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Modeling of structure of gamma-aminobutyric acid transporters as an essential biological and therapeutic target

1. Research project objectives/ Research hypothesis

Study of interactions between bioactive compounds and macromolecules can be carried out using methods of molecular modeling based on the structure of a biological target or constructing appropriate models. In the presented project, the molecular targets for research are transporters for the γ-aminobutyric acid (GABA). They are responsible for the reuptake of this amino acid, and their structure has not been fully recognized so far. GABA transporters are divided into four types and constitute essential grip point for new bioactive compounds.

Scientific objective of the project is to build new homology models of all types of γ‑aminobutyric acid transporters (GAT-1, GAT-2, GAT-3 GAT-4) based on a template of dopamine and serotonin transporters and to understand the reasons for the selectivity of the various ligands. The resulting models will be used to study the binding mode of the selected inhibitors, and in the future they can be applied for the design of novel selective ligands.

2. Research project methodology

In the project molecular modeling methods, adequate to the intended goal, will be applied. The amino acid sequences of mouse and human GABA transporters as well as templates will be retrieved from the UniProt database. Additionally, in the search for other homology proteins Blast program will be utilized. The secondary structure and transmembrane domains for GATs will be predicted using freely available web services (e.g. PredictProtein, TMpred, TMHMM Server, HMMTOP). Crystal structures of templates for homology modeling will be retrieved from the Protein Data Bank. Alignment of the amino acid sequence of the modeled proteins and templates will be performed using programs from the Clustal family (Clustal W, X, Omega). In homology modeling as the main template dopamine and serotonin transporters will be used. On the basis of them any models of GABA transporters have not been published yet. LeuT transporter, combinations of all these templates and other homology proteins, whose structures may appear in the literature during the project, will be also taken into account. Homology modeling will be done using the program Modeller  and web service SwissModel. The all obtained models will be evaluated in the context of correctness of structure (WHAT_CHECK, PROCHECK, Ramachandran plot) and energy (Modeller Objective Function, DOPE Score, BCL :: Score). Structural analysis of the GAT models will be carried out using programs PyMOL and VMD and prepared scripts.

Ligand library will be prepared on the basis of literature data using Maestro software  from Schrodinger Suite package. Docking of ligands into homology models will be performed in the programs GOLD, Glide and Scigress. The results of the docking will be used to choose the best models of GABA transporters.

The structures of transporter - ligand  complexes, obtained in the docking process, will be subjected to detailed analysis using programs PyMOL, VMD and Maestro and prepared scripts to indicate the reasons for the selectivity of the individual ligands.

For selected complexes all-atom molecular dynamics simulations with program NAMD will be performed to study the details of interaction between inhibitors and the transporters GAT. Analysis of the results of the simulations will be carried out using VMD and prepared scripts.

3. Expected impact of the research project on the development of science, civilization and society

Results of the project will have an effect on development of medicinal chemistry. The created models of all mouse GABA transporters, i.e. GAT-1, GAT-2, GAT-3 and GAT-4, commonly used in biological research as well as their human counterparts, i.e. GAT-1, BGT-1, GAT- 2 and GAT-3 will enable to know the more detailed structure of GATs, and the conducted analysis will demonstrate both interspecies differences as well as differences between all types of transporters. In the future information obtained in this way will be useful for the design of new compounds with desired selectivity towards GABA transporters. The presented project raises issues that have not been previously explored. This ensures the publication of results in renowned international journals. The project will contribute to a better understanding of gamma-aminobutyric acid transporters and will provide an important base for research groups looking for new inhibitors of these targets with potential application in the treatment of epilepsy, depression, anxiety or pain.

The project is financially supported by National Science Center, Poland - Grant No. 2016/23/D/NZ7/01172.

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Design of novel compounds, influencing beta-amyloid aggregation and cholinergic
transmission, by application of molecular modelling techniques.

Alzheimer's disease is a progressive neurodegenerative disorder that leads not only to memory decline but also to speech and abstract thinking impairment as well. Developing slowly, it causes problems with the performance of even the most basic activities and leads to death. There are over 200 000 people affected by Alzheimer’s disease in Poland, and about 25 million worldwide. Epidemiological data indicate that the number of patients with this disease is increasing rapidly, and therefore it is particularly important to search for new effective drugs.

In the brain of affected patients the presence of the senile plaques, composed of β-amyloid peptide, can be detected. These senile plaques are responsible, inter alia, for the death of neurons and  intercellular signalling disturbances, which consequently lead to the symptoms of the disease.

The study, being carried out due to the postdoctoral grant, has just become an introduction to the wider research on new, original substances that can modify the course of Alzheimer's disease. It included the design stage which is the first step in a long and costly process of finding new drugs and leads to the selection of hits.

 

The multifactorial nature of Alzheimer's disease requires administration of comprehensively acting drugs in a potential therapy. Therefore, the search for new substances that could simultaneously inhibit formation of amyloid deposits and increase the transmission of signals between cells i.e. cholinergic transmission, was undertaken.

Due to the availability of high-performance computers and useful software it became possible to perform analyses that reflect the processes occurring in the brain of patients with Alzheimer's disease as well as to find new compounds that may stop the adverse effects. β-amyloid is produced from the  precursor protein APP under the influence of two enzymes, known as β-secretase and γ-secretase, and consequently large units called the senile plaques are being formed. Computer simulations allowed us a better understanding of the transition process of individual β-amyloid fragments into larger units as well as the systematic identification of those structural elements  involved in the initiation of that process and stabilization of the formed deposits. Further analyses enabled to understand how the compounds could inhibit that process and to design new, more potent substances. The compounds designed in this way were the subject of further laboratory work. Some of them have been obtained by chemical synthesis, and the first biological tests confirmed their inhibitory activity in the aggregation process. Additionally, we developed the  methods of  designing the substances, that could inhibit the production of simple fragments of b-amyloid from precursor protein just to reduce the initial amount of β-amyloid which could undergo aggregation. These approaches provided  the opportunity to influence the transformation of β-amyloid in several stages - directly as well as through inhibition of β‑secretase or modulation of γ-secretase action. For all new designed compounds, their ability to potentiate signalling between nerve cells by inhibition of cholinesterases was verified in computer tests. Moreover, their theoretical absorption and penetration to the brain and the side effects such as toxicity were assessed.

During the internship, many additional tasks,  such as building a computer model of protein, called presenilin 1, which is a component of γ-secretase enzyme and its mutations lead to a higher incidence of early-onset Alzheimer's disease, were  undertaken. This type of analysis allowed to understand how the  selected mutations influence the development of Alzheimer's disease.

The participation in postdoctoral research grant enabled to cooperate with a great number of scientists from other domestic and foreign institutions, and to use computer simulations to design biologically active substances in a more effective way.

Undoubtedly, the realization of the tasks, scheduled in the postdoctoral project, contributed to a better understanding of the processes, accompanying the development of Alzheimer's disease and enabled to  design  new compounds which are currently subject of further studies and in the future they may contribute to  the effective therapy of this disease as  they may act by new mechanisms, and because of the fact that  the current therapy remains ineffective and is limited only to slowing down the progression of the disease.

The project was financially supported by National Science Center, Poland - Grant No. 2012/04/S/NZ2/00116.

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