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

Computer assisted molecular modeling of thymidine nucleoside analog inhibitors of HIV-1 reverse transcriptase

An active analog approach to receptor mapping was used to identify the three dimensional structural characteristics associated with a series of thymidine nucleoside analog inhibitors of HIV-1 reverse transcriptase (RT) that may be essential for their activity. Atomic substitutions at the 5 and 3\u27 positions of nucleoside analogs confer global structural and electrostatic changes that result in either increased or diminished inhibitory activity. From a structural perspective, the activity differences can be attributed to the presentation of select atoms in three dimensional motifs that are common to all active compounds and absent or distorted in inactive/poorly active compounds. The identification of these characteristics will complement more direct studies of the RT structure by providing a specific three dimensional orientation for substrate and inhibitor molecules at their receptor site. They can also serve as a three dimensional template for the screening of potentially active compounds; thus, aiding in the development and identification of new, more potent and selective inhibitor molecules. The characteristics identified are common to 15 thymidine nucleoside analog inhibitors of RT and have allowed the inference of a three dimensional map of the HIV-1 RT receptor site

The development of sialidase inhibitors using structure-based drug design

The sialidases/neuraminidases represent a family of enzymes whose function is important in the pathogenicity of bacteria and the virulence of influenza. Relenza and Tamiflu represent two drugs that were developed using structure-based drug design (SBDD) and computational-assisted drug design (CADD). These drugs target the active site of the influenza neuraminidase A and B (GH-34 family). Sialidases in the GH-33 family could represent novel drug targets for the treatment of bacterial or parasitic infection. SBDD was employed to develop chemical tools of two GH-33 sialidases, NanB and TcTS. NanB is a potential drug target for S. pneumoniae. The chemical tool developed for NanB follows on from work within the Taylor and Westwood research groups, in which a molecule of CHES and a glycerol were found serendipitously bound within a water channel at an allosteric site. Using this information as a basis for SBDD an allosteric inhibitor of NanB, Optactin was developed. Within this work, synthesis of this inhibitor was achieved and optimised. Optactin was then modified to improve potency. This proceeded through an amide analogue and addition of an arene resulting in a mid- micromolar inhibitor (IC₅₀: 55.4±2.5 µM). Addition of polar substituents improved potency further resulting in a low micromolar inhibitor of NanB, Optactamide (IC₅₀: 3.0±1.7 µM). Application of this tool in vitro demonstrated that NanB and NanA have a role in invasion of S. pneumoniae into lung epithelial cells. TcTS is a potential drug target for the treatment of Chagas disease. A CADD approach using a fragment library was unsuccessful at identifying an allosteric inhibitor of TcTS despite structural similarity with NanB. A re-task of the CADD approach towards the active site was successful in identifying an inhibitor of TcTS and a fragment useful for further development. This work sets the groundwork for the development of a chemical tool targeting TcTS

Structure and Function Relationship of Trans-Sialidases from Trypanosoma congolense

The study presented here addresses structural and functional relations of trans-sialidases (TS) from the African parasite Trypanosoma congolense and their biochemical characterisation. TS are unusual enzymes found in the flagellate protozoan parasite Trypanosoma and catalyse the stereo and region specific transfer of terminal sialic acid from donor sialo-glycoconjugates to terminal galactose residues of suitable acceptor substrates, resulting in alpha-2,3-sialylated glycoconjugates. Major research on trypanosomal TS has been done on Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. However, only little has been known about TS from the African Trypanosoma congolense TS, the prevalent causative agent of African animal Trypanosomiasis (AAT) in livestock and domestic animals also termed nagana

Understanding Glycans and Glycosidases in Infection and Biofilm Formation as a Route to Novel Antimicrobials

Periodontitis is a chronic bacterially induced disease characterized by inflammation of the gingivae and subsequent destruction of the tissues and supporting structures of the periodontium, which can lead to tooth loss. The red complex pathobionts: Porphyromonas gingivalis and Tannerella forsythia are mostly associated with periodontitis and are shown to modulate the host’s innate immune system. These oral pathogens secrete sialidase enzymes that they use to scavenge sialic acids found at the terminus of host glycoprotein chains for nutrition or to evade host immune responses. Activities of these pathogens, therefore, poses a problem to public health as such, necessitates the need to carry out research aimed at understanding this important enzyme as a route for the development of novel inhibitors of periodontitis and other immune-modulatory diseases. Over-expressed P. gingivalis (SiaPG) and T. forsythia (NanH) sialidase enzymes were purified using HisTag low-affinity chromatography, and the sialidase activity was tested using 4-methylumbelliferyl N-acetyl-α-D-neuraminic acid sodium salt (MUNANA), as substrate. Inhibition studies using whole cells and the purified sialidases of T. forsythia (NanH) and P. gingivalis (SiaPg), showed ECG as the best plant-derived inhibitor, while 2e3aDFNeu5Ac9N3 as the most potent of all the screened compounds. Additionally, Palmatine and Berberine chloride synergistically inhibited almost a 100 % NanH sialidase activity, with mechanisms of action (MOA) showing Palmatine, Berberine chloride, and 2e3aDFNeu5Ac9N3, as non-competitive, uncompetitive and competitive inhibitors of NanH sialidase, respectively. Furthermore, sialidase promotes the growth of P. gingivalis and T. forsythia, and supports host-pathogen interactions via adhesion and invasion of oral epithelial cells (H357). In addition, the role of P. gingivalis and T. forsythia sialidases on host innate immune modulation in the presence or absence of 2e3aDFNeu5Ac9N3 was also assessed using flow cytometry. NanH sialidase of T. forsythia appears to upregulate the secretion of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-8, and IL1-β in the cell supernatants, which was abrogated significantly by 2e3aDFNeu5Ac9N3 with minimal cytotoxic effects on the oral epithelial cells. Also, molecular docking of several inhibitors into the active-site pockets of NanH-apo and SiaPg using AutoDock Vina and PyMol suggested that Oseltamivir, Siastatin B, DANA, and 2e3aDFNeu5Ac9N3 coordinate the arginine triad (Arg423, Arg487, Arg212 and Arg194, Arg213, Arg460), of NanH and SIaPg of T. forsythia and P. gingivalis, respectively. Also, the nucleophilic dyad tyrosine and glutamate interact with the anomeric carbon as well as the acid/base aspartate residue Asp237/Asp280 interacting with the N-acetyl group of respective inhibitors. The superior inhibitory properties of 2e3aDFNeu5Ac9N3 on both the purified and whole cell sialidase activities of P. gingivalis and T. forsythia make it a promising compound, and can further be developed as a novel immunomodulatory agent of periodontitis or other inflammatory diseases

Computer-Aided Drug Design of Neuraminidase Inhibitors and MCL-1 Specific Drugs

Ph.DDOCTOR OF PHILOSOPH

Design and synthesis of constrained bicyclic molecules as candidate inhibitors of influenza A neuraminidase

The rise of drug-resistant influenza A virus strains motivates the development of new antiviral drugs, with different structural motifs and substitution. Recently, we explored the use of a bicyclic (bicyclo[3.1.0]hexane) analogue of sialic acid that was designed to mimic the conformation adopted during enzymatic cleavage within the neuraminidase (NA; siali-dase) active site. Given that our first series of compounds were at least four orders of magnitude less active than available drugs, we hypothesized that the new carbon skeleton did not elicit the same interactions as the cyclohexene frameworks used previously. Herein, we tried to address this critical point with the aid of molecular modeling and we proposed new structures with different functionalization, such as the introduction of free ammonium and guanidinium groups and ether side chains other than the 3-pentyl side chain, the characteristic side chain in Oseltamivir. A highly simplified synthetic route was developed, starting from the cyclopropanation of cyclopentenone and followed by an aziridination and further functionalization of the five-member ring. This allowed the efficient preparation of a small library of new bicyclic ligands that were characterized by enzyme inhibition assays against influenza A neuraminidases N1, its H274Y mutant, and N2. The results show that none of the new structural variants synthesized, including those containing guanidinium groups rather than free ammonium ions, displayed activity against influenza A neuraminidases at concentrations less than 2 mM. We conclude that the choice and positioning of functional groups on the bicyclo[3.1.0]hexyl system still need to be properly tuned for producing complementary interactions within the catalytic site