Computational chemistry techniques for the design and the synthesis of molecules with anti-inflammatory and anti-tumor activity

2017 - 2018In the last few years, computational chemistry has played an important role in disclosing novel compounds with relevant biological activity. Inflammation and cancer processes have been recently linked, and the identification of new molecular entities able to interfere with biological targets involved in these pathologies is strongly needed. The analysis of ligand-macromolecule interactions and the evaluation of possible “binding modes” are the starting points in the design and the identification of new and more powerful drugs. Also, in silico Virtual Screening campaigns of large libraries of compounds on a specific target allow the selection of the most promising, leading the identification of new hits. Moreover, the coupling between versatile synthetic approaches and computational protocols is a powerful tool to obtain optimal results with time optimization. Thus, in this thesis both topics are applied at the same time, as they are connected to each other. The research work was mainly focused on mPGES-1 (microsomal Prostaglandin E2 Synthase) as a promising target for the treatment of the pathologies above. This enzyme is a homotrimeric membrane protein involved in the arachidonic acid cascade and it acts as downstream synthase in the cyclooxygenase pathway, catalyzing the conversion of the unstable peroxidic intermediate prostaglandin H2 (PGH2) in prostaglandin E2 (PGE2). Among the three different isoforms of the enzyme, selective inhibition of the inducible mPGES-1 may be considered a valid therapeutic approach to interfere with inflammation-induced PGE2 formation, avoiding to block the biosynthesis of constitutive prostanoids. Also, it is over-expressed in tumor pathologies. In light of this, in order to identify novel molecular platforms able to inhibit the activity of the enzyme strongly, a structure-based multi-step computational protocol was applied starting from selected privileged scaffolds coupled to a synthetic chemical route. Specifically, new libraries of 2-aminobenzothiazole, 2-aminothiadiazole and 2-carboxamidepyrrole-based molecules were generated, and they were docked onto the 3D structure of mPGES-1 (PDB code: 4BPM) crystallized in 2014. Using the interactions with the receptor counterpart as a qualitative filter, a collection of compounds for each library was selected, synthesized and submitted to biological investigation. Finally, cell-free and cell line assays confirmed some of them (benzothiazole derivatives 1, 3, 6, 9 and 13, thiadiazole derivatives 18-20 and 22, pyrrole derivatives 45 and 47) as novel promising mPGES-1 inhibitors. Furthermore, the same computational approach focused on mPGES-1 was applied on substituted saturated N-heterocycles, whose synthesis was performed at the Department of Chemistry and Applied Biosciences at ETH (Zurich). In detail, the libraries were docked onto the crystal structure co-crystallized with the inhibitor 6PW (PDB code: 5K0I), leading to the selection of 21 compounds (62-82). The synthesis was performed using the SnAP (Stannyl Amine Protocol) chemistry, which consists of a reaction between an aminotributylstannane and an aldehyde to form an imine as the first step, followed by an intramolecular cyclization. Biological investigations are in progress on the synthesized compounds. In order to deeply combine computational tools and this versatile and suitable synthetic approach, a large database of about 1,300,000 synthetically accessible compounds was created in silico starting from iSnAP (Iterative SnAP) reagents, a new generation of SnAP reagents bearing a further functional group. The prepared database was then submitted to virtual screening studies on pharmacologically promising targets (mPGES-1 up to now), in order to select the best candidates for the synthesis and the biological evaluation. Finally, the application of multi-step computational protocols including both ligand and structure based approaches took place in the identification of two novel 3-hydroxy-3-pyrrolin-2-one-based hits (compounds 87 and 88) as promising mPGES-1 inhibitors, starting from a large library of commercially available compounds. A second research work regarded Hsp90, a molecular chaperone involved in the development, survival and proliferation of cancer cells, regulating the homeostasis of oncoproteins. Most of the developed Hsp90 inhibitors interact with the N-terminal domain, but this type of modulation induces toxicity issues connected to the induction of the deleterious heat shock response (HSR). Contrariwise, since C-terminus inhibitors do not produce this effect and only a few of them have been developed so far, an aim of this research work consisted of the individuation of novel C-terminus binders. Thus, a biological screening on a small library of 48 commercially available compounds was performed and two novel inhibitors were identified (compounds 100 and 103) able to interact selectively with the C-terminal domain. Starting from these results, a computational rationalization was performed, applying Induced Fit Docking studies and Molecular Dynamic Simulations. In conclusion, in order to perform an optimization campaign, three large libraries of derivatives of compound 103 were generated using the multi-step computational protocol above, and a total number of 21 molecules have been selected for the synthesis and the biological evaluation. [edited by Author]XXXI cicl

Fatigue Damage Identification by a Global-Local Integrated Procedure for Truss-Like Steel Bridges

Civil steel structures and infrastructures, such as truss railway bridges, are often subject to potential damage, mainly due to fatigue phenomena and corrosion. Terefore, damage detection algorithms should be designed and appropriately implemented to increase their structural health. Today, the vast amount of information provided by data processing techniques and measurements coming from a monitoring system constitutes a possible tool for damage identifcation in terms of both detection and description. For this reason, the research activity aims to develop a methodology for a preliminary description of the damage in steel railway bridges induced by fatigue phenomena. Te proposed approach is developed through an integration of global and local pro cedures. At the global scale, vibration-based procedures will be applied to improve a forecast numerical model and, subsequently, to identify the zones most involved in fatigue problems. At the local scale, careful and refned local identifcation will be pursued via image processing techniques whose evidence will be analyzed and described through nonlinear numerical models. A case study of a historical railway bridge in Spain will illustrate the methodology’s performance, potentiality, and critical issue

Three-Dimensional (3D) Printed Silver Nanoparticles/Alginate/Nanocrystalline Cellulose Hydrogels: Study of the Antimicrobial and Cytotoxicity Efficacy

Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against S. aureus and P. aeruginosa was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction

An integrated vibration-image procedure for damage identification in steel trusses

The paper deals with structural damage identification in steel trusses. Classical procedure based on dynamic measurements able to detect flexibility changes are complemented with data predicting cracks and their evaluating by image processing. The procedure proposes first a damage index, the Stiffness Reduction Factor (SRF), evaluated on the basis of the error between the predictive truss model and the experimental modal model. Then, a nonlinear FEM model is used to determine fatigue cracks in the truss nodes which are compared with the observed ones determined by image processing. A real case study, the Quisi bridge located in Spain, is used to show the potentiality of the procedure.This paper is a part of a project that has received funding from the Research Fund for Coal and Steel under grant agreement No 800687. (DESDEMONA EU project

Dynamics and damage in the Quisi steel truss bridge

Quisi Bridge located in Benissa (Alicante) is an historic steel truss structure, which is investigated with different techniques to assess fatigue damages. A method based on vibration measurements, is illustrated to determine defect-induced reduction of stiffness, (combination of damage intensity and extension). Natural frequency change for a structural damage in a truss member is quantified through a classical FEM procedure in which a damaged truss element is defined and implemented. This model is used to produce pseudo-experimental response of a damaged truss which is dynamically excited by white noise. Stochastic Subspace Identification (SSI) has permitted to identify the main modal parameters related to both damaged and undamaged truss system. A damage index depending on Stiffness Reduction Factor (SRF) permits to determine the damage description based on measured quantities

Green Extraction of Cellulose Nanocrystals of Polymorph II from <i>Cynara scolymus</i> L.: Challenge for a “<i>Zero Waste</i>” Economy

The increase of agri-food wastes by agriculture and industries is one of the main causes of environmental pollution. Here we propose the recycling of Cynara scolymus L. wastes to obtain polymorph II cellulose nanocrystals (CNC). Two different extraction procedures are compared: (i) Soxhlet extraction in an ethanol/toluene mixture, and (ii) water boiling of the agricultural waste. Both procedures were followed by purification of cellulose fibers through bleaching treatments and extraction of cellulose nanocrystals by acid hydrolysis. CNCs have been extensively characterized by FTIR spectroscopy, electrophoretic light scattering measurements, X-ray powder diffraction methods, transmission electron microscopy, and thermogravimetric analyses. Extracted CNC are rod-like-shaped polymorph IIs with a good crystallinity index, and they are characterized by high hydrogen bonding intensity. The ELS measurements on samples from both procedures show good results regarding the stability of the CNC II sol (ζ Cynara scolymus L. waste can be used to produce high-quality cellulose nanocrystals as a green alternative to the commonly used synthetic route

Green geopolymer mortars for masonry buildings: effect of additives on their workability and mechanical properties

The fight against climate changes has encouraged the development of most researches on new binders, with the aim of progressively reducing the use of Ordinary Portland Cement, with its huge carbon footprint. Within this context, geopolymer-like binders can represent an eco-friendly alternative, also for the production of mortars for masonry buildings. In this work, a “one-pot” all in powder formulation was developed to the purpose, and special attention was given to explore the influence of different additives on mortar workability and mechanical properties. Different types of adhesives (like rice starch or maize starch, and alginate) were considered, which were separately applied at first, and finally mixed together with water retention additives and superplasticizers. The obtained results highlighted the achievement of an adequate workability and adhesion to the support, at the cost of reduced mechanical performances

Influence of alkali cations on the mechanical properties of geopolymer-like binders based on solid alkali activator.

An increasing attention is currently paid to the possible use of eco-friendly binding materials as alternative to Portland cement, like geopolymers. Geopolymers are synthetized through the alkaline activation of an aluminosilicate source. Usually, activation is achieved by the addition of concentrated alkali metal hydroxide or silicate solutions; however, the harmful and corrosive nature of these alkali solutions make their use quite difficult in the production of binders, especially when they are cast at the construction site. This work focuses on the development of an ambient cured one-part “just add water” geopolymer-like binder to be used for the production of mortars. Metakaolin powder was mixed with different alkali activators (Ca(OH)2 or CaO, and sodium or potassium metasilicate), all in powder form. The reaction kinetic of the so obtained binders was analyzed at different curing times through XRD and SEM techniques. The influence of the alkali cations on the mechanical properties was preliminarily investigated through double punching tests at different times. These results were also integrated with flexural and compressive tests on prismatic specimens, after 7 and 28 days from casting. This preliminary investigation indicates that the use of a dry alkali activator can be very promising for the production of geopolymer-like binders, allowing the reaching of superior mechanical properties with respect to the reference formulation with liquid activato

Anti-inflammatory celastrol promotes a switch from leukotriene biosynthesis to formation of specialized pro-resolving lipid mediators

The pentacyclic triterpenoid quinone methide celastrol (CS) from Tripterygium wilfordii Hook. F. effectively ameliorates inflammation with potential as therapeutics for inflammatory diseases. However, the molecular mechanisms underlying the anti-inflammatory and inflammation-resolving features of CS are incompletely understood. Here we demonstrate that CS potently inhibits the activity of human 5-lipoxygenase (5-LOX), the key enzyme in pro-inflammatory leukotriene (LT) formation, in cell-free assays with IC = 0.19-0.49 µM. Employing metabololipidomics using ultra-performance liquid chromatography coupled to tandem mass spectrometry in activated human polymorphonuclear leukocytes or M1 macrophages we found that CS (1 µM) potently suppresses 5-LOX-derived products without impairing the formation of lipid mediators (LM) formed by 12-/15-LOXs as well as fatty acid substrate release. Intriguingly, CS induced the generation of 12-/15-LOX-derived LM including the specialized pro-resolving mediator (SPM) resolvin D5 in human M2 macrophages. Finally, intraperitoneal pre-treatment of mice with 10 mg/kg CS strongly impaired zymosan-induced LT formation and simultaneously elevated the levels of SPM and related 12-/15-LOX-derived LM in peritoneal exudates, spleen and plasma in vivo. Conclusively, CS promotes a switch from LT biosynthesis to formation of SPM which may underlie the anti-inflammatory and inflammation-resolving effects of CS, representing an interesting pharmacological strategy for intervention with inflammatory disorders