<p>This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from <i>Candida albicans</i> as a tool for discovery of anti-<i>candida</i> drugs. <i>C. albicans</i> causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since <i>C. albicans</i> needs Saps during virulent growth, these enzymes are good targets for drug development.</p><p>In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions.</p><p>Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.</p>

Weak Affinity Chromatography (WAC) is a technology that was developed to analyse weak (KD &gt; 10-5 M) although selective interactions between biomolecules. The focus of this thesis was to develop this method for various applications in the drug development process.   Fragment Based Drug Discovery is a new approach in finding new small molecular drugs. Here, relatively small libraries (a few hundreds to a few thousands of compounds) of fragments (150 – 300 Da) are screened against the target. Fragment hits are then developed into lead molecules by linking, growing or merging fragments binding to different locations of the protein’s active site. However, due to the weakly binding nature of fragments, methods that are able to detect very weak binding events are needed. In this thesis, WAC is presented as a new robust and highly reproducible technology for fragment screening. The technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein-protein interaction (PPI) targets. Comparison of data from fragment screening of 111 fragments by WAC and other more established technologies for fragment screening, such as surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR), validates WAC as a screening technology. It also points at the importance of performing fragment screening by multiple methods as they complement each other.   Other applications of WAC in drug development are also presented. The method can be used for chiral separations of racemic mixtures during fragment screening, which enables affinity measurements of individual enantiomers binding to the target of interest. Further, analysis of crude reaction mixtures is shown. By these procedures, the affinity of the product can be assessed directly after synthesis without any time-consuming purification steps. In addition, a high performance liquid chromatography (HPLC) system for highly efficient drug partition studies was developed by stable immobilization of lipid bilayer disks – lipodisks – on a high performance silica support material. These lipodisks are recognized model membranes for drug partition studies. A WAC system with incorporated membrane proteins into immobilized lipodisks has also been produced and evaluated with the ultimate objective to study affinity interactions between ligands and membrane proteins. / Ett läkemedel utövar sin funktion genom att påverka aktiviteten hos ett protein i kroppen då det binder till dess aktiva säte. Förändringen i aktivitet leder till fysiologiska förändringar i kroppen beroende på vilken funktion proteinet har. Med läkemedelsmolekyl avses här en liten organisk molekyl. Fragment-baserad läkemedelsutveckling är en ny metod for att ta fram nya läkemedel. Metoden fungerar genom att man bygger läkemedelsmolekyler utifrån mindre fragment som binder till målproteinet. Fragmenten hittar man genom att screena hela bibliotek av olika fragment mot samma målprotein för att urskilja de som binder till proteinets aktiva säte. Fördelen med den här metoden är bl. a. att med mindre molekyler som utgångspunkt kan en större del av antalet möjliga kombinationer av atomer representeras med ett mindre antal fragment än för större molekyler. Normalt utgörs ett fragmentbibliotek enbart av några hundra till några tusen substanser. Eftersom fragmenten är små har de få interaktionspunker och binder relativt svagt. De svaga bindningarna är svåra att se och mycket känsliga metoder behövs.   Svagaffinitetskromatografi är en vätskekromatografisk metod som utvecklades för att studera svaga men mycket selektiva bindningar mellan biomolekyler. Den här avhandlingen syftar till att utveckla metoden för olika användningsområden inom läkemedelsutveckling, främst som en ny metod för fragment-screening. Här mäter man interaktionen mellan ett protein och ett fragment. Proteinet kopplas till ett material som sedan packas i en kolonn i formen av en cylinder. När provet pumpas igenom kolonnen kommer de analyter med affinitet till proteinets aktiva säte att fördröjas på kolonnen i relation till hur starkt de interagerar med målproteinet.   I den här avhandlingen presenteras fragment-screening med svagaffinitetskromatografi gentemot ett antal olika typer av målproteiner. Resultatet överensstämmer väl med andra metoder för fragment-screening. Analys av reaktionsblandningar med svagaffinitetskromatografi demonstreras också. Därmed kan bindningen mellan en produkt i en reaktionsblandning och ett målprotein mätas direkt utan föregående uppreningssteg av reaktionsblandningen. Lipodiskar är små diskformade modellmembran som kan användas för att bl. a. mäta hur effektivt läkemedlet tas upp i kroppen vid behandling. Ett system med immobiliserade lipodiskar i en kolonn utvecklades med det framtida målet att kunna arbeta med membranproteiner med svagaffinitetskromatografi.   Detta arbete utgör en del i att utveckla svagaffinitetskromatografi som en lättillgänglig och relativt billig metod för användning inom industrin och akademin för läkemedelsutveckling.

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Nuclear receptors (NRs) are modular ligand-activated transcription factors that control a broad range of physiological processes by regulating the expression of essential genes involved in cell physiology, differentiation, and metabolism. These receptors are implicated in a number of diseases and due to their profound role in development and disease progression and their modularity, much emphasis is being put forth into nuclear receptor based drug discovery and engineering these receptors to bind novel small molecules Chemical Complementation (CC) is a yeast three-hybrid genetic selection system that was developed to aid in the discovery of these engineered receptors by linking the survival of a yeast cell to a small molecules ability to activate the receptor. Due to several advantages, to include faster growth times and higher transformation efficiencies, we have attempted to extend chemical complementation from yeast to E. coli. The bacterial chemical complementation system (BCC) was designed, based on a bacterial two hybrid system, to parallel yeast CC system. However, bacterial chemical complementation did not produce ligand dependent activation due to heterologous protein expression. In a second project designed to further NR based protein engineering and drug discovery, CC was used to evaluate a library of charge reversal variants rationally designed to gain a better understanding of nuclear receptor function and structure and to produce orthogonal ligand receptor pairs. A library of retinoic acid receptor (RARα) variants were developed based on five residues in the binding pocket known to stabilize the natural negatively charged ligand, all-trans retinoic acid (atRA). We altered the binding selectivity of the receptor to bind positively charged retinoid ligands. We were able to engineer two triple variants capable of activating with the positively charged retinoid but not the natural atRA ligand, however they do not activate as well as RARα wild-type does with atRA. In a third project we characterized covalently linked tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. Several dual inhibiting compounds were found to decrease the proliferation of ER positive breast cancer cells better than tamoxifen alone, the HDACi alone, or noncovalently linked HDACi and tamoxifen.

abstract: Relapse after tumor dormancy is one of the leading causes of cancer recurrence that ultimately leads to patient mortality. Upon relapse, cancer manifests as metastases that are linked to almost 90% cancer related deaths. Capture of the dormant and relapsed tumor phenotypes in high-throughput will allow for rapid targeted drug discovery, development and validation. Ablation of dormant cancer will not only completely remove the cancer disease, but also will prevent any future recurrence. A novel hydrogel, Amikagel, was developed by crosslinking of aminoglycoside amikacin with a polyethylene glycol crosslinker. Aminoglycosides contain abundant amount of easily conjugable groups such as amino and hydroxyl moieties that were crosslinked to generate the hydrogel. Cancer cells formed 3D spheroidal structures that underwent near complete dormancy on Amikagel high-throughput drug discovery platform. Due to their dormant status, conventional anticancer drugs such as mitoxantrone and docetaxel that target the actively dividing tumor phenotype were found to be ineffective. Hypothesis driven rational drug discovery approaches were used to identify novel pathways that could sensitize dormant cancer cells to death. Strategies were used to further accelerate the dormant cancer cell death to save time required for the therapeutic outcome. Amikagel’s properties were chemo-mechanically tunable and directly impacted the outcome of tumor dormancy or relapse. Exposure of dormant spheroids to weakly stiff and adhesive formulation of Amikagel resulted in significant relapse, mimicking the response to changes in extracellular matrix around dormant tumors. Relapsed cells showed significant differences in their metastatic potential compared to the cells that remained dormant after the induction of relapse. Further, the dissertation discusses the use of Amikagels as novel pDNA binding resins in microbead and monolithic formats for potential use in chromatographic purifications. High abundance of amino groups allowed their utilization as novel anion-exchange pDNA binding resins. This dissertation discusses Amikagel formulations for pDNA binding, metastatic cancer cell separation and novel drug discovery against tumor dormancy and relapse. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2016

Os receptores ativadores da proliferação de peroxissomos (PPARs) são fatores de transcrição dependentes da ligação de ligantes e possuem um papel chave no controle do metabolismo dos lipídios e da glicose. Existem três isotipos desse receptor: PPAR&alpha;, PPAR&beta; e PPAR&gamma;. O PPAR&gamma; é alvo molecular para os compostos TZDs, os quais são fármacos usados clinicamente no controle da diabetes do tipo 2, aumentando a sensibilidade à insulina. Enquanto que os fibratos são os fármacos que atuam no PPAR&alpha; e são utilizados para diminuir os níveis de triglicerídeos. A maioria dos pacientes que sofrem com a diabetes do tipo 2 apresentam desordens no metabolismo de lipídios. Mesmo com a existência de fármacos capazes de controlar estas desordens metabólicas, a busca de um agonista dual para os PPAR&alpha; e PPAR&gamma; é um grande desafio no controle da síndrome metabólica, uma vez que este composto pode combinar os dois efeitos terapêuticos em uma única molécula. O GL479 é um agonista dual que foi sintetizado com dois grupos farmacóforos, ligando-se tanto ao PPAR&alpha; quanto ao PPAR&gamma;. Dentro desse contexto, este estudo apresenta as bases estruturais de interação do agonista dual GL479 aos PPARs por meio da determinação estrutural dos complexos PPAR&alpha;-LBD:GL479 e PPAR&gamma;-LBD:GL479. A análise detalhada desses complexos revelou diferentes modos de interação do ligante em cada receptor, porém em ambos os casos o GL479 interage com a Tyr da H12. Na estrutura do PPAR&alpha;-LBD, o ligante adquiriu a característica de um agonista total e no caso do PPAR&gamma;-LBD, o GL479 adotou características de um agonista parcial dependente da interação com a H12. Além das analises do agonista dual, 16 compostos foram identificados por docking como ligantes do PPAR&gamma;. Três desses ligantes (8, 10 e 15) foram caracterizados por ThermoFluor e fluorescência de polarização com valores de IC50 menor que 10 &micro;M. Adicionalmente, um dos compostos identificados no docking (16) foi cocristalizado com PPAR&gamma;-LBD. A conformação adotada pelo ligante não permitiu que ele interagisse diretamente com a H12, sugerindo que este composto possa atuar como um agonista parcial independente da H12. Todas estas descobertas podem ser exploradas no desenho de novos moduladores dos PPARs com menores efeitos adversos ou até mesmo na busca de agonistas duais PPAR&alpha; &frasl;&gamma;, que combine os efeitos terapêuticos no tratamento da diabetes do tipo 2 e da dislipidemia. / Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism and they play key roles in the control of glucose and lipid metabolism. There are three different PPAR isotypes: PPAR&alpha;, PPAR&beta; e PPAR&gamma;. PPAR&gamma; is a molecular target of TZD agonists, which are clinically used drugs in the control of type 2 diabetes by increasing insulin sensitivity. Whereas fibrates are drugs that act on PPAR&alpha; and are used to lower serum triglyceride levels. The most patients who have type 2 diabetes also display lipid metabolism disorders. Even with the existence of drugs that can control these metabolic disorders, the search of dual agonist for PPAR&alpha; and PPAR &gamma; is a major challenge in the control of metabolic syndrome, because this compound could combine both therapeutic effects in a single molecule. GL479 is a dual agonist that was synthesized based on a combination of two key pharmacophores, with the ability to bind in the both PPARs, &alpha;, and &gamma;. Thus, this study reveals the structural basis for this dual agonist GL479 by structural determination of the complexes PPAR&alpha;-LBD:GL479 and PPAR&gamma;-LBD:GL479. The detailed analysis of these complexes showed different ligand binding modes for each receptor, however, in the both cases the GL479 interacted with the Tyr of H12. In the PPAR&alpha;-LBD structure the ligand acquired the features of full agonist and in the case of PPAR&gamma;-LBD, GL479 adopted features of a partial agonist dependent of H12 interaction. In addition to the dual agonist analysis, sixteen compounds were identified as PPAR&gamma; ligand by docking. Three of these ligands were characterized by ThermoFluor and fluorescence polarization, which resulted in IC50 values smaller than 10 &micro;M. Additionally, one of the compounds, identified by docking, was co-crystallized with PPAR&gamma;. The ligand conformation adopted would not allow it a direct interaction with the H12. These contacts were mediated by one water molecule, suggesting this compound might also act as a partial agonist, independent of H12 interaction. All these findings may be explored for the design of PPARs novel modulators with lower side effects, as well, in the exploration of dual agonists PPAR&alpha; &frasl; &gamma; that combines the therapeutic effects in the treatment of type 2 diabetes and dyslipidemia.

Mycobacterium tuberculosis (Mtb), a deadly pathogen, has scourged mankind for many centuries and has remained a major threat to global world health. Tuberculosis, the disease caused by this bacterium, is a major cause of death in developing nations and there is potential for its re-emergence in developed countries. An alarming rise in cases of multidrug-resistant and extremely-drug resistant tuberculosis (MDR-TB and XDR-TB) that do not respond to the customary first-line antibiotics necessitates the urgent need for development of new anti-TB drugs. Mtb becomes engulfed in human macrophages post infection of the host, but persists in the harsh environment of the human lungs by utilization of host cholesterol as a carbon source. The P450s CYP125A1, CYP142A1 and CYP124A1 are responsible for catalysing the side-chain degradation of cholesterol, which is critical for cholesterol to be used in the Mtb β-oxidation pathway for energy production. This PhD thesis focuses on understanding the structure/mechanism of the Mtb cholesterol 27-oxidases with the aim of facilitating the development of novel inhibitors of these P450s, which are crucial for Mtb to infect the host and to sustain infection. CYP142A1 and CYP124A1 were purified through three chromatographic steps with contaminating proteins successfully removed to give highly pure forms of these enzymes following the final purification step. Spectrophotometric titrations indicate that CYP142A1 and CYP124A1 bind tightly to cholesterol and cholestenone (and also to branched-chain methyl lipids for CYP124A1), highlighting their physiological roles in sterol and fatty acid metabolism, respectively. Binding analyses with a range of azole antibiotics revealed tight binding to bifonazole, clotrimazole, miconazole and econazole, and weak binding to fluconazole. Studies with compounds from a fragment screening library revealed weak binding to fragment hits for the cholesterol oxidases, but much tighter binding to these enzymes was found for ‘elaborated’ hits from a previous fragment screen on the Mtb cyclodipeptide oxidase CYP121A1, indicative of improved ligand potency achieved via ‘fragment merging’ strategies, and of structural similarities between these diverse Mtb P450s. Light scattering data indicate that CYP142A1 exists in dimeric form in solution, but becomes monomeric when treated with DTT; while CYP124A1 is completely monomeric. Crystal structures of CYP142A1 and CYP124A1 in complex with cholestenone, econazole and fragment library hits were determined. CYP142A1 crystal structures with econazole and fragment hits revealed heme coordination via the heterocyclic nitrogen in an azole group, and provide important data towards design of superior inhibitor drugs. The binding of cholestenone within the active site channels of CYP124A1 and CYP142A1 revealed an alignment favourable for C27 hydroxylation of the cholestenone side chain, which supports the physiological roles of CYP142A1 and CYP124A1 (as well as CYP125A1) in host cholesterol catabolism.

This thesis aimed to identify the trypanocidal mode of action of non-natural chamuvarinin analogs, and to assess the previously uncharacterized secondary alternative oxidase (AOX2) as a possible drug target of the trypanosomatids. The drugs used to treat infections with Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are highly toxic and are increasingly becoming less effective as the parasites develop resistance, therefore new drugs against the diseases are desperately needed. Non-natural analogs of chamuvarinin were tested for trypanocidal activity to determine the structure activity relationships of the compounds against insect-form T. cruzi and Leishmania spp. This identified several potent and selective analogs, which retained good activity against the medically relevant intracellular forms of the parasites. Photoaffinity labeling was utilized to identify the mode of action and protein target(s) of the chamuvarinin analogs. The analogs were shown to deplete ATP levels and to induce mitochondrial dysmorphia and mitochondrial oxidative stress. Photoaffinity labeling confirmed the mitochondrial localization of the protein target(s) of these compounds, however the exact protein target(s) were unable to be identified by protein pull-down and mass spectrometry. The previously uncharacterized secondary alternative oxidases (AOX2) are conserved throughout the human-infective trypanosomatids and are absent from mammalian cells, thus making an attractive drug target if the protein is essential. The AOX2 of T. brucei, T. cruzi and L. major were expressed in Escherichia coli to characterize the enzymatic activity of the proteins. T. brucei AOX2 was successfully purified and shown to be an ubiquinol oxidase, which contains bound iron (III). The role of AOX2 within the trypanosomatids was determined by biochemical phenotyping and genetic manipulation of T. brucei, T. cruzi and L. major, which indicated that AOX2 is an essential mitochondrial oxidase in the three trypanosomatids, with a putative role in energy production, and therefore is an attractive multi-trypanosomatid drug target.

No description available.

No description available.