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Avram S.,University of Bucharest | Milac A.L.,Institute of Biochemistry of the Romanian Academy | Carta F.,University of Florence | Supuran C.T.,University of Florence
Journal of Enzyme Inhibition and Medicinal Chemistry | Year: 2012

Dithiocarbamates (DTC) are promising compounds with potential applications in antitumoral and glaucoma therapy. Our aim is to understand molecular features affecting DTC interaction with carbonic anhydrases (CAs), zinc-containing enzymes maintaining acid-base balance in blood and other tissues. To this end, we generate QSAR models based on a compound series containing 25 DTC, inhibitors of four human (h) CAs isoforms: hCA I, II, IX and XII. We establish that critical physicochemical parameters for DTC inhibitory activity are: hydrophobic, electronic, steric, topological and shape. The predictive power of our QSAR models is indicated by significant values of statistical coefficients: cross-validated correlation q2 (0.55-0.73), fitted correlation r2 (0.75-0.84) and standard error of prediction (0.47-0.23). Based on the established QSAR equations, we analyse 22 new DTC derivatives and identify DTC dicarboxilic acids derivatives and their esters as potentially improved inhibitors of CA I, II, IX and XII. © 2013 Informa UK, Ltd.

Popescu C.-I.,University of Lille Nord de France | Popescu C.-I.,Institute of Biochemistry of the Romanian Academy | Callens N.,University of Lille Nord de France | Trinel D.,Lille University of Science and Technology | And 8 more authors.
PLoS Pathogens | Year: 2011

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly. © 2011 Popescu et al.

Anishkin A.,University of Maryland University College | Milac A.L.,U.S. National Institutes of Health | Milac A.L.,Institute of Biochemistry of the Romanian Academy | Robert Guy H.,U.S. National Institutes of Health
Proteins: Structure, Function and Bioinformatics | Year: 2010

Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs. © 2009 Wiley-Liss, Inc.

Avram S.,University of Bucharest | Mihailescu D.,University of Bucharest | Borcan F.,Victor Babes University of Medicine and Pharmacy Timisoara | Milac A.-L.,Institute of Biochemistry of the Romanian Academy
Monatshefte fur Chemie | Year: 2012

Antimicrobial peptides are an important class of therapeutic agents used against a wide range of pathogens such as gram-negative and -positive bacteria, fungi, and viruses. The minimal inhibitory concentration at the level of the pathogen membrane is a major determinant of the pharmacokinetic behavior and, consequently, it can affect their antimicrobial activity. Here we generated quantitative structure-activity relationship models (3DQSAR- comparative molecular field analysis/comparative molecular similarity indices analysis) using a database of 33 mastoparan analogs, antimicrobial peptides with known experimental activity, and further used these models to predict the minimal inhibitory concentration for 18 new mastoparan analogs, obtained by computational mutagenesis. We discuss two options for structural alignment of mastoparan analogs: superposition of Ca trace atoms or superposition of all backbone atoms. Significant values of the cross-validated correlation q 2 (higher than 0.60) and the fitted correlation r 2 (higher than 0.90) of our models indicate that they are reliable enough for activity prediction in the case of new derivatives. This allows us to identify compounds with possibly enhanced antimicrobial activity against Bacillus subtilis, which are suggested for further experimental studies. © Springer-Verlag 2012.

Maekawa T.,Max Planck Institute For Pflanzenzuchtungsforschung | Cheng W.,Beijing Normal University | Cheng W.,China National Institute of Biological Sciences | Spiridon L.N.,Institute of Biochemistry of the Romanian Academy | And 12 more authors.
Cell Host and Microbe | Year: 2011

Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling. © 2011 Elsevier Inc.

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