Filip Fratev

Structural insight into the UNC-45–myosin complex

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. Proteins 2013; 81:1212–1221.

Combination of Genetic Screening and Molecular Dynamics as a Useful Tool for Identification of Disease-Related Mutations: ZASP PDZ Domain G54S Mutation Case

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 adults clinical data, we propose that ZASP(G54S) 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.

Molecular basis of inactive B-RAF(WT) and B-RAF(V600E) ligand inhibition, selectivity and conformational stability: an in silico study.

The B-RAF kinase plays an important role both in tumor induction and maintenance in several cancers. The molecular basis of the inactive B-RAF(WT) and B-RAF(V600E) inhibition and selectivity of a series of inhibitors was examined with a combination of molecular dynamics (MD), free energy MM-PBSA and local-binding energy (LBE) approaches. The conformational stability of the unbounded kinases and in particular the processes of the B-RAF (V600E) mutant activation were analyzed. A unique salt bridge network formed mainly by the catalytic residues was identified in the unbounded B-RAFs. The reorganization of this network and the restriction of the active segment flexibility upon ligand binding inhibit both B-RAF(WT) and B-RAF (V600E), thus appearing as an important factor for ligand selectivity. A significant correlation between the binding energies of the compounds in B-RAF(WT) and their inhibition effects on B-RAF (V600E) was revealed, which can explain the low mutant selectivity observed for numerous inhibitors. Our results suggest that the interactions between the activation segment and the alpha C-helix, as well as between the residues in the salt bridge network, are the major mechanism of the B-RAF (V600E) activation. Overall data revealed the important role of Lys601 for ligand activity, selectivity and protein stabilization, proposing an explanation of the observed strong kinase activation in the K601E mutated form.

A theoretical study of molecular structure in the excited state and molecular luminescence: III. Types of molecular structure in the excited ππ* singlet states

Abstract The molecular geometry (bond lengths) in the S 1 , and S 2 excited states of a large number of conjugated organic molecules is calculated with the help of the SCF-CI-SC procedure, already described in previous communications. The analysis shows that all excited states studied can be classified in three basic types of structure: (1) with strong conjugation over the whole molecule or along its periphery; (2) with a long main conjugated framework and other shorter conjugated fragments moderately or slightly bonded with the framework and (3) structures consisting of two or more fragments slightly conjugated to each other. An estimation of the role of the possible intramolecular motions of low frequency is done, as well as some other factors contributing to the radiationless deactivation of a given excited state. The presence of structures with both lowest excited singlets of type 1 is shown.

A theoretical study of molecular structure in the excited state and molecular luminescence part II: An SCF—CI calculation of luminescence and molecular structure in equilibrium excited states of some compounds with strong bond alternation

Abstract It is shown that the iterative procedure SCF—CI-SC previously suggested [1] can be successfully used for calculation of the Stokes shift, FC luminescent maxima and equilibrium molecular geometry in excited states of compounds which have strong bond alterna- tion in their ground state. The influence, in the excited state, of molecular geometry on the positions and intensities of the luminescent transitions is discussed. The calculated molecular geometries show that there is a strong conjugation effect in some excited states of compounds with strong, moderate and weak bond alternation; this latter factor probably determines the luminescent properties of these organic compounds.

Structural and Dynamical Insight into PPARγ Antagonism: In Silico Study of the Ligand-Receptor Interactions of Non-Covalent Antagonists

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.

Photoreactions of derivatives of 2-phenylindan-1,3-dione in polar and non-polar solvents

Abstract Two primary photoreactions were observed for derivatives of 2-phenylindian-1,3-dione in various solvents: (1) cleavage of the hydrogen atom in the α position to both keto groups resulting in the formation of 2-arylindan-1,3-dion-2-yl radicals; (2) photoisomerization to benzylidenephthalides as a result of bond rupture via a Norrish type I mechanism. Both processes are possible with different quantum yields from the diketo form (in apolar solvents), from the enol form (in alcohols) and from the enolate form (in alkaline water).

Molecular dynamics simulation of the human estrogen receptor alpha: contribution to the pharmacophore of the agonists

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.

PPARγ non-covalent antagonists exhibit mutable binding modes with a similar free energy of binding: a case study.

The structural and dynamical properties of PPARγ receptor in a complex with either partial or full agonists have been intensively studied but little is known about the receptor antagonistic conformation. A composition of microsecond accelerated molecular dynamics (aMD) simulation show that like partial agonists a non-covalent PPARγ full antagonist can bind in different modes of similar population size and free energies of binding. Four different and periodically exchanging ligand conformations are detected and described. The studied antagonist interacts with different receptor substructures and affects both the co-activator and the Cdk5 phosphorylation sites and, presumably, the natural complex with the DNA. However, no significant changes in the conformational states of the activation helix 12, and in particular an antagonist orientation, have been recorded. Finally, our results show also that the aMD approach can be successfully used in recovering the possible binding modes, considering fully the receptor flexibility, and is not dependent on the starting conformation.

Absorption and emission characteristics of disubstituted 3-phenylmethylene-1(3B)-isobenzofuranones

Abstract The absorption, fluorescence and phosphorescence characteristics of a series of disubstituted 3-phenylmethylene-1(3B)-isobenzofuranones - (X,Y)BPHs - organic luminophores with a stilbene skeleton, have been studied. Depending on the substituents, (X,Y)BPHs fluorescence in the range 400 - 700 nm with a maximal fluorescence quantum yield of 0.65; for some of them a weak phosphorescence in EPA, 77° K is also observed. The changes in the dipole moments upon excitation were measured.