Exploring combined verses single mode of inhibition of Mycobacterium Tuberculosis RNA polymerase as a therapeutic intervention to overcome drug resistance challenges: atomistic perspectives.

Masters Degree. University of KwaZulu-Natal, Westville.The impact of Rifampin resistance on the overall global epidemic of antimicrobial resistance has become very prominent in recent years and is eventually stifling current efforts being made to control tuberculosis drug resistance. Rifampin resistance has significantly contributed to making TB the leading cause of morbidity from an infectious disease globally. The RNA polymerase of Mycobacterium tuberculosis has been extensively explored as a therapeutic target for Rifampin resistance with recent studies exploring synergistic inhibition as an effective approach, by combining Rifampin and other drugs in the TB drug resistance. Apart from the paucity of data elucidating the structural mechanism of action of the synergistic interaction between Rifampin and DAAPI, previous studies did not also utilize the X-ray crystal structure of Mtb RNAP due its unavailability. This thesis used advanced computational tools to unravel molecular insights into the suppression of the emergence of resistance to Rifampin by a novel Nα-aroyl-N-aryl-phenylalaninamides (AAPI) prototype inhibitor, DAAPI, co-bound to Mtb RNAP with Rifampin. Our studies revealed co-binding induced a stable Mtb RNAP protein structure, increased the degree of compactness of binding site residues around Rifampin and subsequently improved the binding affinity of Rifampin. Studies in this thesis further provide an atomistic mechanism behind Rifampin resistance when the recently resolved crystal structure of Mycobacterium tuberculosis RNA polymerase is subjected to a single active site mutation. We also identified and rationalized the structural interplay of this single active site mutation upon co-binding of Rifampin with the novel inhibitor, DAAPI. Our findings report that the mutation distorted the overall conformational landscape of Mycobacterium tuberculosis RNA polymerase, resulting in a reduction of binding affinity of Rifampin and an overall shift in the residue interaction network of Mycobacterium tuberculosis RNA polymerase and upon single binding. Interestingly, co-binding with DAAPI, though impacted by the mutation exhibited improved Rifampin binding interactions amidst a distorted residue interaction network. Findings establish a structural mechanism by which the novel inhibitor DAAPI stabilizes Mycobacterium tuberculosis RNA polymerase upon co-binding with Rifampin, thus suppressing Rifampin resistance. We also provide vital conformational dynamics and structural mechanisms of mutant enzyme-single ligand and mutant enzyme-dual ligand interactions which could potentially shift the current therapeutic protocol of TB infections, thus aiding in the design of novel Mycobacterium tuberculosis RNA polymerase inhibitors with improved therapeutic features against the mutant proteins

Computing the Partition Function for Kinetically Trapped RNA Secondary Structures

An RNA secondary structure is locally optimal if there is no lower energy structure that can be obtained by the addition or removal of a single base pair, where energy is defined according to the widely accepted Turner nearest neighbor model. Locally optimal structures form kinetic traps, since any evolution away from a locally optimal structure must involve energetically unfavorable folding steps. Here, we present a novel, efficient algorithm to compute the partition function over all locally optimal secondary structures of a given RNA sequence. Our software, RNAlocopt runs in time and space. Additionally, RNAlocopt samples a user-specified number of structures from the Boltzmann subensemble of all locally optimal structures. We apply RNAlocopt to show that (1) the number of locally optimal structures is far fewer than the total number of structures – indeed, the number of locally optimal structures approximately equal to the square root of the number of all structures, (2) the structural diversity of this subensemble may be either similar to or quite different from the structural diversity of the entire Boltzmann ensemble, a situation that depends on the type of input RNA, (3) the (modified) maximum expected accuracy structure, computed by taking into account base pairing frequencies of locally optimal structures, is a more accurate prediction of the native structure than other current thermodynamics-based methods. The software RNAlocopt constitutes a technical breakthrough in our study of the folding landscape for RNA secondary structures. For the first time, locally optimal structures (kinetic traps in the Turner energy model) can be rapidly generated for long RNA sequences, previously impossible with methods that involved exhaustive enumeration. Use of locally optimal structure leads to state-of-the-art secondary structure prediction, as benchmarked against methods involving the computation of minimum free energy and of maximum expected accuracy. Web server and source code available at http://bioinformatics.bc.edu/clotelab/RNAlocopt/

NANOHARVESTING AND DELIVERY OF BIOACTIVE MATERIALS USING ENGINEERED SILICA NANOPARTICLES

Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications. To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport). To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites. For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism

Structural and biophysical studies of RNA-Chaperone Hfq from E. coli

Diese Dissertation wurde an der Universität Wien in den Max F. Perutz Laboratories von Mag. Mads Beich-Frandsen von 2005 bis 2011 durchgeführt und betreut von Prof. Kristina Djinović-Carugo vom Department für Strukturbiologie und Computational Biology in Kollaboration mit Prof. Udo Bläsi vom Department für Mikrobiologie, Immunbiologie und Genetik. Der Titel verweist auf die strukturbiologischen Untersuchungen am RNA-bindenden Protein Hfq aus Escherichia coli. Durch die Erkenntnis, dass nur ein Bruchteil des gesamten Genoms für Protein kodiert, verlagerte sich der Forschungsschwerpunkt in der Biologie hin zu RNA-basierter Regulation. Mit der wachsenden Anzahl vollständiger Transkriptomprofile kristallisiert sich das Sm-like Protein Hfq als zentrale Schaltstelle zur Genregulation durch small regulatory RNAs (sRNAs) in Bakterien heraus. In E. coli und anderen gram-negativen Pathogenen ist der konservierte Sm-like Kern von Hfq um eine carboxyterminale Domäne erweitert, deren Länge 30% der Sequenz des gesamten Proteins beträgt. Seine kurze aminoterminale Region ist hingegen in höherem Maße konserviert. Sowohl N- als auch C-Terminus von Hfq, beide gekennzeichnet durch intrinsisches Fehlen einer geordneten Struktur, tragen nachweislich zur Funktionalität des Proteins bei. Der Schlüsselmechanismus der Hfq-vermittelten Regulation besteht darin, transkodierte sRNAs mit ihrer Ziel-mRNA zu hybridisieren. Hfq agiert somit als RNA-Chaperon, welches die Sekundärstruktur von RNA modifiziert. Die Aufgabenstellung des Projekts bestand darin, die Funktion von Hfq aus einer strukturbiologischen Perspektive zu beleuchten. Im Rahmen dieser Forschungsarbeit wurden Röntgenkristallographie, Kleinwinkelstreuung, NMR Spektroskopie, Zirkulardichroismus mit Synchrotronstrahlung, kombiniert mit und integriert in bioinformatische und funktionelle Studien, angewendet. Aus dieser Arbeit gingen zwei Publikationen hervor, die strukturelle Aspekte von Hfq in E. coli beschreiben. Die Analyse jener Ergebnisse geschah im Kontext biophysikalischer und funktioneller Resultate, welche den intrinisch unstrukturierten Termini von E. coli Hfq Funktionalität zuweisen. Es konnte festgestellt werden, dass die Termini, ausgelöst durch die Interaktion mit RNA, Strukturen ausbilden. Die Interpretation dieser Resultate folgt dem „Entropietransfermodell“, welches vorschlägt, dass intrinisch unstrukturierte Sequenzen durch isothermische Enthalpie/Entropie-Kompensation die Entfaltung von Zielstrukturen begünstigen können. Das Zusammenspiel von strukturierter und ungeordeter Sequenz in E.coli Hfq ermöglicht es diesem Protein, mit einer Vielzahl an RNAs zu interagieren und diese zu regulieren. Die zentrale Funktion von Hfq ist hierbei, die RNA in einem ungefalteten Zustand zu erhalten. Sie kann in folgendem Dogma zusammengefasst werden: Bindung fördert Entfaltung – Entfaltung fördert Hybridisierung - Hybridisierung fördert Loslösung von Hfq!This dissertation was conducted at the University of Vienna, Max F. Perutz Laboratories in the years 2005-2011. Here is reported on research performed by Mag. Mads Beich-Frandsen, supervised by Prof. Kristina Djinovic-Carugo at the Department of Structural and Computational Biology, in collaboration with Prof. Udo Bläsi at the Department of Microbiology, Immunobiology and Genetics. The title refers to the biostructural investigations conducted for the RNA-binding protein Hfq from Escherichia coli. Upon the understanding that only a fraction of a genome encodes protein, focus has been shifted to RNA-based regulation in biology. With the increasing number of transcriptome profiles being completed, the Sm-like protein Hfq emerges as the central switchboard of gene regulation, as mediated by small regulatory RNAs (sRNAs) in Bacteria. In E. coli and other gram-negative pathogens, the conserved Sm-like core of Hfq is extended 30% in sequence length, by a C-terminal domain. The short N-terminal region of Hfq is conserved to higher degree. Both the N- and C-terminus of Hfq have been demonstrated of functional importance for the protein, and are characterized as intrinsically disordered. The key mechanism of Hfq-mediated regulation is by annealing trans-encoded sRNAs to target mRNA. Here Hfq acts as an RNA-chaperone, with ability to alter the secondary structure of RNA. The scope of the project was to elucidate the function of E. coli Hfq from the perspective of structural biology. The research presented here employs X-ray crystallography, Small Angle Scattering, Nuclear Magnetic Resonance, Synchrotron-Radiation Circular-Dichroism, in an integrated approach with bioinformatics and functional studies. The work resulted in two publications, reporting on structural aspects of E. coli Hfq. These results were analyzed in context of acquired biophysical and functional results, which annotates function to the intrinsically disordered N- and C-terminus of E. coli Hfq. Interaction with RNA was found to induce structure upon the termini of Hfq. This was interpreted in line of the ‘entropy-transfer’ model, which proposes intrinsically disordered sequence to have a function in unfolding targets by isothermal entropy/enthalpy compensation. The interplay between the structured and disordered sequence in E. coli Hfq provides the protein with the ability to interact with and exert regulation on a wide variety of RNAs. Hfq functions to keep the RNA unfolded, following the dogma: Binding promotes unfolding – unfolding promotes annealing – annealing promotes release of Hfq

RNA Nanotechnology for Next Generation Targeted Drug Delivery

The emerging field of RNA nanotechnology is developing into a promising platform for therapeutically application. Utilizing the state-of-art RNA nanotechnology, RNA nanoparticles can be designed and constructed with controllable shape, size for both RNA therapeutics and chemical drug delivery. The high homogeneity in particle size and ease for RNA therapeutic module conjugation, made it feasible to explore versatile RNA nanoparticle designs for preclinical studies. One vital module for therapeutic RNA nanoparticle design is RNA aptamer, which can enable the RNA nanoparticles find its specific target for targeted drug delivery. A system of screening divalent RNA aptamers for cancer cell targeting was developed. The system utilized a highly stable three way junction (3WJ) derived from phi29 bacteriophage packing RNA (pRNA). Instead of using one random loop for aptamer SELEX as traditionally, the divalent RNA nanoparticle library contains two variable loops for substrate binding, similar to protein antibodies. The presence of two binding sites on one aptamer greatly enhanced its affinity, and the thermodynamically stability of pRNA-3WJ motif enables controllable RNA folding of each loop. The selected RNA antibody against epithelial adhesion molecule (EpCAM) A9-8 can deliver therapeutic anti-miR21 to EpCAM positive cancer cells in vitro. The feasibility of using RNA aptamer for targeted chemical drug delivery is explored. A phosphorothioate bond modified DNA (thio-DNA) aptamer targeting annexin A2 was utilized as ligand to build nucleic acid nanoparticles for ovarian cancer targeted drug delivery. A DNA/RNA hybrid nanoparticle was generated by conjugating the thio-DNA aptamer to pRNA-3WJ motif. The DNA/RNA hybrid nanoparticles showed favorable property for delivering doxorubicin to ovarian cancer cells in vitro, also targeted to ovarian cancer xenograft in bio-distribution study in vivo. Utilizing the spatial orientation of pRNA-3WJ, cholesterol modification on the arrow tail of pRNA-3WJ can display RNA nanoparticle on the surface of exosomes/extracellular vesicles (EV) for active targeting. Taking the advantage of RNA ligand for specific targeting; and exosome for efficient membrane fusion, cytosol homing and functional siRNA delivery; the RNA ligand decorated exosomes were constructed for specific delivery of siRNA to cancer cells. PSMA aptamer-displaying exosomes and encapsulated survivin siRNA (PSMAapt/EV/siSurvivin) showed efficient gene silencing both in cell culture and animal trials. After systemically injection of PSMAapt/EV/siSurvivin to prostate cancer xenograft mice, cancer growth was almost completely blocked. These results suggest the advance of RNA nanotechnology can further drive its way towards clinical application as a novel next generation drug delivery system

Modes and mechanisms of hfq mediated stress regulation in bacteria

To survive bacteria must be able to respond to its ever-changing environmental conditions. sRNAs have been implicated in a variety of stress-response pathways that help bacterial systems modulate gene expression. The RNA binding protein Hfq facilities this process by, helping sRNA to base pair with its target mRNAs to initiate gene regulation. A common feature of Hfq-mediated gene regulation is the network-based organization where a single sRNA can control multiple messages to promote integrated response to stress. Current mechanistic models that are present to describe Hfq functions cannot explain the complexity at which Hfq performs gene regulation. In this work we have used a variety of biophysical, biochemical and biological approaches to understand the nature of Hfq interactions with target mRNAs

The Development Of Peptide Ligands To Target H69 Rrna

ABSTRACT THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69 by DANIELLE NICOLE DREMANN December 2015 Advisor: Prof. Christine S. Chow Major: Chemistry (Biochemistry) Degree: Doctor of Philosophy In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to increase the efficiency of peptide ligand selection in which novel peptides with high affinity and selectivity could be identified for future projects. This assay was done using flow cytometry, instrumentation capable of identifying beads, bound to the target RNA conjugated to a fluorophore, based on fluorophore emission, and sorting them into 96-well plates for MS analysis. The last part of the research focused on aminoglycoside-H69 RNA interactions. ESI MS was used to obtain binding affinities and stoichiometries of 2AP- and A-containing H69 RNAs. The findings revealed that the binding mode had not changed between these two sets of RNAs, which revealed the value for using ESI MS in combination with other techniques, such as fluorescence, to give a complete picture of the binding mode (stoichiometry, affinity, selectivity) in comparison with conformational changes that may occur upon binding. Further exploration of aminoglycoside-H69 RNA interactions took place with the H69 peptide NQVANHQ-NH2 to determine whether a fragment-based drug design approach could be used to create small compounds for future in vivo applications

Laser-assisted single-molecule refolding

Non-coding RNAs must fold into precise secondary and tertiary structures in order to perform the biological functions. Due to the flexibility of RNA, the RNA folding energy landscape can be rugged and full of local minimum (kinetic trap). To provide a means to study kinetically trapped RNAs, we have developed a new technique combining single-molecule FRET detection with laser induced temperature jump. We have calibrated the magnitude of the temperature jump with 1˚C accuracy using gold micro-size sensor. The accuracy of temperature calibration was confirmed by close agreement between single-molecule and bulk DNA duplex melting experiments. HIV 1 DIS RNAs form a kissing complex in the loop region and proceed to the extended duplex structure with the help of enzymes or other cofactors in the later stage of viral replication. The kissing complex itself is very stable, which makes it a unique optimal model system to study kinetically trapped RNAs. The application of LASR to kissing hairpins has allowed us, for the first time, to drive a molecular reaction and monitor the process at the single molecule level. The melting curve for the dissociation and dimerization was used to estimate the thermodynamic properties of the reaction, such as melting temperature, cooperativity, and the enthalpy change. Mutational studies have allowed dissection of the contribution of base pairs to kissing complex stability. And the ratio of the competing reaction pathways was determined. LASR experiments designed to the study of the origin of the memory effect in the hairpin ribozyme folding introduced inter-conversions between the subpopulations of hairpin ribozyme with distinct undocking kinetic rates. This provides strong evidence for the hypothesis that the memory effect is an intrinsic property of hairpin ribozyme folding. Eyring analysis was adopted to fit the enthalpic and entropic height of the inter-conversion barrier. Our results suggest that for inter-conversion to occur, surprisingly, a small number of interactions are broken. However inter-conversion is rare due to a large negative entropic term. Negative entropy indicates a rigid transition state for inter-conversion and an increased free energy barrier height with increased temperature. This explains why there are few inter-conversions even at high temperatures such as 78 ˚C. The entropic barrier may primarily arise from the stretching of the S-turn

Revealing the Mechanism of Thiopeptide Antibiotics at Atomistic Resolution : Implications for Rational Drug Design

For decades drug design has primarily focused on small molecules that bind to well-formed tight binding pockets, such as the catalytic centers of enzymes. Recently, there is increasing interest to design compounds that disrupt or stabilize biomacromolecular interfaces (e.g. protein–protein, protein–DNA, protein–RNA, protein–lipid interfaces). These non-traditional drug targets hold great therapeutic potential as they govern cellular pathways. In contrast to traditional drug targets, where computational methods are now routinely and productively used to complement experiments, the use of computer-based approaches for the study and design of interfacial modulators is still in its infancy. The current thesis is a first detailed study into understanding the effects of modulators of a protein–RNA interface and developing computer-based approaches for their design. This work focuses on the 23S-L11 subunit of the ribosomal GTPase-associated region (GAR), a prototypic protein–RNA interface of high relevance in the development of novel antibacterials. The GAR is the target of naturally occuring thiopeptide antibiotics. These unique molecules are effective inhibitors of bacterial protein synthesis, but are currently unused in human antibacterial therapy due to their low aqueous solubility. Their mechanism of action is explored in the current thesis, enabling the design and proposition of new chemical scaffolds targeting their binding site. The specific challenges associated with the 23-SL11-thiopeptide system, such as the inherent flexibility of the protein–RNA composite environment and the size and structural complexity of the thiopeptide ligands, are addressed by a combination of computational chemistry approaches at different levels of granularity and a steady feedback with experimental data to validate and improve the computational techniques. These approaches range from quantummechanics for deriving optimized intramolecular parameters and partial atomic charges for the thiopeptide compounds, to molecular dynamics simulations accounting for the binding site’s flexibility, to molecular docking studies for predicting the binding modes of different thiopeptides and derivatives. All-atom molecular dynamics simulations were conducted, providing a detailed understanding of the effect of thiopeptide binding at a previously unmet resolution. The findings of this work, coupled with previous experimental knowledge, strongly support the hypothesis that restricting the binding site’s conformational flexibility is an important component of the thiopeptide antibiotics’ mode of action. With the help of an MD-docking-MD workflow and an energy decomposition analysis crucial residues of the binding site and pharmacologically relevant moieties within the ligand structures could be identified. A 4D-pharmacophore model is presented that was derived from a refined 23S-L11-thiopeptide complex and additionally accounts for the dynamic stability of molecular interactions formed between the antibiotic and the ribosomal binding site as the fourth dimension. The results of this thesis revealed, for the first time, a plausable description of the thiopeptide antibiotics’ mode of action, down to the details of their pharmacologically relevant parts and provide a computational framework for the design of new ligands