title: The design, synthesis and biological evaluation of carbohydrate-based probes of proteins
Creators: name: Carroux, Cindy
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Proteins
subject: Carbohydrates
subject: Glycosyl triazoles
subject: Influenza virus

description: Carbohydrates are widely involved in physiological and pathological processes. The development of carbohydrate-based probes to investigate these processes is an important aspect of drug design and drug discovery. A carbohydrate moiety is generally used in drug development as a pharmacophore for a natural biological interaction. Alternatively, a carbohydrate moiety attached to a well-known biologically active pharmacophore (i.e. as a scaffold), can be used to address solubility issues and/or modulate the pharmacokinetic properties of that pharmacophore. The work of this thesis utilises carbohydrates in both of these ways. The research described in this PhD thesis was focused on the development and evaluation of carbohydrate-based probes for two different enzymes: cancer-related carbonic anhydrase isozymes, and influenza virus neuraminidases. A common feature of the development of these carbohydrate-based probes, was the use of glycosyl azides with in copper and ruthenium catalysed Azide-Alkyne Cycloaddition (CuAAC and RuAAC) reactions to respectively form glycosyl 4- and 5-substituted [1,2,3]-triazoles. In Chapter 1, an overview of the significant involvement of carbohydrates in physiological and pathological processes and their use in drug discovery is provided, concentrating on drugs currently on the market or in clinical trials. This overview is followed by a discussion presenting the important role of click chemistry − especially CuAAC and RuAAC reactions − in medicinal chemistry, concentrating on the applications of AAC reactions in glycoscience. The aim of the research described in Chapter 2 was the design of carbonic anhydrase (CA) inhibitors that would specifically target the cancer-related membrane-associated isozymes CA IX and CA XII. CAs are ubiquitous enzymes that are involved in numerous essential biological processes. The various CA isozymes (CA I to XV) are either intracellular or localised on the cell membrane. Two specific membrane-associated isozymes, CA IX and XII, are involved in cancer. Many potent CA inhibitors have been synthesised, however, most of these inhibitors have no specificity for particular isozymes. To create specificity for the cancer-related isozymes, one approach is to design CA inhibitors that would not be able to cross the cell membrane based on their intrinsic physicochemical properties. In the first study described in Chapter 2, glucosyl and galactopyranosyl azides were synthesised, in which the carbohydrate hydroxyl groups were protected with acyl groups of varying chain length. Using CuAAC, glycosyl 4-phenyl-[1,2,3]-triazoles were then prepared. These O-acylated glycosyl triazole derivatives were used as model compounds to study the influence of the O-acylation on in vitro biopharmaceutical properties. Investigation of the metabolic stability, plasma stability, and plasma protein binding of the O-acylated glycosyl triazoles revealed that the derivatives with longer acyl chains were stable whereas the derivatives with smaller acyl chains were more susceptible to metabolism. Overall, these results suggested that relative plasma stability of the various esters may be linked to the susceptibility to plasma esterases, balanced by the binding strength to plasma proteins. Based on the results of the initial study, in the second study described in Chapter 2, O-acylated glycosyl triazoles incorporating a well-known CA inhibitor − a m- or p-substituted aryl sulfonamide − were synthesised by CuAAC as CA probes, using the previously synthesised glycosyl azides. Investigation of the metabolic stability, plasma stability, and plasma protein binding of the new glycosyl triazole sulfonamides revealed similar stability of the acyl chains to that seen for the model glycosyl 4-phenyl-[1,2,3]-triazoles. Biological testing of the O-unprotected (hydroxyl groups free) and the fully O-acylated glycoconjugates against physiologically important CA isozymes (hCA I, II, XIV) and cancer-related isozymes (hCA IX and XII), showed that these compounds were potent (K i micro- to nanomolar) inhibitors. However, no consistent structure-activity relationship trends could be identified across each of the four scaffolds, based on the different acyl groups present. Consequently, this second study suggested that any of the fully acylated compounds could be used as a prodrug for the corresponding O-unprotected glycoconjugate. The aim of the research described in Chapter 3 was the development and evaluation of influenza virus neuraminidase (NA) inhibitors. Influenza viruses cause worldwide seasonal epidemics and sporadic pandemics of influenza. By targeting the viral surface NA, an enzyme that facilitates the release of newly formed virions from the infected cell, structure-based inhibitor design has led to four commercially available anti-influenza drugs − zanamivir, oseltamivir, peramivir and laninamivir. New findings in structure and mechanistic features of influenza virus NA, however, are driving ongoing research into development of new anti-influenza agents. The natural micromolar NA inhibitor Neu5Ac2en and the anti-influenza drug zanamivir both have a glycerol side-chain, which engages in hydrogen-bond interactions with conserved residue Glu276. In contrast, other inhibitors (oseltamivir and peramivir) use a hydrophobic side-chain to bind the glycerol side-chain binding pocket by inducing a reorientation of Glu276, creating a hydrophobic pocket. The 4,5-unsaturated (Δ4-)N-acetylglucosaminuronic acid template, in which a β-glycoside aglycon replaces the glycerol side-chain of Neu5Ac2en, has proved to be a useful template to probe binding interactions within this area of the NA active site. In the first study described in Chapter 3, a multigram-scale synthetic route to the key N-acetylglucosaminuronate glycosyl azide was optimised. Using CuAAC, novel N-acetyl-Δ4-β-D-glucosaminuronyl 4-substituted [1,2,3]-triazoles, with a range of substituents on the triazole ring, were then synthesised. Using an in vitro fluorometric enzyme assay, the NA inhibitory activity of the synthesised compounds was assessed. The best inhibitor, with activity (IC50) equivalent to Neu5Ac2en against an H3N2 NA, was obtained with a phenyl substituent on the triazole group. Further modifications of the aromatic ring did not appear to create additional favourable binding interactions, at least with respect to strength of inhibition. Studies of inhibition against influenza A virus H5N1 and the H5N1–His274Tyr variant, indicated that the inhibitory activity of the triazole derivatives was not adversely affected by the inability of the His274Tyr strain to re-orient Glu276. This is in line with our modelling study, which suggested that the triazole substituent would orient towards the side-pocket lined by Ile222 and Ser246 (or Ala246, depending on the strain), rather than bind in the glycerol side-chain binding pocket. The binding mode of the 4-phenyl triazole derivative, the most potent inhibitor across all tested NAs, will next be assessed in crystallographic studies. In the second study described in Chapter 3, following on from the results of the first study, a basic docking study was undertaken to explore the potential binding mode of N-acetyl-Δ4-β-glucosaminuronyl 5-substituted-[1,2,3]-triazole derivatives. This study suggested that the hydrophilicity, bulkiness and length of the 5-subtituent on the triazole influenced the success of the docking into the NA active site, and that the binding of these compounds might be dependent on the Glu276 orientation. Using, the previously synthesised unsaturated glucosaminuronate glycosyl azide, introduction of a 5-substituted [1,2,3]-triazole group by RuAAC was attempted. Unfortunately, this glycosyl azide showed a lack of reactivity towards functionalised alkynes in the Ru-catalysed reaction, with only phenylacetylene successfully generating the desired 5-phenyl-[1,2,3]-triazole. As suggested by the docking study, this derivative with a bulky triazole substituent had only weak (millimolar) inhibitory activity against the tested influenza A virus NAs. Further method development would be required to provide a series of 5-substituted [1,2,3]-triazole derivatives on the glucosaminuronic acid template to explore the effect of this substitution on influenza virus NA inhibition.