MOLECULAR MODELING OF EBOLA VIRUS INHIBITORS

In this PhD thesis computational methods have been employed in order to study different biologically relevant systems. In the first part of the thesis two Ebola virus proteins were studied, namely Viral Protein 24 (VP24) and Viral Protein 35 (VP35), responsible for the inhibition of the immune response . After a brief theoretical introduction to the main computational methods employed in the thesis, a study of VP35 in complex with small organic molecules is presented. These compounds are able to inhibit the interaction between VP35 and viral nucleoprotein. This study confirms the experimental findings highlighting new important key interactions between the protein the inhibitors. Moreover, an Essential Dynamics analysis points out an interesting collective motion of the apo-form that is hindered by the presence of the ligands. Afterwards, the protein-protein interaction VP24-Karyopherin (KPNA) is studied. An atomistic analysis of the interactions at the interface leads to the design of a nonapeptide with VP24 binding capability. The peptide is derived from a KPNA subsequence and could potentially inhibit the VP24-KPNA interaction. Subsequently an analysis on the pockets present on VP24 surface in different solvents is performed. Once the most promising pocket has been located, a virtual screening on a subset of ZINC database is carried out, leading to the identification of few classes of molecules potentially able to bind VP24. Finally the effect of the osmolytes on VP24 protein structure is studied, pointing out how osmoprotectants and urea have opposite effects on the protein, the former stabilizing the folded state and the latter shifting the equilibrium to the denatured state. In the second part of the manuscript the study of the interaction of an antimicrobial peptide with a lipid membrane is presented. This work was carried out in the University of Groningen under the supervision of Prof. Siewert Jan Marrink in order to deepen the Coarse Grain method

Synthesis and biological evaluation of new 3-amino-2-azetidinone derivatives as anticolorectal cancer agents

Several synthetic combretastatin A4 (CA-4) derivatives were recently prepared to increase the drug efficacy and stability of the natural product isolated from the South African tree Combretum caffrum. A group of ten 3-amino-2-azetidinone derivatives, as combretastatin A4 analogues, was selected through docking experiments, synthesized and tested for their anti-proliferative activity against the colon cancer SW48 cell line. These molecules, through the formation of amide bonds in position 3, allow the synthesis of various derivatives that can modulate the activity with great resistance to hydrolytic conditions. The cyclization to obtain the 3-aminoazetidinone ring is highly diastereoselective and provides a trans biologically active isomer under mild reaction conditions with better yields than the 3-hydroxy-2-azetidinone synthesis. All compounds showed IC50 values ranging between 14.0 and 564.2 nM, and the most active compound showed inhibitory activity against tubulin polymerization in vitro, being a potential therapeutic agent against colon cancer

Halogen bonding in the framework of classical force fields: The case of chlorine

Halogen bonding is nowadays a consolidated tool in chemistry. Only recently, the importance of halogen bonding has been demonstrated also in biological systems, owing to the presence of halogens in drugs. This interaction is due to the anisotropy of the electron density around the halogen that leads to the formation of the \u2018\u3c3-hole\u2019, which is responsible for the interaction with a nucleophile site. Unfortunately, classical force fields used in the study of ligand-receptor systems are not able to describe the \u2018\u3c3-hole\u2019. Here, we propose a pseudo-atom based methodology able to correctly describe halogen bonding involving chlorine using classical force field

A valence bond description of the bromine halogen bond

A theoretical investigation on the nature of the halogen bond through a valence-bond approach has been carried out with two main goals: (a) finding further confirmations of already existing explanations on the physical origins of the halogen bond and (b) possibly enriching the current models with new details. To achieve these goals we have exploited the spin-coupled method and we have performed computations on RBr efNH3 dimers characterized by a different electron withdrawing power of substituent \uf8ffR to the bromine atom. The analysis of typical spin-coupled descriptors (eg, shapes and overlaps of the spin-coupled orbitals, weights of the spin-coupled structures) in the different cases and in function of the distance between the monomers allowed us to draw qualitative conclusions about the formation and the strength of the halogen bonds. In particular, the investigation not only confirmed the validity of already existing models (ie, \u3c3-hole and lump-hole models) but also highlighted interesting new features, such as the fact that the depletion of electron density around the bromine atom does not extend only toward the acceptor of the halogen bond, but also in the opposite direction (toward the substituent of the halogen), thus forming a sort of \u3c3-tunnel, rather than a simple \u3c3-hole

Computer aided design and NMR characterization of an oligopeptide targeting the Ebola virus VP24 protein

The Ebola viral Protein 24 (VP24) inhibits interferon signaling through its interaction with the human protein Karyopherin, thus impairing the immune response of the host against the infection and increasing its rate of diffusion into the organism and its lethality. This makes VP24 a potential pharmacological target, as the inhibition of its interaction with Karyopherin could reduce Ebola virulence. In this work, we carried out an atomic level study of the network of interactions between VP24 and Karyopherin using molecular dynamics and computational alanine scanning. Modeling the VP24-Karyopherin complex allowed us to identify the amino acid residues responsible for protein-protein binding and led to the identification of a nonapeptide with VP24 binding potential. Subsequently, the ability of this peptide to actually bind VP24 in solution has been assayed using Saturation Transfer Difference NMR and Circular Dichroism. Experimental and molecular modeling data concerning the VP24-peptide complex have been compared and putative peptide binding sites and modes are discussed

Molecular dynamics simulations of p97 including covalent, allosteric and ATP-competitive inhibitors

Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of actio

Synthesis of Fully Dense Anatase TiO2 Through High Pressure Field Assisted Rapid

High density bulk nanostructured TiO2 was prepared through densification of anatase nanopowder by the high-pressure field-assisted rapid sintering method in the temperature range 600-1000 degrees C, under a uniaxial pressure of 620 MPa. In these sintering conditions samples characterized by a grain size between 40 and 150 nm have been obtained. Nearly full densification was observed for temperatures above 700 degrees C, while phase transition towards the rutile phase was observed in samples sintered at temperatures >= 800 degrees C. It was therefore possible, for the first time, to completely retain the anatase phase in bulk high-density samples

A kinetic study of the quartz-cristobalite phase transition

Cristobalite is a common silica polymorph in ceramics, as it can crystallize in SiO2-rich systems during high temp. processes. Its occurrence in final traditional ceramic bodies remarkably affects their thermal expansion, thus playing an important role in the shrinkage upon cooling. The quartz-cristobalite transformation kinetics is investigated by in-situ isothermal X-ray powder diffraction expts. and then correlated to the av. particle size (\u3008d\u3009) of the starting quartz using a model here developed. An Avrami-like rate equation, i.e. \u3b1(t) = 1 - exp(- k 7 t)n, in which the n-term is assumed to account for the dependence on the av. particle size, has provided the best fitting of theor. to exptl. data, yielding activation energy values that range from 181 to 234 kJ mol-1, and exponential n-coeffs. from 0.9 to 1.5. Ex-situ observations have demonstrated that the formation of cristobalite from quartz after 50 min, 2, 4 and 6 h at 1200 and 1300 \ub0C, exhibits a remarkable dependence on \u3008d\u3009 of quartz, showing comparable behaviors in the case of \u3008d\u3009 equal to 15.8 and 28.4 \u3bcm, but significant differences for \u3008d\u3009 of 4.1 \u3bcm. The formation of cristobalite is boosted remarkably at temp. higher than 1200 \ub0C, with an increase by wt. even of 500%, with respect to its content at lower temp. The method of sample prepn. (dry powder, wet powder and tablet of compressed dry powder) seems to influence the results only at temp. > 1200 \ub0C and in the case of fine powder

Transport properties in bulk nanocrystalline Sm-doped ceria with doping content between 2 and 30 at.%

Fully dense Sm-doped ceria powders, with doping content between 2 and 30 at.\% and grain size below 20 nm, were consolidated using a High-Pressure Field Assisted Sintering (HP-FAST) apparatus. Using a uniaxial pressure of 600 MPa relative densities above 95\% were achieved at temperatures as low as 600 degrees C with sintering times of only 5 min. Thanks to these exceptionally mild sintering conditions, grain growth during densification was very limited. The samples were characterized using impedance spectroscopy and the results showed a single semicircle, due to an overwhelming contribution of the grain boundary resistance. Thanks to the brick layer model, the present results were compared to literature data on microcrystalline materials and revealed that, contrary to what previously reported by several authors, negligible modification of the specific grain boundary conductivity can be produced through the reduction of the grain size in Sm-doped ceria in the considered doping interval