New techniques of molecular modelling and structural chemistry for the development of bioactive compounds

2011 - 2012Computational chemistry represents today a valid and fast tool for the research of new compounds with potential biological activity. The analysis of ligand-macromolecule interactions and the evaluation of possible “binding modes” have a crucial role for the design and the development of new and more powerful drugs. In silico Virtual Screening campaigns of large libraries compounds (fragments or drug-like) on a specific target allow the selection of promising compounds, leading the identification of new scaffolds. The accurate analysis and the comparison of different bioactive compounds clarify the molecular basis of their interaction and the construction of pharmacoforic models. In parallel, another crucial aspect of pharmacological research is the identification of targets of interaction of bioactive molecules, and this is particularly true for compounds from natural sources. In fact, a wide range of drug tests on a large number of biological targets can represent a useful approach for the study of natural products, but often one of the main problems is their limited availability. Starting from these assumptions, a new computational method named Inverse Virtual Screening is described in details in this thesis. The different works based on this approach were performed considering panels of targets involved in the cancer events, determining the identification of the specific antitumor activity of the natural compounds investigated. Inverse Virtual Screening studies were performed by means of molecular docking experiments on different natural compounds, organized in small libraries or as single compounds. Firstly, a mathematical method for the exclusion of false positive and false negative results was proposed applying a normalization of the predicted binding energies (expressed in kcal/mol) obtained from the docking calculations. Then this approach was applied on a library of 10 compounds extracted from natural sources, obtaining a good validation through in vitro biological tests. Afterwards, another study was performed on the cyclopeptide namalide. Its biological inhibitory activity and selectivity on Carboxipeptidase A target was in accordance with Inverse Virtual Screening results. Virtual Screening topic was also inspected analyzing the efficacy of Molecular Dynamics-based methods for the accurate calculations of the binding affinities. This work was conducted on a library of 1588 compounds (44 ligands + 1544 decoys) extracted from the DUD database on trypsin target, using the Linear Interaction Energy (LIE) method by means of extensive Molecular Dynamics simulations. Four different LIE results obtained combining different scaling factors were compared with docking results, evaluating and comparing ROC and enrichment curves for each of the considered methods. Poor results were obtained with LIE, and further analysis with MM-GBSA and MM-PBSA approaches are under investigation. Moreover, in silico screenings were performed for the detailed study of natural compounds whose activities are known a priori. With this procedure, several binding modes were reported for a library of compounds on PXR target, whose activity or inactivity were rationalized comparing their binding poses with that of Solomonsterol A, used as a reference compound on this receptor. The presence/absence of biological activity of another library of compounds extracted from the marine sponge Plakinastrella Mamillaris on PPAR-γ and for the diterpene oridonin on HSP70 1A are described at a molecular level with molecular docking and Molecular Dynamics simulations. The putative binding modes for the reported molecules was described offering a complete rationalization of docking results, evaluating how ligand target specific interactions (e.g. hydrophobic, hydrophilic, electrostatic contacts) can influence their biological activity. [edited by author]La chimica computazionale rappresenata un valido e rapido strumento per l’identificazione di nuovi potenziali composti bioattivi. L’analisi delle interazioni ligando-target macromolecolare e la valutazione di un possibile “binding mode” sono cruciali per il design e lo sviluppo di nuovi potenziali farmaci. Il Virtual Screening di grandi librerie di composti (fragments o drug-like) condotto in silico su uno specifico recettore può permettere la selezione di composti dalla promettente attività, e parallelamente l’identificazione di nuovi scaffolds molecolari. L’analisi accurata dei modelli di interazione ligando-recettore e il confronto di tali modelli con quelli di composti dalla già nota attività permette la costruzione di un modello farmacoforico, punto di partenza per successivi studi di potenziamento dell’attività farmacologica. Parallelamente, un altro aspetto fondamentale della ricerca farmacologica è rappresentato dall’identificazione dei targets di interazione per composti dalla nota bioattività, e questo risulta particolarmente interessante per i composti di origine naturale. Infatti, per tale classe di molecole sarebbe molto utile effettuare tests biologici su un elevato numero di recettori, ma ciò risulta spesso proibitivo a causa della scarsa quantità di composto disponibile. Partendo da tali presupposti, nella presente tesi è descritto approfonditamente un nuovo metodo computazionale definito Inverse Virtual Screening. I vari lavori basati su questo nuovo tipo di approccio sono stati effettuati considerando pannelli composti da diversi targets coinvolti nello sviluppo del cancro, portando all’identificazione della specifica attività antitumorale dei vari composti naturali investigati. Gli studi basati sull’Inverse Virtual Screening sono stati effettuati attraverso calcoli di Molecular Docking utilizzando diversi composti naturali, raggruppati in piccole librerie o studiati singolarmente. In primo luogo, è stato proposto un metodo matematico con l’obiettivo di escludere i falsi positivi e i falsi negativi applicando una normalizzazione delle affinità di legame predette (espresse in kcal/mol). Successivamente, tale approccio è stato applicato su una libreria di 10 composti di origine naturale, validando l’applicabilità di tale metodo attraverso tests biologici in vitro. Successivamente, un ulteriore studio è stato incentrato su un ciclopeptide definito namalide, la cui attività biologica su Carbossipeptidasi A era in totale accordo con i dati provenienti dallo studio di Inverse Virtual Screening condotto. Il Virtual Screening è stato inoltre studiato anche analizzando l’efficacia dei metodi per il calcolo accurato delle affinità di legame basati su simulazioni di Dinamica Molecolare. Tale studio è stato condotto su una libreria di 1588 composti (44 ligandi + 1544 decoys, estratti dal DUD database) sul target tripsina, utilizzando il metodo LIE (Linear Interaction Energy) attraverso un elevato numero di simulazioni di Dinamica Molecolare. Sono stati ottenuti quattro differenti scale di affinità predetta (attraverso quattro combinazioni di differenti scaling factors) e sono stati confrontati con i risultati derivanti dai calcoli di Molecular Docking, valutando e confrontando curve ROC e di enrichment. Attraverso il metodo LIE sono stati ottenuti risultati non incoraggianti, e quindi ulteriori analisi attraverso metodi MM-GBSA e MM-PBSA sono in corso di studio. Inoltre, screenings in silico sono stati effettuati anche per lo studio dettagliato di altri composti naturali la cui attività era nota a priori. Attraverso questa procedura, sono stati proposti diversi modelli di binding di una libreria di composti sul target PXR, e per tali composti è stata razionalizzata l’attività/inattività confrontando il loro binding mode con quello del Solomonsterol A, utilizzato come composto di riferimento su tale target. La presenza/assenza di attività biologica è stata è stata descritto a livello molecolare per un’altra classe di composti estratti dalla spugna Plakinastrella Mamillaris sul target PPAR-γ e sul diterpene oridonina sul target HSP70 1A attraverso esperimenti combinati di Molecular Docking e Molecular Dynamics. Sono stati proposti e descritti approfonditamente modelli di binding di tali composti, valutando come specifiche interazioni ligando-target macromolecolare (di natura idrofobica, elettrostatica o caratterizzata dalla presenza di specifici legami ad idrogeno) possano influenzare l’attività biologica. [a cura dell'autore]XI n.s

Extensive Molecular Dynamics Simulations Disclosed the Stability of mPGES-1 Enzyme and the Structural Role of Glutathione (GSH) Cofactor

A deep in silico investigation of various microsomal prostaglandin E-2 synthase-1 (mPGES-1) protein systems is here reported using molecular dynamics (MD) simulations. Firstly, eight different proteins models (Models A-H) were built, starting from the active enzyme trimer system (Model A), namely that bound to three glutathione (GSH) cofactor molecules, and then gradually removing the GSHs (Models B-H), simulating each of them for 100 ns in explicit solvent. The analysis of the obtained data disclosed the structural role of GSH in the chemical architecture of mPGES-1 enzyme, thus suggesting the unlikely displacement of this cofactor, in accordance with experimentally determined protein structures co-complexed with small molecule inhibitors. Afterwards, Model A was submitted to microsecond-scale molecular dynamics simulation (total simulation time=10 mu s), in order to shed light about the dynamical behaviour of this enzyme at atomic level and to obtain further structural features and protein function information. We confirmed the structural stability of the enzyme machinery, observing a conformational rigidity of the protein, with a backbone RMSD of similar to 3 angstrom along the simulation time, and highlighting the strong active contribution of GSH molecules due to their active role in packing the protein chains through a tight binding at monomer interfaces. Furthermore, the focused analysis on R73 residue disclosed its role in solvent exchange events, probably excluding its function as route for GSH to enter towards the endoplasmic reticulum membrane, in line with the recently reported function of cap domain residues F44-D66 as gatekeeper for GSH entrance into catalytic site

Inverse Virtual Screening of Antitumor Targets: Pilot Study on a Small Database of Natural Bioactive Compounds

An inverse virtual screening in silico approach has been applied to natural bioactive molecules to screen their efficacy against proteins involved in cancer processes, with the aim of directing future experimental assays. Docking studies were performed on a panel of 126 protein targets extracted from the Protein Data Bank, to analyze their possible interactions with a small library of 43 bioactive compounds. Analysis of the molecular docking results was performed through the use of tables containing energy data organized in a matrix. The application of this approach may facilitate the prediction of the activity of unknown ligands for known targets involved in the development of cancer and could be applied to other models based on different libraries of ligands and different panels of targets

Target identification by structure-based computational approaches: Recent advances and perspectives

The use of computational techniques in the early stages of drug discovery has recently experienced a boost, especially in the target identification step. Finding the biological partner(s) for new or existing synthetic and/or natural compounds by "wet" approaches may be challenging; therefore, preliminary in silico screening is even more recommended. After a brief overview of some of the most known target identification techniques, recent advances in structure-based computational approaches for target identification are reported in this digest, focusing on Inverse Virtual Screening and its recent applications. Moreover, future perspectives concerning the use of such methodologies, coupled or not with other approaches, are analyzed

Inverse Virtual Screening allows the discovery of the biological activity of natural compounds

A small library of phenolic natural compounds belonging to different chemical classes was screened on a panel of targets involved in the genesis and progression of cancer. The re-investigation of their potential activity was achieved through the Inverse Virtual Screening approach. The normalization of the predicted binding energies permitted the selection of promising compounds on definite targets, avoiding the selection of false positive results. In vitro biological tests revealed the inhibitory activity of xanthohumol and isoxanthohumol on PDK1 and PKC protein kinases. This study validates the robustness of the Inverse Virtual Screening in silico approach as a useful tool for the identification of the specific biological activity of a given set of compounds

Insights into the Ligand Binding to Bromodomain-Containing Protein 9 (BRD9): A Guide to the Selection of Potential Binders by Computational Methods

The estimation of the binding of a set of molecules against BRD9 protein was carried out through an in silico molecular dynamics-driven exhaustive analysis to guide the identification of potential novel ligands. Starting from eight crystal structures of this protein co-complexed with known binders and one apo form, we conducted an exhaustive molecular docking/molecular dynamics (MD) investigation. To balance accuracy and an affordable calculation time, the systems were simulated for 100 ns in explicit solvent. Moreover, one complex was simulated for 1 µs to assess the influence of simulation time on the results. A set of MD-derived parameters was computed and compared with molecular docking-derived and experimental data. MM-GBSA and the per-residue interaction energy emerged as the main indicators for the good interaction between the specific binder and the protein counterpart. To assess the performance of the proposed analysis workflow, we tested six molecules featuring different binding affinities for BRD9, obtaining promising outcomes. Further insights were reported to highlight the influence of the starting structure on the molecular dynamics simulations evolution. The data confirmed that a ranking of BRD9 binders using key parameters arising from molecular dynamics is advisable to discard poor ligands before moving on with the synthesis and the biological tests

Una nuova chiesa nel cimitero di Siano

Il Comune di Siano (SA) ha indetto un concorso di idee per individuare la soluzione progettuale più idonea per la riqualificazione dell'area cimiteriale interessata dall'ossario comunale, mediante la costruzione di una Chiesa nel Cimitero Comunale e sistemazione della cripta sottostante con costruzione di loculi e ossari. La finalità dell'intervento è di dotare il civico cimitero di una Chiesa, da edificare sul solaio di copertura della sottostante cripta, in cui dovranno essere previsti loculi e ossari. Si dovrà anche sistemare l'area a stretto ridosso della Chiesa, riqualificando, in tal modo, il contesto cimiteriale. Nella presentazione della proposta progettuale i candidati dovranno tenere conto delle seguenti finalità realizzazione di una chiesa, con struttura portante a scelta del concorrente (c.a. muratura, acciaio, legno), idonea ad essere edificata al di sopra della struttura muraria esistente, con dimensioni in pianta determinate dall'ingombro di quest'ultima. la struttura dovrà garantire la celebrazione di riti religiosi, accogliendo al suo interno un numero di persone commisurato alle dimensioni della superficie di calpestio, determinata dalla struttura sottostante su cui si eleva. dovrà garantire, attraverso idonei sistemi di tamponamento perimetrale, anche scorrevoli, a scelta dei concorrenti, l'espletamento dei riti religiosi celebrati durante le pubbliche ricorrenze ( 2 novembre) l'altezza e l'involucro edilizio dovranno garantire un inserimento paesaggistico armonioso e rispettoso delle strutture esistenti e del contesto in cui è inserito. l'intervento inoltre, dovrà mirare a realizzare, nella parte sottostante, un idoneo numero di loculi e ossari, opportunamente dislocati in aderenza alle murature perimetrali, con accesso dal vano porta esistente, opportunamente ridisegnato al fine di garantirne il libero accesso

Pharmacological evaluation and docking studies of α,β-unsaturated carbonyl based synthetic compounds as inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and proinflammatory cytokines

Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel alpha,beta-unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A(2) (sPLA(2)), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-alpha. Six alpha,beta-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA(2) activity, with IC50 values in the range of 2.19-8.76 mu M. Nine compounds 1-4 and 10-14 displayed inhibition of COX-1 with IC50 values ranging from 0.37 to 1.77 mu M (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-alpha and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3,4, 12,13 and 14 were found strong inhibitors of TNF-alpha and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these alpha,beta-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents. (C) 2014 Elsevier Ltd. All rights reserved