Small-Molecule Inhibitors of Influenza Virus Neuraminidase

Influenza virus has two major surface proteins, hemagglutinin and neuraminidase (see Chapter 34). The hemagglutinin initiates infection by binding to cell-surface sialic acids. The neuraminidase assists virus release by cleaving sialic acids to prevent unwanted retention of newly synthesized virus on the cell surface. Neuraminidase may also function during the invasion phase by removing sialic acids on soluble mucins that would otherwise inhibit cell-surface binding. Because neuraminidase is essential to the viral life cycle, based on the crystal structure of the enzyme a rational drug design program yielded Zanamivir (Relenza). The addition of a bulky guanidino side chain at C-4 of a previously known neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA), markedly increased the affinity for influenza neuraminidase, without affecting host-cell neuraminidases (Figure 57.2; see also Chapter 55). Relenza blocks the influenza virus life cycle by preventing infection and by interrupting the spread of the virus during the early phase of an infection. Due to poor oral availability, Relenza has to be inhaled to work at the mucosal sites of infection in the upper airway. The orally available drug Oseltamivir (Tamiflu) achieves the same effects and has taken over most of the market due to the ease of use. The fear of avian influenza virus (“bird flu”) spreading into human populations has prompted stockpiling of Tamiflu. Fortunately, widespread use of the drug has not become necessary to date. The development of Tamiflu is a textbook example for rational drug design resulting in a powerful drug against a devastating disease.

FIGURE 57.2.

The synthetic influenza neuraminidase inhibitors Relenza and Tamiflu.

FIGURE 57.3.

Glycomimetic E-selectin inhibitors based on sialyl Lewis x.

TABLE 57.1.

Examples of glycan-based drugs, their target diseases, and modes of action

Optimizing Glycans of Therapeutic Glycoproteins for Prolonged Serum Half-Life

Erythropoietin (EPO) is the most successful biotechnology product to date. It is a circulating cytokine that binds to the erythropoietin receptor, inducing proliferation and differentiation of erythroid progenitors in the bone marrow. EPO was developed to treat anemias caused by bone marrow suppression after chemotherapy or lack of erythropoietin (e.g., renal failure). Natural and recombinant forms of erythropoietin carry three sialylated complex N-glycans and one sialylated O-glycan. Although in vitro the activity of deglycosylated erythropoietin is comparable to that of the fully glycosylated molecule, its activity in vivo is reduced by ∼90%, because poorly glycosylated erythropoietin is rapidly cleared by filtration in the kidney. Undersialylated erythropoietin is also rapidly cleared by galactose receptors in hepatocytes and macrophages (see Chapter 31). Fully sialylated chains and increased tetra-antennary branching reduces these problems and increases EPO activity in vivo nearly 10-fold. Addition of an N-glycosylation site also increases half-life and activity in vivo. Covalently linking polyethylene glycol to the protein also reduces clearance by the kidney.

Erythropoietin is unusual because it is small enough to be cleared by the kidney if it is underglycosylated. For most glycoprotein therapeutics, a more important consideration is minimizing clearance by galactose-binding hepatic receptors by ensuring full sialylation of glycans. Because glycans greatly influence the efficacy of these drugs, control over glycosylation during production is very important in light of regulatory requirements for batch-to-batch product consistency. Changes in culture pH, the availability of precursors and nutrients, and the presence or absence of various growth factors and hormones can each affect the extent of glycosylation, the degree of branching, and the completeness of sialylation. Sialidases and other glycosidases that are either secreted or released by dead cells can also cause degradation of the previously intact product in the culture medium. These issues were hotly debated in recent years with the advent of “biosimilars” or generic versions of glycoproteins. The need to proof composition has fueled efforts devoted to glycan analysis and sequencing.

Impact of Glycosylation on Licensing, Patentability, and Prolonged Serum Half-Life of Therapeutic Glycoproteins

Patenting of new therapeutics is typically based on the composition of matter in the claimed molecule. Small molecules of defined structure and nonglycosylated proteins are easily captured in this manner. However, glycoproteins, especially those with multiple glycosylation sites, render it virtually impossible to obtain preparations that contain only a single glycoform. Thus, most biotherapeutic glycoproteins consist of a mixture of glycoforms. Licensing bodies allow for a certain range of variation in glycoforms and the complexity of the mixture. However, the manufacturer and the agency must agree on the extent such variation is acceptable for a given drug formulation. Biopharmaceutical companies therefore spend considerable effort in assuring that their products fall within these defined ranges, once these are approved by licensing bodies. The inherent difficulty in reproducing complex glycoform mixtures also complicates efforts to make generic forms of recombinant glycoprotein drugs. Given the complexities of producing glycotherapeutic agents in mammalian cells, even the smallest changes in growth conditions can have significant effects on the range of glycoforms found in any given product batch. The licensing agencies use consistency in glycoform composition as an indirect measure of the quality of process control in production. Differences in glycosylation can have implications for the patentability of agents in which the polypeptide remains constant. Marked differences in glycosylation have been used to define agents as being uniquely different. However, it is usually necessary to show that the differences in glycosylation being claimed also have a significant effect in changing the functionality of the drug in question. The associated pharmaceutical licensing and legal issues are rapidly evolving to keep pace with scientific advances in this area.

Salvage versus De Novo Synthesis

All monosaccharides needed for cellular glycan synthesis can be obtained from glucose through metabolic interconversions (see Chapter 4). Alternatively, monosaccharides can be derived from the diet or salvaged from degraded glycans. The relative contributions of different sources can vary with the cell type. For instance, even though all mammalian cells use sialic acid, only some contain high amounts of UDP-GlcNAc epimerase/N-acetylmannosamine kinase (GNE), which is required for the de novo synthesis of CMP-sialic acid. But sialic acid salvage from degraded glycans is quite efficient, decreasing the demand on the de novo pathway. Similarly, galactose, fucose, mannose, N-acetylglucosamine, and N-acetylgalactosamine can come from the diet or be salvaged for glycan synthesis, whereas glucuronic acid (GlcA), iduronic acid (IdoA), and xylose cannot. All monosaccharides derived from the diet or degraded glycans can be catabolized for energy, and again, cells vary in their reliance on the different pathways.

The variable contributions of these pathways are important for therapy of some diseases. For instance, patients with congenital disorder of glycosylation type Ib (CDG-Ib), who are deficient in phosphomannose isomerase, benefit greatly from oral mannose supplementation to bypass the insufficient supply of glucose-derived mannose-6-phosphate. A few CDG-IIc patients have been treated with fucose to restore synthesis of sialyl Lewis x on leukocytes (see Chapter 42). Some patients with Crohn's disease show clinical improvement with oral N-acetylglucosamine supplementation, but the mechanism is unknown. Mice deficient in GNE activity have kidney failure, but providing N-acetylmannosamine in the diet prevents this outcome. Clinical trials using N-acetylmannosamine to treat GNE-deficient patients with hereditary inclusion body myopathy type II (HIBM-II) have been conducted but have yielded inconclusive results.

Special Diets

Some monosaccharides and disaccharides can be toxic to humans who lack specific enzymes. For example, people who lack fructoaldolase (aldolase B) accumulate fructose-1-phosphate, which ultimately causes ATP depletion and disrupts glycogen metabolism. Prolonged fructose exposure in these people can be fatal, and fructose-limited diets are critical. Deficiencies in the ability to metabolize galactose (see Chapter 4) are mostly due to a severe reduction in galactose-1-phosphate uridyl transferase activity and cause galactosemia. Although these patients are asymptomatic at birth, ingesting milk leads to vomiting and diarrhea, cataracts, hepatomegaly, and even neonatal death. Low-galactose or galactose-free diets can prevent these life-threatening symptoms. However, even these diets do not prevent unexplained long-term complications, which include speech and learning disabilities and ovarian failure in females with galactosemia.

Infants hydrolyze lactose (Galβ1-4Glc) quite well, but the level of intestinal lactase can be much lower or absent in adults because of down-regulation of lactase gene expression. About two-thirds of the human population has lactase nonpersistence, making milk products a dietary annoyance. Unabsorbed lactose provides an osmotic load and is metabolized by colonic bacteria, causing diarrhea, abdominal bloating, flatulence, and nausea. Lactase persistence has evolved in certain pastoral populations from northwestern Europe, India, and Africa, allowing milk consumption in adult life. However, many adults either avoid lactose-containing foods or use lactase tablets to improve lactose digestion.

Substrate Reduction Therapy

The failure to turn over glycans by lysosomal degradation causes serious problems for patients with lysosomal storage disorders. Deficiencies in individual lysosomal enzymes lead to pathological accumulation of their substrates in inclusion bodies inside the cells (see Chapter 41). One approach to treating these disorders is to inhibit initial glycan synthesis, a strategy termed substrate reduction therapy (SRT). Reduced synthesis of the initial compound decreases the load on the impaired enzyme, and some patients show significant clinical improvement. A small molecule drug used for SRT is N-butyldeoxynojirimycin (or N-butyl-DNJ) (Miglustat, Zavesca), that was approved in 2002 for treatment of Gaucher's disease (glucocerebrosidase deficiency).

Lysosomal Enzyme Replacement Therapy

Another approach for treating lysosomal storage disorders is enzyme replacement therapy (ERT). Unlike most therapeutic glycoproteins that interact with target receptors on the surface of cells, lysosomal enzymes developed for replacement therapy must be delivered intracellularly to lysosomes, their site of action. During the normal biosynthesis of lysosomal enzymes, their N-glycans become modified with mannose-6-phosphate (Man-6-P) residues, which target them to lysosomes using Man-6-P receptors (see Chapter 30). The challenge for ERT is to get the enzymes targeted properly to lysosomes, where they can degrade accumulated substrate. ERT for Gaucher's disease targets the lysosomes of macrophages via the cell-surface mannose receptor (see Chapter 31). The four recombinant enzyme products Imiglucerase (approved in 1995), Velaglucerase (approved in 2010), Taliglucerase alfa (Elelyso, approved in 2012), and Eliglustat (Cerdelga, approved in 2014) are marketed.

The success of glucocerebrosidase treatment stimulated the development of lysosomal enzymes for treatment of other lysosomal storage diseases such as Fabry's disease, mucopolysaccharidoses type I, II, and VI, and Pompe's disease. The replacement therapies clearly have beneficial effects and prolong life but are extremely expensive.

Chaperone Therapy

A third approach for treating lysosomal storage disorders takes advantage of the fact that some genetic defects lead to misfolding of the encoded enzyme in the endoplasmic reticulum (ER). Low-molecular-weight (LMW) competitive inhibitors of some of these enzymes can act as “chaperones” that stabilize the folded enzyme in the ER and effectively rescue the mutation and increase the steady-state concentration of active enzyme in the lysosome. The dose of the inhibitor must be carefully adjusted to ensure that the inhibitory effects on enzyme function do not overshadow beneficial effects on folding. Only a low level of enzyme restoration is needed to significantly reduce the accumulation of undigested glycan substrates, indicating that lysosomal hydrolases are normally present in large catalytic excess.

Glucosamine and Chondroitin Sulfate

Glucosamine (often mixed with chondroitin sulfate [CS]) has been promoted to relieve symptoms of osteoarthritis, which involves the age-dependent erosion of articular cartilage. Cartilage provides a cushion between the bones to minimize mechanical damage, and a net loss of cartilage occurs when the degradation rate exceeds the synthetic rate. A number of clinical trials report that glucosamine improves osteoarthritis symptoms, and some claim to restore partially the structure of the eroded cushion, in particular in the knees. Superficially, this would seem to make sense, because primary glycans of cartilage include hyaluronan (see Chapter 15) and CS, both of which contain hexosamines within their structure (see Chapter 16). Conflicting reports suggest that the outcome may depend on study design and the type and source of material. Nevertheless, veterinarians report positive results after treating animals with glucosamine for more than two decades. Double-blind, placebo-controlled studies in humans have shown a decreased rate of joint space narrowing. Glucosamine might also alter UDP-GlcNAc and potentially UDP-GalNAc levels, thus affecting cellular responses involving major classes of glycans.

Positive effects of CS on osteoarthritis are less well-documented. It remains unclear how the acidic CS polymer can be absorbed and delivered to its proposed site of action. Further studies are needed to determine whether CSs are absorbed by the target tissue, and if they actually lead to changes in cartilage metabolism.

Xylitol and Sorbitol in Chewing Gum

Many studies suggest that chewing gum containing sugar alditols, such as xylitol and sorbitol, can help control the development of dental caries. Mothers who chew xylitol-sweetened gum may even block transmission of caries-causing bacteria to their children. The benefit of these reduced sugars seems to be based on stimulation of salivary flow, but an antimicrobial effect is also possible. Xylitol also inhibits the expression and secretion of proinflammatory cytokines from macrophages and inhibits the growth of Porphyromonas gingivalis, one of the suspected causes of periodontal disease. Children who drank xylitol solutions also had a lower occurrence of otitis media.

Milk Oligosaccharides

Human milk contains about 70 g/L of lactose and 5–10 g/L of free oligosaccharides. More than 130 different glycan species have been identified with lactose at the reducing end, including poly-N-acetyllactosamine units. Some glycans are α2-3- and/or α2-6-sialylated and/or fucosylated in α1-2, α1-3, and/or α1-4 linkages. In contrast, bovine milk, the typical mainstay in human infant formulas, contains much smaller amounts of these glycans. These differences may account for some of the physiological advantages seen for breast-fed versus formula-fed infants. The glycans may also favor growth of a nonpathogenic bifidogenic microflora and/or block pathogen adhesion that causes infections and diarrhea. Surprisingly, a substantial number of human milk oligosaccharides remain almost undigested in the infant's intestine and are excreted intact into the urine. Whether supplementing infant formula with specific, biologically active free glycans enhances infant health is unknown.

Microbial Vaccines

Polysaccharide vaccines consisting solely of glycan components typically elicit poor immunity, especially in infants. Because glycans are T-cell-independent antigens they do not effectively stimulate T-helper-dependent activation and class switching of B-cell-mediated immunity. Conjugate vaccines consisting of glycans coupled to carrier proteins have proven to be highly effective. Three major conjugate vaccines are marketed today: Haemophilus influenzae type b (Hib) causes an acute lower respiratory infection among young children. Children up to this age constitute a high risk group for H. influenzae type B infections. Consequently, the Hib PSV introduced in 1985 was withdrawn from the market in 1988 and replaced by CPS-protein conjugate vaccine formulations. A conjugated form of an Hib-derived oligosaccharide coupled to a protein carrier is part of routine vaccination schedules and has been so successful that infectious diseases caused by this bacterium are nearly eradicated in vaccinated populations. A potent semisynthetic Hib glycoconjugate vaccine marketed in Cuba contains glycan chains with an average length of 16 monosaccharides. Pneumococcal conjugate vaccines have been developed to cover an increasing number of serotypes, and current formulations are 10- (Synflorix, GSK) and 13-valent (Prevnar13, Pfizer). Prevnar 13 provides protection against serotypes that account for >70% of cases of invasive pneumococcal disease worldwide and is the best-selling vaccine exceeding revenues of five billion U.S. dollars in 2015. Conjugate vaccines to protect from Neisseria meningitidis are also very successful on the market. Due to the fast onset and rapid progression of meningococcal infections, vaccination is required to protect against this disease. Several conjugated CPS vaccines are licensed in different parts of the world: the tetravalent serogroup A, C, W, and Y (Menactra, Menveo, and Nimenrix) and a few monovalent vaccines based on serogroup C CPS (Meningitec, Menjugate, NeisVac-C). Two combination vaccines for N. meningitidis and H. influenzae type b (Hib) are available against meningococcal serogroups C/Y (MenHibrix) and against meningococcal serogroup C (Menitorix). A monovalent serogroup A vaccine (MenAfriVac) is widely used in the sub-Saharan meningitis belt of Africa.

Currently, several new vaccines based on synthetic oligosaccharide antigens are being developed to protect children and the elderly from a variety of bacterial infections. Vaccines to protect from hospital acquired infections that are increasingly antibiotic resistant are in preclinical evaluation.

Cancer Vaccines

Several carbohydrate-based cancer vaccines are at different stages of development to treat cancer. Ganglioside immunogens present on certain types of cancer cells, such as gangliosides GM2 and GD2 in melanomas and globo H in breast cancer, are being explored. The shorter glycan sequences such as sialyl-Tn (sialylα2-6GalNAcα-) found on cancer mucins (see Chapter 44) has seen little progress in twenty years. The synthetic GloboH hexasaccharide (see Figure 57.1) resembling the breast and prostate cancer antigen has failed Phase 3 clinical trials in early 2017.

Blocking Infection

As discussed in Chapter 34, many microbes and toxins bind to mammalian tissues by recognizing specific glycan ligands. Thus, small soluble glycans or glycan mimetics can be used to block the initial attachment of microbes and toxins to cell surfaces (or block their release), and thus prevent or suppress infection. Because many of these organisms naturally gain access through the airways or gut, the glycan-based drugs can be delivered directly without being distributed systemically. Milk oligosaccharides are believed to be natural antagonists of intestinal infection in infants (see above) and polymers that will block the binding of viruses such as influenza. Although backed by a strong scientific rationale and robust in vitro studies, such “antiadhesive” therapies have not yet found much practical application.

Inhibition of Selectin-Mediated Leukocyte Trafficking

When specific glycan–protein interactions are responsible for selective cell–cell interactions and a resulting pathology, then administration of small-molecule glycomimetics of the natural ligand is a useful means of intervention. Selectin-mediated recruitment of neutrophils and other leukocytes into sites of inflammation or ischemia/reperfusion injury involves specific selectin–glycan interactions in the vascular system (see Chapter 31). The use of sialyl Lewis x tetrasaccharide derivatives failed due to poor oral availability and a short serum half-life. Glycomimetics that preserve the essential functionality of the parent tetrasaccharide but eliminate unwanted polar functional groups and synthetically cumbersome glycan components have been successful. The design of a monosaccharide glycomimetic starting from sialyl Lewis x is shown in Figure 51.3. First, the sialic acid residue was replaced with a charged glycolic acid group, the N-acetylglucosamine residue was then replaced with an ethylene glycol linker, and finally the galactose residue was replaced with a linker moiety. The resulting glycomimetic had E-selectin binding affinity comparable to sialyl Lewis x.