Dans la cellule, les protéines évoluent dans un environnement très dense et interagissent ainsi avec un grand nombre de partenaires spécifiques et non-spécifiques qui entrent en compétition. L’objectif de ma thèse est de caractériser les propriétés physiques et évolutives des surfaces protéiques pour comprendre comment la pression de sélection s’exerce sur les protéines, façonnant leurs interactions et régulant ainsi cette sévère compétition.Pour cela, j’ai développé une méthodologie permettant de caractériser la propension des protéines à interagir avec les protéines de leur environnement, par des approches de docking. La cartographie moléculaire permettant la visualisation et la comparaison des propriétés de la surface des protéines, j’ai donc mis en place un nouveau cadre théorique basé sur une représentation des paysages énergétiques d'interaction par des cartes d'énergies. Ces cartes (en deux dimensions) reflètent de manière synthétique la propension des surfaces protéiques à engager des interactions avec d’autres protéines. Elles sont donc d’un grand intérêt pratique pour déterminer les régions des surfaces protéiques les plus enclines à engager des interactions avec d’autres molécules.Ce nouveau cadre théorique a permis de montrer que les surfaces des protéines comprennent des régions de différents niveaux d'énergies de liaison (régions chaudes, intermédiaires et froides pour les régions d'interaction favorables, intermédiaires et défavorables respectivement).Une partie importante de la thèse a consisté à caractériser les propriétés physico-chimiques et évolutives de ces différentes régions. L'autre partie a consisté à appliquer cette méthode sur plusieurs systèmes : complexes homomériques, protéines du cytosol de S. cerevisiae, familles d'interologues. Ce travail ouvre la voie à un grand nombre d'applications en bioinformatique structurale, telles que la prédiction de sites de liaison, l’annotation fonctionnelle ou encore le design de nouvelles interactions.En conclusion, la stratégie mise en place lors de ma thèse permet d’explorer la propension d’une protéine à interagir avec des centaines de partenaires d'intérêts, et donc d'investiguer le comportement d’une protéine dans un environnement cellulaire spécifique. Cela va donc au-delà de l'utilisation classique du docking "binaire" puisque notre stratégie fournit une vision systémique des interactions protéiques à l’échelle des "résidus". / In the crowded cell, proteins interact with their functional partners, but also with a large number of non-functional partners that compete with the functional ones. The goal of this thesis is to characterize the physical properties and the evolution of protein surfaces in order to understand how selection pressure exerts on proteins, shaping their interactions and regulating this severe competition.To do this I developed a framework based on docking calculations to characterize the propensity of protein surfaces to interact with other proteins. Molecular cartography enables the visualization and the comparison of surface properties of proteins. I implemented a new theoretical framework based on the representation of interaction energy landscapes by 2-D energy maps. These maps reflect in a synthetic manner the propensity of the surface of proteins to interact with other proteins. These maps are useful from a practical point view for determining the regions of protein’s surface that are more prone to interact with other proteins. Our new theoretical framework enabled to show that the surface of proteins harbor regions with different levels of propensity to interact with other proteins (hot regions, intermediate and cold regions to favorable, intermediate and unfavorable regions respectively).A large part of this thesis work consisted in characterizing the physico-chemical properties and the evolution of these regions. The other part of this thesis work consisted in applying this methodology on several study systems: homomeric complexes, cytosolic proteins from S. cerevisiae, families of interologs. This work opens the way to numerous practical applications in structural bioinformatics, such as binding site prediction, functional annotation and the design of new interactions.To conclude, the strategy implemented in this work enable the exploration of the propensity of a protein to interact with hundred of protein partners. It thus enables the investigation of the behavior of a protein in a crowded environment. This application goes beyond the classical use of protein docking as a, because our strategy provides a systemic point of view of protein interactions at an atomic resolution.

Since the beginning of human civilization, medicinal plants have been used to treat a variety of infectious and non-infectious diseases. The therapeutic properties of phytochemicals have been recognized since ancient human history. The genus Helichrysum Mill. with its attractive flowers consist of an estimated 500‒600 species in the Asteraceae family. In South Africa and Namibia there are about 244‒250 species with tremendous morphological diversity. Several Helichrysum species are widely used by the indigenous population to treat various disorders such as wounds, infections, respiratory conditions, headaches, coughs, colds and fevers. Several of the Helichrysum species exhibit antiviral activity with the most relevant to this study being the discovery of anti-human immunodeficiency virus (anti-HIV) and anti-reverse transcriptase (anti-RT) activity of some species. Drug discovery and development, from the early stages of a promising compound to the final medication, is an intensive, expensive and incremental process. The ultimate goal is to identify a molecule with the desired effect in the human body and to establish its quality, safety and efficacy for treating patients. The ability to combine high-throughput analytical techniques like metabolomic and other experimental approaches with drug discovery will speed up the development of safer, more effective and better-targeted therapeutic agents. The rapidly emerging field of metabolomics and molecular docking analysis provides valuable information on drug activity, toxicity, customized drug treatments and can predict therapeutic outcomes. Extraction of the aerial parts of 32 Helichrysum species was done using polar [methanol (MeOH) 50%: distilled water (dH2O) 50%] and non-polar [hexane (Hex), dichloromethane (DCM) and acetone (Ace)] solvent systems. Anti-human immunodeficiency virus bioassays on the live HI virus revealed that polar extracts of H. mimetes and H. chrysargyrum at 2.5 μg/mL and 25 μg/mL, polar and non-polar extracts of H. infuscum at 25 μg/mL and polar and non-polar extracts of H. zeyheri, H. setosum, H. platypterum and H. kraussii at 2.5 and 25 μg/mL, had higher than 90% inhibitory activity. The polar extract of H. mimetes also exhibited reverse transcriptase (RT) inhibition as a possible indication of the mechanism of action. Proton nuclear magnetic resonance (1H NMR) spectra of the polar extracts exhibited the presence of aromatic compounds and carbohydrate moieties. Principal component analysis (PCA) of the polar extracts showed clustering related to the activity of the extracts with good predictability scores (Q2 > 0.5). However, orthogonal projections to latent structures discriminant analysis (OPLS-DA) predictability of the model was low based on the Q2 at approximately 0.25. Quinic acid (QA), isolated from H. mimetes showed promising anti-RT activity [50% inhibition concentration (IC50) = 53.82 μg/mL] which was comparable to the positive drug control, doxorubicin (IC50 = 40.31 μg/mL). The molecular docking study revealed the probable binding site and conformation of QA within cavity 4, with a docking score of -8.03. The docking score of doxorubicin within cavity 4 was -7.87. With this study, it was shown that metabolomic analysis as a tool to predict anti-HIV activity in Helichrysum species can be valuable to shorten the process. Moreover, the study of molecular docking revealed the mechanism action of quinic acid and doxorubicin against RT. / Thesis (PhD)--University of Pretoria, 2019. / Plant Production and Soil Science / PhD / Unrestricted

The pathogenicity and intractable nature of the microorganism Staphylococcus aureus (SA) has been long documented and highlighted by many health care agencies, with emphasis on its ability to exploit the human coagulation system to deadly effect. Two drugs from a class of inhibitors known as Direct Thrombin Inhibitors (DTI) have been shown to have a substantial effect on the enzyme secreted by SA known as Staphylocoagulase (SC), but up until now the application of this potential treatment has been limited. This paper strives to supply an overview of these clinical studies and propose a novel protocol for testing DTI's on SA in an in vitro setting. Three DTIs have been identified, including two already tested in clinical trials, and computational molecular docking simulations have been applied to elucidate the mechanisms of action for the inhibition. An additional DTI has been developed using these mechanisms as principles and shows promise for future development. After conducting this preliminary protocol, it has been found that running a minimum inhibitory concentration test across several tubes with varying degrees of these DTIs demonstrated varying levels of coagulation consistent with the findings of clinical research papers. It is fair to conclude, then, that after development or discovery of new coagulase inhibitors, they can be quickly and accurately tested against existent DTIs to gauge their efficacy.

No description available.

No description available.

In humans, mammals, and perhaps all vertebrates, sphingolipids exist as a family of cellular signaling molecules and have been shown to be involved in a wide range of biological processes ranging from proliferation to apoptosis. As such, sphingolipid signaling has garnered the attention of numerous researchers as an attractive candidate for pharmacological manipulation. The synthetic pathway of one prominent sphingolipid, sphingosine 1-phosphate (S1P), has been implicated in a variety of disease states such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Formation of S1P is facilitated from the ATP dependent phosphorylation of sphingosine (Sph) through its generative enzyme's sphingosine kinase 1 and 2 (SphK1 and SphK2). Inhibition of SphK1 and SphK2 results in the manipulation of S1P levels, which has been shown to be therapeutic in various animal models of disease. While there are multiple examples of potent SphK1-selective and dual SphK1/2 inhibitors, SphK2-selective inhibitors are scarce. Herein, we describe the design, synthesis and biological testing of SphK2-selective inhibitors. We first describe the discovery that introducing a trifluoromethyl group onto the internal aryl ring of our inhibitor scaffold led to superior selectivity and potency towards SphK2. We demonstrate that the trifluoromethyl moiety is interacting with a previously unknown side cavity in the substrate binding site of SphK2 that is unique and could be exploited in the design of SphK2-selective inhibitors. The synthesis of 21 derivatives with various substituents spanning off the internal aryl ring was completed, therefore characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site. This work led to the development of the most potent SphK2-selective inhibitor known at the time. We then describe the transformation of our SphK2-selective inhibitors into an orally bioavailable drug. We explain how the guanidine functionality on our inhibitor scaffold hinders our compounds from being orally bioavailable. Consequently, a library of 24 derivatives with various modifications to the guanidine functionality was synthesized and evaluated for improved orally bioavailability. Highlighted in this work is the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832), which displays a hSphK2 Ki of 82 nM and 122-fold selectivity for SphK2. Chemical modification and in vivo assessment of 3.14 (SLS1081832) prodrugs was explored. / Doctor of Philosophy / In humans, sphingosine 1-phosphate (S1P) is a signaling molecule that is generated through an ATP dependent reaction of sphingosine (Sph) via sphingosine kinase 1 and 2 (SphK1 and SphK2). Furthermore, S1P has been shown to be implicated in various diseases such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Inhibition of SphK1 and SphK2 has been shown to be therapeutic towards the symptoms of these diseases. Therefore, in order to alleviate these disorders, the concentrations of S1P must be controlled through pharmacological inhibition of SphK1 and SphK2. There are multiple reported examples of potent SphK1-selective and dual SphK1/2 inhibitors; however, SphK2-selective inhibitors are scarce. This work describes the synthesis and biological assessment of 21 compounds for their effectiveness in selectively targeting and inhibiting SphK2. The work led to the discovery of a previously unrecognized side cavity in the binding pocket of SphK2 that enhances inhibitor potency and selectivity towards SphK2. Furthermore, studies characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site led to the development of the most potent SphK2- selective inhibitor known at the time. Building on this work, we next focused on the transformation of our SphK2-selective inhibitors into a drug that could be administered orally. We describe the synthesis of 24 compounds with various modifications to one portion of our scaffold and their effect on improved orally bioavailability. This work led to the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832).

A variety of diseases including Alzheimer's disease, asthma, cancer, fibrosis, multiple sclerosis, and sickle cell disease have been associated with elevated levels of sphingosine-1-phosphate (S1P). S1P, a pleiotropic lipid mediator involved in a broad range of cellular processes, is synthesized solely by the phosphorylation of sphingosine (Sph) and is catalyzed by the two isoforms of sphingosine kinase (SphK1 and SphK2). Therefore, SphKs are a potential therapeutic target; however, the physiological role of SphK2 is still emerging. In order to determine the role of SphK2 in vivo, more potent and selective small molecule inhibitors of SphK2, as well as dual inhibitors are necessary. Herein, explorations and advancements on the second generation SphK2 selective inhibitor SLR080811 are disclosed. Investigations into the lipophilic tail region of the hSphK2 inhibitor SLR080811 are detailed. This investigation highlights the dependency of SphK2 selectivity and potency on overall compound length. More importantly, this study identified the internal aryl ring of SLR080811 as a key pharmacophore of the scaffold. To further probe the significance of the aromatic region, the phenyl ring was replaced by a 2,6-naphthyl ether skeleton. Investigations into the tail region of this scaffold are described in detail. Key discoveries from this structure-activity relationship study include SLC5111312 (hSphK2 Ki = 0.90 μM, dual hSphK inhibitor), SLC5091592 (hSphK2 Ki = 1.02 μM, > 20-fold hSphK2 selective) and SLC5121591 (hSphK2 Ki = 0.61 μM, >16-fold hSphK2 selective). Molecular modeling studies with hSphK2 indicate that the extended aromatic group is able to participate in π-π stacking interactions with Phe548. In silico docking studies indicate that a guanidine hydrogen bond to Asp211 is key for SphK2 selectivity, and incorporation of a 3'-hydroxyl group on the pyrrolidine ring increases hydrogen bonding to Asp308, thereby increasing SphK1 potency and reducing selectivity. Additionally, biological studies employing SLC5111312 have helped to further elucidate the role of SphK2, suggesting that SphK2 has a catalytic role in the regulation of blood S1P levels. The shape of the hSphK2 binding pocket was probed by introducing an indole moiety in place of the naphthyl ring and varying its substitution pattern. One key discovery from this study is SLC5101465 (hSphK2 Ki = 0.09 μM, > 111 fold SphK2 selective), which has a 1,5-indole substitution pattern with an N-nonyl "tail". Molecular docking simulations highlight the importance of rotatable bonds and a relatively linear orientation between the "head group" and "tail group" to maintain essential hydrogen bond interactions to Asp residues with the guanidine moiety while minimizing steric interactions in the middle of the binding pocket. Expanding upon the 1,5-indole scaffold of SLC5101465, a series of aryl tail derivatives are examined. This study confirms the necessity of electron withdrawing groups located at the end of the inhibitor scaffold to optimize binding in the tail region of the SphK2 binding pocket. / Ph. D.

Kinases are involved in a multitude of signaling pathways, such as cellular growth, proliferation, and apoptosis, and have been discovered to be important in numerous diseases including cancer, Alzheimer's disease, cardiovascular health, rheumatoid arthritis, and fibrosis. Due to the involvement in a wide variety of disease types, kinases have been studied for exploitation and use as targets for therapeutics. There are many limitations with developing kinase target therapeutics due to the high similarity of kinase active site composition, making the utilization of new techniques to determine kinase exploitability for therapeutic design with high specificity essential for the advancement of novel drug strategies. In silico approaches have become increasingly prevalent for providing useful insight into protein structure-function relationships, offering new information to researchers about drug discovery strategies. This work utilizes streamlined computational techniques on an atomistic level to aid in the identification of orthologue and isoform exploitability, identifying new features to be utilized for future inhibitor design. By exploring two separate kinases and kinase targeting domains, we found that orthologues and isoforms contain distinct features, likely responsible for their biological roles, which can be utilized and exploited for selective drug development. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design. / Master of Science in Life Sciences / Numerous diseases such as cancer, Alzheimer's disease, cardiovascular disease, rheumatoid arthritis, and fibrosis have been attributed to different cell growth and survival pathways. Many of these pathways are controlled by a class of enzymes called kinases. Kinases are involved in almost every metabolic pathway in human cells and can act as molecular switches to turn on and off disease progression. Due to the involvement of these kinases' in a wide variety of disease types, kinases have been continually studied for the development of new drugs. Developing effective drugs for kinases requires an extensive understanding of the structural characteristics due to the high structural similarity across all kinases. In silico, or computational, techniques are useful strategies for drug development practices, offering new information into protein structure-function relationships, which in turn can be utilized in drug discovery advancements. Utilizing computational methods to explore structural features can help identify specific protein structural features, thus providing new strategies for protein specific inhibitor design. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design.

Peroxisome proliferator-activated receptor γ (PPARγ) is associated with a wide range of diseases, including type 2 diabetes mellitus (T2D). Thiazolidinediones (TZDs) are agonists of PPARγ which have an insulin sensitizing effect, and are therefore used as a treatment for T2D. However, TZDs cause negative side effects in patients, such as weight gain, edema, and increased risk of bone fracture. Partial agonists could be an alternative to TZD-based drugs with fewer side effects. However, there is a lack of understanding of the types of PPARγ partial agonists and how they differ from full agonists. In silico techniques, like virtual screening, molecular docking, and pharmacophore modeling, allow us to determine and characterize markers of varying levels of agonism. An extensive search of the RCSB Protein Data Bank found 62 structures of PPARγ resolved with partial agonists. Cross-docking was performed and found that two PDB structures, 3TY0 and 5TWO, would be effective as receptor structures for virtual screening. By clustering known partial agonists by common pharmacophore features, we found several distinct groups of partial agonists. Interaction and pharmacophore models were created for each group of partial agonists. Virtual screening of FDA-approved compounds showed that the models were able to predict potential partial agonists of PPARγ. This study provides additional insight into the different binding modes of partial agonists of PPARγ and their characteristics. These models can be used to assist drug discovery efforts for intelligently designing novel therapeutics for T2D which have fewer negative side effects. / Master of Science in Life Sciences / The peroxisome proliferator-activated receptor γ (PPARγ) protein is associated with a wide range of diseases, including type 2 diabetes mellitus (T2D). Thiazolidinediones (TZDs) are compounds that activate PPARγ, and increase insulin sensitivity in patients with T2D. However, TZDs cause negative side effects in patients, such as weight gain, increased fluid retention, and increased risk of bone fracture. Partial agonists could be an alternative to TZD-based drugs with fewer side effects. However, there is a lack of understanding of the types of PPARγ partial agonists and how they differ from full agonists. Computational techniques allow us to investigate common features between known partial agonists. An extensive search of the RCSB Protein Data Bank found 62 structures of PPARγ which contained partial agonists. Each known partial agonist was docked into twelve complete PPARγ structures, and it was found that two structure models would be effective as receptor structures for virtual screening. A set of known partial agonists were grouped based on common chemical features, and three distinct groups of partial agonists were found. Binding criteria for each of these three groups were developed. A library of FDA-approved compounds was screened using the criteria for binding to identify potential novel partial agonists. Three potential novel partial agonists were found in the screening. This study provides additional insight into how different compounds activate PPARγ. These methods can be used to assist drug discovery efforts for intelligently designing novel therapeutics for T2D which have fewer negative side effects.

No / Decaprenylphosphoryl-b-d-ribose 20-epimerase (DprE1) is a potential drug target for development of antitubercular agents. Structure based drug discovery approach yielded twenty novel derivatives of benzothiazolylpyrimidine-5-carboxamides (7a–t) which were synthesised by three component one pot reaction involving benzothiazolyl oxobutanamide, thiourea and substituted aromatic benzaldehydes. These derivatives were evaluated for antitubercular activity to determine MIC and compound 7a, 7e, 7f and 7o were found to be potentially active against Mycobacterium tuberculosis (H37Rv). Log P of these compounds was found to be between 2.0 and 3.0 making them suitable for oral dosing. DprE1 selectivity and pharmacokinetic studies were carried out for these compounds of which 7a and 7o were found to be highly selective and bioavailability was found to be above 52% by oral dose. Crystal structure of 7a was studied and molecular packing was determined, it exhibited a triclinic crystal lattice arrangement having hydrogen bonded dimeric arrangement. Drug receptor interactions were studied which exhibited docking in the active site of receptor with hydrogen bonding, hydrophobic interactions, vdW interactions with amino acid residues such as Cys387, Asn385, Lys418, Tyr314, Gln334 and Lys367 respectively. 3D QSAR analysis was carried out by kNN-MFA method to determine and develop theoretical model, best suitable model was found to be based on Simulated Annealing k-Neariest Neighbour Molecular Field Analysis (SA kNN-MFA). The model provided with hydrophobic descriptors in positive side indicating the need of bulky groups, steric and electronegative descriptors in negative coordinates hints with contribution by the electronegative substitutions as favourable and desirable moieties for enhancing the activity. The q2, q2_se and Pred_r2se were found to be 0.5000, 0.6404 and 1.0094 respectively. A pharmacophore model was generated which suggested for necessity of aromatic, aliphatic carbon centre and hydrogen bond donor for development of newer DprE1 selective inhibitors. / Council of Scientific and Industrial Research