Blasco Morozzo della Rocca

Targeting tumor cells through chitosan-folate modified microcapsules loaded with camptothecin.

Poly(vinyl alcohol) microcapsules have been tailored as carriers to deliver camptothecin, an anticancer drug poorly soluble in water. The capsules have been reacted with a chitosan--folate complex in order to selectively target cancer cells overexpressing the folic acid receptor. Microcapsules decorated with the chitosan--folate complex have been characterized in their uptake and release of camptothecin, following the absorption band at λ = 370 nm diagnostic of the drug molecule. The selectivity of chitosan-folate microcapsules in targeting cancer cells has been demonstrated by fluorescence microscopy using HeLa cells, overexpressing the folate receptor and NIH3t3 fibroblasts as a negative control. The chitosan--folate microcapsules loaded with camptothecin significantly reduce the proliferation of HeLa tumor cells, while they have a negligible effect on fibroblasts. This work demonstrates that the chitosan--folate microcapsules represent a promising system to selectively target hydrophobic drugs, such as camptothecin, to tumor cells.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.MethodsThe three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.ResultsThe three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.ConclusionsThese results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier : a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.

A Simple and Fast Semiautomatic Procedure for the Atomistic Modeling of Complex DNA Polyhedra

A semiautomatic procedure to build complex atomistic covalently linked DNA nanocages has been implemented in a user-friendly, free, and fast program. As a test set, seven different truncated DNA polyhedra, composed by B-DNA double helices connected through short single-stranded linkers, have been generated. The atomistic structures, including a tetrahedron, a cube, an octahedron, a dodecahedron, a triangular prism, a pentagonal prism, and a hexagonal prism, have been probed through classical molecular dynamics and analyzed to evaluate their structural and dynamical properties and to highlight possible building faults. The analysis of the simulated trajectories also allows us to investigate the role of the different geometries in defining nanocages stability and flexibility. The data indicate that the cages are stable and that their structural and dynamical parameters measured along the trajectories are slightly affected by the different geometries. These results demonstrate that the constraints imposed by the covalent links induce an almost identical conformational variability independently of the three-dimensional geometry and that the program presented here is a reliable and valid tool to engineer DNA nanostructures.

ADP/ATP mitochondrial carrier MD simulations to shed light on the structural-dynamical events that, after an additional mutation, restore the function in a pathological single mutant.

Molecular dynamics simulations of the wild type bovine ADP/ATP mitochondrial carrier, and of the single Ala113Pro and double Ala113Pro/Val180Met mutants, embedded in a lipid bilayer, have been carried out for 30ns to shed light on the structural-dynamical changes induced by the Val180Met mutation restoring the carrier function in the Ala113Pro pathologic mutant. Principal component analysis indicates that, for the three systems, the protein dynamics is mainly characterized by the motion of the matrix loops and of the odd-numbered helices having a conserved proline in their central region. Analysis of the motions shows a different behaviour of single pathological mutant with respect of the other two systems. The single mutation induces a regularization and rigidity of the H3 helix, lost upon the introduction of the second mutation. This is directly correlated to the salt bridge distribution involving residues Arg79, Asp134 and Arg234, hypothesized to interact with the substrate. In fact, in the wild type simulation two stable inter-helices salt bridges, crucial for substrate binding, are present almost over all the simulation time. In line with the impaired ADP transport, one salt interaction is lost in the single mutant trajectory but reappears in the double mutant simulation, where a salt bridge network matching the wild type is restored. Other important structural-dynamical properties, such as the trans-membrane helices mobility, analyzed via the principal component analysis, are similar for the wild type and double mutant while are different for the single mutant, providing a mechanistic explanation for their different functional properties.

Mapping multiple potential ATP binding sites on the matrix side of the bovine ADP/ATP carrier by the combined use of MD simulation and docking

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier—AAC—was crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). The protein consists of a six-transmembrane helix bundle that defines the nucleotide translocation pathway, which is closed towards the matrix side due to sharp kinks in the odd-numbered helices. In this paper, we describe the interaction between the matrix side of the AAC transporter and the ATP4− molecule using carrier structures obtained through classical molecular dynamics simulation (MD) and a protein–ligand docking procedure. Fifteen structures were extracted from a previously published MD trajectory through clustering analysis, and 50 docking runs were carried out for each carrier conformation, for a total of 750 runs (“MD docking”). The results were compared to those from 750 docking runs performed on the X-ray structure (“X docking”). The docking procedure indicated the presence of a single interaction site in the X-ray structure that was conserved in the structures extracted from the MD trajectory. MD docking showed the presence of a second binding site that was not found in the X docking. The interaction strategy between the AAC transporter and the ATP4− molecule was analyzed by investigating the composition and 3D arrangement of the interaction pockets, together with the orientations of the substrate inside them. A relationship between sequence repeats and the ATP4− binding sites in the AAC carrier structure is proposed.

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling.

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an α-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.

A new nitrobenzoxadiazole-based GSTP1-1 inhibitor with a previously unheard of mechanism of action and high stability

Abstract Context: The nitrobezoxadiazole derivative NBDHEX is a potent inhibitor of glutathione transferase P1-1 (GSTP1-1) endowed with outstanding anticancer activity in different tumor models. Objective: To characterize by in vitro biochemical and in silico studies the NBDHEX analogues named MC2752 and MC2753. Materials and methods: Synthesis of MC2752 and MC2753, biochemical assays and in silico docking and normal-mode analyses. Results: The presence of a hydrophobic moiety in the side chain of MC2753 confers unique features to this molecule. Unlike its parent drug NBDHEX, MC2753 does not require GSH to trigger the dissociation of the complex between GSTP1-1 and TRAF2, and displays high stability towards the nucleophilic attack of the tripeptide under physiological conditions. Discussion and conclusion: MC2753 may represent a lead compound for the development of novel GSTP1-1 inhibitors not affected in their anticancer action by fluctuations of cellular GSH levels, and characterized by an increased half-life in vivo. Graphical Abstract