Mechanism of RGD-conjugated nanodevice binding to its target protein integrin αVß3 by atomistic molecular dynamics and machine learning.

Active targeting strategies have been proposed to enhance the selective uptake of nanoparticles (NPs) by diseased cells, and recent experimental findings have proven the effectiveness of this approach. However, no mechanistic studies have yet revealed the atomistic details of the interactions between ligand-activated NPs and integrins. As a case study, here we investigate, by means of advanced molecular dynamics simulations (MD) and machine learning methods (namely equilibrium MD, binding free energy calculations and training of self-organized maps), the interaction of a cyclic-RGD-conjugated PEGylated TiO2 NP (the nanodevice) with the extracellular segment of integrin αVß3 (the target), the latter experimentally well-known to be over-expressed in several solid tumors. Firstly, we proved that the cyclic-RGD ligand binding to the integrin pocket is established and kept stable even in the presence of the cumbersome realistic model of the nanodevice. In this respect, the unsupervised machine learning analysis allowed a detailed comparison of the ligand/integrin binding in the presence and in the absence of the nanodevice, which unveiled differences in the chemical features. Then, we discovered that unbound cyclic RGDs conjugated to the NP largely contribute to the interactions between the nanodevice and the integrin. Finally, by increasing the density of cyclic RGDs on the PEGylated TiO2 NP, we observed a proportional enhancement of the nanodevice/target binding. All these findings can be exploited to achieve an improved targeting selectivity and cellular uptake, and thus a more successful clinical outcome.

Redox Potentials of Small Inorganic Radicals and Hexa-Aquo Complexes of First-Row Transition Metals in Water: A DFT Study Based on the Grand Canonical Ensemble.

The potentials of redox systems involving nitrogen, oxygen, and metal ions of the first-row transition series have been computed according to the general approach of the grand canonical ensemble, which leads to the equilibrium value of the reduction potential via a (complete) sampling of configuration space at a given temperature. The approach is a single configuration approach in the sense that identical molecular structures are sampled for both the oxidized and reduced species considered in water solution. In this study, the solute and a cluster of 11-12 water molecules are treated explicitly at the same level of theory and embedded in a continuum solvent. The molecular energies are computed in the framework of the density functional theory. Our approach is basically different from the approach based on the ThermoDynamic Cycle involving gas-phase calculations of the electron affinity of the oxidized species, corrected by the differential hydration energy (obtained from continuum solvent models only) between oxidized and reduced forms. The calculated redox potentials are in agreement with the available experimental data much closer than other results so far presented in the literature. Our results are very satisfactory also in the case of the 3+/2+ redox states of the first-row transition metals, i.e., systems with a high positive charge for which enhanced effects of the solvent are expected.

Structure prediction and functional analysis of KdsD, an enzyme involved in lipopolysaccharide biosynthesis.

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide and the O-antigen. The inner core region is highly conserved and contains at least one residue of 3-deoxy-D-manno-octulosonate (Kdo). The first committed step of Kdo biosynthesis is the aldol-keto isomerisation of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by arabinose 5-phosphate isomerase encoded in Escherichia coli by the kdsD gene. KdsD contains an N-terminal sugar isomerase (SIS) domain commonly found in phosphosugar isomerases but its three-dimensional structure is unknown. The structure of the KdsD SIS domain has been predicted by homology modeling using the hypothetical 3etn protein as a template. Moreover by sequence alignments, comparison with other sugar isomerases structurally related to KdsD, and site-directed mutagenesis we implicated four residues in KdsD activity or substrate recognition. A possible role of these residues in the catalysis is discussed.

CK2 regulates in vitro the activity of the yeast cyclin-dependent kinase inhibitor Sic1.

We have previously demonstrated that the cyclin-dependent kinase inhibitor (Cki) Sic1 of Saccharomyces cerevisiae is phosphorylated in vitro by the CK2 kinase on Ser(201) residue. Moreover, we have collected evidence showing that Sic1 is functionally and structurally related to mammalian Cki p27(Kip1) and binds to the mammalian Cdk2/cyclin A complex with a similar mode of inhibition. In this paper, we use SPR analysis to investigate the binding of Sic1 to the catatytic and regulatory subunits of CK2. Evidence is presented showing that phosphorylation of Sic1 at the CK2 consensus site QES(201)EDEED increases the binding of a Sic1-derived peptide to the Cdk2/cyclin A complex, a functional homologue of the yeast Cdk1/Clb5,6. Moreover, Sic1 fully phosphorylated in vitro on Ser(201) by CK2 is shown to be a stronger inhibitor of the Cdk/cyclin complexes than the unphosphorylated protein. Taken together, these data disclose the possibility that CK2 plays a role in the regulation of Sic1 activity.