**11. Glycosidases and related enzymes**

er transcripts and alteration of the reading frames in mRNAs, defense against viruses via hypermutation-based inactivation, and somatic hypermutation or class switching of antigen

Adenosine deaminase (ADA) is an enzyme present in all organisms and catalyzes the irre‐ versible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine (Fig. 26). Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology. For example it plays a vital role in regulating T-cell coactiva‐ tion. Deficiency of this enzyme in humans causes severe combined immunodeficiency. In‐ creased serum activity of this enzyme have been found in many infectious diseases caused by microorganisms infecting the macrophages, in leprosy, brucellosis, HIV infections, viral hepatitis, infectious mononucleosis, liver cirrhosis and tuberculosis. Its extended transition state inhibitor – conformycin was isolated from *Nocardia interforma* and *Streptomyces kanihar‐ aensis*. Analogs of conformycin (Fig. 26) are proposed as an antineoplastic synergists and im‐

The wide potential of these inhibitors may be illustrated by the fact that deaminoformycin was recently applied to evaluate mechanisms responsible for lethality caused by genetic and herbicide-based activity of adenosine deaminase [Sabina et al., 2007], as well as identifica‐ tion of highly selective inhibitor of purine salvage pathway in malaria parasites [Tyler et al., 2007]. This is because of a unique feature of *Plasmodium falciparum* enzyme that catalyzes the

Guanine deaminase is an enzyme that hydrolyzes guanine to form xanthine that is unsuita‐ ble for DNA/RNA buildup. This enzyme has been found in normal or transformed human

receptor genes in vertebrates [Iyer et al., 2011].

348 Drug Discovery

munosuppressants [Wolfenden, 2003].

**Figure 26.** Inhibitors of adenosine deaminase.

deamination of both adenosine and 5'-methylthioadenosine.

Glycoside hydrolases, the enzymes catalyzing hydrolysis of the glycosidic bond in di-, oligoand polysaccharides, and glycoconjugates, are ubiquitous in Nature and fundamental to ex‐ istence. The extreme stability of the glycosidic bond caused that they have evolved into highly proficient catalysts, with an estimated 1017 fold rate enhancement over the uncata‐ lysed reaction. Such rate enhancements mean that enzymes bind the substrate at the transi‐ tion state with extraordinary affinity [Gloster & Davies, 2010].

In the most cases of glycoside hydrolysis, the short-lived transition state possesses substan‐ tial oxocarbenium character (Fig. 27) resembling classical SN1 reaction intermediate. Under these conditions the anomeric carbon possesses trigonal character, which causes sp2 hybridi‐ sation predominantly along the bond between the anomeric carbon and endocyclic oxygen and significant relative positive charge accumulation on the pyranose ring [Lee et al., 2004; Biarnés et al., 2011; Davies at al., 2012].

The quest for potent and selective inhibitors of glycosidases is extremely active at present. This results from the involvement of glycosidases in lysosomal storage disorders, cancer, vi‐ ral infections, diabetes and many others. Consequently a plethora of glycosidase inhibitors have been already synthesized and evaluated. The number of them is continually growing. It is outside the scope of this chapter to mention all of them in detail. One of the most ap‐ pealing ways to design a transition state analog would be to incorporate both the features of geometry and charge present at the transition state. Distortion of the ring to generate com‐ pounds which may resemble the geometry of the transition state can be done by introducing a double bond in the pseudo-glycoside ring itself, whereas introduction of the charge might be done by application of sulfonium or ammonium ions [Rempel & Withers, 2008; Gloster & Davies, 2010; Sumida et al., 2012].

cleavage of terminal sialic acid residues from sialylated oligosaccharides, glycoproteins, and glycolipids. Aberrant expression of different human sialidases was found to associate with various pathological conditions, including lysosomal storage diseases such as sialidosis and galactosialidosis. Non-specific transition-state analog of sialidase, 2-deoxy-2,3-dehydro-*N* acetylneuraminic acid (DANA, Fig. 30) is a good starting point for the synthesis of specific

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Influenza viruses, in particular those of type A that can infect animals and humans, continue to represent a major threat to public health and animal health worldwide. The social and economic burden associated with a pandemic is substantial. Two viral surface glycoproteins, the sialoside-hydrolysing neuraminidase and the sialic acid-binding hemagglutinin, have become important targets for such approach. Most likely, the function of flu virus neurami‐ nidase is to remove sialic acid receptors for the virus from the host cells, and also, perhaps more importantly, from the newly formed virus particles themselves [Nelson & Holmes, 2007; Medina & Garcia-Sastre, 2011]. Three inhibitors of neuraminidase have been success‐ fully introduced as anti-influenza drugs, all of them being transition state analog inhibitors. They were designed by systematic reduction of DANA structure using crystallographic data and computer-aided methods [Wei, et al., 2006]. Relenza (zanamivir) was the first inhibitor to be synthesized which specifically inhibited neuraminidases of both Type A and Type B influenza viruses and is effective in controlling influenza infections. In people is given as a powder by oral inhalation [Palese et al., 1974]. Interestingly, it is weaker inhibitor of neura‐ midase than DANA, however, DANA inhibited influenza virus replication in tissue culture

inhibitors of human enzymes [Streicher & Busse, 2006; Li et al., 2011].

**Figure 30.** Chemical structures of neuraminidaze inhibitors.

**Figure 28.** Mechanism of β-glucosidase action [after Vasella et al., 2002].

Some representatives, which fulfills these requirements are: salicinol, one of the active prin‐ ciples in the aqueous extracts of *Salacia reticulata* that is traditionally used in Sri Lanka and India for the treatment of diabetes [Ghavami et al., 2001] and its structurally variable ana‐ logues [Liu et al., 2006; Bhat et al., 2007; Mohan & Pinto, 2008].

**Figure 29.** Salicinol and its analogs.

Sialic acids play an important role in a variety of biological processes. They are usually at‐ tached to the terminal positions of glycoproteins, glycolipids and oligosaccharides. From more than 100 different sialic acids, *N-*acetylneuraminic acid (NeuAc) is the most abundant one. Sialidases or neuraminidases are a family of exo-glycoside hydrolases that catalyze the cleavage of terminal sialic acid residues from sialylated oligosaccharides, glycoproteins, and glycolipids. Aberrant expression of different human sialidases was found to associate with various pathological conditions, including lysosomal storage diseases such as sialidosis and galactosialidosis. Non-specific transition-state analog of sialidase, 2-deoxy-2,3-dehydro-*N* acetylneuraminic acid (DANA, Fig. 30) is a good starting point for the synthesis of specific inhibitors of human enzymes [Streicher & Busse, 2006; Li et al., 2011].

**Figure 30.** Chemical structures of neuraminidaze inhibitors.

geometry and charge present at the transition state. Distortion of the ring to generate com‐ pounds which may resemble the geometry of the transition state can be done by introducing a double bond in the pseudo-glycoside ring itself, whereas introduction of the charge might be done by application of sulfonium or ammonium ions [Rempel & Withers, 2008; Gloster &

Some representatives, which fulfills these requirements are: salicinol, one of the active prin‐ ciples in the aqueous extracts of *Salacia reticulata* that is traditionally used in Sri Lanka and India for the treatment of diabetes [Ghavami et al., 2001] and its structurally variable ana‐

Sialic acids play an important role in a variety of biological processes. They are usually at‐ tached to the terminal positions of glycoproteins, glycolipids and oligosaccharides. From more than 100 different sialic acids, *N-*acetylneuraminic acid (NeuAc) is the most abundant one. Sialidases or neuraminidases are a family of exo-glycoside hydrolases that catalyze the

Davies, 2010; Sumida et al., 2012].

350 Drug Discovery

**Figure 28.** Mechanism of β-glucosidase action [after Vasella et al., 2002].

logues [Liu et al., 2006; Bhat et al., 2007; Mohan & Pinto, 2008].

**Figure 29.** Salicinol and its analogs.

Influenza viruses, in particular those of type A that can infect animals and humans, continue to represent a major threat to public health and animal health worldwide. The social and economic burden associated with a pandemic is substantial. Two viral surface glycoproteins, the sialoside-hydrolysing neuraminidase and the sialic acid-binding hemagglutinin, have become important targets for such approach. Most likely, the function of flu virus neurami‐ nidase is to remove sialic acid receptors for the virus from the host cells, and also, perhaps more importantly, from the newly formed virus particles themselves [Nelson & Holmes, 2007; Medina & Garcia-Sastre, 2011]. Three inhibitors of neuraminidase have been success‐ fully introduced as anti-influenza drugs, all of them being transition state analog inhibitors. They were designed by systematic reduction of DANA structure using crystallographic data and computer-aided methods [Wei, et al., 2006]. Relenza (zanamivir) was the first inhibitor to be synthesized which specifically inhibited neuraminidases of both Type A and Type B influenza viruses and is effective in controlling influenza infections. In people is given as a powder by oral inhalation [Palese et al., 1974]. Interestingly, it is weaker inhibitor of neura‐ midase than DANA, however, DANA inhibited influenza virus replication in tissue culture but failed to prevent disease in flu-infected animals. In order to produce a neuraminidase inhibitor, which was orally bioavailable and which was taken orally in capsules or as a sus‐ pension, Tamiflu (oseltavimir) was developed in 1997 [Kim et al.]. Third drug, which has been authorized for the emergency use of treatment of certain hospitalized patients with known or suspected 2009 H1N1 influenza, is permavir [Chand, et al., 2005]. Structures of these drugs are presented in Figure 30.

The focus on transition states for a family of *N*-ribosyltransferases roots from physiologic importance of these enzymes. Similarly as in the case of glycosidases, most sugar transferas‐ es form transition states with cationic charge at the anomeric carbon. The geometry is al‐

planargeometry) at the transition state (Fig. 32) [Schramm, 2002; Murkin et al., 2007; Silva et

Newborns with a genetic deficiency of purine nucleoside phosphorylase are normal, but ex‐ hibit a specific T-cell immunodeficiency during the first years of development. All other cell and organ systems remain functional. Human purine nucleoside phosphorylase degrades deoxyguanosine, and apoptosis of T-cells occurs as a consequence of the accumulation of deoxyguanosine in the circulation. Thus, control of T-cell proliferation is desirable in T-cell cancers, autoimmune diseases, and tissue transplant rejection. The search for powerful in‐ hibitors of these enzymes as anti-T-cell agents has culminated in the discovery of immucil‐ lins. The atomic replacements between inosine and immunocilin H make an insignificant change in atomic size, but a dramatic change in the molecular electrostatic potential surface (Fig. 33). Thus, analysis of the molecular electrostatic potential surface similarity between transition state and immucilin confirmed utility of this simple approach in helping to design

Evolution of immucilin structure, performed using standard structural analogy techniques, enabled to obtain new inhibitors of purine nucleotide phosphorylase of nano- to picomolar affinities to the enzyme (Fig. 34) [Evans et al., 2008; Edwards et al., 2009; Ho et al. 2010; Rej‐

*Plasmodium* parasites (causative agents of malaria) are purine auxotrophs and require pre‐ formed purine bases for synthesis of nucleotides, cofactors, and nucleic acids. The purine phosphoribosyltransferases catalyze transfer the 5-phosphoribosyl group from 5-phosphoα-*D*-ribofuranosyl-1-pyrophosphate to salvage hypoxanthine, guanine, or xanthine to form intracellular nucleosides. Purine salvage in *Plasmodium falciparum* uses hypoxanthine formed in erythrocytes or in parasites by the sequential actions of adenosine deaminase and purine

(tetrahedral geometry) in the reactant sugar to sp2 (trigonal

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tered at this center from sp3

**Figure 32.** Course of reaction catalyzed by *N*-ribosyltransferases.

effective inhibitor [Schramm, 2002; Schramm, 2007].

man et al., 2012].

nucleoside phosphorylase

al., 2011].

All three drugs soon became lead structures for the design and preparation of new, presum‐ ably more effective ones. Syntheses and eveluation of phosphinic analogs and significantly simplified analogs of permavir (Fig. 31) have been recently described [Kati et al., 2001; Bian‐ co et al., 2005; Shie et al., 2007; Udommaneethanakit et al., 2009].

**Figure 31.** Second generation of influenza neuraminidase inhibitors.

Modified phosphonic analogs of oseltamivir were used to functionalize gold nanoparticles and were found to bind strongly and selectively to all seasonal and pandemic influenza vi‐ rus strains, and thus could serve as prototypes for novel virus sensors. This may be helpul in fast influenza diagnosis [Stanley et al., 2012].
