Mechanistic Elucidation of Protease–Substrate and Protein–Protein Interactions for Targeting Viral Infections

Viral infections represent an old threat to global health, with multiple epidemics and pandemics in the history of mankind. Despite several advances in the development of antiviral substances and vaccines, many viral species are still not targeted. Additionally, new viral species emerge, posing a menace without precedent to humans and animals and causing fatalities, disabilities, environmental harm, and economic losses. In this thesis, we present rational modeling approaches for targeting specific protease-substrate and protein-protein interactions pivotal for the viral replication cycle. Over the course of this work, antiviral research is supported beginning with the development of small molecular antiviral substances, going through the modeling of a potential immunogenic epitope for vaccine development, towards the establishment of descriptors for susceptibility of animals to a viral infection. Notably, all the research was done under scarce data availability, highlighting the predictive power of computational methods and complementarity between in-silico and in-vitro or in-vivo methods

The in silico investigation of pharmacological targets of the zika virus : insights into the structural characteristics of the NS5 and NS3 proteins from atomistic molecular simulations.

Doctor of Philosophy in Pharmacological Science. University of KwaZulu-Natal, Durban 2017.The re-emerging Zika virus has evolved into a catastrophic epidemic during the past year, with an estimated 1.5 million reported cases of Zika infections worldwide, since the 2015 outbreak in Brazil. The virus has received considerable attention during 2016 with a flood of new discoveries, from evolving modes of viral transmission to viral-linked neurological disorders, unique specificity to host cells and increasing mutation rates. However, prior to the devastating 2015 outbreak in Brazil, the virus was classified as a neglected pathogen similar to Dengue and the West Nile virus. Despite the wide-scale research initiative, there is still no cure for the virus. There are currently vaccine clinical trials that are on-going but there has not been a breakthrough with regard to small molecule inhibitors. A lot of experimental resources have been allocated to repuposing FDA-approved drugs as possible inhibitors, however, even some of the most potent flavivirus inhibitors have adverse toxic effects. The first crystal structure of the zika virus was released in May 2016 and since then, six viral protein structures have been made available. Due to this lack in structural information, there is little known regarding the structural dynamics, active binding sites and the mechanism of inhibition of ZIKV enzymes. This study delves into the structural characteristics of three of the most crucial enzymatic targets of the zika virus, the NS5 RNA-dependent RNA polymerase and Methyltransferase as well as the NS3 Helicase. With emerging diseases, such as ZIKV, computational techniques including molecular modeling and docking, virtual screening and molecular dynamic simulations have allowed chemists to screen millions of compounds and thus funnel out possible lead drugs. These in silico approaches have warranted Computer-Aided Drug Design as a cost-effective strategy to fast track the drug discovery process. The The above techniques, amongst numerous other computational tools were employed in this study to provide insights into conformational changes that elucidate potential inhibitory mechanisms, active site identification and characterization and pharmacophoric features leading to promising small molecule inhibitor cadidates. The first study (Chapter 4), provided a comprehensive review on potential host/viral targets as well as provided a concise route map depicting the steps taken toward identifying potential inhibitors of drug targets when no crystal structure is available. A homology model case study, of the NS5 viral protein, was also demonstrated. The second study (Chapter 5) used the validated NS5 homology model to investigate the active sites at both the RNA-dependent RNA polymerase and Methyltransferase domains and subsequently employ a generated pharmacophore model to screen for potential inhibitors. Chapter 6 reports the third study, which investigates the structural dynamics and in turn, the possible mechanism of inhibition of the ZIKV NS3 Helicase enzyme when bound to ATP-competitive inhibitor, NITD008. The study also provides insight on the binding mode at the ATPase active site, thus assisting in the design of effective inhibitors against this detrimental viral target. Chapter 7 maps out the binding landscape of the ATPase and ssRNA site by demonstrating the chemical characteristics of potent flavivirus lead compounds, Lapachol, HMC-HO1α and Ivermectin at the respective NS3 Helicase binding sites. This study offers a comprehensive in silico perspective to fill the gap in drug design research against the Zika virus, thus giving insights toward the structural characteristics of pivotal targets and describing promising drug candidates. To this end, the work presented in this study is considered to be a fundamental platform in the advancements of research toward targeted drug design/delivery against ZIKV

Crystallographic Studies for Structure-Based Drug Design

Structure-based drug design is an iterative design cycle reliant upon lead fragments and a known target. The result is two separate avenues of structure-based drug design, explored in this thesis: speculative, with no defined target (in the form of peptoid precursors) and rational, with a characterized target (in the form of EthR inhibitors). Peptoids are N-substituted glycine molecules, able to mimic peptide structure and function with notable advantages for drug delivery and independent function. Here, the structures of two precursor molecules are presented as part of a wider aim to produce a tool-box of peptoid monomers for a fragment-based approach. The intramolecular interactions made by the two precursors can be extrapolated for the final peptoid. EthR is a transcriptional repressor from Mycobacterium tuberculosis, a part of the activation pathway for the second-line drug ethionamide. It has been shown that inhibition of EthR results in a conformational change which renders the protein inactive, resulting in an increase in the bioactivation of ethionamide and so increased drug efficiency. Inhibitors for EthR have been designed, able to effect this response in vitro and in vivo. This thesis details the crystallisation and structural study of peptoid precursors for use as lead fragments or monomers for peptoid click chemistry; and EthR inhibitors for improving the bioactivation and efficacy of ethionamide, with evaluation by molecular docking analysis in AutoDock Vina. The results show that shorter ligands, capable of engaging certain residues in the ligand-binding channel, are rated highest and so indicated to be the more effective inhibitors. The two approaches highlight the diversity of tactics for new therapeutics and the powerful advantage of three-dimensional crystal structures, acquired through X-ray crystallography, to the drug design process

Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection

In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van’t Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases

Virus Purification Framework And Enhancement In Aqueous Two-Phase System

Viral infections regularly pose detrimental health risks to humans. Preventing viral infections through global immunization requires the production of large doses of vaccines. The increasing demand for vaccines, especially during pandemics such as COVID-19, has challenged current manufacturing strategy to develop advanced unit operations with high throughput capability. Over the decade, the upstream processing responsible for synthesizing viral products in cell cultures has shown significant success in yielding high titers of viruses and virus-like particles. The progress in the upstream stage has now shifted the bottleneck to the downstream processing (DSP). Overlooked for decades, the DSP responsible for viral product purification from the cell culture contaminants requires a makeover with the development of new purification strategies and an upgrade in the traditional unit operations. The current DSP train employing chromatography and filtration methods have been suboptimal in efficiently processing comparatively complex and fragile viral particles. Thus, the lack of platform technology for viral vaccine and biotherapeutic DSP has led to a search for alternative and innovative methods that have not only high-throughput capabilities but also have potential for continuous operation. In the pool of potential technologies, aqueous two-phase system (ATPS) has shown to be a promising candidate with the numerous advantages over conventional methods. However, an unambiguous and complex biomolecule partitioning mechanism has required a large experimental setup for optimizing virus purification. This work focused on a framework utilizing a phase diagram of a rationalized polyethylene glycol-citrate system to optimize virus purification. The partitioning behavior of two non-enveloped viruses, porcine parvovirus (PPV) and human rhinovirus-14 (HRV), were studied in various system compositions. A tie-line length framework was utilized to define the systems and relate the partitioning behavior of viruses with different surface physicochemical characteristics

Aptamers for Diagnostics with Applications for Infectious Diseases

Aptamers are in vitro selected oligonucleotides (DNA, RNA, oligos with modified nucleotides) that can have high affinity and specificity for a broad range of potential targets with high affinity and specificity. Here we focus on their applications as biosensors in the diagnostic field, although they can also be used as therapeutic agents. A small number of peptide aptamers have also been identified. In analytical settings, aptamers have the potential to extend the limit of current techniques as they offer many advantages over antibodies and can be used for real-time biomarker detection, cancer clinical testing, and detection of infectious microorganisms and viruses. Once optimized and validated, aptasensor technologies are expected to be highly beneficial to clinicians by providing a larger range and more rapid output of diagnostic readings than current technologies and support personalized medicine and faster implementation of optimal treatments

Viral vector platforms within the gene therapy landscape

Throughout its 40-year history, the field of gene therapy has been marked by many transitions. It has seen great strides in combating human disease, has given hope to patients and families with limited treatment options, but has also been subject to many setbacks. Treatment of patients with this class of investigational drugs has resulted in severe adverse effects and, even in rare cases, death. At the heart of this dichotomous field are the viral-based vectors, the delivery vehicles that have allowed researchers and clinicians to develop powerful drug platforms, and have radically changed the face of medicine. Within the past 5 years, the gene therapy field has seen a wave of drugs based on viral vectors that have gained regulatory approval that come in a variety of designs and purposes. These modalities range from vector-based cancer therapies, to treating monogenic diseases with life-altering outcomes. At present, the three key vector strategies are based on adenoviruses, adeno-associated viruses, and lentiviruses. They have led the way in preclinical and clinical successes in the past two decades. However, despite these successes, many challenges still limit these approaches from attaining their full potential. To review the viral vector-based gene therapy landscape, we focus on these three highly regarded vector platforms and describe mechanisms of action and their roles in treating human disease

Interactions of the HIV-1 nef virulence factor with host cell tyrosine kinases of the src and tec families

Current antiretroviral therapies effectively slow AIDS progression and lengthen the life of AIDS patients but sadly, cannot completely cure HIV-positive individuals. The development of drug-resistance in HIV often renders the current anti-HIV therapeutic regimens ineffective and even contraindicated in some cases. Thus there exists an urgent need to identify alternate targets for the discovery and development of newer anti-HIV drugs. A promising approach lies in targeting an underexplored yet critical accessory factor in HIV pathogenesis – Nef, which promotes AIDS progression by binding to a plethora of host cell factors leading to altered cell signaling. Identifying novel host factors that are direct effectors for HIV-1 Nef will enable future drug discovery directed against this key HIV virulence factor. In the first part of my dissertation study, I developed a novel, cell-based approach to explore the scope of Nef-SH3 interactions. Particularly, I explored the interaction of Nef with Tec-family kinases and their relevance to HIV biology. This assay allowed direct visualization of protein-protein interactions between Nef and three Tec family members – Bmx, Btk and Itk in live cells. Interaction occurred between the SH3 domains of the kinases and a conserved polyproline motif on Nef. Allelic variants of Nef representing all the M-group HIV-1 subtypes interacted strongly with Itk demonstrating the highly conserved nature of this interaction. Interaction with Nef induced Itk activation which was reversed by treatment with an Itk inhibitor that also potently blocked Nef-dependent HIV replication. These results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family, and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle. In the second part of this study, I validated the biological relevance of a newly determined high resolution crystal structure of Nef in complex with its best characterized kinase binding partner, Hck. Using human and yeast cell-based systems, I have shown by mutagenesis studies that the newly recognized intercomplex contact between Nef R105 and E93 in the RT loop of the SH3 domain is critical to complex formation and function. These results renew our perception of the Nef:Hck binding interface by offering new insight into possible conformations for the active Nef:Hck complex, which is essential for Nef function and further establishes it as a valid druggable target for HIV-1. Taken together, the studies presented in this dissertation deepen our understanding of the interaction between the HIV-1 virulence factor Nef and the Src family kinase, Hck; identify additional novel cytoplasmic tyrosine kinases that are direct SH3-based effectors of HIV-1 Nef and validate a novel virus:host cell interaction as a potential target for therapeutic intervention. Thus, my results not only have a strong public health significance and advance the field of HIV research, but also offer a step forward in our combat against what remains as one of the most relevant public health menaces of today – HIV/AIDS

Massively-Parallel Computational Identification of Novel Broad Spectrum Antivirals to Combat Coronavirus Infection

Philosophiae Doctor - PhDGiven the significant disease burden caused by human coronaviruses, the discovery of an effective antiviral strategy is paramount, however there is still no effective therapy to combat infection. This thesis details the in silica exploration of ligand libraries to identify candidate lead compounds that, based on multiple criteria, have a high probability of inhibiting the 3 chymotrypsin-like protease (3CUro) of human coronaviruses. Atomistic models of the 3CUro were obtained from the Protein Data Bank or theoretical models were successfully generated by homology modelling. These structures served the basis of both structure- and ligand-based drug design studies. Consensus molecular docking and pharmacophore modelling protocols were adapted to explore the ZINC Drugs-Now dataset in a high throughput virtual screening strategy to identify ligands which computationally bound to the active site of the 3CUro . Molecular dynamics was further utilized to confirm the binding mode and interactions observed in the static structure- and ligand-based techniques were correct via analysis of various parameters in a IOns simulation. Molecular docking and pharmacophore models identified a total of 19 ligands which displayed the potential to computationally bind to all 3CUro included in the study. Strategies employed to identify these lead compounds also indicated that a known inhibitor of the SARS-Co V 3CUro also has potential as a broad spectrum lead compound. Further analysis by molecular dynamic simulations largely confirmed the binding mode and ligand orientations identified by the former techniques. The comprehensive approach used in this study improves the probability of identifying experimental actives and represents a cost effective pipeline for the often expensive and time consuming process of lead discovery. These identified lead compounds represent an ideal starting point for assays to confirm in vitro activity, where experimentally confirmed actives will be proceeded to subsequent studies on lead optimization