Development of Novel Heparan Sulfate Analogues as Antiviral Agents

Abstract

Heparan sulfate (HS) is a large, poly-anionic, linear polysaccharide that is made up of repeating disaccharide units of alternating glucosamine and gluco or ido uronic acid residues, and that features distinct patters of O- and N-sulfation. In mammalian biology, HS is the most prominent glycosaminoglycan (GAG) and in the form of heparan sulfate proteoglycans (HSPGs) is a major constituent of the extracellular matrix. As such, HSPGs are inherently involved in an enormous diversity of biological functions. Extracellular HSPGs are almost ubiquitously expressed in mammalian systems and therefore many viral, bacterial, and parasitic human pathogens initiate infection through recognition of and attachment to HSPGs on the surface of host cells. In microbial pathogenesis, HSPGs primarily function as initial, low affinity co-receptors that provide a scaffold to mediate the interaction of microbes with the host cell. Concentration of the pathogen at the cell surface, then allows enhanced binding to specific secondary receptors that facilitate host cell entry. Many viruses adopt HSPGs as cellular receptors to facilitate attachment and entry, including enteroviruses (e.g. enterovirus A71, EV71) and pneumoviruses (human respiratory syncytial virus, hRSV, and human metapneumovirus, hMPV). EV71 is a major cause of hand, foot, and mouth disease, and hMPV is a leading cause of acute respiratory tract infections in children. It has been shown that poly-anionic carbohydrate-based agents (natural polysulfated oligosaccharides, as well as the highly sulfated oligomannoside HS mimetic PI-88) show antiviral activity. However, despite the extensive work carried out there remains no specific antivirals available for the treatment of EV71 and hMPV infections. Due to the increasing understanding of how HSPGs are involved in viral pathogenesis, we sought to investigate how the interactions of HS GAGs could be exploited for drug discovery purposes against a number of viral targets including EV71 and hMPV. Chapter 2 presents the investigation of HS disaccharide fragments and mimetics as inhibitors of EV71 in vitro infection. To begin the study, a series of HS disaccharide fragments were synthesised that possessed varied sulfation patterns to broadly represent the chemical diversity of HS disaccharides. Utilising saturation transfer difference (STD) NMR, and screening against EV71 cell infection, we were able to identify an increase in EV71 binding affinity for fragments that feature higher levels of sulfation. Armed with this information, we then rationally designed and synthesised a series of highly sulfated HS disaccharide mimetics that feature additional sulfation to those of naturally occurring HS disaccharide fragments. Furthermore, we were able to utilise the structurally simple D-maltose, in the place of natural HS disaccharide scaffolds, without any significant loss of potency. An aryl aglycone was found to increase antiviral potency over the simple methyl glycoside, leading to synthesis of a series b-maltosyl 4-aryl-1,2,3-triazole derivatives. The most active compounds, which incorporate a sulfate group on the aryl ring of the aglycone, provide sub-micromolar inhibition of EV71 infection across a range of different cell lines. These functionalised and more densely sulfated HS mimetic disaccharides were found to significantly more potent than the known inhibitors HS and heparin. Data from functional biological assays, including assessment of the effect of compound addition at different stages of viral infection indicated that the compounds were most effective before, or as the virus bound to the cell, and that the observed binding events occur specifically at the viral surface. We therefore proposed that the sulfated HS mimetic disaccharides interfere with virus-cell interactions by acting as decoy receptors to block EV71 infection. In addition, the compounds were assessed for their anticoagulant activity, and importantly unlike HS and heparin, did not affect plasma clotting time even at very high concentrations. The compounds described in this chapter demonstrate potent inhibition of EV71 infection and that poly-sulfated HS mimetic disaccharides can serve as scaffolds for the development of novel EV71 inhibitors. Chapter 3 presents the inhibitory activity of poly-sulfated HS mimetic disaccharides against an alternative HS-recognising viral target, hMPV. HSPGs have been identified as an essential attachment factor for initiation of hMPV infection. Targeting this initial stage of cell infection, we investigated the effect of poly-sulfation of small-molecule, carbohydrate-based compounds on inhibition if hMPV in vitro infection. In addition to the series of sulfated disaccharides described in Chapter 2, an alternative scaffold in N-acetylneuraminic acid (Neu5Ac) was also explored. The poly-sulfated disaccharides and Neu5Ac a-glycoside derivatives were evaluated against hMPV in vitro infection of rhesus monkey kidney epithelial (LLC-MK2) cells. The poly-sulfated HS mimetic disaccharides (maltosides), and in particular those that bear a negatively charged aglycone unit, were found to produce inhibition of hMPV in vitro infection at low micromolar concentrations, demonstrating efficient antiviral activity and an improvement over the known small-molecule hMPV inhibitors ribavirin and suramin. This proof of concept study demonstrated that sulfated maltosides and Neu5Ac derivatives can serve as new templates for the development of novel inhibitors of hMPV infection. Finally, Chapter 4 presents extensive additional work that was carried out for the EV71 project, as well as some preliminary studies against a third viral target, influenza A virus. For the development of EV71 inhibitors, we explored the effects of changing the size of the inhibitor footprint for both HS fragments and mimetics, as well as the use of different linkers between the carbohydrate scaffold and the aglycone. It was found that for HS fragments, a tetrasaccharide is more efficacious than that of a disaccharide. Yet for densely sulfated carbohydrates, the disaccharides were most efficient at blocking EV71 infection. Furthermore, the sulfated hydroxy-phenyl motif, similar in nature to sulfated tyrosines that are found on the side-chains of the EV71 receptor PSGL-1, when bound to sulfated maltosides seemed to endow more effective binding, even compared to display of poly-sulfated maltosyl triazole dimers. This chapter also features an integrated discussion of future research directions and presents the overall conclusions for the body of work described in the thesis.