Pietro Cozzini

Target Flexibility: An Emerging Consideration in Drug Discovery and Design†

Department of General and Inorganic Chemistry, UniVersity of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy, National Institute for Biosystems and Biostructures, Rome, Italy, Department of Medicinal Chemistry and Institute for Structural Biology & Drug DiscoVery, Virginia Commonwealth UniVersity, Richmond, Virginia 23298-0540, Department of Pharmaceutics, UniVersity of Parma, Via GP Usberti 27/A, 43100 Parma, Italy, High Performance Systems, CINECA Supercomputing Centre, Casalecchio di Reno, Bologna, Italy, Dulbecco Telethon Institute, Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy, Department of Mathematics and Natural Sciences, Pharmaceutical Institute, Christian-Albrechts-UniVersity, Gutenbergstrasse 76, 24118 Kiel, Germany, Departments of Biochemistry & Molecular Biology, Computer Science & Engineering, and Physics & Astronomy, Michigan State UniVersity, East Lansing, Michigan 48824-1319, Department of Molecular Biology, MB-5, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037-1000, Molecular Modeling and Bioinformatics Unit, Institute of Biomedical Research, Scientific Park of Barcelona, Department of Biochemistry and Molecular Biology, UniVersity of Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, Department of Experimental Medicine, UniVersity of Parma, Via Volturno, 39, 43100, Parma, Italy, Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, UniVersity of Piemonte Orientale “Amedeo AVogadro”, Via BoVio 6, 28100 NoVara, Italy, Institute of Pharmacy and Food Chemistry, UniVersity of Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Hydrazones of 1,2-benzisothiazole hydrazides: synthesis, antimicrobial activity and QSAR investigations.

A series of hydrazones of 1,2-benzisothiazole hydrazides 1a-m, 2a-m, 3a-m, 4a-m, 5a-m as well as their cyclic (1 and 4) and acyclic (2, 3 and 5) 1,2-benzisothiazole parent hydrazides, were synthesised and evaluated as antibacterial and antifungal agents. All of the 2-amino-1,2-benzisothiazol-3(2H)-one derivatives, belonging to series I and IV, showed a good antibacterial activity against Gram positive bacteria. Most of them were active against yeasts too. Compounds 1 and 4, together with 1l, proved to be the most effective compounds. Quantitative structure-activity relationship (QSAR) investigation with a 2D-QSAR analysis was applied to find a correlation between different experimental or calculated physicochemical parameters of the compounds studied. A 3D-QSAR study was performed, applying Comparative Molecular Similarity Indices Analysis (CoMSIA) method, to derive quantitative models relating the structural features of 1,2-benzisothiazole derivatives 1, 1a-m and 4, 4a-m and their antimicrobial activity against Bacillus subtilis, resulted the most sensitive micro-organism.

Free Energy of Ligand Binding to Protein: Evaluation of the Contribution of Water Molecules by Computational Methods

One of the more challenging issues in medicinal chemistry is the computation of the free energy of ligand binding to macromolecular targets. This allows for the screening of libraries of chemicals for fast and inexpensive identification of lead compounds. Many attempts have been made and several algorithms have been developed for this purpose. Whereas enthalpic contributions are evaluated using methods and equations for which there is a reasonable consensus among researchers, the entropic contribution is evaluated using very different, and, in some cases, very approximate methods, or it is entirely ignored. Entropic contributions are of primary importance in the formation of many ligand-protein complexes, as well as in protein folding. The hydrophobic interaction, associated with the release of water molecules from the protein active site and the ligand, plays a significant role in complex formation, predominantly contributing to the total entropy change and, in some cases, to the total free energy of binding. There are distinct approaches for the evaluation of the contribution of water molecules to the free energy of binding based on Newtonian mechanics force fields, multi-parameter empirical scoring functions and experimental force fields. This review describes these methods -- discussing both their advantages and limitations. Particular emphasis will be placed on HINT (Hydropatic INTeractions), a natural force field that takes into account in a unified way enthalpic and entropic contributions of all interacting atoms in protein-ligand complexes, including released and structured water molecules. As a case-study, the contribution of water molecules to the binding free energy of HIV-1 protease inhibitors is evaluated.

Robust classification of "relevant" water molecules in putative protein binding sites.

A statistically validated protocol to identify relevant water molecules in protein binding sites using HINT score and a geometric descriptor termed Rank is described. In training, conservation/nonconservation was modeled for 86% of the waters. For the test set, 87% of waters were correctly classified (92% when crystallographic resolution was <or=2.0 A). Conserved waters make at least two hydrogen bonds with protein and gain 0.6-2.0 kcal mol(-1) more binding energy than nonconserved waters.

Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature

The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing l-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. Four-hundred MNXXI pentapeptides were generated in silico, docked into OASS active site using GOLD, and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4 and, to a lesser extent, at position P3 also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptide-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.

Energetics of the protein-DNA-water interaction

BackgroundTo understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions) computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species.ResultsAn initial correlation between the calculated HINT scores and the experimentally determined binding free energies in the protein-DNA system exhibited a relatively poor r2 of 0.21 and standard error of ± 1.71 kcal mol-1. However, the inclusion of 261 waters that bridge protein and DNA improved the HINT score-free energy correlation to an r2 of 0.56 and standard error of ± 1.28 kcal mol-1. Analysis of the water role and energy contributions indicate that 46% of the bridging waters act as linkers between amino acids and nucleotide bases at the protein-DNA interface, while the remaining 54% are largely involved in screening unfavorable electrostatic contacts.ConclusionThis study quantifies the key energetic role of bridging waters in protein-DNA associations. In addition, the relevant role of hydrophobic interactions and entropy in driving protein-DNA association is indicated by analyses of interaction character showing that, together, the favorable polar and unfavorable polar/hydrophobic-polar interactions (i.e., desolvation) mostly cancel.

Bound Water at Protein-Protein Interfaces: Partners, Roles and Hydrophobic Bubbles as a Conserved Motif

Background There is a great interest in understanding and exploiting protein-protein associations as new routes for treating human disease. However, these associations are difficult to structurally characterize or model although the number of X-ray structures for protein-protein complexes is expanding. One feature of these complexes that has received little attention is the role of water molecules in the interfacial region. Methodology A data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å) X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on the HINT forcefield and hydrogen-bonding geometry. A metric termed Relevance was used to classify the general roles of the water molecules. Results The water molecules were found to be involved in: a) (bridging) interactions with both proteins (21%), b) favorable interactions with only one protein (53%), and c) no interactions with either protein (26%). This trend is shown to be independent of the crystallographic resolution. Interactions with residue backbones are consistent for all classes and account for 21.5% of all interactions. Interactions with polar residues are significantly more common for the first group and interactions with non-polar residues dominate the last group. Waters interacting with both proteins stabilize on average the proteins interaction (−0.46 kcal mol−1), but the overall average contribution of a single water to the protein-protein interaction energy is unfavorable (+0.03 kcal mol−1). Analysis of the waters without favorable interactions with either protein suggests that this is a conserved phenomenon: 42% of these waters have SASA ≤ 10 Å2 and are thus largely buried, and 69% of these are within predominantly hydrophobic environments or “hydrophobic bubbles”. Such water molecules may have an important biological purpose in mediating protein-protein interactions.

Identification of Xenoestrogens in Food Additives by an Integrated in Silico and in Vitro Approach

In the search for xenoestrogens within food additives, we have analyzed the Joint FAO-WHO expert committee database, containing 1500 compounds, using an integrated in silico and in vitro approach. This analysis identified 31 potential estrogen receptor alpha ligands that were reduced to 13 upon applying a stringent filter based on ligand volume and binding mode. Among the 13 potential xenoestrogens, four were already known to exhibit an estrogenic activity, and the other nine were assayed in vitro, determining the binding affinity to the receptor and biological effects. Propyl gallate was found to act as an antagonist, and 4-hexylresorcinol was found to act as a potent transactivator; both ligands were active at nanomolar concentrations, as predicted by the in silico analysis. Some caution should be issued for the use of propyl gallate and 4-hexylresorcinol as food additives.

In silico approach to evaluate molecular interaction between mycotoxins and the estrogen receptors ligand binding domain: A case study on zearalenone and its metabolites

An association of virtual screening, docking and a good rescoring procedure is a well known technique to discover and design new lead compounds in medicinal chemistry. We have demonstrated that the study on the interactions of unsuspected molecules with estrogen receptors, using the same technique applied in medicinal chemistry could be a valuable choice to discover new hypothetical xenoestrogens. The same approach can be applied to a wide set of chemicals found in food and seed. We propose this approach using as a case study on the zearalenone family to food safety. Zearalenone and its reductive metabolites are a well known set of mycotoxins able to bind estrogen receptors (ERs) thereby interfering with the endogenous estrogenic response. Their endocrine disrupting behavior is tightly related to their capability to competitively bind the ligand binding pocket (LBP) and to stabilize at least one of the functionally active conformational assets of the ligand binding domain (LBD). Altought proposing an interactive model for three-dimensional complexes not yet solved, this kind of computational aided analysis is a potentially intriguing tool to predict the binding event and to evaluate the xenoestrogenic role of any kind of chemicals and derivatives.

Energy-based prediction of amino acid-nucleotide base recognition

Despite decades of investigations, it is not yet clear whether there are rules dictating the specificity of the interaction between amino acids and nucleotide bases. This issue was addressed by determining, in a dataset consisting of 100 high‐resolution protein‐DNA structures, the frequency and energy of interaction between each amino acid and base, and the energetics of water‐mediated interactions. The analysis was carried out using HINT, a non‐Newtonian force field encoding both enthalpic and entropic contributions, and Rank, a geometry‐based tool for evaluating hydrogen bond interactions. A frequency‐ and energy‐based preferential interaction of Arg and Lys with G, Asp and Glu with C, and Asn and Gln with A was found. Not only favorable, but also unfavorable contacts were found to be conserved. Water‐mediated interactions strongly increase the probability of Thr‐A, Lys‐A, and Lys‐C contacts. The frequency, interaction energy, and water enhancement factors associated with each amino acid–base pair were used to predict the base triplet recognized by the helix motif in 45 zinc fingers, which represents an ideal case study for the analysis of one‐to‐one amino acid–base pair contacts. The model correctly predicted 70.4% of 135 amino acid–base pairs, and, by weighting the energetic relevance of each amino acid–base pair to the overall recognition energy, it yielded a prediction rate of 89.7%.