Use of Structure-And Ligand-Based Drug Design Tools for the Discovery of Small Molecule Inhibitors of Cysteine Proteases for the Treatment of Malaria and Sars Infection

A wide array of molecular modeling tools were utilized to design and develop inhibitors against cysteine protease of P. Falciparum Malaria and Severe Acute Respiratory Syndrome (SARS). A number of potent inhibitors were developed against cysteine protease and hemoglobinase of P. falciparum , referred as Falcipains (FPs), by the structure-based virtual screening of the focused libraries enriched in soft-electrophiles containing compounds. Twenty one diverse, non-peptidic, low micromolar hits were identified. A combined data mining and combinatorial library synthesis approach was performed to discover analogs of virtual screening hits and establish the structure-activity relationships (SAR). However, the resulting SAR of the identified hits was unusually steep in some cases and could not be explained by a traditional analysis of the interactions (electrostatics, van der Waals or H-bond). To gain insights, a statistical thermodynamic analysis of explicit solvent in the ligand binding domain of FP-2 and FP-3 was performed that explained some of the complex trends in the SAR. Furthermore, the moderate potency of a subset of FP-2 hits was elucidated using quantum mechanics calculations that shoreduced reactivity of the electrophilic center of these hits. In addition, solvent thermodynamics and reactivity analysis also helped to elucidate the complex trends in SAR of peptidomimetic inhibitors of FP-2 and FP-3 synthesized in our laboratory. Multi nanosecond explicit solvent molecular dynamics simulations were carried out using the docking poses of the known inhibitors in the binding site of SARS-3CLpro, a cysteine protease important for replication of SARS virus, to study the overall stability of the binding site interactions as well as identify important changes in the interaction profile that were not apparent from the docking study. Analysis of the simulation studies led to the identification of certain protein-ligand interaction patterns which would be useful in further structure based design efforts against cysteine protease (3CLpro) of SARS

The future of molecular dynamics simulations in drug discovery

Molecular dynamics simulations can now track rapid processes—those occurring in less than about a millisecond—at atomic resolution for many biologically relevant systems. These simulations appear poised to exert a significant impact on how new drugs are found, perhaps even transforming the very process of drug discovery. We predict here future results we can expect from, and enhancements we need to make in, molecular dynamics simulations over the coming 25 years, and in so doing set out several Grand Challenges for the field. In the context of the problems now facing the pharmaceutical industry, we ask how we can best address drug discovery needs of the next quarter century using molecular dynamics simulations, and we suggest some possible approaches

An integrated bioinformatics and computational biophysics approach to enterovirus surveillance and research

This PhD thesis examines the integration of complex computational methodologies with the surveillance and research of a genus of viruses implicated in a wide variety of clinical conditions, ranging from asymptomatic infection to death. These viruses, known as the enteroviruses, are some of the most studied viruses in history and as a result are represented by a vast body of literature. The fact that enterovirus research and surveillance rests upon such an extensive foundation of published material, makes enteroviruses a perfect candidate for the experimental application of modern computational methods, or in-silico experimentation. The hypothesis that computational power currently available can be utilised for multiple stages of virus study incorporating identification, epidemiology and atomic structure prediction forms the basis of this thesis. Fundamental to the understanding of virus behaviour is the determination of molecular structure and function, a fact which applies not only to viruses, but to biological entities in general. Extensive work was performed during the course of this thesis in adapting classical molecular dynamics techniques to the large scale simulation of a prototype poliovirus, using millions of simulated atoms. The successful application of these techniques has resulted in microsecond-timescale, atomistic simulations of complete virus particles. These simulations represent the first published instance of the simulation of a biologically complete pathogenic microorganism, incorporating the encoding genetic information. This thesis also examines the use of bioinformatics methods in the development and application of an advanced quantitative multiplex real-time reverse-transcription polymerase chain reaction (qRT-PCR) methodology, for the primary screening of samples from patients suffering acute flaccid paralysis (AFP), which is one of the most debilitating presentations of enterovirus infection. The application of this novel qRT-PCR method reduces the initial screening time of samples derived from a symptomatic patient from 4-5 days using virus culture, to four hours using the novel qRT-PCR. This novel qRT-PCR method can be rapidly scaled-up in response to an outbreak situation. The ability to screen large numbers of samples during an outbreak situation is important and is hampered when using virus culture methods exclusively. In Australia and the Western Pacific region over the last decade, the rate at which non-polio enteroviruses in cases of AFP have been identified, is on average 18%. With the introduction of PCR screening methods, a number of non-cultivable enteroviruses were identified, along with newly described and a previously undescribed enterovirus. Little is known about these newly described and novel enteroviruses. This thesis aimed to investigate the identification of viruses that may represent a significant public health threat and to then use their genetic sequence information to recreate major virus structural components in-silico. This reconstruction process was achieved by exploiting advances in comparative protein modelling and molecular dynamics simulation methods. In order to apply these methods to the reconstruction of previously undescribed viruses for which no structural data exist, validation of different comparative protein modelling techniques was required. The predictive in-silico methods generated reliable atomic coordinates, representing structures suitable for the reconstruction of virus capsid models for further study

Computer-aided approaches in drug design: the exigent way forward: dynamic perspectives into the mechanistic activities of small molecule inhibitors toward antiviral, antitubercular and anticancer therapeutic interventions.

Doctoral Degree. University of KwaZulu-Natal, Durban.The crucial role of CADD in the drug design process is now indisputable and has proven over the years that it can accelerate the discovery potential drug candidates while reducing the associated cost. Using knowledge and information about biological target or knowledge about a ligand with proven bioactivity, CADD, and its techniques can influence various drug discovery pipeline stages. The ability CADD approaches to elucidate drug-target interactions at the atomistic level allows for investigations of the mechanism of drugs' actions, revealing atomistic insights that influence drug design and improvement. CADD approaches also seek to augment traditional in vitro and in vivo experimental techniques and not replace them since CADD approaches can also allow modeling complex biological processes that hitherto seemed impossible to explore using experimental methods. According to the World Health Organization (WHO), featuring prominently in the top ten causes of death are cancer, lower respiratory tract infection, tuberculosis (TB), and viral infections such as HIV/AIDS. Collectively, these diseases are of global health concerns, considering a large number of associated deaths yearly. Over the years, several therapeutic interventions have been employed to treat, manage, or cure these diseases, including chemotherapy, surgery, and radiotherapy. Of these options, small molecule inhibitors have constituted an integral component in chemotherapy, thereby undoubtedly playing an essential role in patient management. Although significant success has been achieved using existing therapeutic approaches, the emergence of drug resistance and the challenges of associated adverse side effects has prompted the need for the drug design processes against these diseases to remain innovative, including combining existing drugs and establishing improved therapeutic options that could overcome resistance while maintaining minimal side effects to patients. Therefore, an exploration of drug target interactions towards unraveling mechanisms of actions as performed in the reports in this thesis are relevant since the molecular mechanism provided could form the basis for the design and identification of new therapeutic agents, improvement of the therapeutic activity of existing drugs, and also aid in the development of novel therapeutic strategies against these diseases of global health concern. Therefore the studies in this thesis employed CADD approaches to investigates molecular mechanisms of actions of novel therapeutic strategies directed towards some crucial therapeutics implicated in viral infections, tuberculosis, and cancer. Therapeutic targets studied included; SARS-CoV-2 RNA dependent RNA polymerase (SARS-CoV-2 RdRp), Human Rhinovirus B14 (HRV-B14) and human N-myristoyltransferases in viral infections, Dihydrofolate reductase (DHFR) and Flavin-dependent thymidylate synthase (FDTS) in TB, human variants of TCRCD1d, and Protein Tyrosine Phosphatase Receptor Zeta (PTPRZ) in cancer. The studies in this thesis is divided into three domains and begins with a thorough review of the concept of druggability and drug-likeness since the crux of the subsequent reports revolved around therapeutic targets and their inhibitions by small molecule inhibitors. This review highlights the principles of druggability and drug-likeness while detailing the recent advancements in drug discovery. The review concludes by presenting the different computational, highlighting their reliability for predictive analysis. In the first domain of the research, we sought to unravel the inhibitory mechanism of some small molecule inhibitors against some therapeutic targets in viral infections by explicitly focusing on the therapeutic targets; SARS-CoV-2 RdRp, HRV-B14, and N-myristoyltransferase. Therapeutic targeting of SARS-CoV-2 RdRp has been extensively explored as a viable approach in the treatment of COVID-19. By examining the binding mechanism of Remdesivir, which hitherto was unclear, this study sought to unravel the structural and conformational implications on SARS-CoV-2 RdRp and subsequently identify crucial pharmacophoric moieties of Remdesivir required for its inhibitory potency. Computational analysis showed that the modulatory activity of Remdesivir is characterized by an extensive array of high-affinity and consistent molecular interactions with specific active site residues that anchor Remdemsivir within the binding pocket for efficient binding. Results also showed that Remdesivir binding induces minimal individual amino acid perturbations, subtly interferes with deviations of C-α atoms, and restricts the systematic transition of SARS-CoV-2 RdRp from the “buried” hydrophobic region to the “surface exposed” hydrophilic region. Based on observed high-affinity interactions with SARS-CoV-2 RdRp, a pharmacophore model was generated, which showcased the crucial functional moieties of Remdesivir. The pharmacophore was subsequently employed for virtual screening to identify potential inhibitors of SARS-CoV-2 RdRp. The structural insights and the optimized pharmacophoric model provided would augment the design of improved analogs of Remdesivir that could expand treatment options for COVID-19. The next study sought to explore the therapeutic targeting of human rhinoviruses (HRV) amidst challenges associated with the existence of a wide variety of HRV serotypes. By employing advanced computational techniques, the molecular mechanism of inhibition of a novel benzothiophene derivative that reportedly binds HRV-B14 was investigated. An analysis of the residue-residue interaction profile revealed of HRV upon the benzothiophene derivative binding revealed a distortion of the hitherto compacted and extensively networked HRV structure. This was evidenced by the fewer inter-residue hydrogen bonds, reduced van der Waals interactions, and increased residue flexibility. However, a decrease in the north-south wall's flexibility around the canyon region also suggested that the benzothiophene derivative's binding impedes the “breathing motion” of HRV-B14; hence its inhibition. The next study in the first domain of the research investigated the structural and molecular mechanisms of action associated with the dual inhibitory activity of IMP-1088. This novel compound reportedly inhibits human N-myristoyltransferase subtypes 1 and 2 towards common cold therapy. This is because it has emerged that the pharmacological inhibition of Nmyristoyltransferase is an efficient non-cytotoxic strategy to completely thwart the replication process of rhinovirus toward common cold treatment. Using augmentative computational and nanosecond-based analyses, findings of the study revealed that the steady and consistent interactions of IMP-1088 with specific residues; Tyr296, Phe190, Tyr420, Leu453, Gln496, Val181, Leu474, Glu182, and Asn246, shared within the binding pockets of both HNMT subtypes, in addition to peculiar structural changes account for its dual inhibitory potency. Findings thus unveiled atomistic and structural perspectives that could form the basis for designing novel dualacting inhibitors of N-myristoyltransferase towards common cold therapy. In the second domain of the research, the mechanism of action of some small molecule inhibitors against DHFR, FDTS, and Mtb ATP synthase in treating tuberculosis is extensively investigated and reportedly subsequently. To begin with, the dual therapeutic targeting of crucial enzymes in the folate biosynthetic pathway was explored towards developing novel treatment methods for TB. Therefore, the study investigated the molecular mechanisms and structural dynamics associated with dual inhibitory activity of PAS-M against both DHFR and FDTS, which hitherto was unclear. MD simulations revealed that PAS-M binding towards DHFR and FDTS is characterized by a recurrence of strong conventional hydrogen bond interactions between a peculiar site residue the 2-aminov decahydropteridin-4-ol group of PAS-M. Structural dynamics of the bound complexes of both enzymes revealed that, upon binding, PAS-M is anchored at the entrance of hydrophobic pockets by a strong hydrogen bond interaction while the rest of the structure gains access to deeper hydrophobic residues to engage in favorable interactions. Further analysis of atomistic changes of both enzymes showed increased C-α atom deviations and an increase C-α atoms radius of gyration consistent with structural disorientations. These conformational changes possibly interfered with the enzymes' biological functions and hence their inhibition as experimentally reported. Additionally, in this domain, the therapeutic targeting of the ATP machinery of Mtb by Bedaquiline (BDQ) was explored towards unravelling the structures and atomistic perspectives that account for the ability of BDQ to selectively inhibits mycobacterial F1Fo-ATP synthase via its rotor c-ring. BDQ is shown to form strong interaction with Glu65B and Asp32B and, consequently, block these residues' role in proton binding and ion. BDQ binding was also revealed to impede the rotatory motion of the rotor c-ring by inducing a compact conformation on the ring with its bulky structure. Complementary binding of two molecules of BDQ to the rotor c-ring, proving that increasing the number of BDQ molecule enhances inhibitory potency. The last study in this research domain investigated the impact of triple mutations (L59V, E61D, and I66M) on the binding of BDQ to Mtb F1F0 ATP-synthase. The study showed that the mutations significantly impacted the binding affinity of BDQ, evidenced by a decrease in the estimated binding free energy (ΔG). Likewise, the structural integrity and conformational architecture of F1F0 ATP-synthase was distorted due to the mutation, which could have interfered with the binding of BDQ. The third domain of the research in this thesis investigated some small molecule inhibitors' inhibitory mechanism against some therapeutic targets in cancer, specifically PTPRZ and hTCRvi CD1d. Studies in the third domain of the research in the thesis began with the investigation of the investigation of the inhibitory mechanism of NAZ2329, an allosteric inhibitor of PTPRZ, by specifical investigating its binding effect on the atomic flexibility of the WPD-loop. Having been established as crucial determinant of the catalytic activity of PTPRZ an implicated protein in glioblastoma cells, its successfully therapeutic modulation could present a viable treatment option in glioblastoma. Structural insights from an MD simulation revealed that NAZ2329 binding induces an open conformation of the WPD-loop which subsequently prevents the participation of the catalytic aspartate of PTPRZ from participating in catalysis hence inhibiting the activity of PTPRZ. A pharmacophore was also created based of high energy contributing residues which highlighted essential moieties of NAZ2329 and could be used in screening compound libraries for potential inhibitors of PTPRZ. A second study in this domain sought to explore how structural modification could improve a therapeutic agent's potency from an atomistic perspective. This study was based on an earlier report in which the incorporation of a hydrocinnamoyl ester on C6’’ and C4-OH truncation of the sphingoid base of KRN7000 generated a novel compound AH10-7 high therapeutic potency and selectivity in human TCR-CD1d and subsequently results in the activation of invariant natural killer T cells (iNKT). The hydrocinnamoyl ester moiety was shown to engage in high-affinity interactions, possibly accounting for the selectivity and higher potency of AH10-7. Molecular and structural perspectives provided could aid in the design of novel α-GalCer derivatives for cancer immunotherapeutics. Chapter 3 provides theoretical insights into the various molecular modeling tools and techniques employed to investigate the various conformational changes, structural conformations, and the associated mechanism of inhibitions of the studied inhibitors towards viral, tuberculosis, and cancer therapy. Chapter 4 provided sufficient details on druggability and drug-likeness principles and their recent advancements in the drug discovery field. The study also presents the different computational tools and their reliability of predictive analysis in the drug discovery domain. It thus provides a comprehensive guide for computational-oriented drug discovery research. Chapter 5 provides an understanding of the binding mechanism of Remdesivir, providing structural and conformational implications on SARS-CoV-2 RdRp upon its binding and identifying its crucial pharmacophoric moieties. Chapter 6 explains the mechanism of inhibition of a novel benzothiophene derivative, revealing its distortion of the native extensively networked and compact residue profile. Chapter 7 unravels molecular and structural bases behind this dual inhibitory potential of the novel inhibitor IMP-1088 toward common cold therapy using augmentative computational and cheminformatics methods. The study also highlights the pharmacological propensities of IMP- 1088. Chapter 8 unravels the molecular mechanisms and structural dynamics of the dual inhibitory activity of PAS-M towards DHFR and FDTS. Chapter 9 reports the structural dynamics and atomistic perspectives that account for the reported ability of BDQ to halt the ion shuttling ability of mycobacterial c-ring. Chapter 10 presents the structural dynamics and conformational changes that occur on Mtb F1F0 ATP-synthase binding as a result of the triple mutations using molecular dynamics simulations, free energy binding, and residue interaction network (RIN) analyses. Chapter 11 explored the impact of NAZ2329, a recently identified allosteric inhibitor of Protein Tyrosine Phosphatase Receptor Zeta (PTPRZ), on the atomic flexibility of the WPD-loop, an essential loop in the inhibition of PTPRZ. The study also presents the drug-likeness of NAZ2329 using in silico techniques and its general inhibitory mechanism. Chapter 12 provides atomistic insights into the structural dynamics and selective mechanisms of AH10-7 for human TCR-CD1d towards activating iNKT cells. The studies in this thesis collectively present a thorough and comprehensive in silico perspective that characterizes the pharmacological inhibition of some known therapeutic targets in viral infections, tuberculosis, and cancer. The augmentative integration of computational methods to provide structural insights could help design highly selective inhibitors of these therapeutic targets. Therefore, the findings presented are fundamental to the design and development of next generation lead compounds with improved therapeutic activities and minimal toxicities

Structural studies on members of the picornavirus superfamily

The pathogenic members of the picornavirus superfamily have adverse effects on humans, their crops and their livestock. As structure is related to function, detailed structural studies on these viruses are important not only for fundamental understanding of the viral life cycle, but also for the rational design of vaccines and inhibitors for disease control. These viruses have positive sense, single-stranded RNA genomes enclosed in a protein capsid. X-ray crystallography and cryo-electron microscopy studies have revealed that the isometric members of this group have icosahedrally-symmetric capsids made up of 60 copies of each of the structural proteins. The members that infect animal cells often employ one or more cellular receptors to facilitate cell entry which in some cases is known to initiate the uncoating sequence of the genome. The nature of the interactions between individual viruses and alternative cellular receptors has rarely been probed. The capsid assembly of the members of the picornavirus superfamily is considered to be cooperative and the interactions of RNA and capsid proteins are thought to play an important role in orchestrating virus assembly. The major aims of this thesis were to solve the structures of blackcurrant reversion virus (BRV), human parechovirus 1 (HPEV1) and coxsackievirus A7 (CAV7), as well as the structure of HPEV1 complexed with two of its cellular receptors using cryo-electron microscopy, three-dimensional image reconstruction and homology modeling. Each of the selected viruses represents a taxonomic group where little or no structural data was previously available. The results enabled the detailed comparison of the new structures to those of known picornaviruses, the identification of surface-exposed epitopes potentially important for host interaction, the mapping of RNA-capsid protein interactions and the elucidation of the basis for the specificity of two different receptor molecules for the same capsid. This work will form the basis for further studies on the influence of RNA on parechovirus assembly as a potential target for drug design.Pikornavirusten superperheen patogeeniset jäsenet aiheuttavat haittaa ihmisille, viljelykasveille ja karjalle. Koska rakenne on yhteydessä toimintaan, yksityiskohtaiset rakenteelliset tutkimukset ovat tärkeitä niin virusten elämänkierron ymmärtämisen kuin rokotteiden ja inhibiittorien rationaalisen suunnittelunkin tähden. Näillä viruksilla on yksijuosteinen, positiivinen RNA genomi suljettuna proteiinikapsidin sisään. Röntgenkristallografia ja kryo-elektroni mikroskopia tutkimukset ovat paljastaneet että tämän ryhmän isometrisillä jäsenillä on ikosaedraalisesti symmetriset kapsidit, jotka koostuvat jokaisen rakenteellisen proteiinin 60:stä kopiosta. Ne jäsenet jotka infektoivat eläinsoluja usein käyttävät yhtä tai useampaa solun reseptoria päästäkseen sisään soluun. Joissain tapauksissa solun reseptori käynnistää tapahtumasarjan joka johtaa genomin vapautumiseen. Yksittäisen viruksen ja sen vaihtoehtoisten reseptorien vuorovaikutuksia on harvoin tutkittu. Picornavirusten superperheen jäsenien kapsidin rakentumista pidetään RNA:n ja rakenteellisten proteiinien yhteistyönä. Tämän työn päätavoitteina oli selvittää blackcurrant reversion virus (BRV), human parechovirus 1 (HPEV1) and coxsackievirus A7 (CAV7) rakenteet, samoin kuin kahden HPEV1-reseptori kompleksin rakenteet käyttäen kryo-elektroni mikroskopiaa, kolmiulotteista rekonstruktio menetelmää ja homologia mallinnusta. Jokainen valituista viruksista edustaa taksonomista ryhmää, josta rakenteellista tietoa ei ole ollut laisinkaan tai on ollut tarjolla niukalti. Tulokset mahdollistivat uusien rakenteiden yksityiskohtaisen vertailun jo tunnettuihin pikornavirusten rakenteisiin. Lisäksi tunnistimme kapsidien pinnalta epitooppeja jotka voivat olla merkittäviä viruksen ja isäntäsolun vuorovaikutuksessa, kartoitimme RNA:n ja rakenteellisten proteiinien vuorovaikutuksia ja selvitimme kahden eri reseptorin vuorovaikutuksia saman kapsidin pinnalla. Tämä työ muodostaa pohjan jolta jatkotutkimuksia RNA:n vaikutukseen parechoviruksen kapsidin muodostuksessa ja mahdollisena lääkeaineiden suunnittelun kohteena voidaan jatkaa

In-vivo and in silico studies of the receptor binding specificity of human rhinoviruses

Humane Rhinoviren (HRV) sind verantwortlich für rund die Hälfte aller Erkältungen beim Menschen. Die zur Familie der Picornaviren gehörigen Rhinoviren besitzen ein ikosaedrisches Kapsid, das aus den 4 viralen Strukturproteinen (VP1, VP2, VP3 und VP4) aufgebaut ist. Der Durchmesser dieses Kapsids beträgt 30 nm. Die mehr als hundert verschiedenen Virustypen können anhand ihrer Fähigkeit an zelluläre Rezeptoren zu binden eingeteilt werden. Die weitaus größere Gruppe der beschriebenen Rhinoviren binden an den ICAM-1 Rezeptor um in die Wirtszelle zu gelangen. Eine kleine Gruppe von Rhinoviren, genannt „minor group“ Viren binden an LDL-Rezeptoren, wie LDLR, LRP und VLDLR. Ein Ziel dieser Arbeit war es neue Einblicke in die Details der Interaktion von Virus und Rezeptor zu erhalten. Vorhergehende Untersuchungen dieser Interaktion zeigten, dass nur ein virales Protein in diese Wechselwirkung involviert ist. Dieses virale Protein 1 (VP1 genannt), und im speziellen die Oberflächen-„loops“ sind an der Wechselwirkung mit den LDL Rezeptoren beteiligt. Sequenzanalysen der LDLR bindenden Rhinoviren („minor group“ viruses) zeigten, dass lediglich ein Aminosäurerest, ein Lysin, strikt konserviert ist. Dieses Lysin befindet sich in der Mitte des HI-„loop“. Man nahm an, dass dieses Lysin verantwortlich sei für die Bindung an LDL Rezeptoren. Es wurden jedoch einige Rhinovirus Typen gefunden, die an der gleichen Stelle ebenfalls ein Lysin aufweisen. Diese Gruppe von Viren (K-Typen) können anhand ihrer Sequenzmerkmale nicht von „minor group“ Viren unterschieden werden, jedoch können sie den LDL Rezeptor nicht für die Infektion verwenden. Im ersten Teil meiner Diplomarbeit ging es um die Entwicklung eines Bioinformatischen Klassifizierungsverfahrens. Mit diesem sollte es möglich sein alle bekannten Rhinovirus Typen gemäß ihrer Rezeptor Spezifikation einzuteilen. Die Methode beruht auf der Hypothese, dass bindende Typen eine zum Rezeptor komplementäre Ladungsverteilung an ihrer Oberfläche aufweisen, die es ihnen ermöglicht den Rezeptor zu binden. Gemäß dieser Hypothese sollten die Bindungsaffinitäten der bindenden Typen höher sein als die der nicht bindenden Typen. Die 3D Strukturen des VP1 Proteins wurden durch „homology modelling“ erhalten. 3D Koordinaten, die der determinierten Röntgenstruktur von HRV2 mit gebundenen V3 entnommen wurden, dienten als Matrize für alle die Anordnung aller anderen Rhinoviren-Rezeptor Komplexe. Nach einem Energie Minimisierungsschritt wurden die Modelle aller Rhinoviren mit gebundenem Rezeptor hinsichtlich ihrer Bindungsaffinität analysiert. Eine der verwendeten Methoden, war tatsächlich in der Lage alle Rhinoviren korrekt zu klassifizieren. Die Gruppe der K-Typen hatten generell höhere Affinitäten zu dem verwendeten Rezeptor (V3), als andere „major group“ Viren. Im anschließenden Teil der Arbeit wurde ein „major group“ Virus (HRV14) so mutiert, das er einen gänzlich veränderten HI-loop aufweist. Die Sequenz des am stärksten in der Virus-Rezeptor Interaktion involvierten Oberflächen“loop“ (HI-loop) wurde gegen die Sequenz eines „minor group“ Virus mittels Zielgerichteter Mutagenese ausgetauscht. RNA Klone der mutierten Sequenz waren jedoch nicht infektiös. Es gelang lediglich einen infektiösen Klon der nur eine Punktmutation im HI-„loop“ aufwies herzustellen.Major group HRVs bind intercellular adhesion molecule 1 (ICAM1) and minor group HRVs bind members of the low-density lipoprotein receptor (LDLR) family for cell entry. Whereas the former share common sequence motives in their capsid proteins, in the latter only a lysine residue within the binding epitope in VP1 is conserved; this lysine is also present in ten "K-type" major group HRVs which fail to bind LDLR. A bioinformatic approach based on the available VP1 sequences three-dimensional models of VP1 of all HRVs were built and binding energies, with respect to module 3 of the very-low density lipoprotein receptor, were calculated. Based on the predicted affinities, K-type HRVs and minor group HRVs were correctly classified. With the intention to find conserved binding patterns the energy tables that indicate the interacting binding partners were transformed into heatmaps. In addition to the heatmaps a bar diagram that shows the interaction energy of the different receptor residues of all minor group and K-type viruses was made. In further improvements the module 3 of VLDLR was replaced by the ligand binding repeat 5 of human and mouse LDLR. To examine the predictive power of the in silico application two non-classified field isolates were analyzed. In a site directed mutagenesis experiment the HI-loop of HRV14 (major group) was changed into the sequence of the HI-loop of HRV2 (minor group). The newly created chimeric virus (HRV14_HI2) was not infective. Also a revision of the experiments under optimized conditions could not create an infective virus. The only chimeric virus that could be produced was HRV14_K. In this virus a histidine to lysine mutation at position 232 was successfully accomplished. The properties of this artificial K-Type virus to bind LDLR were checked in infection assays using RD cells

Viral infection in a murine model of allergic airways inflammation: actions of corticosteroids

Viral respiratory infection exacerbates asthma symptoms in almost all patients with allergic asthma. Asthma symptoms in viral associated asthma exacerbation are often severe and require urgent care as well as hospitalisation. Corticosteroids are the mainstay treatment for asthma. However, they are less effective in treating virus associated asthma exacerbation. The main aim of the thesis is to determine the role of virus infection in airway allergic inflammation and then define the effects of corticosteroids in virus associated exacerbations of airway allergic inflammation. Mice sensitised and challenged with ovalbumin demonstrated most of the main features of asthma including lung cellular inflammation with eosinophilia, early phase asthmatic responses (EAR), late phase asthmatic responses (LAR), and airway hyperresponsiveness (AHR) to methacholine provocations. Treatment with either systemic (dexamethasone: DEX) or inhaled (fluticasone propionate: FP) corticosteroids in the murine ovalbumin allergic airways inflammation model attenuated inflammatory cells influx and eosinophilia, LAR, and the AHR. Influenza A (H1N1/PR8) is the most infective to mice compared to human parainfluenza virus type 3 (HPIV3), and a synthetic dsRNA, poly (I:C). Influenza infection in mice caused a significant increase of inflammatory cell influx in the airways with marked neutrophilia, and AHR. Ovalbumin challenge in the acute course of influenza infection on a murine model of allergic airways inflammation exacerbated the inflammatory cells influx, LAR, and AHR. Treatment with either DEX or FP attenuated the airway cellular inflammation, LAR, but not the AHR. Mice only infected with influenza were resistant to the corticosteroids (DEX and FP) treatment. DEX but not FP showed antiviral activity against HPIV3 and influenza A in vitro. These data suggest that influenza infection in a murine model of allergic airways inflammation exacerbates the inflammation and alters the sensitivity toward corticosteroids. It is also suggested that some elements in the influenza associated exacerbation of murine model of allergic airways inflammation are refractory or not regulated by corticosteroid treatment

Structural Studies of Non-Enveloped Viruses Associated with Human Diseases

Non-enveloped viruses encompass many human pathogens which are responsible for a broad range of diseases that have very significant impacts on human health. By studying the structures of such viruses, insights can be gained into aspects of their lifecycle, including receptor attachment, genome uncoating and capsid assembly. This information can then serve as a structural platform for the design of targeted antivirals and vaccines. This thesis aimed to use cryo-electron microscopy (cryo-EM) to structurally characterise the structures, and receptor binding, of two important human pathogens, BK polyomavirus (BKV) and Coxsackievirus A24v (CV-A24v). BKV causes polyomavirus-associated nephropathy and haemorrhagic cystitis in immunosuppressed patients. These are diseases for which we currently have limited treatment options. Initially, a modest resolution structure (~7 Å) of BKV is used to investigate the organisation of the viral genome and minor capsid proteins. Subsequently, high-resolution structures of BKV alone (3.8 Å) and in complex with the receptor fragment of GT1b ganglioside (3.4 Å) and heparin (3.6 Å) were determined. Collectively, these structures provide insights into capsid assembly, rationalise how GT1b enhances infection over smaller gangliosides studied previously and provide the first structural clues for glycosaminoglycan binding to BKV. CV-A24v is responsible for large outbreaks of acute haemorrhagic conjunctivitis (AHC), a painful, contagious eye disease. Here, ICAM-1 is identified as an essential receptor for CV-A24v and the high-resolution cryo-EM structure (3.9 Å) of the virus–ICAM-1 complex is presented, which reveals the critical ICAM-1–binding residues within the capsid. These data could help identify a possible conserved mode of receptor engagement among ICAM-1–binding enteroviruses. In addition, structures of the uncoating intermediates of CV-A24v are presented which describe the molecular basis of capsid expansion. Furthermore, the molecular details of a branched pocket factor binding site in CV-A24v are described which is unique amongst currently structurally characterised human enteroviruses

Synthesis and biological evaluation of nitrogen heterocycle systems as potential antiviral agents

Viruses are obligate intracellular parasites that consist of either double- or single-stranded DNA or RNA enclosed in a protein coat called capsid. Some viruses also possess a lipid envelope that, like the capsid, may contain antigenic glycoproteins. Most of them contain or encode enzymes essential for their replicative cycle inside host cells, sometimes usurping their metabolic machinery. Traditional therapeutic approaches have mostly focused on targeting specific viral components or enzymes. This pathogen-directed strategy, while successful in numerous cases, in many others results ineffective due to the emergence of drug-resistance. A different approach, addressed to target host-factors essential for viral replication, has recently draw an increasing attention. My PhD project aimed at synthesizing new nitrogen heterocycle systems, designed especially against RNA viruses, such as those belonging to Flaviviridae, Orthomyxoviridae and Paramyxoviridae families. Among them there are, respectively, pathogens responsible for diseases with a high epidemiological impact, as BVDV in cattle and HCV in humans, influenza A and B viruses and respiratory syncytial virus (RSV). The project has been organized into the following phases: 1. Chemical synthesis of the novel compound series. During my PhD I designed and synthesized diverse chemical series of different chemotypes, in order to obtain new antiviral agents: the acridine nucleus, the dihydrotriazine scaffold, the benzimidazole ring as well as anilino and benzenesulfonamide derivatives. Previous studies performed by the research group where I develop my Ph.D. thesis identified some prototypes for the different classes endowed with intrinsic antiviral activity; thus, during my Ph.D. research work I explored various possibilities of functionalisation with the aim of increasing their potency and selectivity profiles towards the respective antiviral target. 2. Characterization of the new compounds. Each newly synthesized compound have been characterised by spectroscopic methods (such as UV, IR, 1H-NMR and 13C-NMR) and elemental analysis. 3. Evaluation of cytotoxicity, antiviral activity in vitro, enzymatic assays and computational studies have been performed in collaboration with several national and international research groups, to assess the biological activity and to identify/confirm the respective molecular targets

Long range interaction networks within 3Dpol and the roles they play in picornavirus genome replication and recombination

Includes bibliographical references.2020 Summer.Picornaviruses contain a single-stranded positive sense RNA genome approximately 7.5kb in length. The genome encodes for a single polyprotein that can future be divided into three functional regions; the P1 region containing the viral capsid proteins, the P2 region whose proteins function primarily in membrane rearrangement during viral replication, and the P3 region which contains four protein responsible for RNA replication. The final protein in the P3 region is 3Dpol, an RNA-dependent RNA polymerase (RdRP) whose structure is analogous to a "right hand" with fingers, palm and thumb domains, and around which this dissertation will be centered. Section one of this work investigates the roles three regions within the fingers domain play in the catalytic cycle of 3Dpol: "The kink" located within the index finger, "the gateway" found on the pinky, and "the sensor", which bridges the two beta-strands of the middle finger. This study demonstrates that the kink residues are involved in RNA binding as mutations to these residues result in decreased initiation time and elongation complex lifetime. The gateway residues are found to act as a molecular stop against which the template-RNA strand positions itself post-translocation, eventually resetting the active site for the next round of nucleotide incorporation. Lastly the sensor residues serve two key functions: 1) A final checkpoint to determine the correct nucleotide has entered the active site, and 2) As a possible source for proton donation to the pyrophosphate leaving group formed during catalysis. The inter-connected nature of the residues investigated in this section give rise to the idea that it is not individual residues alone that control major steps during the catalytic cycle, but instead that long ranging interaction networks within the different polymerase domains are ultimately responsible for controlling different actions carried out by the polymerase. Section two of this work looks at the long-range interaction networks within 3Dpol by dissecting the roles each polymerase domain plays in catalytic cycle. Through generation of chimeric polymerases it was determined that the pinky finger, with some influence by the fingers domain, controls RNA binding, the palm domain dictates nucleotide discrimination, and nucleotide capture and active site closure rates. It was also established that the thumb domain controls translocation, and an interaction between the palm and thumb domains was needed to generate a viable virus, supporting the idea of interface I, a protein-protein interface that was discovered in the first 3Dpol crystal structure. What is most striking about these findings is that unlike other single subunit polymerases that perform translocation by using a large swinging motion within the fingers domain, viral RdRPs use an entirely different domain altogether. The last section of this work deals with viral recombination, an event that is carried out at a low frequency during virus replication. Recombination is proposed to be a mechanism by which mutations can be purged from the genome independent of polymerase fidelity. This study carries out a mechanistic investigation into how mutation of residue 420 from a leucine to an alanine affects polymerase replication kinetics. It also takes a look at the mutation of residue 64 from a glycine to a serine, a previously identified mutation that results in a high-fidelity polymerase, in the presence and absence of L420A. This work revealed that mutations L420A and G64S operate independently of each other by affecting different steps in the catalytic cycle with G64S increases in fidelity predominately from monitoring nucleosugar positioning while L420A affects nucleobase positioning and polymerase grip on the product RNA strand