title: Investigation of human parainfluenza virus type 1 haemagglutinin-neuraminidase as a target for inhibitor discovery
Creators: name: Eveno, Tanguy
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Human parainfluenza virus type 1
subject: Haemagglutinin-neuraminidase
subject: Inhibitor discovery
subject: Respiratory tract illness

description: Viral pathogens are of special significance in the infection of infants, children, immunocompromised patients and the elderly worldwide and are associated with a substantial economic burden. Human parainfluenza viruses (hPIV) are the second most prevalent cause of viral respiratory tract illness after human respiratory syncytial virus and of the four hPIV types, hPIV type 1 is the second most frequent. hPIV-1 infection is commonly associated with respiratory tract symptoms resulting in the syndrome croup. To date, neither antiviral treatments nor vaccines are clinically available to treat or prevent parainfluenza virus infection. The scope of the investigation presented in this Thesis is focused on the multifunctional hPIV-1 haemagglutinin-neuraminidase (HN) protein and the discovery of novel HN inhibitors. hPIV-1 HN is a glycoprotein anchored in the envelope of the virus and is significantly involved in the viral replication cycle. In the early stages of the cycle, it is responsible for recognition of the host cell by binding to sialic acid-containing cellular receptors. It is also implicated in triggering the fusion of the viral envelope with the host membrane by interacting with the fusion protein of hPIV. Finally, in the later stages of the replication cycle, it mediates the release of progeny virions through its neuraminidase activity that cleaves terminal sialic acids from the host cell receptors. The multiple functions of hPIV-1 HN and its involvement in the virus replication cycle make it an ideal target for the development of small molecule inhibitors. The first part of this work presents two structure-activity relationship studies of hPIV-1 HN inhibitors, which were designed using observations inferred from a homology model of hPIV-1 HN. The first study was focused on the alkylamide moiety at the C-5 position on a 2-deoxy-2,3-didehydro-D-N-acteylneuraminic acid (Neu5Ac2en) template. Molecular dynamics simulations highlighted that the active site of hPIV-1 HN could accommodate larger alkylamides at the C-5 position of Neu5Ac2en. A series of compounds with varying C-5 substituents was synthesised and screened against hPIV-1 to evaluate their inhibition potency of the virus neuraminidase and hemagglutination functions. The results demonstrated the highest antiviral activity for the C-5 isobutyramido function from the benchmark compound BCX 2798. The effect of the incorporation of this moiety at the C-5 position was evaluated together with a series of well-known C-4 modifications from diverse sialidase inhibitors. The 4-azido substituent of the benchmark BCX 2798 was proven to be the most potent. The second study used molecular dynamics simulations to investigate the flexibility around the hPIV-1 HN active site and compare it with hPIV-3 HN. While a hPIV-3 HN flexible loop (216-loop) was identified, the same loop in hPIV-1 was found to be predominantly closed in the simulations. This had an impact on the size of the C-4 substituent on the Neu5Ac2en scaffold that could be accommodated in the hPIV-1 HN active site. The results showed a decrease in potency against hPIV-1 HN for compounds with a large and hydrophobic C-4 moiety. On the other hand, the relatively smaller 4-methoxymethyltriazole in compounds 3 and 4 (N.B. the numbering refers to that particular chapter) improved the compounds’ potencies. Compound 4 showed inhibition activities against hPIV-1 HN functions comparable to the benchmark compound BCX 2798 making it one of the most potent hPIV-1 inhibitors to date. The second part of this investigation describes the evaluation of mechanism-based inhibitors of hPIV-1 HN. Several sialidases have been successfully targeted using compounds that were fluorinated at the C-2 and C-3 position on the sialic acid scaffold. They were shown to form a covalent bond with a tyrosine residue in the active site, therefore inhibiting the enzyme’s catalytic activity. This study used a difluoro derivative, namely compound 3 (N.B. the numbering refers to that particular chapter), of the benchmark compound BCX 2798 to investigate this new template as an hPIV-1 HN inhibitor. 1H nuclear magnetic resonance spectroscopy was used to evaluate blockade of hPIV-1 HN by compound 3 and showed a sustained inhibition of HN’s neuraminidase activity. The effects of this blockade on inhibitor potency were determined using several virological methods. Taken together, these results showed that compound 3 is the most potent hPIV-1 HN inhibitor described since BCX 2798. The final part of this work focuses on attempts to solve the three-dimensional structure of hPIV-1 HN by X-ray crystallography. A protein expression system in insect cells was established to generate the ectodomain of hPIV-1 HN. Following a two-step purification procedure based on affinity and size exclusion chromatography, highly pure and active protein was obtained. A combination of commercially available and designed screens were set to find optimal conditions for hPIV-1 HN crystallisation. Two conditions that produced small protein crystals were identified but could not provide an X-ray diffraction pattern, nor could they be improved. To reduce the protein sample heterogeneity, several options were evaluated to remove N-glycosylation from the protein with one option proving successful, providing an active deglycosylated protein. The screening for crystallisation conditions using this protein sample did not yield any positive outcomes. The work presented in this Thesis broadens the knowledge on hPIV-1 HN crystallisation and provides new scaffolds for the design of potent, next generation hPIV-1 HN inhibitors.