Dissertations / Theses on the topic 'Biophysical screening'

MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene. MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the ubiquitination (and degradation) of p53. Several proteins are responsible for the ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential for ubiquitination, the protein-protein recognition site is a potential drug target. Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues 386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these results showed that the MDM2RING construct formed a concentration-dependent oligomeric structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations. Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified. Various biophysical techniques were used to study its interaction with MDM2 as well as with potential small molecule inhibitors as in principle, small molecules which disrupt the interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53. The dimerisation of MDM2 is important for its E3 activity and the C8-binding site potentially provides a second druggable site. In this work, peptide 9, which has the same sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2 E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and electrospray mass spectrometry were used to investigate the interaction between two inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to search for small molecules which can affect MDM2-dependent ubiquitination. Three potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were identified using our virtual screening database-mining and docking programs that were shown to affect MDM2-dependent ubiquitination of p53. In terms of understanding the complex biochemical mechanism of MDM2 this work provides two interesting and functionally relevant observations: (i) the MDM2 RING△C construct is a dimer as this would not be expected form the existing studies, and has less E3 ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding site enhanced E3 ligase activity. One model to explain these observations is that binding of small molecule activators family to the RING induces a change in the conformation of the Cterminal tail residues which may enhance E2 binding.

In this work high resolution crystals were produced of full length human eIF4E complexed to m7GTP and the 4E-BP1 motif peptide. The interactions of eIF4E with the cap structure were also analysed by mass spectrometry, revealing a requirement of eIF4E for a guanine monophosphates derivative with a positive delocalised charge on the m7G string. Potential inhibitors for screening were either isolated using virtual screening techniques or synthesised to produce a series of cap mimicking molecules containing a positive delocalised charge. Mass spectrometry identified a series of N7 substituted CMP derivatives that bound to eIF4E in the gas-phase. These compounds were then used in co-crystallisation trials with full length human eIF4E complexed with a 4E-BP1 motif peptide. The co-crystal structures of eIF4E with N7 benzyl derivatives revealed a flipping of the tryptophan 102 to accommodate the bulky N7 group and the expulsion of two structured waters. It also showed that if a para-fluoro group is located on the benzyl modification, then interactions also occur with structured water and an arginine, which explains its increased binding to eIF4E. The flipping of the tryptophan reveals the inherent flexibility in the cap-binding site. The structural information, revealing that the cap-binding site of eIF4E undergoes a conformational change in binding N7 derivatives of GMP with large bulky groups, provides us with valuable insight that can be used in future drug design efforts. The mass spectrometry assay coupled with a clear structure activity relationship, developed on the basis of various “cap-like” ligands studied in the work, gives an excellent starting point for the development of cap-analogue mimics for anti-cancer therapeutics.

En cancérologie, la radiothérapie est une des armes essentielles pour éradiquer les cellules tumorales. Les cassures des deux brins de l'ADN dites "double-brin" qu'elle induit sont particulièrement toxiques et constituent la principale cause de mort cellulaire. La NHEJ (Jonction d'Extrémités Non-Homologues) est la voie métabolique majeure de réparation de ces cassures double-brin de l'ADN et par ce mécanisme, les cellules humaines adoptent une résistance à la radiothérapie. Ce mécanisme de réparation constitue donc une cible de choix pour un traitement anticancéreux combiné en vue d'augmenter la sensibilité des cellules cancéreuses aux rayons ionisants (radiosensibilisation). Au cours du mécanisme NHEJ, la ligature finale des extrémités d'ADN est assurée par le complexe protéique tripartite: XRCC4/ADN Ligase IV/Cernunnos-XLF. Les interfaces protéiques concernées représentent toutes des cibles potentielles dans une stratégie rationnelle d'isolement de molécules inhibitrices, guidée par les structures tridimensionnelles de chaque protéine. A travers des expériences de criblage virtuel et de validation à la fois biophysique et biochimique, nous avons isolé les premières molécules capable de prévenir in vitro les interactions protéine-protéine pour les complexes XRCC4/Lig4 et XRCC4/Cer-XLF, respectivement. Ces composés sont des points de départ pour l'élaboration d'inhibiteurs potentiels de plus haute affinité grâce à l'apport de la biologie structurale, en vue d'un effet radiosensibilisant cellulaire
Radiotherapy is a major weapon used against cancer. Radio-induced DNA double strand breaks (DSB) are the main lesions responsible for cell death. Non-homologous end-joining (NHEJ) is a predominant DSB repair mechanism which contributes to cancer cells resistance to radiotherapy. NHEJ is thus a good target for strategies which aim at increasing the radio-sensitivity of tumors. Through in silico screening and biophysical and biochemical assays, our objective was to find specific ligands for the XRCC4/Lig4 and XRCC4/Cer-XLF protein-protein interactions involved in NHEJ. Here, we isolated the first compounds able to prevent their interaction in vitro. These early stage inhibitors are promising tools for cancer therapy with the hope to develop more specific compounds for cellular assays through the 3D structure of the protein/inhibitor complexes

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

In this thesis, molecular dynamics simulations, molecular docking, and homology modeling methods have been used in combination to design possible inhibitors as well as to study the structural changes and function of target proteins related to diseases that today are in the spotlight of drug discovery. The inwardly rectifying potassium (Kir) channels constitute the first target in this study; they are involved in cardiac problems. On the other hand, tensin, a promising target in cancer research, is the second target studied here. The first chapter includes a brief update on computational methods and the current proposal of the combination of MD simulations and docking techniques, a procedure that is applied for the engineering of a new blocker for Kir2.1 ion channels and for the design of possible inhibitors for Tensin. Chapter two focuses in Kir ion channels that belong to the family of potassium-selective ion channels which have a wide range of physiological activity. The resolved crystal structure of a eukaryotic Kir channel was used as a secondary structure template to build the Kir-channels whose crystallographic structures are unavailable. Tertiapin (TPN), a 21 a.a. peptide toxin found in honey bee venom that blocks a type of Kir channels with high affinity was also used to design new Kir channel blockers. The computational methods homology modeling and protein-protein docking were employed to yield Kir channel-TPN complexes that showed good binding affinity scores for TPN-sensitive Kir channels, and less favorable for Kir channels insensitive to TPN block. The binding pocket of the insensitive Kir-channels was studied to engineer novel TPN-based peptides that show favorable binding scores via thermodynamic mutant-cycle analysis. Chapter three is focused on the building of homology models for Tensin 1, 2 and 3 domains C2 and PTP using the PTEN X-ray crystallographic structure as a secondary structure template. Molecular docking was employed for the screening of druggable small molecules and molecular dynamics simulations were also used to study the tensin structure and function in order to give some new insights of structural data for experimental binding and enzymatic assays. Chapter four describes the conformational changes of FixL, a protein of bradyrhizobia japonicum. FixL is a dimer known as oxygen sensor that is involved in the nitrogen fixation process of root plants regulating the expression of genes. Ligand behavior has been investigated after the dissociation event, also the structural changes that are involved in the relaxation to the deoxy state. Molecular dynamics simulations of the CO-bound and CO-unbound bjFixL heme domain were performed during 10 ns in crystal and solution environments then analyzed using Principal Component Analysis (PCA). Our results show that the diffusion of the ligand is influenced by internal motions of the bound structure of the protein before CO dissociation, implying an important role for Arg220. In turn, the location of the ligand after dissociation affects the conformational changes within the protein. The study suggests the presence of a cavity close to the methine bridge C of the heme group in agreement with spectroscopic probes and that Arg220 acts as a gate of the heme cavity.

Drug-resistant pathogenic and opportunistic bacteria are increasing in occurrence and prevalence, and pose a dangerous threat to human health. In the search for novel antibiotics with which to combat this threat, plants, specifically those used in traditional medicine with ascribed antibacterial properties, offer a promising and potentially vast source of such therapeutic compounds. The purpose of this study was therefore to screen chemical extracts created from various plant species for antibacterial properties versus pathogenic bacterial species. In the course of these antibacterial assays, we successfully identified a methanol extract derived from Artemisia tridentata tridentata plant material as capable of inhibiting the growth of the opportunistic pathogen Staphylococcus aureus. Three sub-fractions were created using hexane, ethyl acetate and water solvents. Each of these extracts displayed significant antibacterial activity versus a wild-type strain over a period of six hours, at concentrations as low as 62.5 µg/ml. The extracts also demonstrated an enhancement of antibiotic effects when combined with ampicillin, G418 sulfate or amikacin, for a period of up to twelve hours. Though the efficacy of the extracts was lessened when tested against an ampicillin-resistant strain, significant enhancement of the efficacy of this antibiotic was still observed. Gas chromatography-mass spectrometry analysis of these three extracts revealed the sesquiterpene lactone achillin as present in each. Column chromatography of the hexane extract resulted in a fraction retaining its antibacterial activity, and still containing this compound, further implicating it as responsible for the antibacterial activity of this plant. The results of serial dilution and plating of extract-treated samples, along with those of ethidium bromide assays and transmission electron microscopy analysis, indicated a bacteriostatic mechanism of action involving disruption of the bacterial membrane, which is in agreement with the literature on the antibacterial properties of this plant, and those of sesquiterpene lactones, respectively. We therefore conclude that achillin, likely produced as a secondary metabolite by Artemisia tridentata tridentata, possesses growth inhibitory properties versus Staphylococcus aureus, and should be isolated and studied further for the purposes of evaluating its potential use, either as a stand-alone antibiotic, or as an adjunctive therapeutic, in the treatment of drug-resistant bacterial pathogens.

Individuals under 20 years old have the highest risk of developing type 1 diabetes because their beta cells are destroyed at a faster rate than any other age group. Previous studies have looked at delaying and slowing down the rate of beta cell destruction through the use of anti-CD3 antibody treatments. Specifically, Teplizumab and Otelixizumab drug therapies have been used to treat individuals within 12 weeks of diagnosis. Previous studies done with Teplizumab and Otelixizumab have focused on individuals between 12 and 40 years old; however, there is little research done the effects of these treatments on individuals under 12 years old. Since type 1 diabetes is primarily diagnosed in children the purpose of this proposal is to further the knowledge of the preservation of beta cells in children with the use of Teplizumab and Otelixizumab drugs over 2 years. This study will also focus on the delay of onset type 1 diabetes with the use of Teplizumab in high-risk individuals under 20 years of age. The high-risk individuals will be determined by using genetic screening on individuals with an affected immediate family member. Individuals will also be tested to see if they carry insulin autoantibodies (IAA), protein tyrosine phosphate-related IA-2 molecule (IA-2A), islet cell antibodies (ICA), and glutamic acid decarboxylase (GADA). Individuals that test positive for carrying the DR3/DR4 alleles in the genetic screening and that have two or more autoantibodies present in their immune systems will be selected to participate. The purpose of this proposal is to further understand the impact of anti-CD3 antibody treatment on young individuals diagnosed with type 1 diabetes and to further understand if treatment in young individuals with anti-CD3 antibodies can delay or prevent the onset of type 1 diabetes.

En el presente trabajo, utilizamos técnicas de microfabricación, simulaciones numéricas, experimentos de electrofisiología para explorar la viabilidad en me- jorar la interface ordenador-neurona a través de microcanales, y la biofísica para la generación de señales en los dispositivos con microcanales. También demos- tramos que los microcanales pueden ser usados como una técnica prometedora con alto rendimiento en el muestreo automático de canales iónicos a nivel subce- lular. Finalmente, se ha diseñado, fabricado y probado el micropozo-microcanal como modificación adicional a los arreglos de multielectrodos, permitiendo una alta ganancia en la relación señal/ ruido (en inglés Signal to Noise Ratio SNR), y el registro de múltiples-lugares en poblaciones de baja densidad de redes neu- ronales del hipocampo in vitro. Primero, demostramos que son de alto rendimiento los microcanales de bajo costo con interface neurona-electrodo, para el registro extracelular de la activi- dad neuronal con baja complexidad, por periodos estables de larga duración y con alta ganancia SNR. En seguida, se realiza un estudio mediante experimentos y simulaciones nu- méricas de la biofísica para la generación de las señales obtenidas de los dispositi- vos con microcanales. Basados en los resultados, racionalizamos y demostramos como es que la longitud del canal (siendo 200 μm) y la sección transversal del microcanal (siendo 12 μm2) canaliza a los potenciales de acción para estar dentro del rango de milivolts. A pesar del bajo grado de complexidad envuelto en la fabricación y aplicación, los dispositivos con microcanales otorgan una sola media de valor SNR de 101 76, lo cual es favorablemente comparable con la SNR que se obtiene de desarrollos recientes que emplean electrodos curados con CNT y Si-NWFETs. Más aún, nosotros demostramos que el microcanal es una técnica promete- dora para el alto rendimiento del muestro automático de canales iónicos a nivel subcelular: (1) Información experimental y simulaciones numéricas sugieren que las señales registradas sólo afectan los parches membranales localizados dentro del microcanal o alrededor de 100 μm de las entradas del microcanal. (2) La transferencia de masa de los componentes químicos en los microcanales fue ana- lizada por experimentos y simulaciones FEM. Los resultados muestran que los microcanales que contienen glía y tejido neuronal pueden funcionar como barre- ra de fluido/química. Los componentes químicos pueden ser solamente aplicados a diferentes compartimentos a nivel subcelular. Finalmente, basado en simulaciones numéricas y resultados experimentales, se propone que del micropozo-microcanal, obtenido de la modificación de MEA (MWMC-MEA), la longitud óptima del canal debe ser 0,3 mm y la posición 1 óptima del electrodo intracanal, hacia la entrada más cercana del microcanal, debe ser 0,1 mm. Nosotros fabricamos un prototipo de MWMC-MEA, cuyo hoyo pasante sobre las películas de Polydimethylsiloxane (PDMS) fue microtrabajado a través de la técnica de grabados reactivos de plasma de iones. La baja densidad del cultivo (57 neuronas /mm2) en el MWMC-MEAs permitió que las neuronas vivieran al menos 14 días, con lo que la señal neuronal con la máxima SNR obtenida fue de 142. 2
In this present work, we used microfabrication techniques, numerical simulations, electrophysiological experiments to explore the feasibility of enhancing neuron-computer interfaces with microchannels and the biophysics of the signal generation in microchannel devices. We also demonstrate the microchannel can be used as a promising technique for high-throughput automatic ion-channel screening at subcellular level. Finally, a microwell-microchannel enhanced multielectrode array allowing high signal-to-noise ratio (SNR), multi-site recording from the low-density hippocampal neural network in vitro was designed, fabricated and tested. First, we demonstrate using microchannels as a low-cost neuron-electrode interface to support low-complexity, long-term-stable, high SNR extracellular recording of neural activity, with high-throughput potential. Next, the biophysics of the signal generation of microchannel devices was studied by experiments and numerical simulations. Based on the results, we demonstrate and rationalize how channels with a length of 200 μm and channel cross section of 12 μm2 yielded spike sizes in the millivolt range. Despite the low degree of complexity involved in their fabrication and use, microchannel devices provided a single-unit mean SNR of 101 76, which compares favourably with the SNR obtained from recent developments employing CNT-coated electrodes and Si-NWFETs. Moreover, we further demonstrate that the microchannel is a promising technique for high-throughput automatic ion-channel screening at subcellular level: (1) Experimental data and numerical simulations suggest that the recorded signals are only affected by the membrane patches located inside the microchannel or within 100 μm to the microchannel entrances. (2) The mass transfer of chemical compounds in microchannels was analyzed by experiments and FEM simulations. The results show that the microchannel threaded by glial and neural tissue can function as fluid/chemical barrier. Thus chemical compounds can be applied to different subcellular compartments exclusively. Finally, a microwell-microchannel enhanced MEA (MWMC-MEA), with the optimal channel length of 0.3 mm and the optimal intrachannel electrode position of 0.1 mm to the nearest channel entrance, was proposed based on numerical simulation and experiment results. We fabricated a prototype of the MWMCMEA, whose through-hole feature of Polydimethylsiloxane film (PDMS) was micromachined by reactive-ion etching. The low-density culture (57 neurons/mm2) were survived on the MWMC-MEAs for at least 14 days, from which the neuronal signal with the maximum SNR of 142 was obtained.

SAS-6 is the structural core of the forming centriole - a cylindrical protein complex, which is an essential component of the centrosome. Oligomerisation of SAS-6 is crucial for successful centriole duplication and is achieved through two dimerisation domains in the SAS-6 protein; a long C-terminal coiled-coil domain and a globular N-terminal dimerisation domain. As core components of the centrosome, centrioles help facilitate various cellular functions. They are involved in the anchoring of flagella and cilia to the membrane and in coordinating the spindle apparatus during chromosome segregation. A deeper insight into the molecular mechanisms at play in the centriole duplication process would have implications on our understanding of fundamental cell division processes and a number of related diseases. Here the involvement of an unstudied loop region in the C. elegans SAS-6 N-terminal domain dimerisation is described. Combining structural biology, biophysical and computational techniques, the molecular interactions of this loop were explored, contributing to the oligomerisation of SAS-6 at the N-terminal dimer interface. Furthermore, the screening and testing of small molecule inhibitors of the SAS-6 N-terminal domain dimerisation is described, targeting a hydrophobic pocket in the domain. Two candidate compounds are presented as a result of the screens and next steps towards structure based compound design are suggested, based on computational analysis. The search for inhibitory compounds includes a set-up of an in-house virtual screening pipeline, as well as in vitro screening efforts and a new crystallographic structure of the H. sapiens SAS-6 N-terminal domain. By investigating the making and breaking of the SAS-6 N-terminal domain dimerisation, light is shed on so far neglected details of this essential protein-protein interaction and advancements towards a SAS-6 oligomerisation inhibitor described, which could ultimately be used for new approaches in cell cycle research and might open up new avenues for medical research by binding a disease relevant target.

With the continued need for drug discovery and the quest to understand disease and treatment, there remains a requirement for improved methods to study protein-protein interactions and to identify potential drug leads for protein targets. We sought to develop a new approach to directly link genotype and phenotype to use as a probe for the identification of binding partners of proteins. The method creates millions of water-in-oil emulsions, each of which functions as a micro-environment for the amplification of a library of random peptide-encoding DNA molecules, which covalently bind to a bead. Subsequently the emulsions are broken and the bead-DNA complexes are recovered, which subsequently form another emulsion with in vitro transcription and translation components and incubated under suitable protein synthesis conditions. The synthetic peptide is designed with tags that link to the same bead which it is translated from. In chapter 3, the detailed design and optimisation of the method will be discussed. Cross-clade neutralising antibodies specific to HIV-1 are rare, partly because glycosylation restricts access to conserved backbone residues of gp120. In chapter 4, we hypothesized that peptides may have greater access than relatively large antibody structures, and so used our method to display random peptides on beads using a protein domain scaffold. Using a single round of selection, we identified 22 gp120-binding peptides, 4 of which were able to inhibit HIV-1 replication in vitro. One of the inhibitory peptides was found to bind the CCR5/CXCR4-binding site of gp120 and was able to inhibit clade B and C CCR5-tropic isolates of HIV-1. We have identified HIV-1 cross-clade neutralising peptides using a novel in vitro bead display library. Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. We reasoned that a bead-based display high-throughput approach may overcome these challenges. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to β-2-microglobulin on beads, we refolded with exogenous wild-type HLA-A*0201 or CD8-null A*0201 (domains 1 and 2 of HLA-A*0201 and domain 3 of Kb with mutated residues 226A/227L). The HLA bead libraries were used to probe HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. High-throughput sequencing was used to rank sequences of identified peptides. Compared to pre-selection sequences, we observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells. In summary, we combine the convenient handling of beads, the homogeneity of micro-environment in emulsion, and next-generation sequencing to create an attractive alternative to identify ligands of protein targets and antigenic peptides.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. The discovery and characterization of an engineered nanopore with diode-like properties is enabling the construction of DIB networks capable of biochemical computing. Moreover, DIB networks might be used as model systems for the study of membrane-based biological phenomena. We develop and experimentally validate an electrical modeling approach for DIB networks. Electrical circuit simulations will be important in guiding the development of increasingly complex DIB networks. In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. Towards this end, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both α-helical and β-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores. Even with the small volumes (~100 nL) that can be used to form DIBs, the separation between two adjacent bilayers in a DIB network is typically still hundreds of microns. In contrast, dual-membrane spanning proteins require the bilayer separation to be much smaller; for example, the bilayer separation for gap junctions must be less than 5 nm. We designed a double bilayer system that consists of two monolayer-coated aqueous spheres brought into contact with each side of a water film submerged in an oil-lipid solution. The spheres could be brought close enough together such that they physically deflected without rupturing the double bilayer. Future work on quantifying the bilayer separation and studying dual-membrane spanning proteins with the double bilayer platform is planned.

Small for gestational age (SGA) is common in pregnancy and it has been associated with an increase in adverse perinatal outcomes, predisposition for neurological and cognitive delay in childhood and cardiovascular and endocrine diseases in adulthood. The classification is not consensual, having been defined in different studies as estimated fetal weight, abdominal circumference or birthweight below the 10th, 5th or 3rd percentiles, with the prevalence varying with the definition that is used. The increased risk of perinatal mortality and morbidity can be substantially reduced in cases identified prenatally, as close monitoring, timely delivery and prompt neonatal care can be undertaken, in comparison to those cases detected after birth. Over time, several screening methods have been introduced, in order to optimize the detection rate for SGA. These approaches range from abdominal palpation, symphyseal-fundal height measurement, fetal biometries, uterine artery doppler assessment and, more recently, biochemical markers. The aim of this thesis is to develop a model for prediction of SGA neonates in the absence of pre-eclampsia, based on maternal characteristics, clinical history, fetal biometry, uterine pulsatility index (Ut PI), mean arterial blood pressure (MAP) and serum biochemical markers (serum placental growth factor: PlGF; Soluble fms-like tyrosine kinase-1: sFlt-1) at 35-37 gestational weeks. This was a prospective screening project for detection of SGA neonates, in women attending for their third-trimester hospital visit in pregnancy at King's College Hospital (London) and Medway Maritime Hospital (Kent). The project comprised three studies. The first study included biophysical measurements of 5515 pregnant women, including 278 that delivered SGA (<5th) neonates. A SGA predictive model was developed based on the combination of maternal factors, clinical history and estimated fetal weight. In the second study, a subset of 5121 pregnant women was evaluated, 245 of which had SGA (<5th) newborns. A model was developed based on the combination of maternal factors, clinical history, estimated fetal weight, mean arterial pressure and uterine artery 9 dopplers. It was found that the additional use of mean arterial pressure and pulsatility index of the uterine arteries did not significantly improve the performance of screening for delivery of SGA neonates in comparison to the first study. In the third study, a subset of 3859 pregnant women was evaluated, comprising 158 SGA newborns. The SGA prediction model combined the following parameters: maternal factors, estimated fetal weight and biochemical values (serum placental growth factor, PlGF; fms-like soluble tyrosine kinase-1, sFlt-1). It was found that sFlt-1, when combined with maternal factors and fetal biometries, did not remain an independent factor in this predictive model. Additionally, serum PlGF only marginally improved the SGA screening performance when compared to the model of the first study. Hence, based on the findings, the best prediction was provided by the combination of maternal factors, estimated fetal weight and serum placental growth factor (PlGF). This combined screening predicted, at a 10% false positive rate, 88, 96 and 94% of SGA neonates with birth weight below the 10th, 5th and 3rd percentiles delivering at <2 weeks following assessment. The respective values for SGA delivering ≥37 weeks were 64, 75 and 80%. In conclusion, combined screening by maternal factors, biophysical and biochemical markers at 35-37 weeks can identify a high percentage of pregnancies that will deliver SGA neonates.
European Union 7th Framework Programme - FP7-HEALTH-2013-INNOVATION-2 (ASPRE Project #601852)
The Fetal Medicine Foundation (Charity No: 1037116)
Roche Diagnostics Limited

Cationic antimicrobial peptides (AMPs) are present in many organisms and can kill a broad range of microorganisms by membrane permeabilization. Consequently short cationic peptides may serve as a new generation of antibiotics, especially in light of the alarming increase in microbial resistance to the conventional antibiotics. Membrane pore-forming peptides and known AMPs share a similar mechanism of action in lipid bilayers in vitro. In an effort to develop a novel strategy to screen for new peptide antibiotics, we used combinatorial chemistry and high-throughput screening. We selected potent pore-forming peptides from a combinatorial library of 16,000 putative short, cationic amphipathic beta-strand peptides of 9-15 residues using our screening assay. We selected 10 water-soluble potent pore-forming peptides, less than 0.1% of library population. These pore-forming peptides were soluble in buffer but bind strongly to lipid vesicles and induce rapid transient leakage of encapsulated contents. All of the peptides were beta-sheets in membranes. We found that these peptides release the contents of liposomes in all-or-none leakage mechanism. These pore-forming peptides have potent, broad-spectrum antimicrobial activity ranging from 2 to 4 muM minimum sterilization concentrations (MSC). But they have little lytic activity against erythrocytes or living human cells. In an attempt to select AMPs directly from the library, we developed a bioassay, and compared the results with the liposome-based screening assay. We found that the bioassay may allow us to screen for species-specific peptides while the vesicle-based assay gives broad-spectrum AMPs. Here, we propose that a rational combinatorial design coupled with a function-based screening assay is a powerful method to select peptides that are not only potent membrane-active, but also broad-spectrum antibiotics
acase@tulane.edu

Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.

This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.

The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.

To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.

Dissertation

Les cellules épithéliales des voies aériennes respiratoires sécrètent du Cl- via le canal CFTR. La fibrose kystique est une maladie génétique fatale causée par des mutations de ce canal. La mutation la plus fréquente en Amérique du Nord, ∆F508, met en péril la maturation de la protéine et affecte les mécanismes d’activation du canal. Au cours des dernières années, plusieurs molécules ont été identifiées par criblage à haut débit qui peuvent rétablir l’activation de protéines CFTR mutées. Ces molécules sont nommées potentiateurs. Les canaux K+ basolatéraux, dont KCa3.1, jouent un rôle bien documenté dans l’établissement d’une force électromotrice favorable à la sécrétion de Cl- par CFTR dans les cellules épithéliales des voies aériennes respiratoires. Il a par exemple été démontré que l’application de 1-EBIO, un activateur de KCa3.1, sur des monocouches T84 résulte en une augmentation soutenue de la sécrétion de Cl- et que cette augmentation était réversible suite à l’application de CTX, un inhibiteur de KCa3.1(Devor et al., 1996). Dans le cadre d’une recherche de potentiateurs efficaces en conditions physiologiques et dans un contexte global de transport trans-cellulaire, il devient essentiel de considérer les effets des potentiateurs de CFTR sur KCa3.1. Une caractérisation électrophysiologique par la méthode du patch clamp et structurelle via l’utilisation de canaux modifiés par mutagenèse dirigée de différents potentiateurs de CFTR sur KCa3.1 fut donc entreprise afin de déterminer l’action de ces molécules sur l’activité de KCa3.1 et d’en établir les mécanismes. Nous présentons ici des résultats portant sur les effets sur KCa3.1 de quelques potentiateurs de CFTR possédant différentes structures. Un criblage des effets de ces molécules sur KCa3.1 a révélé que la genisteine, le SF-03, la curcumine et le VRT-532 ont des effets inhibiteurs sur KCa3.1. Nos résultats suggèrent que le SF-03 pourrait agir sur une protéine accessoire et avoir un effet indirect sur KCa3.1. La curcumine aurait aussi une action inhibitrice indirecte, probablement via la membrane cellulaire. Nos recherches sur les effets du VRT-532 ont montré que l’accessibilité au site d’action de cette v molécule est indépendante de l’état d’ouverture de KCa3.1. L’absence d’effets inhibiteurs de VRT-532 sur le mutant constitutivement actif V282G indique que cette molécule pourrait agir via l’interaction CaM-KCa3.1 et nécessiter la présence de Ca2+ pour agir. Par ailleurs, un autre potentiateur de CFTR, le CBIQ, a des effets potentiateurs sur KCa3.1. Nos résultats en canal unitaire indiquent qu’il déstabilise un état fermé du canal. Nos travaux montrent aussi que CBIQ augmente la probabilité d’ouverture de KCa3.1 en conditions sursaturantes de Ca2+, ainsi que son affinité apparente pour le Ca2+. Des expériences où CBIQ est appliqué en présence ou en absence de Ca2+ ont indiqué que l’accessibilité à son site d’action est indépendante de l’état d’ouverture de KCa3.1, mais que la présence de Ca2+ est nécessaire à son action. Ces résultats sont compatibles avec une action de CBIQ déstabilisant un état fermé du canal. Finalement, des expériences en Ba2+ nous ont permis d’investiguer la région du filtre de sélectivité de KCa3.1 lors de l’action de CBIQ et nos résultats pointent vers une action de CBIQ dans cette région. Sur la base de nos résultats nous concluons que CBIQ, un potentiateur de CFTR, aurait un effet activateur sur KCa3.1 via la déstabilisation d’un état fermé du canal à travers une action sur sa ‘gate’ au niveau du filtre de sélectivité. De plus, les potentiateurs de CFTR ayant montré des effets inhibiteurs sur KCa3.1 pourraient agir via la membrane ou via une protéine accessoire du canal ou sur l’interaction CaM-KCa3.1. Dans l’optique de traitements potentiels de la fibrose kystique, nos résultats indiquent que le CBIQ pourrait être un potentiateur efficace pusiqu’il est capable de trimuler à la fois KCa3.1 et CFTR. Par contre, dans les cas du VRT-532 et du SF-03, une inhibition de KCa3.1 pourraient en faire des potentiateurs moins efficaces.
Airway epithelial cells are the site of Cl- secretion through CFTR. Cystic fibrosis is a fatal genetic disease caused by mutations in CFTR. The most frequent mutation in North America (∆F508) results in impaired maturation and altered channel gating of the protein. In the last years, several small molecules were identified by high throughput screening that could restore mutated CFTR function. Compounds addressing CFTR gating defects are referred to as potentiators. The basolateral K+ channel KCa3.1 has been documented to play a prominent role in establishing a suitable driving force for CFTR-mediated Clsecretion in airway epithelial cells. It has been shown, for example, that the application of 1-EBIO on T84 monolayers results in a sustained increase of Clsecretion and that this current can be reversed by application of CTX, a KCa3.1 inhibitor (Devor et al., 1996). Thus, in a global approach of transepithelial transport, the research for physiologically relevant CFTR potentiators should also consider their effects on the KCa3.1 channel. Electrophysiological patch clamp measurements and channel structural modification by site directed mutagenesis were used to characterize the action of CFTR potentiators on KCa3.1 and study their molecular mode of action. In this work we present results on the effects on KCa3.1 of several CFTR potentiators of different structures. We observed that the CFTR potentiators genistein, curcumin, SF-03 and VRT-532 could inhibit KCa3.1 activity at concentrations known to activate CFTR. Our results suggest that SF- 03 could act indirectly on KCa3.1 through a mechanism involving an accessory protein. Curcumin would also have an indirect inhibitory effect, probably mediated by the plasma membrane, as documented for other ion channels. A detailed study of VRT-532 revealed that this molecule has access to its binding site in a state independent manner, and is poorly effective on the V282G mutant of KCa3.1, which is constitutively active. These results suggest that VRT-532 could act through the CaM/KCa3.1 complex and require the presence of Ca2+ to inhibit channel activity. In contrast, CBIQ, another CFTR potentiator, succeeded to activate KCa3.1. Our results in single channel show that CBIQ vii destabilizes a non conducting state of the channel. We also showed that this molecule increases the apparent Ca2+ affinity as well as the channel open probability, even in saturating Ca2+ conditions. Experiences in which Ba2+ was used as a probe were also performed to determine if the action mechanism of CBIQ involves an effect on the selectivity filter. Our results showed that Ba2+ could displace CBIQ from its interacting site, suggesting that the increases in channel activity induced by CBIQ could result from a change in the energetics of the channel at the level of the selectivity filter. On the basis of our results, we conclude that CBIQ, a CFTR potentiator, could activate KCa3.1 by destabilizing a non conducting state of the channel, probably through an action near the selectivity filter region. Also, CFTR potentiators having an inhibitory effect on KCa3.1 are likely to act through the plasmic membrane, the CaM/KCa3.1 interaction or an accessory protein of the channel. In a perspective of future treatments for CF, our results indicate that CBIQ could be an efficient potentiator since this product stimulates KCa3.1 as well as CFTR. Conversly, the VRT-532 and SF-03 could be less efficient than on CFTR alone, due to their inhibition of KCa3.1.

Le projet de recherche a été réalisé en collaboration avec Professeur Jean-François Masson du Département de Chimie de l'Université de Montréal. The research project was made in collaboration with Professor Jean-François Masson from the Chemistry department of University of Montreal.
L'objectif du projet de recherche est de développer une méthode de criblage d’inhibiteurs basée sur une technologie émergente, soit un dispositif portatif utilisant la résonance des plasmons de surface (SPR). La cible du criblage est la dihydrofolate réductase R67 (DHFR R67), une enzyme qui confère une résistance bactérienne à l'antibiotique triméthoprime. Ici, l'enzyme cible est immobilisée sur une surface d'or mince avec des propriétés plasmoniques (optiques) spécifiques qui varient en fonction de la masse des molécules se liant à cette surface. Cette technique permet de suivre les événements de liaison de molécules à la DHFR R67 immobilisée, et ainsi peut permettre l'identification d'inhibiteurs potentiels. Cependant, la masse molaire des inhibiteurs typiquement utilisés lors de criblages préliminaires (i.e. 500-1000 g/mol) est trop faible pour générer un signal SPR détectable. Afin de contrer cette lacune, ce mémoire a pour objet de développer un essai compétitif indirect qui mettra en jeu des molécules de masse supérieure. D’abord, une nanoparticule d'or portant un analogue de substrat se liera à la DHFR R67 immobilisée à la surface d’or, générant ainsi un signal SPR important en raison de la masse molaire élevée de la nanoparticule. Ensuite, lors du criblage d'inhibiteurs potentiels, les nanoparticules liées seront déplacées de l'enzyme cible si la molécule criblée fournit une affinité suffisante. Ainsi, il sera possible de suivre indirectement la liaison d'un inhibiteur à la cible. Ce projet vise donc à tester et à valider l'approche de criblage SPR appliquée à la DHFR R67.
The objective of the research project is to develop a method for inhibitor screening based on a portable Surface Plasmon Resonance (SPR) device, an emerging technology. The target is R67 dihydrofolate reductase (R67 DHFR), an enzyme that confers bacterial resistance to the antibiotic trimethoprim. Here, the target enzyme is linked to a thin gold surface having specific plasmonic (optical) properties that vary as a function of the mass of bound molecules. This allows monitoring binding to the surface-linked R67 DHFR, and thus permits identification of inhibitors. However, the mass of the low-affinity inhibitors typically identified in early stages of screening (i.e. 500-1000 g/mol) is too low to produce a significant SPR signal. To address this shortcoming, a competitive assay will be developed: a gold nanoparticle carrying a substrate analog will bind the surface-immobilized R67 DHFR, resulting in a strong SPR signal due to its high mass. Then, upon screening for potential inhibitors, the bound nanoparticle will be displaced from the target enzyme if a molecule provides sufficient affinity. By those means, it will be possible to indirectly monitor the binding of an inhibitor to the target. This goal of this project is to test and validate the SPR screening approach applied to R67 DHFR.