Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, inhibitors have with the binding pocket, optimization efforts can be focused on maximizing interactions with “hot spots” within the pocket, thus leading to larger, high affinity inhibitors. A similar optimization technique can also be applied to known inhibitors. By examining the interactions of a known inhibitor with the binding site, new compounds can be designed to target “hot spots” in the binding pocket using the known inhibitors core structure as a starting point. The affinity of the newly designed compounds can then be compared to the affinity of the original inhibitor, and further rounds of optimization can be carried out. While simple in design, there are many challenges associated with structure based drug design studies, and there is no guarantee novel inhibitors will be found, but ultimately, it is an extremely powerful methodology that results in a much higher hit rate than other, similar, techniques. The work herein describes the use of structure based drug design to target several different proteins involved in bacterial antibiotic resistance, and a protein that has been implicated in the development of Alzheimer’s disease. The goal of the first project was to design a new PBP inhibitor based upon an existing scaffold, and to better understand the binding mechanism and molecular interactions between penicillin binding proteins and their inhibitors. PBPs are a group of proteins that catalyze the last steps of bacterial cell wall formation, and are the targets of the β-lactam antibiotics. Two compounds were designed which conjugated a ferrocene or ruthenocene group to 6-aminopenicillinic acid, and their antibiotic properties were tested against a range of bacterial strains. To get a better understanding of how the 6-APA organometallic compounds interacted with the PBP active site, a CTX-M-14 β-lactamase model system was used for X-ray crystallographic studies. CTX-M-14 was chosen as its active site shares many key catalytic features with PBPs, and it easily, and reproducibly, yields crystals capable of diffracting to sub-atomic (< 1.0 Å) resolution. I determined a 1.18 Å structure of 6-APA-Ru in complex with CTX-M-14 E166A β-lactamase and was able to gain unprecedented details of the interactions of the ruthenocene group with the CTX-M active site. This structure also revealed that the compound bound in the CTX-M active site was actually the decarboxylated and hydrolyzed product, which was the first time a decarboxylated product had been captured in the CTX-M active site. A second, 0.85 Å, structure of CTX-M in complex with 6-APA-Ru was determined and shed light on how the hydrogen bonding network in the CTX-M active site changes in response to the 6-APA-Ru product binding. A final, 1.30 Å, structure captured the carboxylated and hydrolyzed 6-APA-Ru product in complex with CTX-M, which was the first time the carboxylated product had been captured in the CTX-M active with the catalytic Ser70 residue intact. The results show the potential of the ruthenocene group in improving antibiotic potency, and help to better elucidate the changes that occur in the CTX-M active site upon inhibitor binding, while at the same time, telling us what changes could occur in the active site of PBPs. The next project was focused on novel inhibitor discovery against several different PBPs. PBPs have been successfully inhibited by β-lactam antibiotics for decades, but the alarming rise of bacteria resistant to these antibiotics has placed increased urgency on the discovery of novel PBP inhibitors. A fragment based molecular docking approach was employed to virtually screen millions of small compounds for interactions with the targeted active sites, and then high scoring compounds were selected for visual inspection and inhibitory testing. Virtual screening was first done against Staphylococcus aureus monofunctional transglycosylase, a type of PBP. MTG provided a good binding pocket for virtual screening, but proved challenging to purify and crystallize. However, through great effort MTG crystals were eventually obtained. After repeated rounds of virtual screening against MTG, multiple compounds were selected for inhibition testing, and testing is currently ongoing. Virtual screening was also done against Pseudomonas aeruginosa PBP5 and PBP1a. Purification and crystallization of these proteins proved to be easier than MTG, and both yielded diffraction quality crystals. The final project focused on virtual screening against a protein implicated in the development of Alzheimer’s disease, Slingshot Phosphatase 1. The brains of AD patients have been found to contain elevated levels of active Cofilin, and these elevated levels of active Cofilin may lead to the overproduction of amyloid β. Aβ overproduction, and its resulting accumulation, is believed to be one of the pathways that lead to AD symptoms. Cofilin is activated when it is dephosphorylated by SSH1, and inhibiting this activation may decrease the production of Aβ and the development of AD symptoms. There is no known structure of SSH1, so to perform virtual screening a SSH1 homology model was constructed using the homolog SSH2 as a starting point. Virtual screening was then performed using the SSH1 homology model and many compounds were selected for inhibition testing. Initial testing found several compounds that could prevent Cofilin dephosphorylation at levels > 10μM. However, three compounds were found to be exceptionally active, and could prevent Cofilin dephosphorylation at both 1 and 10 μM. One of these three compounds was tested directly against purified SSH1 and found to inhibit its activity, and reduce Aβ production. Crystallization of purified SSH1, and SSH2, was attempted in order to get complex structures with the three best compounds. SSH2 crystals were obtained which diffracted to 1.91 Å, and several initial hits were found for SSH1. Optimization of crystals for both proteins is currently ongoing. The SSH1 inhibitor, along with the two other highly active compounds, provides an excellent starting point for the development of highly potent SSH1 inhibitors.

This study aimed to calculate physicochemical descriptors for a class of 22 naphtoimidazolic derived compounds that possess microbicidal, bactericidal, fungicidal, trypanocidal activity and so on. The descriptors were also calculated for benznidazole, a drug used in the treatment of Chagas disease and the ravuconazole, which shows itself effective in all strains of the parasite. After calculation of the descriptors found at which of these would be responsible for the separation of compounds into active and inactive. To do so, the structures were pre-optimized through chemical calculations of molecular mechanics and optimized by semi empirical calculations, based on Austin Model 1. After the molecular optimization, multivariate chemometric calculations to make the separation of active and inactive compounds were performed and verify which of descriptors were calculated for separating the relevant class of compounds. Were made 4 chemometric analysis of principal component analysis and hierarchical cluster analysis, where information was available and the similarities of groups formed for the compounds and the descriptors. In the last chemometric analysis it was found that the most active compounds were FSE-1, FSE-2, FSE-13 and FSE-14 and the descriptor responsible for the observed activity would be ELUMO while that would be responsible for the inactivity was AE. Through calculations of the LUMO frontier orbitals, we found that the benzene ring and the imidazole ring are regions in structures that favor the formation of molecular interaction. For benzene of naphthalene, it was observed that the para and ortho positions are also regions of probable formation of molecular interaction. The electrostatic potential maps calculated showed that clusters with concentration of negative charges do not encourage biological activity of naphttimidazolic derivatives. To calculate the molecular anchoring it was used the cruzain enzyme from T. cruzi due its importance in all stages of the parasite life cycle. Realized molecular anchoring it was found that the most relevant interactions are stacking interactions and interactions with the imidazole ring in the ortho and para positions of the naphthalene ring of FSE derivatives. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este trabalho teve por objetivo calcular descritores físico-químicos para uma classe de 22 compostos de derivados naftoimidazólicos que possuem atividade microbicida, bactericida, fungicida, tripanocida dentre outras. Os descritores também foram calculados para o benzonidazol, fármaco usado no tratamento da doença de Chagas e para o ravuconazol, fármaco que se mostra efetivo em todas as cepas do parasito. Após o cálculo dos descritores verificou-se quais destes seriam responsáveis pela separação destes compostos em ativos e inativos. Para tanto, as estruturas foram pré-otimizadas através de cálculos químicos da mecânica molecular e otimizadas em cálculos semi-empíricos, na base Austin Model 1. Após a otimização molecular, foram realizados cálculos quimiométricos multivariados para fazer a separação dos compostos em ativos e inativos e, verificar quais dos descritores calculados foram relevantes para a separação da classe de compostos. Foram efetuadas 4 análises quimiométricas de análise de componentes principais e análise hierárquica de agrupamentos, onde foi possível obter informações dos grupamentos e das similaridades formadas para os compostos e para os descritores. Na última análise quimiométrica foi verificado que os compostos mais ativos foram FSE-1, FSE-2, FSE-13 e FSE-14 e que o descritor responsável pela atividade observada seria o ELUMO enquanto que aquele responsável pela inatividade seria o AE. Através dos cálculos dos orbitais de fronteira LUMO, foi possível verificar que o anel benzênico e o anel imidazol são regiões nas estruturas que favorecem a formação de interação molecular. Para o benzeno do naftaleno, observou-se que as posições para e orto, também são regiões de provável formação de interação molecular. Os mapas de potencial eletrostático calculados mostraram que grupamentos com concentração de cargas negativas, não favorecem a atividade biológica dos derivados naftoimidazólicos. Para o cálculo do ancoramento molecular foi utilizada a enzima cruzaína de T. cruzi, devido sua importância em todos os estágios do ciclo de vida do parasito. Realizado o ancoramento molecular verificou-se que as interações mais relevantes são as de empilhamento, as interações com anel imidazol e interações nas posições orto e para do anel naftaleno para os derivados FSE.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-11-10T10:37:56Z No. of bitstreams: 2 Dissertação - Uessiley Ribeiro Barbosa - 2016.pdf: 3901383 bytes, checksum: f52c92a81d289db26f933d75a30e8788 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-11-10T17:48:40Z (GMT) No. of bitstreams: 2 Dissertação - Uessiley Ribeiro Barbosa - 2016.pdf: 3901383 bytes, checksum: f52c92a81d289db26f933d75a30e8788 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-10T17:48:40Z (GMT). No. of bitstreams: 2 Dissertação - Uessiley Ribeiro Barbosa - 2016.pdf: 3901383 bytes, checksum: f52c92a81d289db26f933d75a30e8788 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-04 / The Paracoccidioides brasiliensis (Pb) is a thermo-dimorphic fungus described as the etiological agent of paracoccidioidomycosis (PCM), an important systemic mycosis in Latin America. The isocitrate lyase (ICL) is an enzyme involved in glyoxylate cycle, an alternative pathway to Krebs cycle, which has been described in fungi, bacteria and plants. The absence of this enzyme in mammals makes it an interesting target for design of specific antifungal compounds for PCM. In this work, we use in silico methods like homology modeling, molecular dynamics and virtual screening, aiming the development of inhibitors compounds for PbICL enzyme in the absence and presence of cofactor and positive control. From a molecular docking protocol, it was possible to select promising compounds by criteria of affinity and efficiency based on screening of natural products. Two regions were selected for molecular docking, one involving a region already known by the binding of argentilactona inhibitor and another region involving the cofactor Mg 2+, a possible catalytic site of ICL. All compounds selected by affinity criteria had more than 80% of success rate in achieving lower energy and allowed to describe common residues within the protein interaction for the target sites. The structural quality parameters are significantly improved after 100 ns of simulation. The structure of PbICL and PbICL with magnesium show all quality parameters as acceptable to define them as highresolution structures. All selected compounds show aromatic chains with the possibility of being a pharmacophore, which is essential for biological activity. Another interesting aspect is that, through the selected compounds, it was possible to describe structural patterns related to the ligand specificity, that might be promising for a basic chemical sketch for rational drug design. / O Paracoccidioides brasiliensis (Pb) é um fungo termo-dimórfico descrito como agente etiológico da paracoccidioidomicose (PCM), uma micose sistêmica importante na América Latina. A Isocitrato liase (ICL) é uma enzima envolvida no ciclo do glioxilato, uma via do ciclo Krebs já descritos em fungos, bactérias e plantas. Sua ausência em mamíferos faz dessa enzima, um alvo interessante para o desenho de compostos antifúngicos específicos para a PCM. Neste trabalho, utilizamos métodos in silico como modelagem por homologia, dinâmica molecular e triagem virtual na busca de compostos inibidores para enzima isocitrato liase de P brasilienses (PbICL) na ausência e presença do cofator e com a seleção de controle positivo. A partir de um protocolo de ancoragem molecular, foi possível selecionar compostos promissores por critérios de afinidade e eficiência a partir de triagens virtuais com produtos naturais. Duas regiões foram selecionadas para a ancoragem molecular, uma envolvendo uma região já conhecida pela ligação do inibidor argentilactona e outra envolvendo a região de ligação do cofator Mg2+ , posicionada no possível sítio catalítico da enzima. Todos os compostos selecionados pelo critério de afinidade tiveram mais do que 80% de sucesso em alcançar a mais baixa energia e permitiram descrever resíduos frequentes na interação proteína-inibidor para sítios alvo da enzima. Os parâmetros de qualidade da estrutura são sensivelmente melhorados após 100 ns de simulação. As estruturas de PbICL e PbICL com Mg2+ apresentam todos os parâmetros dentro do aceitável para defini-la como estrutura de alta resolução. Todos os compostos selecionados apresentaram cadeias aromáticas com possibilidade de ser um grupo farmacofórico, o qual é essencial para a atividade biológica. Outro aspecto interessante é que através dos compostos selecionados foi possível descrever padrões estruturais relacionados à especificidade do composto, os quais podem vir a ser promissores para um esqueleto básico no desenho racional de fármacos.

In this dissertation a novel approach to protein-ligand docking is presented. First an existing method to predict putative active sites is employed. These predictions are then used to cut down the search space of an algorithm that uses the fast Fourier transform to calculate the geometrical and electrostatic complementarity between a protein and a small organic ligand. A simplified hydrophobicity score is also calculated for each active site. The docking method could be applied either to dock ligands in a known active site or to rank several putative active sites according to their biological feasibility. The method was evaluated on a set of 310 protein-ligand complexes. The results show that with respect to docking the method with its initial parameter settings is too coarse grained. The results also show that with respect to ranking of putative active sites the method works quite well.

Parmi les 57 cytochromes P450 identifiés lors du séquençage complet du génome humain, on en dénombre environ 15 dont on ne connaît pratiquement rien de leurs rôles physiologiques, de leurs substrats, et de leurs structures, d’où le nom de «P450 orphelins». Le CYP2U1 est l’un des cytochromes P450 les plus fortement exprimé au niveau du cerveau et du cervelet mais c’est aussi l’un des plus conservé parmi les différentes espèces du règne animal. Ce travail de thèse a tout d’abord consisté à optimiser les conditions d’expression du CYP2U1 sous une forme active. Un premier système d’expression dans la levure Saccharomyces Cerevisiae a permis une production d’un complexe CYP2U1-P450 réductase catalytiquement actif permettant des études de recherche de substrat. Un second système d’expression dans Escherichia Coli devrait permettre d’obtenir de plus grandes quantités d’enzyme soluble destinée à des études structurales. Dans un second temps, une recherche de substrats a été effectuée à l’aide d’analyse d’incubats par chromatographie liquide couplée à une détection par spectrométrie de masse. A ce jour, un screening dirigé de plus de soixante-dix molécules, substrats de P450s de la famille 2, a permis d’identifier les premiers substrats exogènes du CYP2U1, les analogues de terfénadone et la débrisoquine. D’autre part, une étude par modélisation moléculaire de la structure du CYP2U1 a été effectuée. Cette étude montre que le CYP2U1 diffère de tous les autres P450s par la présence d’un insert très spécifique dans son domaine N-terminal. Des modèles par homologie basés sur les structures cristallographiques des P450s de la famille 2 ont été construits. Ces modèles ont été validés par dynamique moléculaire et ont permis de proposer un mode d’interaction avec la membrane, d’identifier la position des canaux d’accès ainsi que de déterminer la topologie du site actif. Enfin, un docking des premiers substrats exogènes au sein du site actif du CYP2U1 a permis de confirmer la régioselectivité des hydroxylations catalysées par le CYP2U1. / Among the 57 human cytochrome P450 genes that have been identified; substrates, structure and physiologic role of 15 of them is practically unknown. They are called orphan. One of them, CYP2U1 is one of the most expressed cytochrome P450 in the brain and in the cerebellum but also one of the most conserved isoform in the all animal kingdom. This manuscript first describes the optimization of the heterologous expression of an active form of CYP2U1. Expression in a eukaryotic host, yeast Saccharomyces Cerevisiae first allows the production of a catalytic active CYP2U1-P450 reductase complex needed for substrate screening. Another expression system in a prokaryote host Escherichia Coli will allow higher production rate of a truncated and soluble form of the protein which will permit structural studies. Then a directed substrate screening was performed with the liquid chromatography – mass spectrometry analysis of CYP2U1 incubations. To date, 70 molecules, CYP2 family substrates, were tested that allow the identification of the two first exogenous CYP2U1 substrates: débrisoquine and terfenadone analogs. A structural study was achieved using a homology tridimensional model of the enzyme. We have found that CYP2U1 is longer than the other human CYPs, with an N-terminal 20 amino acids insertion, located after the  helical membrane spanning domain. Structural models were built using six crystallized human CYP2s as templates. Molecular dynamics experiments in membrane suggested a specific interaction with the membrane. The active site topology and the access channels were also determined and a docking of the two first exogenous CYP2U1 substrates was performed in order to confirm the regioselective hydroxylation activities observed in vitro.

Computational methods have been extensively developed at various levels of approximation in recent years to model biomolecular interactions and for rational drug design. This research work aims to explore the feasibility of using quantum mechanical (QM) methods within the two broad categories of in silico ligand-based and structure-based drug design. First, density functional theory at the M06L level of theory was employed to examine structure-activity relationships of boron-based heterocyclic compounds, anti-inflammatory inhibitors targetting the interleukin-1β (IL-1β) cytokine. Our findings from computed energies and shapes of the molecular orbitals provide understanding of electronic effects associated with the inhibitory activity. We also found that the boron atom, specifically its electrostatic polarity, appears to be essential for the anti-IL-1β activity as evidenced by the biological assay of non-boron analogues selected from the ligand-based virtual screening results. Secondly, we aimed to dock ligands at the active sites of zinc-containing metalloproteins with reasonable computational cost and with accuracy. Therefore, an in-house docking scheme based on a Monte Carlo sampling algorithm using the semiempirical PM6/AMBER force field scoring function was compiled for the first time within the Gaussian 09 program. It was applied to four test cases, docking to cytidine deaminase and human carbonic anhydrase II proteins. The docking results show the method’s promise in resolving false-positive docking poses and improving the predicted binding modes over a conventional docking scheme. Finally, semiempirical QM methods which include dispersion and hydrogen-bond corrections were assessed for modelling conformations of β-cyclodextrin (βCD) and their adsorption on graphene. The closed in vacuo βCD cccw conformer was found to be in the lowest energy, in good agreement with previous ab initio QM studies. DFTB3, PM6-DH2 and PM7 methods were applied to model the intermolecular interactions of large βCD/graphene complexes, over a thousand atoms in size. We found that the binding preference of βCD on graphene is in a closed conformation via its C2C3 rim, agreeing with reported experimental and computational findings.

No description available.

Department of Chemistry / MSc (Chemistry) / Due to the increasing incidence of Plasmodium strains that are resistant to current frontline antimalarial drugs, malaria remains a global public health challenge. In recent years, the emergence of resistance to frontline antimalarial drugs including the more recently discovered artemisinin class drugs has become one of the greatest challenges of controlling malaria incidence and mortality. There is, therefore, an urgent need to develop novel targets and antimalarial drugs that are effective against drug-resistant malarial parasites. Recent studies have demonstrated that calcium dependent protein kinases (CDPKs) regulate a variety of biological processes in the malaria parasite Plasmodium falciparum and that CDPK4 is important for parasite development. The gene disruption of CDPK4 in Plasmodium berghei, which results in major defects in sexual differentiation of the parasite has highlighted the importance of CDPK4 in Plasmodium biology and suggests that it may be used as a target for therapeutic drugs. PfCDPK4 is expressed in the gamete/gametocyte stage, and this could make PfCDPK4 an essential target for malaria drug discovery. The structure of PfCDPK4 was used as a template in the discovery of malaria drug leads and in designing chemical compounds or inhibitors that will show anti-parasitic activity against the target molecule. The model structure of PfCDPK4 was generated through homology modelling, and model structure validation confirmed that the model structure of PfCDPK4 is of stereochemical quality. The molecular modelling approach of in silico screening was utilized in this research, wherein a large library of chemical compounds, some natural chemical compounds, and clinically approved kinase inhibitors were screened against the target molecule PfCDPK4. In silico screening of the Bio-Focus library against PfCDPK4 resulted in twenty-six compounds being identified; in vitro single screening at a concentration of 5 μM confirmed that three compounds exhibit moderate antimalarial activity against the NF54 strain of Plasmodium falciparum, with the percentage inhibition ranging between 42% and 47%.

Flies are a widely distributed pest insect that poses a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis all have the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extracellular binding site of a G protein-coupled receptor causes its activation. In this thesis, the structure of Phote-HrTH in SDS micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be reasonably rigid, with an S 2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis were used to construct two models for each receptor: Drome-AKHR, Sarcr-AKHR, and Bacdo-AKHR. It is proposed that these two models represent the active and inactive state of the receptor. The models based on the crystal structure of the β-2 adrenergic receptor were found to bind Phote-HrTH with a predicted binding free energy of –107 kJ mol–1 for Drome-AKHR, –102 kJ mol–1 for Sarcr-AKHR and –102 kJ mol–1 for Bacdo-AKHR. Under molecular dynamics simulation, in a POPC membrane, the β-2AR receptor-like complexes transformed to rhodopsin-like. The identification and characterisation of the ligand-binding site of each receptor provide novel information on ligand-receptor interactions, which could lead to the development of species-specific control substances to use discriminately against these pest flies.

La nécroptose est une mort cellulaire régulée impliquée dans les pathologies inflammatoires, ischémiques et dégénératives. RIPK1 serait une cible thérapeutique intéressante car son activité kinase serait à l’origine de leur initiation ou aggravation. Nous avons caractérisé les effets biologiques de deux nouveaux inhibiteurs de RIPK1. La sibiriline protège les souris d’une hépatite autoimmune et 6E11 protège les cellules endothéliales de l’aorte de la mort par ischémie froide/réoxygénation, suggérant des propriétés intéressantes pour ces inhibiteurs. Ces composés diffèrent par leur mode d’action, d’interaction et de sélectivité, et restent à optimiser en vue d’une utilisation thérapeutique. / Necroptosis is a regulated cell death pathway involved in inflammatory, ischemic or degenerative diseases. RIPK1 would be an interesting therapeutic target since its kinase activity is probably responsible of their initiation or aggravation. We have characterized the biological effects of two new RIPK1 inhibitors. Sibirilin protects mice from autoimmune hepatitis and 6E11 protects endothelial aortic cells from death due to cold ischemia/reoxygenation, suggesting interesting properties for these inhibitors. These compounds differ in their mode of action, interaction and selectivity, and still need to be optimized for therapeutic use.