reversible inhibitor histamine (HIS), differing only in a single methyl group distant from the reactive ethylamino center. Our results help in rationalizing the fact that HIS is not at all a physiological MAO B substrate, but has to be N-methylated to NMH before the enzymatic conversion by showing that a dominant selectivity contribution is offered by a lower activation free energy for NMH by 2.6 kcal/mol, in excellent agreement with the experimental value of 1.4 kcal/mol, together with more favourable both reaction exergonicity and active-site binding. Our results also confirmed the hydrophobic nature of the MAO B active site and underlined the important role of Ile199, Leu171, and Leu328 residues in properly orienting substrates for the reaction. In addition, we determined the molecular mechanism of the irreversible MAO A inhibition by the antidepressant clorgyline, and showed that both the calculated activation parameters and the structure of the final adduct are in a good agreement with experiments. We also demonstrated that clorgyline is a more efficient drug than typical MAO B inhibitors, antiparkinsonian drugs rasagiline and selegiline, in line with experiments. All of these insights might turn useful in rational modification of the MAO reactivity to offer opportunities to exploit this enzyme in biotechnology and protein engineering, and in providing guidelines for designing more potent and selective MAO inhibitors that are all clinically employed in treating a variety of neuropsychiatric and neurodegenerative conditions.

In order to shed some light on the ability of receptors to discriminate between their agonists and antagonists, being of primary fundamental interest for the pharmaceutical industry, we conducted a combined experimental and computational study in which we monitored the effects of deuteration on the binding affinities of the histamine H2 receptor towards antagonist 3H-tiotidine and three agonists, namely histamine, 2- and 4-methylhistamine. Deuteration-induced changes in the length and strength of the hydrogen bonds did not cause any statistically significant difference in the binding of the antagonist, while, in contrast, the affinities of all agonists, histamine in particular, towards histamine H2 receptor binding sites were changed, confirming the relevance of hydrogen bonding in the process of agonist-receptor binding. The computational study involved the construction of a homology model of the H2 receptor and quantum chemical modelling of the binding free energies with included empirical quantization of the proton motion in order to assess the influence of hydrogen isotopes on the histamine binding. We predicted that the overall binding of histamine and its deuterated analogue is stronger by 0.51 kcal/mol for the latter, in line with the experimental value of 0.73 kcal/mol. Our computational analysis also revealed a new mechanism of histamine binding, which underlined an important role of the Tyr250 residue. This work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future.

The gained insight into the structure and function of the mentioned biological systems together with a collected large set of kinetic parameters for chemical processes responsible for the development and progression of neurodegeneration, allowed us to propose a gross scheme of neurodegeneration on the molecular level based on two pathways. This analysis can be employed in developing strategies for the prevention and treatment of neurodegeneration, and, hopefully, aid in the building of an overall kinetic molecular model of neurodegeneration itself.

All of the achieved objectives go beyond the current state-of-the-art and should help improve the efficiency and reduce adverse effects of commercially available drugs used nowadays to treat neurodegeneration, and should suggest to both researchers and pharmaceutical industries novel classes of active compounds as transition state analogues, thus being of considerable interest to both academic and industrial communities. The impact of this research is such that it is, in general, highly interdisciplinary with the promise of industrial applications, since it bridges the gap between fundamental understanding of biological systems and preclinical medicine combining modern methods of computational biochemistry with pharmacological approaches.

During the project realization, Dr. Vianello co-authored 11 research papers, 1 broad review article and 1 book chapter directly related to the current project, together with 7 other research papers acknowledging the project. He held invited or plenary lectures at 11 European conferences and events, and a significant number of popular presentations about research results for the postgraduate students, or about the success in obtaining competitive FP7 Marie Curie grants for the potential applicants within researchers from Croatia and Slovenia. All of the specified objectives of the CompBAND project are achieved as planned without any deviations or delays. It is safe to conclude that the knowledge and expertise gained through this project would enable Dr. Vianello to continue this leading–edge research, defining forefront research topics for his students, increasing the role of the computational simulations of enzymes and receptors at his home institution, continuing collaboration with world’s best experts and enhancing his participation in other competitive international projects. Overall, competencies and goals acquired through CompBAND would help Dr. Vianello to reach a position of professional maturity, strengthen his independent research position at the RBI, and facilitate his further professional integration within Europe in both medium- and long-terms. In conclusion, we could safely say that the CompBAND project is fully realized in accordance with what was specified in the project proposal, and neither deviations nor delays are identified.


Dr. Robert Vianello, Principal Investigator
Computational Organic Chemistry and Biochemistry Group
Rudjer Boskovic Institute, Zagreb, Croatia