Computational structure‐based drug design: Predicting target flexibility

The role of molecular modeling in drug design has experienced a significant revamp in the last decade. The increase in computational resources and molecular models, along with software developments, is finally introducing a competitive advantage in early phases of drug discovery. Medium and small companies with strong focus on computational chemistry are being created, some of them having introduced important leads in drug design pipelines. An important source for this success is the extraordinary development of faster and more efficient techniques for describing flexibility in three‐dimensional structural molecular modeling. At different levels, from docking techniques to atomistic molecular dynamics, conformational sampling between receptor and drug results in improved predictions, such as screening enrichment, discovery of transient cavities, etc. In this review article we perform an extensive analysis of these modeling techniques, dividing them into high and low throughput, and emphasizing in their application to drug design studies. We finalize the review with a section describing our Monte Carlo method, PELE, recently highlighted as an outstanding advance in an international blind competition and industrial benchmarks.We acknowledge the BSC-CRG-IRB Joint Research Program in Computational Biology. This work was supported by a grant from the Spanish Government CTQ2016-79138-R.J.I. acknowledges support from SVP-2014-068797, awarded by the Spanish Government.Peer ReviewedPostprint (author's final draft

Understanding and design of molecular dynamics based simulation methods.

Several new strategies for employing modified molecular dynamics (MD) protocols for finding energy minima were developed developed. First, we derived a new method of searching for the lowest energy conformation by employ employing the results of our work to explain "mean mean-field" molecular dynamics simulation methods. Called ensembles extracted by atomic coordinate transformations (EXACT), the method allows simulations to be performed with a variable degree of approximation. Then, we examine the previously published locally enhanced sampling (LES) approximation. The method makes copies of a small part of interest in a larger system, and allows the dynamics to unfold. The method works by making the copies invisible to each other and allowing them to interact only with the remainder of the system, called the bath. The bath, on the other hand, interacts with an averaged representation of the copied part. This averaged interaction allows the copied particles to move into geometries they might not visit in a conventional MD simulation, which allows a greater variety of structures to be sampled. We derive the algorithm by copying the entire system, and then by employing holonomic constraints between the bath particles between the various systems. Using this new approach, we explore several issues previously noted in the literature. We also use the EXACT approximation method to illustrate the nature of the LES approximation. Finally, we present another optimization method, which add adds pressure along with temperature in analogue with simulated annealing. The method is tested against simulated annealing for condensed phase systems of argon, monoglyme, and tetraglyme. The method noticeably improves the results for the glyme systems, but does not seem to hurt the results for the argon system

De Novo Designed Metallopeptides to Investigate Metal Ion Homeostasis, Electron Transfer, and Redox Catalysis.

Protein design is a powerful way to interrogate the basic requirements for function of metal sites by systematically incorporating elements important for function. Single-stranded three-helix bundles with either thiolate-rich sites for spectroscopic characterization and electron transfer, or histidine-rich sites for redox catalysis are described. Using a previous design, two constructs were designed to incorporate a fourth cysteine residue to investigate thiolate-rich sites involved in metal ion homeostasis and electron transfer. Rational re-design replaced a putative coordinating histidine with a cysteine. A second construct embedded a CXXC binding motif into the helical scaffold. These two constructs show different UV-visisble, 113Cd NMR, and 111mCd PAC, which indicate that they form different proportions of CdS3O and CdS4. The spectroscopy of these sites sheds light on how Cd(II) bindis to CadC and suggests a dynamic site in fast exchange with the solvent. Previous attempts at the design of a rubredoxin site have focused on reproducing the peptide fold around or using flexible loop regions to define the site in addition to canonical CXXC motifs. However, the use of CXXC motifs embedded in an α-helical scaffold produces a rubredoxin site that reproduces the Mössbauer, MCD, and EPR of rubredoxin without the use of loop regions. This successful design is the largest deviation from consensus rubredoxin and zinc finger folds reported. Electron transfer rates through a de novo designed scaffold were studied by the design and synthesis of a ruthenium trisbipyridine derivative appended to an exterior cysteine residues. A redox-active tyrosine in the 70th position is implicated as a relay amino acid from the iron center and absence of the tyrosine decreases the rate of electron transfer from the metal site. This is the first photo-generated tyrosine radical in a designed protein. A construct, which was previously reported for CO2 hydration, is substituted with copper and its spectroscopic and nitrite reductase activity are studied. This is the first demonstration of nitrite reductase activity in a single-stranded designed peptide. This thesis provides insight into designed proteins and their applications and lays the groundwork for further studies to progress towards a unified multifunctional redox protein.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113513/1/agtebo_1.pd

Realistische Modellierung von Elektrophorese in freier Lösung : eine Studie über geladene Makromoleküle

Im Rahmen der vorliegenden Doktorarbeit aus dem Forschungsgebiet der sogenannten "Weichen Materie" (soft matter) wird im Besonderen der Einfluss hydrodynamischer Wechselwirkungen auf die Elektrophorese linearer Polyelektrolyte in freier Lösung, d. h. in Abwesenheit eines trennenden Gels, untersucht. Hierzu wird eine vergröberte Molekulardynamiksimulation verwendet, die die vollständigen elektrostatischen und hydrodynamischen Wechselwirkungen zwischen allen Komponenten des Systems berücksichtigt. Ein besonderes Gewicht liegt auf der Analyse der Rolle der Hydrodynamik für das elektrophoretische Verhalten geladener Makromoleküle. Diese Arbeit untersucht die Längenabhängigkeit der hydrodynamischen Wechselwirkungen, den Einfluss der Kettenflexibilität und der das Polyelektrolyt umgebenden Gegenionen, sowie ihre gemeinsame Wirkung auf die dynamischen Transportkoeffizienten, die Diffusion und die elektrophoretische Mobilität. Diese Problemstellung wird mit Hilfe umfangreicher Computersimulationen bearbeitet, deren Ergebnisse quantitativ mit experimentellen Resultaten verglichen werden können. Dabei werden bestehende Theorien sorgfältig analysiert und durch die in dieser Arbeit ermittelten Erkenntnisse erweitert.This thesis contributes to the field of soft matter research and studies the importance of hydrodynamic interactions during free-solution electrophoresis of linear polyelectrolytes by means of coarse-grained molecular dynamics simulations including full electro-hydrodynamic interactions. The center of attention is the specific role of hydrodynamic interactions on the electrophoretic behaviour of charged macromolecules. Points of interest are the dependence of hydrodynamic interactions on the chain length, the chain flexibility and the surrounding counterions, and their combined influence on important observables such as the static chain conformations and the dynamic transport coefficients, i.e., the diffusion and the electrophoretic mobility. These problems are addressed by extensive computer simulations that are quantitatively matched with experimental results. Existing theoretical predictions are carefully examined and are augmented by the observations in this thesis

MITOCHONDRIAL VDAC AND BACTERIAL PORA/C1: ION PERMEATION AND SELECTIVITY

VDAC and PorA/C1 are large diameter channels with properties reminiscent of those found in narrow channels. VDAC, located in the mitochondrial outer membrane, shows high selectivity for ATP over comparably sized ions. VDAC is characterized by a single open state with anionic selectivity and multiple cation-selective closed states. PorA/C1 from Neisseria meningitidis achieves high cationic selectivity and large conductance. VDAC has multiple functions in cellular processes and the most important one is the regulation of metabolite flow across the outer membrane. A variety of functions could be achieved by the existence of different isoforms. In this thesis I summarized the electrophysiological properties of VDAC-like proteins from Drosophila Melanogaster encoded by genes CG17137, CG17139 and CG17140. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137, CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 did not form channels. The special selectivity of VDAC for large anions was explored using the mutant of the mouse isoform 2. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for the wildtype. Also we determined that the intact outer membrane containing the mutant is permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from anion to cation preference does not substantially influence the flux of negatively charged metabolites. However, this finding supports the previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall. The third part of my thesis represents experimental data supporting the theoretical model for the PorA/C1 structure. This model explains the almost ideal cationic selectivity of the channel and high level of rectification. These properties are proposed to arise from a high density of charges in the channel that results in both high selectivity and high ionic flux

Computational modeling of biological nanopores

Throughout our history, we, humans, have sought to better control and understand our environment. To this end, we have extended our natural senses with a host of sensors-tools that enable us to detect both the very large, such as the merging of two black holes at a distance of 1.3 billion light-years from Earth, and the very small, such as the identification of individual viral particles from a complex mixture. This dissertation is devoted to studying the physical mechanisms that govern a tiny, yet highly versatile sensor: the biological nanopore. Biological nanopores are protein molecules that form nanometer-sized apertures in lipid membranes. When an individual molecule passes through this aperture (i.e., "translocates"), the temporary disturbance of the ionic current caused by its passage reveals valuable information on its identity and properties. Despite this seemingly straightforward sensing principle, the complexity of the interactions between the nanopore and the translocating molecule implies that it is often very challenging to unambiguously link the changes in the ionic current with the precise physical phenomena that cause them. It is here that the computational methods employed in this dissertation have the potential to shine, as they are capable of modeling nearly all aspects of the sensing process with near atomistic precision. Beyond familiarizing the reader with the concepts and state-of-the-art of the nanopore field, the primary goals of this dissertation are fourfold: (1) Develop methodologies for accurate modeling of biological nanopores; (2) Investigate the equilibrium electrostatics of biological nanopores; (3) Elucidate the trapping behavior of a protein inside a biological nanopore; and (4) Mapping the transport properties of a biological nanopore. In the first results chapter of this thesis (Chapter 3), we used 3D equilibrium simulations [...]Comment: PhD thesis, 306 pages. Source code available at https://github.com/willemsk/phdthesis-tex

Structure-based drug design for the discovery of new treatments for trypanosomiasis

Human African trypanosomiasis (HAT) and Chagas disease are caused by infection with the protozoan parasites Trypanosoma brucei and T. cruzi, respectively. There has historically been a lack of investment into measures to control these diseases. As a result, few drugs are available to treat HAT and Chagas disease, and there is an urgent need for novel alternatives. The enzyme L-threonine 3-dehydrogenase (TDH) initiates the conversion of L-threonine into acetyl-coenzyme A and glycine. This pathway has been shown to play a vital role in T. brucei, particularly in fatty acid synthesis. Exposure of T. brucei in culture to a potent TDH inhibitor, has been shown to be lethal(1) and dual blockade of the TDH pathway and a second pathway for acetyl-coenzyme A production, terminated by pyruvate dehydrogenase, completely inhibits the growth of T. brucei(2,3). Multiple three-dimensional structures of TDH, alone and in complex with ligands, were determined by X-ray crystallography. In parallel, enzyme assays were carried out to investigate the kinetic behaviour of TDH and the modes of action of known TDH inhibitors. The structural information on TDH was used in a virtual screen to predict the binding interactions between the enzyme and a library of around 3000 ligands. These ligands were mainly selected for their diversity and for their inhibition of proteins related to TDH. Subsequently, an in vitro screen was performed to test compounds identified by virtual screening, along with small molecules and fragments from commercial libraries. In total, 27 compounds were identified as TDH inhibitors. Of these compounds, four were found to potently inhibit T. brucei growth. This study has demonstrated the effectiveness of combining structural and functional data in rational drug discovery. Novel aspects of TDH have been discovered, in addition to novel inhibitors that will aid in the design of a new class of antitrypanosomal drugs

Extracção supercrítica de óleo de grainha de uva combinada com pré-tratamentos enzimáticos

Doutoramento em Engenharia QuímicaNeste trabalho estudou-se a extracção supercrítica do óleo de grainha de uva, usando dióxido de carbono, e combinou-se este processo com um prétratamento enzimático da semente para aumentar o rendimento global da extracção. A qualidade dos extractos obtidos foi avaliada pelo seu conteúdo em triacilglicerídeos, perfil de ácidos gordos e capacidade antioxidante. Realizaram-se também alguns estudos exploratórios sobre a aplicação de um pré-tratamento de alta pressão (HPP) à grainha da uva. Adicionalmente, efectuou-se o estudo da extracção, fraccionamento e caracterização estrutural das procianidinas da grainha da uva, bem como a avaliação da sua capacidade antioxidante. A extracção de procianidinas da grainha da uva foi efectuada sequencialmente com metanol e acetona/água, tendo sido posteriormente fraccionadas por adição sucessiva de misturas metanol/clorofórmio progressivamente mais concentradas em clorofórmio. A caracterização das procianidinas foi feita por HPLC-UV e LC–MS, antes e depois de sujeitar as amostras a uma tiólise, e também por ESI-MS e ESI-MS/MS. Este estudo permitiu reportar, pela primeira vez, a ocorrência de procianidinas do tipo-A galoiladas na grainha da uva. Os resultados de HPLC-UV permitiram determinar o grau médio de polimerização das procianidinas e a sua composição monomérica em (+)- catequina, (-)-epicatequina e (-)-epicatequina-O-galato. Mostrou-se que a (+)- catequina é o flavan-3-ol terminal mais abundante e a (-)-epicatequina predomina largamente como unidade de extensão. No caso de procianidinas do tipo A, a ligação interflavânica C2-C7 encontra-se essencialmente nas unidades terminais. O grau médio de polimerização das diversas fracções varia entre 1.0 e 10.8. A sua capacidade antioxidante, medida pelo método espectrofotométrico de DPPH•, mostrou-se ser equivalente à de uma amostra comercial de (+)-catequina usada como referência. A partir dos graus médios de polimerização experimentais e das análises de FTIR das fracções correspondentes foi possível obter um modelo preditivo O-PLS com apenas uma variável latente. O pré-tratamento enzimático justificou-se pelo conhecimento existente acerca do uso de enzimas específicas que destroem parcialmente as paredes celulares. Atendendo à composição das paredes celulares da grainha da uva preparou-se uma suspensão contendo protease, xilanase, pectinase e celulase. Para determinar as condições experimentais do pré-tratamento que maximizam o rendimento da extracção, estudou-se o efeito do tempo de reacção, temperatura, pH, diâmetro médio das partículas de grainha moída e a concentração das enzimas. Os incrementos do rendimento da extracção de óleo observados atingiram 163.2%. O estudo da extracção supercrítica (SFE) do óleo da grainha de uva tratada e não-tratada permitiu obter as curvas de extracção correspondentes, bem com analisar a influência das condições operatórias sobre o seu andamento. Montou-se uma instalação laboratorial onde se realizaram experiências com dióxido de carbono a 160, 180, 200 e 220 bar e temperaturas de 313.15 e 323.15 K. Os rendimentos obtidos por SFE foram semelhantes aos de Soxhlet com n-hexano. As curvas de extracção medidas compreendem um primeiro período de extracção, onde se remove cerca de 92-97% do óleo disponível, e um segundo período, essencialmente difusional, com pouco impacto no rendimento final. Os vários extractos recolhidos e o óleo global obtido foram caracterizados para avaliar a sua qualidade e relacioná-la com as condições operatórias de SFE. Determinaram-se o conteúdo total em triacilglicerídeos, o seu perfil de ácidos gordos e a capacidade antioxidante (AOC). Os resultados mostraram que a AOC aumenta com a elevação da pressão e, acentuadamente, com o acréscimo da temperatura. Ao longo da curva de extracção, a AOC é mais pronunciada nos extractos iniciais, nomeadamente nos primeiros 30 a 40% da extracção. A modelação efectuada considerou que o óleo extractável se reparte entre células rompidas, predominantes na periferia da semente, e células intactas, mais interiores. Admitiu-se que o transporte de massa ocorre em série, i.e. das células intactas para as rompidas e destas para o solvente; mostrou-se que a dispersão axial era desprezável. Os balanços materiais à fase fluida e aos volumes de células rompidas e intactas, combinados com os fluxos interno, externo e a relação de equilíbrio foram resolvidos numericamente pelo método das linhas combinado com diferenças finitas atrasadas. O modelo reproduziu bem as curvas experimentais e permitiu simular curvas de eluição e os três perfis de concentração no leito.The supercritical extraction of grape seed oil with carbon dioxide has been studied and combined with an enzymatic pre-treatment of the seed to enhance the global extraction yield. The quality of the attained extracts has been assessed by evaluating the content in triacylglycerides, fatty acids profile, and antioxidant capacity. An exploratory study with the application of a high pressure pre-treatment (HPP) to the seed has also been considered. Additionally, the extraction, fractionation, and structural characterization of grape seed procyanidins, as well as the evaluation of their antioxidant capacity, has been carried out. The procyanidins were sequentially extracted with methanol and acetone/water from defatted seed, and further fractionated by precipitation in graded methanol/chloroform solutions. The procyanidins analysis included HPLC-UV and LC–MS, before and after thiolysis, as well ESI-MS e ESI-MS/MS. Such study allowed to report, for the first time, the occurrence of type-A galloylated procyanidins. The average degree of polymerization and the monomeric composition in (+)-catechin, (-)-epicatechin and (-)-epicatechin-O-gallate, were obtained using HPLC-UV. The results, showed (+)-catechin as the most abundant flavan-3-ol terminal unit, and the high predominance of (-)-epicatechin as the extension unit. In type-A procyanidins the C2-C7 interflavanic linkage appears essentially in the terminal units. The collected fractions presented an average degree of polymerization range from 1.0 to 10.8. Their antioxidant capacity, assessed by the DPPH• spectrophotometric method, presented a similar response to a commercial standard of (+)-catechin. A predictive O-PLS method, with one latent variable, was obtained using the experimental average degree of polymerization determined and combined with FTIR analyses. The enzymatic pre-treatment approach was based on the knowledge that partial hydrolysis of the cell walls can occur by means of appropriate enzymes. With the given composition of grape seed walls four types of enzymes were considered, namely protease, xylanase, pectinase, and cellulase as the enzymatic cocktail. The effect of reaction time, temperature, pH, grape seed average particle sizes and enzymes concentration were considered in the search for extraction yield improving conditions. The increments in the extraction yield attained 163.2%. The supercritical fluid extraction (SFE) curves of grape seed oil for both treated and untreated seed were obtained and characterized on the influence of the operatory conditions on them. The supercritical extraction experiments were performed with carbon dioxide at 160, 180, 200, and 220 bar and 313.15 and 323.15 K in a semi-continuous apparatus built and assess at the University of Aveiro. The yields obtained by SFE were similar to those of Soxhlet using nhexane. The extraction curves present a first period of extraction, where about 92-97% of the available oil is removed, and a second diffusional period, with no significant impact in the final yield. The quality of the individual SFE extracts and global oil was evaluated and related to the SFE operating conditions. The total triacylglycerides content, fatty acids profile, and antioxidant capacity (AOC) of the extracts were measured. Results showed the AOC increases with increasing pressure and noticeably with rising temperature. Along extraction curves, the AOC is more pronounced on the oil collected during the first stages of the process, where 30-40% of the total oil is extracted. The modelling assumed the hypothesis of the existence of broken (peripherical) and intact cell (core) fractions in the seed. A series mass transport was assumed, i.e., from intact cells to broken cells, and from those to the solvent; axial dispersion was shown to be neglectable. The resulting mass balances to the fluid phase, broken, and intact cells, combined with internal fluxes, and the equilibrium relationship were numerically solve by applying the method of lines combined with backward finite differences. The experimental extraction curves were well reproduced by the model which allowed the simulation of the elution curves and the three concentration profiles in the bed

THE INFLUENCE OF SURFACTANTS ON THE SORPTION OF PHENANTHRENE IN NATURAL WATERS

Polycyclic aromatic hydrocarbons (PAHs) are widely recognised as harmful, persistent organic pollutants, whilst surfactants are more easily degraded but their ubiquitous use both domestically and industrially ensures their presence in natural waters. Owing to both their hydrophobic and hydrophilic properties, surfactants are capable of both adsolubilisation and solubilisation of co-contaminants, hence, their presence in natural waters is considered extremely significant. The purpose of this research was to determine how surfactants and PAHs influence each other in estuaries where both are co-disposed. This study is the first to employ environmentally realistic concentrations of surfactants and to use natural particles in order to determine how changes in surfactant behaviour impinge on the sorption of a representative PAH to sediment. The experimental method employed involved tracing the solubility and sorption of a '''C-labelled PAH (phenanthrene) in the presence of representative surfactants from the three main groups, i.e., anionic, nonionic and cationic. Initially the four compounds were examined in isolation at two temperatures (8°C and 20°C), salinities and particle concentrations. The next stage involved incorporating each surfactant with phenanthrene in the same experiment and repeating the range of variables. Solubility and adsorption of phenanthrene in the presence of Triton X-100, sodium dodecyl sulphate (SDS) or hexadecyltrimethylammonium iodide (HDTMA) proved to be highly complex. The adsorption, expressed as a Freundlich coefficient, KF was generally nonlinear and varied according to surfactant type, salinity, temperature, and contact sequence. The overriding influence, however, was sediment particulate matter (SPM) concentration. On some occasions the surfactants in isolation displayed unusual particle concentration effects (PCE), such as an increase of surfactant in the aqueous phase as the SPM concentration increased. Despite this, they all generally exaggerate the more typical PCE displayed by phenanthrene, where the particle-water partition coefficient is reduced as particle concentration increases. This effect is then modified by temperature and contact sequence, which has important environmental implications with regard to removal or release of compounds stored within sediments. However, overall, at environmentally relevant concentrations, the surfactants appear to enhance sorption, rather than solubility, with HDTMA the most effective surfactant at removing phenanthrene from the aqueous phase.BMT Marine Information Systems Limited Southampton and AstraZeneca Brixha