Methods for the Analysis of Matched Molecular Pairs and Chemical Space Representations

Compound optimization is a complex process where different properties are optimized to increase the biological activity and therapeutic effects of a molecule. Frequently, the structure of molecules is modified in order to improve their property values. Therefore, computational analysis of the effects of structure modifications on property values is of great importance for the drug discovery process. It is also essential to analyze chemical space, i.e., the set of all chemically feasible molecules, in order to find subsets of molecules that display favorable property values. This thesis aims to expand the computational repertoire to analyze the effect of structure alterations and visualize chemical space. Matched molecular pairs are defined as pairs of compounds that share a large common substructure and only differ by a small chemical transformation. They have been frequently used to study property changes caused by structure modifications. These analyses are expanded in this thesis by studying the effect of chemical transformations on the ionization state and ligand efficiency, both measures of great importance in drug design. Additionally, novel matched molecular pairs based on retrosynthetic rules are developed to increase their utility for prospective use of chemical transformations in compound optimization. Further, new methods based on matched molecular pairs are described to obtain preliminary SAR information of screening hit compounds and predict the potency change caused by a chemical transformation. Visualizations of chemical space are introduced to aid compound optimization efforts. First, principal component plots are used to rationalize a matched molecular pair based multi-objective compound optimization procedure. Then, star coordinate and parallel coordinate plots are introduced to analyze drug-like subspaces, where compounds with favorable property values can be found. Finally, a novel network-based visualization of high-dimensional property space is developed. Concluding, the applications developed in this thesis expand the methodological spectrum of computer-aided compound optimization

X-ray microtomography measurements of bioactive glass scaffolds in rabbit femur samples at multiple stages of bone regeneration : reduction of image artefacts and a preliminary segmentation

A series of x-ray microtomography (micro-CT) measurements was performed on a set of rabbit femur bone samples containing artificial scaffolds of bioactive glass BAG-S53P4, implanted into an intentionally induced defect, i.e. a gap, in the femur. The scaffolds, some additionally enveloped in PLGA, were supportive structures composed of small granules of bioactive glass, intended to enhance, stimulate and guide the healing and regeneration of bone. The 34 samples were harvested from the rabbits at three different stages of healing and bone regeneration: 2 weeks, 4 weeks and 8 weeks. In addition to 27 samples that contained scaffolds of BAG-S53P4 or BAG-S53P4-PLGA, which had been implanted into the femur of a rabbit, 3 scaffolds of BAG-S53P4(-PLGA) that were not implanted and 7 control samples containing inert PMMA-implants were also included in the measurements for comparison. During the healing process the bioactive glass granules are gradually dissolved into the surrounding bodily fluids and a thin reaction layer composed of silica gel forms onto the surfaces of the granules. Subsequently an additional surface layer composed of HCA, a material that closely resembles natural hydroxyapatite, is formed onto the granules. As the healing process to regenerate the bone in the gap progresses, a complex three-dimensional network of newly formed trabecular bone grows in between the granules, attaching onto the surface layers and eventually enveloping the gradually dissolving granules entirely. Ultimately, the scaffold is intended to degrade completely, and a structure of regenerated, remodeled cortical bone is expected to be formed into the volume of the initial defect. As the thicknesses of both the surface layers of the granules and the individual trabeculae of the newly formed bone are in the micrometre range, x-ray microtomography was employed to evaluate and assess the complex three-dimensional structure, consisting of trabecular bone intertwined with granules at varying stages of dissolution. By evaluating the rate of formation of these structures at three different stages, i.e. time points, of regeneration, valuable information on the effectiveness of the bioactive glass BAG-S53P4(-PLGA) for the regeneration of defected bone can be obtained. The measurements were performed at University of Helsinki’s Laboratory of Microtomography using its Nanotom-apparatus with 80kV voltage, 150microA current and a voxel size of 15micrometres. 1000 projection images per sample were used in 37 reconstructions utilizing the FBP-algorithm. Subsequent image processing to analyze and compare the samples was conducted using ImageJ. A procedure to reduce image artefacts – due to metal parts in the samples – was developed, utilizing Gaussian filtering, as well as a preliminary image segmentation scheme, utilizing Morphological filtering, to automatically separate the bone from the granules and their surface layers

Multi-faceted Structure-Activity Relationship Analysis Using Graphical Representations

A core focus in medicinal chemistry is the interpretation of structure-activity relationships (SARs) of small molecules. SAR analysis is typically carried out on a case-by-case basis for compound sets that share activity against a given target. Although SAR investigations are not a priori dependent on computational approaches, limitations imposed by steady rise in activity information have necessitated the use of such methodologies. Moreover, understanding SARs in multi-target space is extremely difficult. Conceptually different computational approaches are reported in this thesis for graphical SAR analysis in single- as well as multi-target space. Activity landscape models are often used to describe the underlying SAR characteristics of compound sets. Theoretical activity landscapes that are reminiscent of topological maps intuitively represent distributions of pair-wise similarity and potency difference information as three-dimensional surfaces. These models provide easy access to identification of various SAR features. Therefore, such landscapes for actual data sets are generated and compared with graph-based representations. Existing graphical data structures are adapted to include mechanism of action information for receptor ligands to facilitate simultaneous SAR and mechanism-related analyses with the objective of identifying structural modifications responsible for switching molecular mechanisms of action. Typically, SAR analysis focuses on systematic pair-wise relationships of compound similarity and potency differences. Therefore, an approach is reported to calculate SAR feature probabilities on the basis of these pair-wise relationships for individual compounds in a ligand set. The consequent expansion of feature categories improves the analysis of local SAR environments. Graphical representations are designed to avoid a dependence on preconceived SAR models. Such representations are suitable for systematic large-scale SAR exploration. Methods for the navigation of SARs in multi-target space using simple and interpretable data structures are introduced. In summary, multi-faceted SAR analysis aided by computational means forms the primary objective of this dissertation

Bioactive conformational ensemble server and database. A public framework to speed up in silico drug discovery.

Modern high-throughput structure-based drug discovery algorithms consider ligand flexibility, but typically with low accuracy, which results in a loss of performance in the derived models. Here we present the Bioactive Conformational Ensemble (BCE) server and its associated database. The server creates conformational ensembles of drug-like ligands and stores them in the BCE database, where a variety of analyses are offered to the user. The workflow implemented in the BCE server combines enhanced sampling molecular dynamics with self-consistent reaction field quantum mechanics (SCRF/QM) calculations. The server automatizes all the steps to transform 1D or 2D representation of drugs into three dimensional molecules, which are then titrated, parametrized, hydrated and optimized before being subjected to Hamiltonian replica-exchange (HREX) molecular dynamics simulations. Ensembles are collected and subjected to a clustering procedure to derive representative conformers, which are then analyzed at the SCRF/QM level of theory. All structural data is organized in a noSQL database accessible through a graphical interface and in a programmatic manner through a REST API. The server allows the user to define a private workspace and offers a deposition protocol as well as input files for "in house" calculations in those cases where confidentiality is a must. The database and the associated server are available at https://mmb.irbbarcelona.org/BC

Development and in vitro characterization of three dimensional biodegradable scaffolds for peripheral nerve tissue engineering

Tissue engineering emerges nowadays to seek new solutions to damaged tissues and/or organs by replacing or repairing them with engineered constructs or scaffolds. In nerve tissue engineering, scaffolds for the repair of peripheral nerve injuries should act to support and promote axon growth following implantation. It is believed that substantial progress can be made by creating scaffolds from biomaterials, with growth-promoting molecules and spatially-controlled microstructure. To this end, this research aims to develop three dimensional (3D) scaffolds for peripheral nerve tissue regeneration by focusing on studies on the axon guidance, development and characterization of a novel 3D scaffold, and visualization of scaffolds by means of synchrotron-based diffraction enhanced imaging (DEI). Axon guidance is one of crucial considerations in developing of nerve scaffolds for nerve regeneration. In order to study the axon guidance mechanism, a two dimensional (2D) grid micropatterns were created by dispensing chitosan or laminin-blended chitosan substrate strands oriented in orthogonal directions; and then used in the in vitro dorsal root ganglion (DRG) neuron culture experiments. The results show the effect of the micropatterns on neurite directional growth can preferentially grow upon and follow the laminin-blended chitosan pathways. A novel 3D scaffold was developed for potential applications to peripheral nerve tissue engineering applications. The scaffolds were fabricated from poly L-lactide (PLLA) mixed with chitosan microspheres (CMs) by using a rapid freeze prototyping (RFP) technique, allowing for controllable scaffold microstructure and bioactivities protein release. The scaffold characterization shows that (1) the mechanical properties of the scaffolds depend on the ratio of CMs to PLLA as well as the cryogenic temperature and (2) the protein release can be controlled by adjusting the crosslink degree of the CMs and prolonged after the CMs were embedded into the PLLA scaffolds. Also, the degradation properties of the scaffolds were investigated with the results showing that the addition of CMs to PLLA can decrease the degradation rate as compared to pure PLLA scaffolds. This allows for another means to control the degradation rate. Visualization of polymer scaffolds in soft tissues is challenging, yet essential, to the success of tissue engineering applications. The x-ray diffraction enhanced imaging (DEI) method was explored for the visualization of the PLLA/CMs scaffolds embedded in soft tissues. Among various methods examined, including conventional radiography and in-line phase contrast imaging techniques, the DEI was the only technique able to visualize the scaffolds embedded in unstained muscle tissue as well as the microstructure of muscle tissue. Also, it has been shown that the DEI has the capacity to image the scaffolds in thicker tissue, and reduce the radiation doses to tissues as compared to conventional radiography. The methods and results developed/obtained in this study represent a substantial progress in the development and characterization of 3D scaffolds. This progress forms a basis for the future tests on the scaffolds as applied for peripheral nerve injuries

Cell adhesion evaluation of laser-sintered HAp and 45S5 bioactive glass coatings on micro-textured zirconia surfaces using MC3T3-E1 osteoblast-like cells

Laser texturing is a technique that has been increasingly explored for the surface modification of several materials on different applications. Laser texturing can be combined with conventional coating techniques to functionalize surfaces with bioactive properties, stimulating cell differentiation and adhesion. This study focuses on the cell adhesion of laser-sintered coatings of hydroxyapatite (HAp) and 45S5 bioactive glass (45S5 BG) on zirconia textured surfaces using MC3T3-E1 cells. For this purpose, zirconia surfaces were micro-textured via laser and then coated with HAp and 45S5 BG glass via dip coating. Afterwards, the bioactive coatings were laser sintered, and a reference group of samples was conventionally sintering. The cell adhesion characterisation was achieved by cell viability performing live/dead analysis using fluorescence stains and by SEM observations for a qualitative analysis of cell adhesion. The in vitro results showed that a squared textured pattern with 100μm width grooves functionalized with a bioactive coating presented an increase of 90% of cell viability compared to flat surfaces after 48h of incubation. The functionalized laser sintered coatings do not present significant differences in cell viability when compared to conventionally sintered coatings. Therefore, the results reveal that laser sintering of HAp and 45S5 BG coatings is a fast and attractive coating technique.publishe

Metodologias para produção, extração e caracterização de compostos antibacterianos

This doctoral project is based on two parts, one using text and data mining and another in laboratory providing strategies based on advanced analytical methodologies for the discovery of bioactive compounds (BC), with potential applications on pharmaceutical and biotechnological tools from fungi organisms. The primary goal of this project was using a text mining technique for identifying the topics of a relevant scientific text related to fungi bioactive compounds and evolutionary connection among these topics, as well as using visualization tools for presenting both the topics and the association among them, as a convenient way to help users to determine relevant topics. In the second part of this thesis, firstly, fungi species were cultivated in lab using a batch reactor. Then, these fungi organisms were extracted by using environmentally friendly extraction techniques, such as High-Pressure Extraction (HPE). Secondly, the extracted compounds were characterized using elemental analysis (CNHS), nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). Thirdly, the bioactivity was evaluated through antimicrobial assays based on European pharmacopeia. Finally, the project findings will provide relevant data to stakeholders involved in the economic recovery plan through the sustainable resource management.Este trabalho de doutoramento é baseado em duas partes, uma usando mineração de dados e texto e outra em laboratório, fornecendo estratégias baseadas em metodologias analíticas avançadas para a descoberta de compostos bioativos (CB), com potenciais aplicações em ferramentas farmacêuticas e biotecnológicas de organismos fúngicos. O objetivo principal deste projeto foi usar uma técnica de mineração de texto para identificar os tópicos de um texto científico relevante relacionado com compostos bioativos de fungos e a conexão evolutiva entre esses tópicos, bem como usar ferramentas de visualização para apresentar os tópicos e a associação entre eles, como uma maneira conveniente de ajudar os utilizadores a determinar tópicos relevantes. Na segunda parte desta tese, inicialmente, as espécies de fungos foram cultivadas em laboratório usando um reator descontínuo. Em seguida, esses organismos fúngicos foram extraídos usando técnicas de extração verdes, como a Extração de Alta Pressão (HPE). Em segundo lugar, os compostos extraídos foram caracterizados por análise elementar (CNHS), ressonância magnética nuclear (RMN) e espectroscopia de infravermelho por transformada de Fourier (FTIR). Em terceiro lugar, a bioatividade foi avaliada através de ensaios antimicrobianos baseados na farmacopeia europeia. Finalmente, as conclusões do projeto fornecerão dados relevantes às partes interessadas e envolvidas no plano de recuperação económica através da gestão de recursos sustentáveis.Programa Doutoral em Químic