La realidad nacional:

With the diverse role that oligosacchaiides play in biological functions it is apparent that the prevention of carbohydrate recognition may have potentially important therapeutic benefits. The selectin - caibohydrate ligand interaction has been targeted for possible therapeutic intervention because of the over recruitment of leukocytes, resulting in tissue damage and swelling. In fact, at present several companies are testing sialyl Lewis* antigen in clinical trials for reperfusion injury, and there are currently some fifteen carbohydrate drugs undergoing clinical trials for ailments including cancer, diarrhoea, thrombosis, diabetes, ulcers, epilepsy, and Parkinson’s disease (reviewed by McAuliffe and Hindsgaul, 1997). Carbohydrate based drugs may also have added benefits such as low toxicity and immunogenicity. In the treatment of microbial infections, carbohydrate-based drugs only prevent the adhesion of microbes, thus there is less evolutionary pressure for mutation.

Chapterl: Introduction 22

reducing the likelihood of resistance. The mode of action of these caibohydrate drugs is varied:

L Inhibition o f protein attachment

The most common approach is to design a drug based upon the natural carbohydrate receptor which would bind iixeversibly and competitively with the natural substrate, thus preventing cellular* adhesion. This method is the current approach for* mimics to the sialyl Lewis* - selectin interaction for* the treatment of reperfusion injury, where the accumulation of leukocytes in tissues after heart attack and surgery can lead to tissue damage. The clearance of bacteria and their toxins from the gastrointestinal tract with oligosaccharides is used in nature. The high levels of oligosaccharides present in mammalian milk, often found on the surface of the gut, is believed to protect infants from infection. Mimics of this process include the development of a Gbg conjugate linked to an insoluble powder* to prevent verotoxin adhesion.

2. Alteration of carbohydrate biosynthesis

Many diseases are associated with defects in oligosaccharide metabolism, such as diabetes. Azasugars (nitrogen analogous of carbohydrates) are believed to be a possible treatment by inhibiting sucrase and amylase, responsible for the breakdown of polysaccharides in the gut, and therefore prevent the adsorption of glucose into the bloodstream. Inhibition of the catabolism of Neu5Ac ternrinating oligosaccharides has been successfully used in the treatment of influenza (von Itzstein et a l, 1993).

3. Eliciting an immune response

Cells undergoing oncogenesis often express a different range of glycoproteins due to defects in the sugar* metabolic pathways with higher densities of smaller gangliosides present at the expense of more complex gangliosides (reviewed by Oettgen, 1989). The treatment of cancer patients by stimulating anti-ganglioside antibodies has had some limited success (Livingston et a l, 1987). The present treatment consists of injecting patients with a glycopeptide hapten found on the surface of these cancer* cell lines which elicits an immune response against these cells. This treatment is being applied successfully to the treatment of lung, breast, colon, and ovarian cancers.

Chapter!: Introduction 23

1.3.1 Rational Drug Design

A prerequisite for the rational design of drugs is a detailed understanding of the interaction of a carbohydrate with its receptor. A high resolution three dimensional structure of the complex is required, and several crystallographic studies have been described recently which illustrate in detail the precise nature of certain carbohydrate-protein interactions. The most celebrated example of model based drug design was the work performed in von Itzstein’s laboratory on the development of an influenza sialidase inhibitor (von Itzstein et a l, 1993). Sialidase is critical in the release of newly synthesised influenza virions from infected cells by the cleavage of terminal NeuSAc residues from host cell-surface glycans. Inhibition of this enzyme limits the establishment and progression of infection. Computer-aided molecular modelling was used to analyse the active site and predict the functional group changes which would increase the binding affinity of the sialic acid residue. Modelling studies predicted that the substitution of the 4-OH group with an amino or guanidynl group would produce an increase in overall binding. Results show that the 4-guanidino derivative has an inhibition constant, for various strains of influenza, in the 10'^° range. Additionally the affinity of this compound against human sialidases is a million times lower, therefore offering excellent selectivity for vii-us sialidases.

Detailed x-ray crystal structures of biologically important oligosaccharide complexes are rare, as the relatively flexible oligosaccharide often results in poor electron densities for the carbohydrate residues, and when observed they are often stabilised by crystal packing forces which may orient the sugar in an unnatural conformation. Many lectins only weakly bind isolated oligosaccharide analogues of their receptor glycans, and so may not be co­ crystallised (Stein et ah, 1992). An additional problem is that carbohydrate binding sites in the well ordered solid state may not be active in the less well ordered solution state (c./. Wright, 1992, with Wright and Kellog, 1996, and reviewed by Rossi, 1992).

In contrast, high resolution structural studies of glycan protein interactions by nuclear magnetic resonance spectroscopy can offer complimentary information on the structure of the carbohydrate in sugar-protein interactions. Solution studies also have an important potential advantages since a comparison of the solution structure of the free and bound conformations of the ligand is more meaningful, and moreover, the dynamics of the system are accessible.

Chapterl: Introduction 24