Micar21 Ltd.

Sofia, Bulgaria

Micar21 Ltd.

Sofia, Bulgaria
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Jereva D.,Bulgarian Academy of Science | Fratev F.,Bulgarian Academy of Science | Fratev F.,Micar21 Ltd. | Tsakovska I.,Bulgarian Academy of Science | And 3 more authors.
Mathematics and Computers in Simulation | Year: 2015

Human estrogen receptor alpha (ERα) is one of the most studied targets for in silico screening of bioactive compounds. The estrogenic activity of a vast number of chemicals has been studied for their potentially adverse effects on the hormone regulation of the endocrine system. The commonly accepted presentation of the ERα agonist pharmacophore includes terminal phenolic groups and a hydrophobic rigid backbone. In this study we report on molecular dynamics (MD) simulations of ERα to get a deeper structural insight into the agonist-receptor interactions and the pharmacophore pattern of compounds with agonistic activity. We rely on a crystallographic structure of a complex of ERα (PDB ID 2P15) with an agonist of picomolar affinity. As the X-ray structure has a mutation next to a key structural element for ERα agonistic activity (helix H12, Y537S), a series of MD simulations have been performed on the mutated and on the wild type receptor to prove the stability of the agonist-receptor interactions. No significant difference in the ligand-protein interactions has been detected between the studied proteins implying that the Y537S mutant structure can be used for refinement of the pharmacophore model of the ERα agonists. The results suggest that the pharmacophore of compounds with ERα agonistic activity can be extended by a feature that occupies a free hydrophobic region of the binding pocket. The extended pharmacophore model has been evaluated by a pharmacophore-based virtual screening of databases of ERα binders and decoys. The results also imply that MD simulations are a powerful in silico tool for both protein dynamics and structure investigation, especially when mutations are available that can potentially disturb the protein structure and functions. © 2015 International Association for Mathematics and Computers in Simulation (IMACS).

Fratev F.,Bulgarian Academy of Science | Fratev F.,Micar21 Ltd.
Physical Chemistry Chemical Physics | Year: 2015

In recent years, the nuclear receptors (NR) dynamics have been studied extensively by various approaches. However, the transition path of helix 12 (H12) to an agonist or an antagonist conformation and the exchange pathway between these states is not clear yet. A number of accelerated molecular dynamics (aMD) runs were performed on both an ERα monomer and a homodimer with a total length of 2.2 μs. We have been able to sample reasonably well the H12 conformational landscape to reproduce precisely both the agonist and the antagonist conformations, starting from an unfolded position, and to describe the transition path between them, even in the presence of an agonist ligand. These conformations were the most prevalent, suggesting that the extended H12 state is not likely to exist and that the natural ERα H12 position might exist in both the agonist and antagonist states. Remarkably, the H12 transition occurs and is regulated only in a dimer form and the proper agonist or antagonist H12 conformation can be achieved solely in one of the dimer subunits. These results clearly demonstrate that clusters of the two well-known H12 states exist by themselves in the protein free energy landscape, i.e. they are not constituted directly by the ligands, and dimerization favors the switch between them. Conversely, in a monomer, no transitions have been observed. Thus, the dimer formation helps the constitution of populations of discrete H12 conformational states and reshapes the conformational landscape. Further analyses have shown that these observations can be explained by specific interface and long range protein-protein interactions, resulting in conformational fluctuations in helices 5 and 11. Based on these results, a new ERα activation/deactivation mechanism and a sequence of binding events during receptor activity modulation have been suggested according to which ligands control the H12 conformation via alterations of the inter-dimer interactions. These findings agree with the HDX and fluorescence experiments and provide an explanation on a structural basis of these data, demonstrating that the dynamics of H12 are not altered greatly upon ligand binding and large fluctuations at the end of H11 are present. © 2015 the Owner Societies.

Fratev F.,Bulgarian Academy of Science | Fratev F.,Micar21 Ltd | Tsakovska I.,Bulgarian Academy of Science | Al Sharif M.,Bulgarian Academy of Science | And 2 more authors.
International Journal of Molecular Sciences | Year: 2015

The structural and dynamical properties of the peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptor have been broadly studied in its agonist state but little is known about the key features required for the receptor antagonistic activity. Here we report a series of molecular dynamics (MD) simulations in combination with free energy estimation of the recently discovered class of non-covalent PPARγ antagonists. Their binding modes and dynamical behavior are described in details. Two key interactions have been detected within the cavity between helices H3, H11 and the activation helix H12, as well as with H12. The strength of the ligand-amino acid residues interactions has been analyzed in relation to the specificity of the ligand dynamical and antagonistic features. According to our results, the PPARγ activation helix does not undergo dramatic conformational changes, as seen in other nuclear receptors, but rather perturbations that occur through a significant ligand-induced reshaping of the ligand-receptor and the receptor-coactivator binding pockets. The H12 residue Tyr473 and the charge clamp residue Glu471 play a central role for the receptor transformations. Our results also demonstrate that MD can be a helpful tool for the compound phenotype characterization (full agonists, partial agonists or antagonists) when insufficient experimental data are available. © 2015 by the authors; licensee MDPI, Basel, Switzerland.

Fratev F.,Bulgarian Academy of Science | Fratev F.,Micar21 Ltd. | Mihaylova E.,Micar21 Ltd. | Pajeva I.,Bulgarian Academy of Science
Journal of Chemical Information and Modeling | Year: 2014

Cypher/ZASP (LDB3 gene) is known to interact with a network of proteins. It binds to α-actinin and the calcium voltage channels (LTCC) via its PDZ domain. Here we report the identification of a highly conserved ZASP G54S mutation classified as a variant of unknown significance in a sample of an adult with hypertrophic cardiomyopathy (HCM). The initial bioinformatics calculations strongly evaluated G54S as damaging. Furthermore, we employed accelerated and classical molecular dynamics and free energy calculations to study the structural impact of this mutation on the ZASP apo form and to address the question of whether it can be linked to HCM. Seventeen independent MD runs and simulations of 2.5 μs total were performed and showed that G54S perturbs the α2 helix position via destabilization of the adjacent loop linked to the β5 sheet. This also leads to the formation of a strong H-bond between peptide target residues Leu17 and Gln66, thus restricting both the α-actinin2 and LTCC C-terminal peptides to access their natural binding site and reducing in this way their binding capacity. On the basis of these observations and the adult's clinical data, we propose that ZASPG54S and presumably other ZASP PDZ domain mutations can cause HCM. To the best of our knowledge, this is the first reported ZASP PDZ domain mutation that might be linked to HCM. The integrated workflow used in this study can be applied for the identification and description of other mutations that might be related to particular diseases. © 2014 American Chemical Society.

Fratev F.,Micar 21 Ltd. | Osk Jonsdottir S.,Technical University of Denmark | Pajeva I.,Bulgarian Academy of Science
Proteins: Structure, Function and Bioinformatics | Year: 2013

The UNC-45 chaperone protein interacts with and affects the folding, stability, and the ATPase activity of myosins. It plays a critical role in the cardiomyopathy development and in the breast cancer tumor growth. Here we propose the first structural model of the UNC-45-myosin complex using various in silico methods. Initially, the human UNC-45B binding epitope was identified and the protein was docked to the cardiac myosin (MYH7) motor domain. The final UNC45B-MYH7 structure was obtained by performing of total 630 ns molecular dynamics simulations. The results indicate a complex formation, which is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosin-actin complex. A significant interspecies difference in the myosin binding epitope is observed. Our results reveal the structural basis of MYH7 exons 15-16 hypertrophic cardiomyopathy mutations and provide directions for drug targeting. © 2013 Wiley Periodicals, Inc.

Kouskoumvekaki I.,Technical University of Denmark | Petersen R.K.,BioLigands | Petersen R.K.,Copenhagen University | Fratev F.,Technical University of Denmark | And 12 more authors.
Journal of Chemical Information and Modeling | Year: 2013

Full agonists to the peroxisome proliferator-activated receptor (PPAR)γ, such as Rosiglitazone, have been associated with a series of undesired side effects, such as weight gain, fluid retention, cardiac hypertrophy, and hepatotoxicity. Nevertheless, PPARγ is involved in the expression of genes that control glucose and lipid metabolism and is an important target for drugs against type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In an effort to identify novel PPARγ ligands with an improved pharmacological profile, emphasis has shifted to selective ligands with partial agonist binding properties. Toward this end we applied an integrated in silico/in vitro workflow, based on pharmacophore-and structure-based virtual screening of the ZINC library, coupled with competitive binding and transactivation assays, and adipocyte differentiation and gene expression studies. Hit compound 9 was identified as the most potent ligand (IC50 = 0.3 μM) and a relatively poor inducer of adipocyte differentiation. The binding mode of compound 9 was confirmed by molecular dynamics simulation, and the calculated free energy of binding was -8.4 kcal/mol. A novel functional group, the carbonitrile group, was identified to be a key substituent in the ligand-protein interactions. Further studies on the transcriptional regulation properties of compound 9 revealed a gene regulatory profile that was to a large extent unique, however functionally closer to that of a partial agonist. © 2013 American Chemical Society.

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