the enantioselectivity of enzymes involved in current

Enzymes are enantiomerically pure catalysts and are thuslargely responsible for the conservation of chirality in livingorganisms. The enantioselectivity of enzymes – or moreprecisely the enantioselectivity in enzymatic catalysis – hasbeen exploited in organic synthesis, for example in kineticresolution of racemic mixtures as well as in the synthesis ofchiral building blocks from achiral precursors. It has gener-ally been assumed (especially by biologists) that enzymesmaintain a high degree of enantioselectivity as far as natu-ral substrates are concerned. However, organic chemistsprobing the selectivities of enzymes often used in synthesiswith large series of substrate analogues of variable struc-

tures soon realized that many enzymes often display moreor less relaxed enantioselectivities depending on the struc-ture of the candidate substrate (Drauz & Waldman, 1995;Faber, 1995; Popple & Novak, 1992).

This tendency is well illustrated in the series of nucleo-sides and nucleotides in which it was generally acceptedthat enzymes that catalyse the transformation of nucleo-sides and their analogues, were enantioselective and pre-ferred natural D-enantiomers. Indeed, some of theseenzymes have been successfully used in organic synthesis toresolve racemic mixtures of carbocyclic nucleosides or otheranalogues (Herdewijn et al., 1985; Secrist III et al., 1987;

Antiviral Chemistry & Chemotherapy 11:165–190

Review

The enantioselectivity of enzymes involved in currentantiviral therapy using nucleoside analogues: a newstrategy?Georges Maury

UMR 5625 du CNRS, Case Courrier 006, Université Montpellier II, Place Bataillon, Montpellier, France

For correspondance: Tel: +33 04 6714 3316; Fax: +33 04 6704 2029; E-mail: [email protected]

This review is primarily intended for syntheticbio-organic chemists and enzymologists who areinterested in new strategies in the design of virusinhibitors. It is an attempt to assess the importanceof the enzymatic properties of L-nucleosides andtheir analogues, particularly those that are activeagainst viruses such as human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), herpes simplexvirus (HSV), etc. Only data obtained with purifiedenzymes have been considered and discussed. Theexamined enzymes include nucleoside- ornucleotide-phosphorylating enzymes, catabolicenzymes, viral target enzymes and cellular poly-merases. The enantioselectivities of these enzymeswere determined from existing data and are sig-nificant only when a sufficient number of enan-tiomeric pairs of substrates could be examined.The reported data emphasize the weak enantiose-lectivities of cellular or viral nucleoside kinases andsome viral DNA polymerases. Thus, cellular deoxy-cytidine kinase has a considerably relaxed enan-tioselectivity with respect to a large number ofnucleosides or their analogues, and it occupies astrategic position in the intracellular activation of

the compounds. Similarly, HIV-1 reverse transcrip-tase often has a relatively weak enantioselectivityand can be inhibited by the 5-triphosphates of alarge series of L-nucleosides and analogues. In con-trast, degradation enzymes, such as adenosine orcytidine deaminases, generally demonstrate strictenantioselectivities favouring D-enantiomers andare used by chemists in asymmetric syntheses. Theweak enantioselectivities of some enzymesinvolved in nucleoside metabolism are more or lesspronounced, and one enantiomer or the other isfavoured depending on the substrate. This sug-gests that the low enantioselectivity is fortuitousand does not result from evolutionary pressure,since these enzymes do not create or modify asym-metric centres in substrates. The combined enan-tioselectivities of the enzymes examined in thisreview strongly suggest that the field of L-nucleo-sides and their analogues should be systematicallyexplored in the search for new virus inhibitors.

Keywords: enantioselectivity; nucleoside metab-olizing enzymes; viral enzymes; virus inhibitors; L-nucleosides

Introduction

165©2000 International Medical Press 0956-3202/00/$17.00

Hutchinson, 1990; Mahmoudian et al., 1993). Lately, how-ever, the enantioselectivity of some of these enzymes hasbeen questioned and recognized as an important factor forthe recently disclosed antiviral activity of several L-nucleo-side analogues, especially against human immunodeficien-cy virus (HIV) or hepatitis B virus (HBV) (Furman et al.,1995). It is now accepted that these activities may result –at least in part – from the relaxed enantioselectivities of thetarget viral enzymes or the cellular enzymes which activateor deactivate the nucleoside analogues. Thus, a new strate-gy in antiviral therapy seems to have emerged based on thefavourable enantioselectivities of some key enzymesinvolved in the inhibition process. In this short review, theenantioselectivities of some of these enzymes will bereported and discussed in relation to the use of L-nucleo-side analogues as antiviral agents. Only data obtained withpurified enzymes will be considered.

Enantioselectivity in enzyme catalysis

Enzymatic enantioselectivity may be defined as the abilityof an enzyme to distinguish between two enantiomericsubstrates, or by extension two enantiotopic ligands or facesof a substrate. Let us consider a simple three-step mecha-nism of an irreversible enzymatic reaction involving twoenantiomers A and B of a substrate:where K

mand K′

mare the Michaelis constants for A and B,

respectively, and kcat

and k′cat

are the apparent first order

constants for conversion to products of the complexesbetween the enzyme and A and B, respectively. From theexpression of the reaction rate under steady-state kinetics(Chen et al., 1982; Price & Stevens, 1989; Wong &Whitesides, 1994):

vP=d[A]/dt=(k

cat/K

m)[A][Enz]

(d[A]/dt)/(d[B]/dt)=(kcat

[A]/Km)/(k′

cat[B]/K′

m)=(V

m[A]/K

m)/(V′

m[B]/K′

m)

where [Enz] is the concentration of the free enzyme,[Enz

o] the concentration of total enzyme and

Vm=k

cat[Enz

o] or V′

m=k′

cat[Enz

o] are the maximum rates.

By integration:

(Log([A]/[Ao])/(Log([B]/[B

o])=(V

m/K

m)/(V′

m/K′

m)=E

Therefore, the discriminating capacity of the enzyme isquantitatively measured by the ratio E of the correspondingspecificity constants V

m/K

m(or k

cat/K

m). For a given

enzyme, the E coefficient depends on the nature of the

substrate and the conditions in which the kinetic parame-ters (apparent or not) have been determined. Relating thepseudo second order kinetic constants k

cat/K

mand k′

cat/K′

mto the free energy of activation ∆G≠ yields:

E=(kcat

/Km)/(k′

cat/K′

m)=e-(∆∆G≠/RT)

This indicates that the enantioselection occurs because theenzyme and the enantiomeric substrates form diastereoiso-meric transition states that differ in energy owing to differ-ent binding interactions. As a corollary, if enzyme/ligandinteractions concern mainly the non-asymmetric parts ofthe two enantiomers of a substrate bound to an enzyme,then the enantioselectivity of the enzyme may be expectedto be low with respect to this particular substrate.

The preceding treatment is related to simple, irreversibleenzymatic reactions involving a single substrate. The enan-tioselectivity of enzymes catalysing multi-substrate reac-tions has been examined and found to require additionalselectivity parameters ( Jongejan & Duine, 1996). However,the above treatment can still be applied to two-substratereactions, provided one of the reactants is at saturating con-centration.

Each step of enzyme catalysis, either ligand binding orsubstrate chemical transformation, can be enantioselective.In particular, the complexations of two enantiomericinhibitors to an enzyme correspond to different energiesand may result in different inhibition efficiencies depend-ing on the affinities of the enantiomers for the enzyme.These affinities may be measured by the dissociation con-stant K

dof the corresponding enzyme/ligand complex.

L-Nucleoside analogues with antiviral(anti-HIV or anti-HBV) or anti-cancer activities

To date, only six nucleoside analogue inhibitors of HIVreplication have been approved by the American Food andDrug Administration, and they are currently clinically usedeither alone or in conjunction with other inhibitors(Balzarini et al., 1998) (Figure 1). Among these six com-pounds, five have the D-stereochemistry (AZT, ddI, ddC,d

4T and ABV) and one has the L-stereochemistry (3TC).The search for antiherpetic drugs has also been produc-

tive and has led to the discovery of several active nucleosideanalogues, mostly acyclic 2′-deoxyguanosine derivatives or5-substituted 2′-deoxyuridines, some of which have beenapproved for clinical use (Balzarini et al., 1998; Kulikowski,1994) (Figure 2).

3TC, or (2R,5S)-1-[2-(hydroxymethyl)-1,3-oxathio-lan-5-yl]cytosine is derived from an heterocyclic analogueof β-L-2′,3′-dideoxyribose. It has the advantage of beingless cytoxic than the corresponding D-enantiomer. In addi-tion to its anti-HIV properties, this very important com-

G Maury

166 ©2000 International Medical Press

Enzyme enantioselectivity in current antiviral therapy

pound also presents a powerful activity against the hepatitisB virus, and it is currently administered in therapy againstthis virus (Balzarini et al., 1998; Schinazi et al., 1994).

In addition to 3TC, a number of L-nucleoside analoguespresent interesting anti-HIV, anti-HBV or other antiviralactivities, although they have not been fully tested and arenot approved by the Food and Drug Administration(Figure 3). The most noteworthy are β-L-2′,3′-dideoxycy-tidine (L-ddC), the fluoro derivatives (2R,5S)-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (FTC),β-L-2′,3′-dideoxy-5-fluorocytidine (L-FddC), 2′,3′-dide-hydro-2′,3′-dideoxy-β-L-5-fluorocytidine (L-Fd4C), and2′-fluoro-5-methyl-β-L-arabinofuranosyluracil (FMAU)(Gosselin et al., 1994; Lin et al., 1994; Martin et al., 1997;Pai et al., 1996; Zhu et al., 1998). Unnatural enantiomers ofthe uridine analogues β-L-5-iodo-2′-deoxyuridine (L-IdU)and β-L-(E)-5-(2-bromovinyl)-2′-deoxyuridine (β-L-BVdU) have been reported to be active against herpes sim-plex virus type 1 (HSV-1), being less active but also lesstoxic than the D-enantiomers (Spadari et al., 1995b). L-

ddA (β-L-2′,3′-dideoxyadenosine) also presents a fairlyimportant anti-HBV activity in infected cell systems(Bolon et al., 1996). The enantiomers of the guanosine ana-logue carbovir (CBV) have different levels of activity, thenatural β-D-enantiomer being active against HIV and theβ-L-enantiomer against HBV (Furman et al., 1995).Finally, only one L-nucleoside analogue with anti-cancerproperties has been reported: β-L-dioxolane cytidine(OddC) displays anti-cancer activities against prostate andcolon tumours, hepatocellular carcinoma and leukaemiacells in vitro and in mice in vivo (Grove et al., 1995).

Enantioselectivities of enzymes involved inthe inhibition of virus replication by nucleoside analogues

The preceding L-enantiomers must be activated in vivo totheir 5′-triphosphate derivatives, to be able to inhibit thevirus. This transformation is sequential involving threeenzyme-catalysed phosphorylation steps (an esterification

Antiviral Chemistry & Chemotherapy 11:3 167

Figure 1. Nucleoside analogues approved as anti-HIV drugs

O

HO

N

HN

O

O

CH3

N3

O

HO

N

NHN

N

O

O

HO

N

N

O

NH2

O

HO

N

HN

O

O

CH3

HO

N

NN

N NH2

HN

S

O

HO

N

N

O

NH2

and two successive anhydride formations) yielding the cor-responding 5′-mono-, di- and triphosphates, consecutively.The corresponding enzymes are the nucleoside- ornucleotide kinases and 5′-nucleotidases. In particular, thenucleoside kinases catalyse the transfer of the γ-phosphorylgroup of ATP (or another nucleoside triphosphate) to the5′-position of a nucleoside or its analogues. This is oftenrecognized as the most important step in the activation tothe triphosphate. Some viruses encode specific enzymes forphosphorylation (herpesvirus) but most depend on enzy-matic resources of the host.

The triphosphates of the nucleoside analogues formedintracellularly must then inhibit a critical viral enzyme nec-essary for the development of the virus, for example a DNApolymerase in the case of HIV and HBV. HIV-1 DNA

polymerase has a reverse transcriptase activity (HIV-1 RT)since it catalyses the conversion of the single-stranded RNAretroviral genome into a double-stranded linear DNAwhich is subsequently integrated into the host DNA (Nanniet al., 1993). The mechanism of HBV DNA polymerase isless well known and somewhat more complex than that ofRT (Hu & Seeger, 1996; Wang & Seeger, 1993). Thetriphosphates of nucleoside analogues may also inhibit cel-lular DNA polymerases and DNA replication and repair incells (Ono, 1987), thus generating toxicity phenomena.

Before reaching its target, a nucleoside or a nucleotideanalogue must retain its structural integrity. Consequently,it must resist the enzymes catalysing its decomposition orimpairing its conversion to the triphosphate and decreasingits efficiency. The most active enzymes in the deactivation

G Maury


Figure 2. Nucleoside analogues with anti-herpetic activity


of nucleosides or nucleotides and their analogues are gen-erally the deaminases and the phosphorylases. Thus,adenosine deaminase or cytidine deaminase catalyse thedeamination of adenosine and cytidine derivatives, respec-tively, to the corresponding inosine or uridine analoguesthat often prove devoid of activity.

It is clear that the understanding of virus inhibition byL-nucleoside analogues requires the study of the enantios-electivities of at least all preceding categories of enzymes.

Enzyme enantioselectivity in the phospho-rylation of nucleosides, nucleotides andtheir analogues

Cellular nucleoside kinasesThere are four deoxynucleoside kinases in mammalian cellsthat are involved in the salvage pathway of natural nucleo-

side biosynthesis. Two of these enzymes are cytosolic:thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK);and two are mitochondrial: thymidine kinase 2 (TK2) anddeoxyguanosine kinase (dGK). Adenosine kinase (AK) isanother cellular kinase that catalyses the monophosphory-lation of adenosine and its analogues. The main function ofthis enzyme seems to be to regulate the extracellular levelsof adenosine and preserve the intracellular levels of adeny-late, rather than to participate in the salvage of nucleotides,since it has only a limited capacity to phosphorylate 2′-deoxyadenosine (Hurley et al., 1985). The main features ofthe cellular deoxyribonucleosides kinases are presented inFigure 4 (Arner & Eriksson, 1995). It should be added thatTK1 is active in proliferating cells in S-phase, whereasdCK, TK2 and dGK do not seem to be cell cycle regulat-ed, their phosphorylating capacities depending on thenature of the tissues containing the kinases. Each nucleo-


Figure 3. L-Nucleoside analogues presenting important antiviral activities

O

HO N

N

O

H2N

O

HO N

N

O

NH2

F

O

HO

OH

F N

HN

O

O

CH3

S

O

HO N

N

O

F

NH2

O

HO

OH

N

HN

O

I

O

O

HO N

NN

N

NH2

O

O

HO N

N

O

NH2

O

HO N

N

O

NH2

F

HO N

NHN

N

O

NH2

side kinase has distinct substrate properties with respect tonatural deoxynucleosides as well as their analogues (Hurleyet al., 1985). For example, the antiviral drug AZT is a sub-strate for TK1 and TK2, whereas dCK catalyses the phos-phorylation of ddC and 3TC (Shewach et al., 1993; VanDraanen et al., 1994). Structure–activity relationships indi-cate that the introduction of a moderately bulky substituent(particularly a fluor atom) at position 5 of deoxycytidinederivatives does not generally suppress the substrate pro-perties with respect to dCK. dGK tolerates changes both inthe base and in the sugar but requires a 3′-hydroxyl groupfor activity. TK2, dCK and dGK catalyse the phosphoryla-tion of a number of arabinofuranosyl nucleosides with sur-prising efficiency ( Johansson & Eriksson, 1996).

The sequences of human deoxyribonucleoside kinaseshave recently been determined. It is remarkable that threeof the preceding kinases, namely TK2, dCK and dGK,present important sequence homologies and share thisproperty with thymidine kinase of the HSV-1, thus imply-ing a common origin (Eriksson & Wang, 1997; Harrison etal., 1991; Johansson & Karlsson, 1996; Wang et al., 1996).In contrast, the sequence of TK1 is different and presentshomologies with poxviral TK and bacterial TK suggesting

that they belong to a distinct family. The recently deter-mined primary structure of human adenosine kinase indi-cates that it is not structurally related to the other nucleo-side kinases but rather to microbial ribokinases and fruc-tokinases (Spychala et al., 1996). This has been confirmedby the determination of the crystal structure of human AK(Mathews et al., 1998), the only cellular nucleoside kinasewhose 3D-structure is known.

To date, the enantioselectivity of dCK has been the mostextensively studied and shown to be low with practically allsubstrates. Human dCK purified from MOLT-4 T lym-phoblasts has been reported to catalyse the phosphoryla-tion of 3TC (E

L/D=4.5) and of FTC (E

L/D=4.6) (Shewach et

al., 1993), and both enantiomers of β-ddC are substrates ofdCK from calf thymus (E

L/D=7.7) (Van Draanen et al.,

1994). Human dCK catalyses the phosphorylation of D-and L-dC with similar efficiencies as shown by HPLCanalysis of reaction media (Verri et al., 1997a). 2′,3′-dide-hydro-2′,3′-dideoxy-β-L-5-fluorocytidine (L-Fd4C) andL-FMAU are also substrates of dCK, but only the lattercompound was recognized by human mitochondrialdeoxypyrimidine nucleoside kinase (Dutschman et al.,1998; Kukhanova et al., 1998b; Liu et al., 1998; Yao et al.,

G Maury


Figure 4. Main properties of cellular deoxyribonucleoside kinases

HOB

OH R

+ P

OO O

O

P

OO

O

P

OO

OO

OH OH

N

NN

N

NH2

O

- - -

O

PO O

OO

B

OH R

+

O

PO O

O

P

OO

OO

OH OH

N

NN

N

NH2

- - -

- -

Thymidine kinase 1 (TK1): Cytosolicenzyme.Dimer (55 kDa) or tetramer (110 kDa).Natural substrates: dT, dU.Important substrate analogues: AZT.Main phosphate donor: ATP.

Thymidine kinase 2 (TK2): Mitochondrialenzyme.Monomer (30 kDa).Natural substrates: dT, dC, dU.Important substrate analogues: AZT, araT,FIAU.Main phosphate donor: ATP, CTP.

Deoxycytidine kinase (dCK): Cytosolicenzyme.Dimer (60 kDa), complex kinetic mechanism.Natural substrates: dC, dA, dG.Important substrate analogues: araC, 5-azadC, ddC, 3TC.Main phosphate donor: UTP and/or ATP.

Deoxyguanosine kinase (dGK):Mitochondrial enzyme.Dimer (56 kDa).Natural substrates: dG, dA, dI.Important substrate analogues: araG,araHx.Main phosphate donor: ATP or other nucleoside triphosphates.


1996). A more complete study of the enantioselectivity ofhuman recombinant dCK in the presence of saturatingamounts of ATP and cytidine derivatives of variable sugarstructures, has shown that the enzyme enantioselectivity isalways low, independently of the stuctures of the substrates,and generally favours the L-enantiomer (Shafiee et al.,1998). Furthermore, under the same conditions, the sameenzyme accepts L-enantiomers of various adenosine orguanosine derivatives as substrates, again with low enan-tioselectivity (Gaubert et al., 1999; Pelicano et al., 1997)(Figure 5). The enzyme from MOLT-4 cells phosphory-lates both enantiomers of the carbocyclic analogue of β-dG(E

L/D=5.3) (Bennett et al., 1998). The determination of the

affinities of D- or L-substrates for human dCK using thevariation of the intrinsic fluorescence of the enzyme yieldsbiphasic titration curves corresponding probably to twoenzyme states, one with high affinity and the other withlow affinity. Similar K

dvalues for both enantiomers were

obtained in the high affinity binding state of the enzyme.In contrast, the enzyme binds to both enantiomers of ATPwith monophasic titration curves and with higher affinityfor the natural enantiomer. These results suggest that thesubstrate binding steps in the dCK mechanism are notenantioselective (Shafiee et al., 1999). L-ATP, L-dATP, L-

dGTP and L-dTTP are accepted as phosphate donors bymouse dCK with 15–30% of the activity of the correspon-ding D-enantiomers, and the enzyme does not discriminateD- and L-dCTP as feedback inhibitors (Tomikawa et al.,1997). Similarly, human dCK recognized both D-ATP andL-ATP as phosphate donors with apparently comparableefficiencies (Verri et al., 1999a). Such a complete lack ofenantioselectivity of dCK may imply that the stericdemands of the sugar moiety of the nucleoside moleculebound into the active site are not specific and are negligiblecompared to those of the nucleic base or the phosphatedonor.

In sharp contrast to dCK, the other human cytosolicenzyme, TK1, is much more strict in substrate specificity(Shewach et al., 1993) as well as enantioselectivity, since itdoes not recognize L-dT even at high concentrations and itdoes not accept L-ATP as a phosphate donor (Spadari etal., 1992,1995b; Verri et al., 1999b). However, L-FMAUhas been recently reported to be an atypical substrate ofboth TK1 and dCK (Liu et al., 1998).

The enantioselectivities of mitochondrial nucleosidekinases dGK and TK2 have not been as extensively studiedas that of dCK. Nevertheless, the existing investigations dosuggest that they possess relaxed enantioselectivities.


Figure 5. Lack of enantioselectivity of human deoxycytidine kinase expressed by EL/D

coefficients in the cytidine, adenosine and guanosine series

O

HO

OH

CyO

HO

CyO

HO5FCy

O

HO

OH

Cy

HO

O

HO

Cy

OH OH

O

HO

Ad

OH

O

HO

Ad

O

HO

Ad

O

HO

Ad

OH OH

O

HO

Ad

OH

HO

O

HO

Gu

OH

O

HO

Gu

OH

Gaubert et al. (1999); Pelicano et al. (1997); Shafiee et al. (1998).

Human dGK from CEM cells catalyses the phosphoryla-tion of both enantiomers of the carbocyclic analogue of dG,with a marked preference for the L-enantiomer (E

L/D=35)

(Bennett et al., 1998). More recently, it has been reportedthat human recombinant dGK accepts both D- and L-enantiomers of dG or dA with enantioselectivity factors Edepending on the concentrations of the fixed substrateATP and the range of concentrations of the variable sub-strate (Wang et al., 1999). Such dependence on substrateconcentration has also been observed for human TK2 fromHeLa cells, which catalyses the phosphorylation of D- andL-thymidine with weak enantioselectivities, both at lowsubstrate concentrations (E

L/D=0.16) and at high concen-

trations (EL/D

=1.3) (Verri et al., 1997b). In the presence ofa non-saturating ATP concentration, human recombinantTK2 phosphorylates both enantiomers of dT with almostequal activities (Wang et al., 1999).

The relaxed enantioselectivities of the three cellularnucleotide kinases dCK, dGK, and TK2 probably derivefrom their previously mentioned structural homologies(Eriksson & Wang, 1997; Johansson & Karlsson, 1996;Wang et al., 1996).

The enantioselectivity of adenosine kinase is even lessknown than that of TK2. β-L-Adenosine has been report-ed to be neither a substrate nor an inhibitor of rat brain AK(Gu et al., 1991). Bovine liver AK catalyses the phosphory-lation of β-L-adenosine with a lower efficiency than that ofits natural enantiomer and it displays only a very weakaffinity for β-L-dA (Gaubert, 1999b).

Viral nucleoside kinasesOf the various viral thymidine kinases, the enantioselectiv-ity of the HSV-1 TK is the most studied (Gentry, 1992).The existing sequence homologies between HSV-1 TK andcellular kinases dCK, dGK and TK2 (Harrison et al., 1991)suggest that the viral enzyme should also display a relaxedenantioselectivity. Indeed, HPLC analysis of reactionmedia indicated that D- and L-dT are substrates of the viralenzyme, apparently with similar efficiencies (Spadari et al.,1992,1995b). Furthermore, the lack of enantioselectivityextends to enzyme inhibition, and HSV-1 TK is competi-tively inhibited by L-dT, L-dG, L-dU, L-dC and L-dA withaffinities decreasing in this order (Spadari et al., 1992).Similarly, pseudorabies virus thymidine kinase catalyses D-and L-dT phosphorylation with comparable efficiencies(E

L/D=0.62) (Maga et al., 1993). HSV-1 TK also has a

nucleotide phosphorylating activity, but it is apparentlyenantioselective, and β-D-dTMP is a substrate of theenzyme that does not catalyse the phosphorylation of β-L-dTMP (Maga et al., 1994; Spadari et al., 1992). Finally,neither HSV-1 TK nor HSV-2 TK accepts L-ATP asphosphate donor (Verri et al., 1999a).

β-L-5-Iodo-2′-deoxyuridine (L-IdU) and β-L-(E)-5-(2-

bromovinyl)-2′-deoxyuridine (L-BVDU), antipodes of theantiherpetic drugs IdU and BVdU, have been reported tobe substrates of HSV-1 TK with efficiencies comparable tothat of the corresponding D-enantiomers, and also to beinhibitors of this enzyme using D-dT as a substrate(Spadari et al., 1995a,b). Moreover, neither L-enantiomerinhibits human TK1 (Spadari et al., 1995a). The carbo-cyclic analogues of the (+)- and (–)-enantiomers of IdU (C-IDU) or BVdU (C-BVDU) are both active against HSV-1, and they inhibit HSV-1 TK with similar efficiencies [Ki0.09 and 0.19 µM for (+)-C-BVDU and (+)-C-IDU,respectively, and 0.16 and 0.19 µM for (–)-C-BVDU and(–)-C-IDU, respectively] (Balzarini et al., 1990).

HSV TK also demonstrates a lack of enantiomeric selec-tivity with respect to guanosine analogues, either cyclic oracyclic. The fact that HSV-1 TK can catalyse the phospho-rylation of both pyrimidine and purine nucleosides hasrecently received an explanation based on the comparisonof the 3D-structures of this enzyme and that of thymidy-late kinase (Lavie et al., 1998). The D- and L-enantiomersof the carbocyclic analogue β-CdG are phosphorylatedwith similar efficiencies by HSV-1 TK (E

L/D=0.66) and they

both inhibit the enzyme in the phosphorylation of D-dT(Bennett et al., 1993). The analogues of β-CdG with athree- or a four-membered carbocyclic ring are also sub-strates of viral TK. (1S,2R)-9-{[1,2-Bis(hydroxy-methyl)cycloprop-1-yl]methyl}guanine (Figure 6) haspotent activity against HSV-1, HSV-2, varicella-zostervirus (VZV) and human cytomegalovirus, and it was foundto be phosphorylated at the 2-hydroxylmethyl group in thepresence of HSV-1 TK or VZV TK (Ono et al., 1998).(1R)(1α ,2β,3α )-9-[2,3-Bis(hydroxymethyl)cyc lo-butyl]guanine (R-BHCG) (Figure 6) is also a potentinhibitor of HSV whereas the antipode has no activity.Surprisingly, R-BHCG is a markedly poorer substrate ofHSV TK than the 1-S enantiomer (E

S/R=16 or 30), both

enantiomers being phosphorylated at the 3-hydroxymethylgroup, which is then homologous to the 4′-hydroxymethylgroup of β-dG (Terry et al., 1991) (Figure 6). This appar-ent contradiction is due to the enantioselective propertiesof HSV-1 DNA polymerase which has a much strongeraffinity for R-BHCGTP than for the antipode (see ‘Otherviral DNA polymerases’ section below).

Ganciclovir (GCV) and penciclovir (PCV) are acyclicsubstrates of HSV TK with a prochiral carbon (Figure 6),and they are phosphorylated in infected cells to chiralmonophosphates with a marked preference for the pro-Shydroxymethyl group yielding a majority of the correspon-ding (S)-monophosphate (Vere Hodge, 1993). This prochi-ral enantioselectivity probably results from the fact that theS-monophosphates are more structurally related to D-dGMP than the (R)-monophosphates. Experiments withpurified HSV-1 TK have confirmed these results, and the

G Maury



phosphorylation of GCV catalysed by this enzyme gaveonly (S)-GCV monophosphate (Karkas et al., 1987), where-as the phosphorylation of PCV was found by NMR analy-sis of the reaction medium to give 75% (S)- and 25% (R)-

PCV monophosphates (Vere Hodge et al., 1993). HSV-1TK phosphorylates the primary hydroxyl group of both the(R)- and (S)-enantiomers of the antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (Karkas et al., 1986).


Figure 6. Enantioselectivity of the enzymatic phosphorylation of guanosine and guanosine monophosphateanalogues

HO

HO

Gu

pO

HO

Gu

Gu OH

HO

Gu Op

HO

HO

OH

X

Gu

pO

HO

X

Gu

pO

HO

O

Gu

ppO

HO

O

Gu

S

pO

HO

Gu

ppO

HO

Gu

pO O

OH

Gu

ppO O

OH

Gu

Recently, the crystal structures of HSV-1 TK complexedwith various ligands [D-dT and ATP (Wild et al., 1995), D-dT or GCV (Brown et al., 1995), acyclovir or PCV, ADPor D-dTMP (Champness et al., 1998), and D-dT or 5-IdUMP (Wild et al., 1997)] have been disclosed. In thecomplex GCV/HSV-1 TK, the hydroxymethyl groupsattached to the prochiral carbon atom of GCV are differ-entiated. The pro-S hydroxymethyl group of bound GCVand PCV apparently mimics the 5’-hydroxyl group interac-tions of thymidine with the enzyme, thus explaining theenantioselectivity of the phosphorylation (Champness etal., 1998). However, a more definite justification of theenantioselectivity of HSV-1 TK requires the study of ter-nary complexes with ATP. One can also predict that thedetermination of the 3D-structure of the complex of HSV-1 TK/L-dT/ATP may reveal the structural causes of thelack of enzyme enantioselectivity by comparison with itscomplex with D-dT/ATP (Wild et al., 1995).

5′-NucleotidasesAlong with nucleoside kinases, 5′-nucleotidases may pres-ent a phosphotransferase activity that allows them to phos-phorylate a number of nucleosides or their analogues.These so-called ‘high K

mnucleotidases’ (Spychala et al.,

1988) use IMP as a phosphate donor and catalyse the phos-phorylation of purine nucleosides or analogues as β-D-dA,β-D-dG, β-D-ddG, carbovir, the carbocyclic analogue ofdG and acyclovir (Bennett et al., 1998; Johansson &Eriksson, 1996). The human enzyme is thought to beresponsible for the 5′-phosphorylation of 2′,3′-dideoxyino-sine (β-D-ddI) in the metabolism of this anti-HIV drugapproved for clinical treatment ( Johnson & Fridland,1989). Recently, two human cytosolic nucleotidases havebeen isolated that have a phosphotransferase activity spe-cific for pyrimidine nucleosides, contrary to the precedinghigh K

mnucleotidases (Amici et al., 1994,1997). These

pyrimidine nucleotidases are apparently enantioselectiveand did not catalyse the phosphorylation of β-L-dC, β-L-ddC or β-L-araC (Gaubert, 1999b). In contrast, studies ofthe enantioselectivity of 5′-nucleotidases active on purinenucleosides have yielded divergent results. The enzymefrom human placenta phosphorylates the ‘D’-enantiomer ofcarbovir and not the ‘L’-enantiomer (Miller et al., 1992),but the 5′-nucleotidase from Hep-2 cells has been report-ed to catalyse the phosphorylation of both enantiomers ofthe carbocyclic analogue of dG with a preference for the‘D’-enantiomer (E

L/D=0.4) (Bennett et al., 1998). It has also

been observed that L-FMAU is not a likely substrate ofcytosolic 5′-nucleotidase (Liu et al., 1998).

The enantioselectivity of another type of 5′-nucleoti-dase, which catalyses the hydrolysis of nucleoside 5′-monophosphates or their analogues, has been used to pre-pare enantiomerically pure nucleoside analogues. Thus, the

enantiospecific hydrolysis of racemic 3TC 5′-monophos-phate in the presence of the 5′-nucleotidase of Crotalusatrox venom favoured the natural enantiomer and yieldedonly the corresponding pure (+)-D-nucleoside and (–)-L-nucleotide (Storer et al., 1993). This implies that (–)-aris-teromycin (Herdewijn et al., 1985) and (+)-carbocyclic-9-(2 ′ -deoxy-2 ′ -β- f luoroarabinofuranosy l )guanine(Borthwick et al., 1988), previously obtained using thesame method, had the ‘D-configuration’ and suggests thatthis type of 5′-nucleotidases have strict enantioselectivitiesfavouring natural enantiomers (Figure 10).

Nucleotide kinasesNucleoside monophosphate kinases catalyse the phospho-rylation of nucleoside 5′-monophosphates or analogues toform the corresponding 5′-diphosphates in the presence ofATP or other nucleoside triphosphates. These enzymes areessentially different to the nucleoside kinases, since theycatalyse the formation of an anhydride bond instead of anester. They are generally considered to be less specific andthe second phosphorylation less critical than the first,although fewer structure–activity relationships using puri-fied enzymes have been established compared to nucleosidekinases (Navé et al., 1996).

Thymidylate kinase (dTMPK) catalyses the phosphory-lation of AZTMP, which is the rate-determining step inthe formation of AZTTP in cells (Balzarini et al., 1989;Daluge et al., 1994; Furman et al., 1986). This has been jus-tified by comparing the 3D-structure of dTMPK com-plexed with either dTMP or with AZTMP (Lavie et al.,1998; Lavie et al., 1997). The enantioselectivity of thisenzyme has apparently not been studied.

Human deoxycytidylate kinase (dCMPK) has been pro-posed to be the enzyme responsible for the phosphorylationof the two enantiomers of 3TCMP or FTCMP in cells(Furman et al., 1995). This is likely in view of the fact thatboth D- and L-FTCMP are substrates of calf thymusdCMPK with a preference for the L-enantiomer (E

L/D=8.1)

(Furman et al., 1992). No study of the interaction ofdCMPK with β-ddCMP or β-FddCMP has been reported.

Carbovir-5′-monophosphate having the natural stereo-chemistry of β-D-nucleoside monophosphates has beenshown to be a much better substrate of pig brain guanylatekinase (GMPK) than the L-enantiomer (E

D/L=7500)

(Miller et al., 1992). The high value of the ED/L

coefficientin this case demonstrates that the enzyme is enantioselec-tive and suggests that it catalyses the synthesis of the D-enantiomer of the diphosphate metabolite in cells. Thestudy of L-CdG metabolism in cells indicates that thiscompound is a mediocre substrate of GMPK, thus explain-ing its relatively low antiviral activity (Bennett et al., 1998).Acyclic guanine nucleoside monophosphate analogues aresubstrates of GMPK, which also displays fairly strict enan-

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tioselectivities (Figure 6). Thus, the S-enantiomer of ganci-clovir monophosphate, (S)-GCVMP (the stereoisomermost resembling the corresponding D-nucleosidemonophosphate) is a better substrate of GMPK than theR-enantiomer (E

S/R=62) (Karkas et al., 1987; Tolman,

1989). The same enzyme also favours the S-enantiomers ofseveral analogues of ganciclovir monophosphate includingpenciclovir monophosphate, (S)-PCVMP (E

S/R=64)

(Figure 5) (Tolman, 1989).Nucleoside diphosphate kinase (NDPK) catalyses the

last phosphorylation step to the triphosphate, and the 3D-structure of the human enzyme is known (Morera et al.,1995). The enzyme is independent of the nature of the baseand thus displays little specificity with respect to the nucle-oside diphosphate and the natural triphosphate donor,although discrimination with respect to 2′,3′-dideoxynucle-oside triphosphate donors has been reported (Bourdais etal., 1996; Deville-Bonne et al., 1996; Schneider et al., 1998).The enantioselectivity of the enzyme has not been studiedexcept in the case of beef liver nucleoside diphosphatekinase, which phosphorylates both enantiomers of carbovirwith a preference for the natural enantiomer (Miller et al.,1992). It should be added that other enzymes not directlyconnected with nucleosides or nucleotides (pyruvate kinase,creatine kinase, phosphoglycerate kinase) are also able tocatalyse the phosphorylation of both enantiomers of car-bovir diphosphate (Miller et al., 1992). This suggests thatenantioselectivity, as substrate selectivity, does not need tobe strict in the last step of nucleoside triphosphorylation.

Enantioselectivities of viral or cellular DNApolymerases

The activity of L-nucleosides and their analogues is alsofundamentally dependent on the enantioselectivity of theDNA polymerases which have been selected in principle torecognize natural D-nucleoside. Two classes of DNA poly-merases should be examined in each case, the viral targetenzyme to be inhibited by nucleoside analogues and thecellular polymerases necessary to the organism, which ide-ally should not be perturbed by the analogues.

Viral DNA polymerases

HIV-1 reverse transcriptase. Most of the existing enantios-electivity studies concerning viral DNA polymerases havebeen aimed at HIV-1 RT. This enzyme may be inhibitedeither by direct binding of a deoxyribonucleoside triphos-phate analogue into the substrate active site or by catalysingthe incorporation of a modified nucleotide chain terminator(without 3′-hydroxyl group) at the 3′-end of a primeroligodeoxynucleotide bound to the viral RNA template, thusterminating the elongation of the primer (Reardon, 1993).

A simplified mechanism of HIV-1 RT involves: thesequential formation of a binary complex between RT anda template/primer duplex TP

n; a ternary complex with the

subsequent binding of a nucleoside triphosphate dNTP;the formation of a covalent bond between the α-phospho-rus of the triphosphate group and the oxygen of the termi-nal 3′-hydroxyl group of the primer; and, finally, the disso-ciation of the elongated duplex TP

n+1 from the enzyme

(Figure 7) (Reardon, 1993). Depending on the substrate,the rate-determining step has been reported to be the cova-lent bond formation, a precatalytic conformational change,or the dissociation of the enzyme from its substrate (Hsiehet al., 1993; Reardon, 1993; Rittinger et al., 1995).

The enantioselectivity of HIV-1 RT has been investi-gated using two different experimental conditions forsteady-state kinetics with either homopolymeric tem-plate/primer duplexes, or templates and primers of definedsequences. In the first series of experiments, D- and L-enantiomers of nucleoside triphosphate analogues wereused as inhibitors of the HIV-1 RT catalysed elongation ofa homopolymeric primer (for example, polyrA/(dT)n) withthe corresponding natural deoxyribonucleotide triphos-phate (for example, β-D-dTTP) as substrate. Under theseconditions, the overall inhibition of primer elongation wascompetitive in all cases with respect to the natural sub-strate, and the corresponding constants K

iwere generally

similar for both enantiomers of a pair suggesting that HIV-1 RT essentially lacks the ability to discriminate the enan-tiomeric nucleoside triphosphate analogues (Figure 7)(Faraj et al., 1994; Focher et al., 1995; Maga et al., 1999;Miller et al., 1992; Skalski et al., 1993; Yamaguchi et al.,1994). However, the introduction of a fluoro substituent atthe 3′-position of β-dTTP or β-dCTP resulted in suppres-sion of the inhibitory capacity of L-enantiomers againstHIV-1 RT contrary to the corresponding D-enantiomers(von Janta-Lipinski et al., 1998). The K

ivalues of mutant

M184V HIV-1 RT for β-L-nucleoside triphosphate ana-logues were increased 30–500-fold for both RNA- andDNA-directed synthesis compared to native HIV-1 RT,whereas the K

iof the corresponding β-D-enantiomers were

enhanced only 1.1–5.4-fold (Wilson et al., 1996). Recently,the enantioselectivities of native HIV-1 RT and six of itsmutants (with the substitutions L100I, K103N, V106A,V179D, Y181I or Y188L) have been determined withrespect to the enantiomers of β-dTTP, β-dCTP, β-ddCTPand β-FddCTP. The substitution Y181I was the onlymutation which allowed the discrimination of the enan-tiomers of β-dTTP and β-dCTP, but this effect did notextend to the 2′,3′-dideoxy series (Maga et al., 1999).

Alternatively, the use of oligonucleotides of definedsequences and a nucleoside triphosphate complementary tothe first free nucleotide on the template seems more appro-priate to study the enantioselectivity of the enzyme, since it


allows the step-by-step calculation of kinetic parameters ofindividual substrates. Pre-steady-state and steady-statekinetic studies of the HIV-1 RT catalysed incorporation ofD- and L-enantiomers of ddTTP or d4TTP to a tem-plate/primer duplex of defined sequence, showed that bothenantiomers were substrates of the enzyme but the ratiosk

cat/K

mfor D-enantiomers were 50–70-fold larger than the

values for the L-enantiomers (Van Draanen et al., 1992).Another study concerning D- and L-3TCTP (Feng &Anderson, 1999) was in accordance with previous investi-gations (Gray et al., 1995; Hart et al., 1992; Skalski et al.,1993), and showed that the efficiency of incorporation of

D-3TCTP in a RNA/DNA template/primer duplex washigher than that of L-3TCTP. It also showed that theaffinities of the enantiomeric triphosphates for the enzymewere similar. It is important to note that D-3TCMP-ter-minated DNA or L-3TCMP-terminated DNA obtainedafter incorporation were substrates of a 3′- to 5′- exonucle-ase purified from human leukemic cells, with a two to sixtimes faster excision rate for D-3TCMP compared to L-3TCMP. This explains in part the differences of anti-HIVefficiency between D- and L-3TCTP (Skalski et al., 1993).Under comparable conditions, both enantiomers of a carbocyclic β-2′ ,3′-didehydro-2′ ,3′-dideoxyadenosine

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Figure 7. Enantioselectivity of the inhibition of HIV-1 reverse transcriptase using nucleoside triphosphate analogues

O

pppO N

N

O

µ µ

O

pppO N

N

O

F

µ µ

S

O

pppO N

N

O

µ µ

H2N H2N H2N

S

O

pppO N

N

O

H2N

F

µ µ

O

N

HN

O

O

CH3

OH

µ µ

O

N

HN

O

O

CH3

µ µ

O

N

HN

O

O

CH3

µ µ

N

NHN

N

O

NH2

µ µ

pppO pppO

pppO pppO

Faraj et al. (1994); Focher et al. (1995); Maga et al. (1999); Miller et al. (1992); Skalski et al. (1993); Yamaguchi et al. (1994).


triphosphate analogue were accepted as substrates, but thecorresponding α-L stereoisomer was not (Semizarov et al.,1994). Following this work, it was shown that both enan-tiomers of α-dTTP and α-dATP were not substrates ofHIV RT (Semizarov et al., 1997). Finally, a comparativestudy of the interaction of β-L-FddCTP or β-L-Fd4CTPwith HIV-1 RT showed that the latter compound is amuch better substrate than the former compound suggest-ing that the flattening of the sugar ring induced by theunsaturation may increase the affinity for the enzyme(Kukhanova et al., 1998a).

To investigate the apparently different conclusions ofexperiments with homopolymeric template/primer duplex-es or with duplexes of defined sequences, and to separatelyevaluate the affinity of the substrates for the enzyme andthe rate of incorporation, methods based on fluorescencewere used (Pelicano et al., unpublished results). The incor-poration of a fluorescently labelled derivative of β-D-dCTPat the 3′-end of a 36/18 DNA/DNA duplex in the presenceof HIV-1 RT was kinetically followed using the variationof fluorescence intensity (Figure 8). The fluorescence of thelabelled duplex was then similarly measured to study the


Figure 8. Kinetics of the HIV-1 catalysed incorporation of enantiomeric nucleoside triphosphate analogues atthe 3′-end of a template/primer duplex using fluorescence spectrometry

O

OH

N

N

O

NH 2 X

X =

3-OS

N+

CH3

CH3

N

CH3

SO3-

(CH2)5CONHCH2

O 3O9-4

P

H3CH3C

(i) Principle: Primer/ 5′ TCC CTG TTC GGG CGC CACTemplate 3′ AGG GAC AAG CCC GCG GTG GCG ATC TCT AAA AGG TGT 5′

Fluorescent marker:

O

pppO N

N

O

NH2

O

N

N

O

NH2

F

O

pppO N

NH

O

O

CH3

N3

O

pppO N

NN

N

NH2

pppO

(ii) Results: Apparent 2nd order rate constants (M–1.sec–1):

Pelicano et al. (unpublished results)

subsequent incorporation of several non-fluorescentnucleotides, including L-enantiomers. Although L-nucleo-side triphosphate analogues were substrates of HIV-1 RT,they were incorporated much more slowly than the corre-sponding D-enantiomers (Figure 8). Using the intrinsictryptophan fluorescence of the enzyme (Rittinger et al.,1995), the titration of the complex between HIV-1 RT anda modified 36/18 DNA/DNA duplex by the enantiomericnucleoside triphosphate analogues allowed the determina-tion of their affinities for the complex (Pelicano et al.,unpublished results). The resulting dissociation constantsof enantiomeric triphosphates calculated from the titrationcurves were similar. The results of these experiments sug-gest that in this case HIV-1 RT has a relaxed enantioselec-tivity in the binding step of the nucleoside triphosphateanalogue to the enzyme/duplex complex, but a more strin-gent enantioselectivity in the chemical incorporation of thechain terminator nucleotide.

The biologically active form of HIV-1 RT is a het-erodimer. The crystal structures of the unliganded HIV-1RT p66/p51 heterodimer (Rodgers et al., 1995) or of theheterodimer complexed with several ligands including theinhibitor nevirapine (Kohlstaedt et al., 1992; Ren et al.,1995), a DNA/DNA duplex and a monoclonal antibodyFab fragment ( Jacobo-Molina et al., 1993), and recently β-D-dTTP (Huang et al., 1998), have been determined.Computer modelling of the interaction of D- or L-3TCTPwith the HIV-1 RT active site has been attempted to inter-pret their kinetic behaviour (Feng & Anderson, 1999).However, any real interpretation of the apparent relaxedenantioselectivity of the enzyme must await the compari-son of the 3D-structures of ternary complexes with D- or L-deoxyribonucleoside triphosphates or their analogues.

HBV DNA polymerase. Similar to HIV DNA polymerase,HBV DNA polymerase has multiple functions includingRNA-dependent DNA synthesis, DNA-dependent DNAsynthesis and RNase-H activity (Seeger et al., 1991). Theenzyme mechanism is rather complex, and, for example,the reverse transcriptase of duck hepatitis B virus (DHBVRT) uses the hydroxyl group of a tyrosine residue to primeDNA synthesis in incorporating first dGMP and thenother nucleotides in a template-dependent manner(Dannaoui et al., 1997; Wang & Seeger, 1993). Comparedto HIV RT, the difficulty of obtaining pure samples of theenzyme has probably hampered the inhibition studies withnucleoside triphosphate analogues. In vitro assays ofenzyme activity generally use human HBV particles isolat-ed from chronic producer lines (for example, 2.2.15 cells) asthe source of enzyme (Davis et al., 1996; Saw et al., 1996;von Janta-Lipinski et al., 1998; Zhu et al., 1998), althoughassays for the expression of enzymatically active duck HBVRT and human HBV polymerase were recently developed

(Aguesse-Germon et al., 1998; Li & Tyrrell, 1999).Mechanistic studies of HBV polymerase inhibition by L-nucleoside triphosphate analogues have seldom been per-formed but, at least in the case of L-3TCTP, it has beensuggested that both competitive inhibition by binding toDHBV RT and DNA chain termination occur (Severini etal., 1995). Another study of DHBV inhibition has shownthat (R)-PCVTP or (S)-PCVTP inhibit hepadnavirusreverse transcription through the inhibition of the synthe-sis of the short DNA primer (Dannaoui et al., 1997).

The L-enantiomers of nucleoside triphosphate ana-logues tested against HBV DNA polymerase have emergedas effective inhibitors of the enzyme, with IC

50coefficients

ranging from 0.0007 to 10.4 µM. Moreover, in most com-parative studies, the L-enantiomer presented more efficientinhibitory properties against human HBV DNA poly-merase than the corresponding D-enantiomer (Figure 9)(Chang et al., 1992b; Davis et al., 1996). However, in theseries of 2′,3′-dideoxynucleoside triphosphates, the intro-duction of a fluorine atom at position 2′ of the ribosereversed this tendency (Figure 9) (von Janta-Lipinski et al.,1998). The (R)-enantiomer of PCVTP is a more potentinhibitor of human or duck HBV DNA polymerases thanthe (S)-enantiomer (Dannaoui et al., 1997; Saw et al.,1996). Finally, the interaction of β-L-FMAUTP (Aguesse-Germon et al., 1998; Pai et al., 1996), β-L-FddCTP(Zoulim et al., 1996), or β-L-Fd4CTP (Zhu et al., 1998)with HBV or DHBV DNA polymerases resulted in stronginhibitions of the enzymes in all cases.

Other viral DNA polymerases. Few reports exist on thethe stereoselectivity of the interaction of nucleoside triphos-phate analogues and HSV- or human cytomegalovirus(HCMV) DNA polymerases. (S)-PCVTP is a competitiveinhibitor of HSV-1 and HSV-2 DNA polymerases withrespect to β-D-dGTP, whereas the (R)-enantiomer has lit-tle inhibitory activity (Vere Hodge & Cheng, 1993). HSV-1 DNA polymerase catalyses the incorporation of GCVMP(presumably the S-enantiomer) at the 3′-end of the tem-plate/primer duplex but then allows the further incorpora-tion of only one nucleotide (Terry et al., 1991). L-FMAUTP was not a substrate of Epstein–Barr virus (EBV)DNA polymerase, contrary to D-FMAUTP. The L-antipode of FMAUTP was reported to inhibit the elonga-tion reaction and the 3′ to 5′ exonuclease activity associatedwith this DNA polymerase, presumably by binding at a sitedifferent from the sites of the natural dNTP or the tem-plate/primer duplex (Kukhanova et al., 1998b; Yao et al.,1996). Contrary to what would be expected from the enan-tioselectivity of HSV-1 or -2 TK that favour the 1S-enan-tiomer of BHCG (Figure 6), (1R)-BHCG is a betterinhibitor of HSV-1 growth than its antipode. This can beexplained by the triphosphate of (1R)-BHCG being a much

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better inhibitor of HSV-1 DNA polymerase than (1S)-BHCGTP [K

i(R)/K

i(S)=4.10–5]. Moreover, only the 1R

enantiomer of BHCGTP is accepted as a substrate of thepolymerase which then allows only a limited extension ofthe primer after incorporation of (1R)-BHCGMP (Terry etal., 1991).

Cellular DNA polymerases.L-Nucleoside triphosphate analogues may interact withDNA polymerases α, δ, ε, the main replicative polymeras-es in nuclei, DNA polymerase β which is believed to func-tion in DNA repair and recombination, and DNA poly-merase γ which is in charge of the replication of the mito-chondrial genome (Ono, 1987). As with HIV RT, the inhi-bition of these enzymes by D- or L-nucleoside triphosphateanalogues may occur either competitively at the active siteor through incorporation at the 3′-terminus of the primerstrand of double stranded DNA. However, the 3′ to 5′exonuclease activity associated with any cellular DNApolymerase may catalyse the excision of the chain termina-tor, thus restoring the conditions for normal synthesis ofDNA. This 3′ to 5′ exonuclease activity may in turn beinhibited by D- or L-nucleoside triphosphate analogues or

template/primer duplexes terminated by the correspondingD- or L- monophosphate residues, but this eventuality hasseldom been investigated.

Table 1 shows a concise report of the published substrateand inhibition properties of cellular DNA polymerases withrespect to some D- and L-nucleoside triphosphate ana-logues. No kinetic parameter has been added in this Table,as the experimental conditions for the determination ofthese parameters were too variable. Most of these qualitativedata are related to human DNA polymerases, but studieswith animal enzymes have also been reported, which mayyield different results (see β-dTTP). Owing to the variety inthe structures of the nucleotide analogues and in the prop-erties of cellular DNA polymerases, it would be surprisingto find any clear pattern emerging from the data in Table 1.In almost all cases, the replicative polymerases α, δ and ε,not unexpectedly, present similar tendencies. The data showeither that they are insensitive to inhibition by the D- or L-nucleoside triphosphate analogues presented in Table 1, orthat the enantioselectivity of the substrate or inhibitionproperties is generally low. For example, the corresponding(K

i)

L/(K

i)

Dratios seldom differ significantly from unity

(Chang et al., 1992b; Hart et al., 1992; Kukhanova et al.,


Figure 9. Inhibition of human hepatitis B DNA polymerase with D- or L-enantiomers of nucleoside triphosphate analogues: ratios of IC

50coefficients

O

pppO

Cy

S

O

pppO

Cy

S

O

pppO

FCy

O

pppO

Th

pppO

Gu

O

pppO

Th

F

O

pppO

Ur

F

O

pppO

Cy

F

O

pppO

MeCy

F

pppO

Gu

OH

1995). The lack of enantioselectivity of the replicativehuman DNA polymerases α, β, ε is particularly apparent inthe case of the dioxolane-cytidine derivatives OddCTP and5-FOddCTP, and concerns both their inhibitor and sub-strate properties (Kukhanova et al., 1995). However, thereare exceptions, and the enantioselectivity in the substrate orinhibition properties of these enzymes appears to be fairlyhigh with respect to the enantiomers of β-FMAUTP, β-dTTP, β-dCTP (Table 1) and BHCGTP (Terry et al.,1991). The substrate properties and inhibition of DNApolymerase β are apparently enantioselective, with the D-

enantiomers generally better recognized than the L-enan-tiomers, except for dioxolane-cytidine analogues (Chang etal., 1992b; Hart et al., 1992; Kukhanova et al., 1995; vonJanta-Lipinski et al., 1998) (Table 1). Apart from β-L-FdTTP, β-L-ddCTP or β-L-FdUTP, all L-nucleotide ana-logues of Table 1 are inhibitors of DNA polymerase γ,which appears to be particularly sensitive to this type oftriphosphate analogue. The acyclic guanosine analogues (S)-GCVTP and (S)-PCVTP have been reported to be morepotent inhibitors of the replicative human DNA polymeraseδ than of human polymerases α and ε (Ilsley et al., 1995).

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Table 1. Substrate properties and inhibition of cellular* DNA polymerases with respect to D- and L- nucleosideanalogues

DNA polymeraseCompound α β γ δ ε Reference

β-dTTP† D – – – – – Yamagushi (1994);L S,I NS,NI NS,I NS,I NS,I Focher (1995); von Janta L

(1998); Semizarov (1997)

β-dCTP D – – – – – Semizarov 1997L NS NS – – NS

β-FdTTP D NI I I NI‡ NI‡ von Janta L (1998)L NI NI NI NI‡ NI‡

β-FdCTP D NI I I NI‡ NI‡ von Janta L (1998)L NI NI Poor I NI‡ NI‡

β-FMedCTP D NI I I NI‡ NI‡ von Janta L (1998)L NI NI Poor I NI‡ NI‡

β-ddCTP D NS, NI S, I S, I Poor I Poor I Kukhanova (1995,1998a)L NS, NI NS, NI NS, NI NS, NI INS, poor I

β-FddCTP D NI I I NI NI Kukhanova (1995,1998a)L NS, NI S, poor I S, poor I NS, NI Poor S, NI

β-Fd4CTP D – – – – – Kukhanova (1998a)

L Poor S, poor I S, I S, I – NS, NI

β-OddCTP D S, I S, I S, I S, I S, I Kukhanova (1995)L S, I S, I S, I S, I S, I

β-FOddCTP D S, I S, I S, I S, I S, I Kukhanova (1995)L S, I S, I S, I S, I S, I

β-FdUTP D NI I I NI‡ NI‡ von Janta L (1998)L NI NI NI NI‡ NI‡

β-FMAUTP D S S S S – Kukhanova (1998b);L NS, poor I NS, poor I NS, poor I NS NS Yao (1996)

β-CBVTP D I – – – – Miller (1992)L I – – – –

β-3TCTP D I S, I Poor S, I I – Hart (1992); Gray (1995);L NS, I NS, I S, I NS, I – Chang (1992)

PCVTP S Poor I – – I Poor I Vere Hodge (1993);R I – – – – Ilsley (1995)

S: Substrate, I: Inhibitor, NS: Non-substrate, NI: Non-inhibitor.*Human DNA polymerases unless otherwise mentioned.†Animal DNA polymerases yield different results.‡DNA polymerases from animals.


Terminal deoxynucleotidyl transferase (TdT), a cellularDNA synthase which does not use a template, has beenreported to be inhibited by β-L-dTTP (Focher et al., 1995)and by both enantiomers of an analogue of carbocyclicdideoxyadenosine (Theil et al., 1998). It also utilizes α-D-dTTP and α-D-dATP as substrates but not the correspon-ding α-L-enantiomers (Semizarov et al., 1997).

β-L-ATP has been shown to be a competitive inhibitorof T4 DNA ligase or of human DNA primase with β-D-ATP as substrate, thus indicating that these enzymes areable to recognize both enantiomers (Verri et al., 1999a).

Enantioselectivities of nucleoside deaminases and phosphorylases

The importance of adenosine deaminase (ADA), cytidinedeaminase (CDA) and purine nucleoside phosphorylase(PNP) in the catabolism of antiviral or anticancer nucleo-side analogues, and therefore in the development of thesedrugs, has been recognized for a long time. Efforts havebeen made to establish structure–activity relationships, butfew concern the enantioselectivity of these enzymes. Therecent crystal structure determinations of these enzymesmay help to understand their selectivities. The 3D-struc-ture of monomeric murine ADA complexed with the tran-sition-state analogue 6-hydroxy-1,6-dihydropurineribonucleoside has shown that the (6S)-enantiomer of theligand is held in place by coordination of the 6-hydroxylgroup to a zinc ion and by at least six hydrogen bondsbetween the ligand and the enzyme (Sharff et al., 1992;Wilson et al., 1991). This and a subsequent study concern-ing ADA complexed with 1-deazaadenosine (Wilson &Quiocho, 1993) suggest that the deamination mechanismis either a SN

2or a stereoselective two-step addition–elim-

ination process. Although CDA is dimeric or tetramericand has an entirely different architecture to ADA, bothenzymes present similar active sites (Carter, 1995). Thecrystal structures of E. coli CDA complexed with 5-fluo-ropyrimidin-2-one riboside (fluorozebularine, FZEB)(Betts et al., 1994), with 3-azacytidine (Xiang et al., 1996)or with uridine (Xiang et al., 1997) have been solved. Aswith ADA, the transition state analogue is bound as acovalent hydrate with its hydroxyl group interacting with azinc ion buried deeply in the active site. It has also beenshown that hydration of FZEB catalysed by CDA resultsin a covalent hydrate of opposite chirality (4R) comparedto the hydrate obtained in adenosine analogue deamina-tion with ADA as a catalyst (Betts et al., 1994). The 3D-stucture of human erythrocytic PNP (Ealick et al., 1990)and that of E. coli PNP (Koellner et al., 1998; Mao et al.,1997) have also been solved and used to design newinhibitors of PNP.

The enantioselectivities of ADA and CDA have been

determined either directly from kinetic studies of enan-tiomeric substrate and inhibitors, or indirectly from the re-solution of racemic mixtures of substrates in the synthesisof enantiomerically pure compounds.

β-L-2′,3′-Dideoxyadenosine and β-L-2′,3′-didehydro-2′ ,3′-dideoxyadenosine are very poor substrates andinhibitors of calf intestinal ADA contrary to the naturalenantiomers (Pelicano et al., 1997). Several sources havereported that β-L-adenosine is not a substrate of ADA(Gu et al., 1991; Phadtare & Zemlicka, 1989) and that β-L-2′-deoxyadenosine is a very poor substrate compared tothe D-enantiomer (Gaubert, 1999b). Inhibition of ADAalso proceeds with high enantioselectivity, since the(2S,3R) enantiomer of erythro-9-(2-hydroxy-3-nonyl)ade-nine (EHNA) is a 250-fold more potent inhibitor ofhuman erythrocyte ADA than the opposite enantiomer(Bessodes et al., 1982). β-L-Adenosine is only a weakinhibitor of rat brain ADA when the natural enantiomer isused as substrate (Gu et al., 1991). The high enantioselec-tivity of deamination cata-lysed by ADA finds its use inthe resolution of racemic adenosine analogues in the pres-ence of ADA: the enantiomer with the natural or pseudo-natural configuration is always rapidly deaminated where-as the antipode is resistant. Thus, several enantiomericallypure carbocyclic nucleoside analogues (Gala &Schumacher, 1992; Hutchinson, 1990; Secrist III et al.,1987) or other unusual adenine or hypoxanthine deriva-tives (Belleau et al., 1993; Katagiri et al., 1993; Megati etal., 1992; Qiu et al., 1998; Van Draanen & Koszalka, 1994)have been prepared by this method (Figure 10).

As expected from the similarities in the structures of theactive sites of the two deaminases, the enantioselectivity ofCDA follows the same trend as that of ADA. Thus, the β-L-enantiomer of dC, or the α-L- and β-L-enantiomers ofriboC, araC, xyloC and lyxoC were resistant to humanCDA (Shafiee et al., 1998). Similarly, the β-L-enantiomersof 3TC (Chang et al., 1992a), FTC (Furman et al., 1992),FddC (Martin et al., 1997) and Fd4C (Dutschman et al.,1998) were not substrates or weak substrates of CDA,contrary to the corresponding β-D-enantiomers. E. coliCDA has been used to develop a process for the produc-tion of β-L-3TC in multikilogram amounts, which isbased on the exclusive deamination of the D-enantiomerfrom the racemic mixture (Mahmoudian et al., 1993)(Figure 10).

The enantioselectivity of PNP is apparently unknown,apart for the fact that β-L-2′,3′-dideoxyinosine is neither asubstrate nor an inhibitor of the human enzyme, contraryto the natural antipode (Gaubert, 1999b). The sameenzyme does not recognize the two enantiomers of β-2′,3′-dideoxyadenosine or of β-2′,3′-didehydro-2′,3′-dideoxy-adenosine as substrates or inhibitors (Pelicano et al., 1997).


G Maury


Figure 10. Use of the enantioselectivities of catabolic enzymes to prepare enantiomerically pure nucleosideanalogues

pO

OH OH

Ad

HOAd

OH OH

+ pOOH OH

Ad

OH

Gu

F

HO

Gu

OH

+OH

Gu F

F

S

O

Cy

S

OHO

Cy

+ S

O

Cy

S

OHO

Cy

S

OHO

Ur

+ S

O

HO

Cy

HO

OH OH

Ad

HO

Hyp

OH OH

+ HOOH OH

Ad

pO

pO

pO

pO

+−

+−

+−

+−

+−

HO HO

Gu

+ HO

HO HO

Gu

+ HO

OH OH

OH

O

SHO

Ad

O

SHO

Hyp

+ O

S

HO

Ad

HOAd

HOAd

+ HO

Hyp

HO HO

OH

C

H

CH2OH

Ad

H

C

H

CH2OH

H

Hyp

+C

H

CH2OH

Ad

H

[2,6-DiNH2]-Pu

[2,6-DiNH2]-Pu

[2,6-DiNH2]-Pu

[2,6-DiNH2]-Pu

+−

+−

+−

+−

+−


Discussion and conclusion

Apart from amino acid racemases in prokaryotes and a li-mited number of enzymes that catalyse the formation ofopposite enantiomeric forms (Walsh, 1979), the vastmajority of enzymes create and preserve the natural config-uration of molecules. This does not necessarily imply thatthe enantioselectivity of all these enzymes must always behigh with respect to natural substrates or analogues.Enzymatic enantioselectivity depending on the structuresof enzyme and substrate; it is eventually possible that thehigh enantioselectivity required in the enzymatic formationof a natural biomolecule with creation of a chiral centremay be largely decreased when a substrate analogue is used(Popple & Novak, 1992). Moreover, if the substrate of agiven enzyme in a biosynthetic chain of reactions is presentonly as a pure enantiomer undergoing a chemical transfor-mation neither creating nor modifying chiral centres, theproduct will have the right stereochemistry even if theenantioselectivity of the enzyme is low with respect to thisnatural substrate. This raises the possibility that the evolu-tionary selection of enzymes catalysing this type of reac-tions may have retained proteins with mediocre enantiose-lective properties if, in contrast, other selectivities needed incatalysis were high.

Enzymes that catalyse the transformation of pureenantiomers of nucleosides with conservation of theasymmetry of the sugar and without introduction of a newchiral centre may obviously belong to this category. Forexample, the enzymes of nucleoside, nucleotide oroligonucleotide metabolism considered in this review mayhave variable enantioselectivities without in theory affect-ing the stereochemistry of the product DNA. It is a pos-sible explanation of the relaxed enantioselectivities pre-sented by several of these enzymes. This situation offersan opportunity to exploit the fortuitous differences in theenantioselectivities of the various metabolic enzymes forthe design of new potentially active nucleoside analoguesin antiviral chemotherapy.

The comprehensive evaluation of the data reported in thepreceding section leads to the conclusion that perhaps somebroad tendencies may be found in the enantioselectivities ofthe enzymes examined in this section. Part of the recentsuccess of L-nucleoside analogues as antiviral compoundsmay, for example, derive from the relaxed enantioselectivi-ties of several nucleoside kinases. Among them, dCKappears to be of major strategic importance, since it acceptsdC, dA and dG analogues as substrates and also demon-strates a marked lack of enantioselectivity with almost anysubstrate considered. Similarly, mitochondrial dGK andTK2 display relaxed enantioselectivities with dG, dA anddT derivatives respectively, although only a limited numberof analogues have been tested with TK2. Only TK1 appears

to be strictly enantioselective. The few published data con-cerning 5′-nucleotidase phosphoryl transferases are infavour of medium to high enantioselectivity. It follows thatL-nucleoside analogues stand a better chance of being phos-phorylated by cellular kinases if they belong to the cytidine,or at least the adenosine and guanosine series, instead of thethymidine series. In contrast, previous work suggests thatantiherpetic candidate L-nucleoside analogues derived fromdeoxythymidine, deoxyuridine and deoxyguanosine may bephosphorylated by poorly enantioselective HSV thymidinekinases. The few existing data concerning nucleotide kinaseenantioselectivity indicate that deoxycytidylate kinase mayhave a relaxed enantioselectivity and that deoxyguanylatekinase has a strong preference for natural enantiomers. Thismay constitute a ‘bottleneck’ in the activation of L-guano-sine analogues to the triphosphates.

The efficiency of a L-nucleoside antiviral candidate isalso directly dependent on the substrate or inhibition enan-tioselectivities of the target DNA polymerases. A survey ofthe known enantioselective properties of the viral enzymessuggests that HIV- and HBV-DNA polymerases (RT)stand a good chance of being inhibited by L-nucleosidetriphosphates in their β-anomeric stereochemistry,although the inhibition strength and mechanism may bevariable. In contrast, available data concerning herpesvirusDNA polymerases seem in favour of higher enantioselec-tivities. The situation concerning cellular DNA polymeras-es is more complex and elusive. These enzymes presentdiverse sensitivities towards L-nucleoside triphosphates andtheir analogues with perhaps a more frequently observedinhibition of DNA polymerase γ by L-nucleoside triphos-phates compared to other cellular polymerases. Other rela-ted and less studied enzymatic processes may be influencedby L-nucleoside triphosphates (for example the 3′ to 5′exonuclease activity), increasing the risk of toxicity andadding to the difficulty of evaluating the interest of theseanalogues as antiviral agents.

A major argument in favour of using L-nucleoside ana-logues (especially cytidine and adenosine derivatives) asantiviral compounds is their resistance to degradingenzymes such as nucleoside deaminases, which are widelydistributed in tissues and decrease the efficiency of nucleo-sidic drugs. All studies of the enantioselectivity of adenosineor cytidine deaminases have established the high sensitivityof D-enantiomers to deamination compared to theantipodes. More results are needed in the case of purine orpyrimidine phosphorylases. Nevertheless, the existing datasuggest that L-nucleosides are more resistant to degradationby these enzymes than the natural enantiomers. The knowncrystal structures of several deaminases and phosphorylasesmay also be useful in understanding the poor substrateproperties of the enzymes with respect to L-enantiomers.

At first sight, the overall trends in the enantioselectivities


of the preceding enzymes favour the use of L-nucleosides ortheir analogues in antiviral therapy. An ‘ideal scenario’ fromthese data would be one where L-nucleosides are conve-niently phosphorylated, inhibit viral DNA polymerases(HIV, HBV) and resist deaminases and phosphorylases.However, reality is much more complex. The enantioselec-tivity of an enzymatic process depends on both the enzymeand the substrate structures. It is always possible that sub-strate analogues may be found that contradict the tendencyestablished with other substrates. This is particularly expect-ed when enzyme enantioselectivity has been studied with alimited set of compounds. Enzymatic enantioselectivity in acellular medium may also differ from that determined inexperiments using purified samples of enzymes, owing to thedifferent conditions and the possible interactions with manyligands in cells. L-nucleosides or their analogues may bepotential substrates or effectors of enzymes not considered inour survey (Patanella & Walsh, 1992) with the risk of alter-ing other cellular processes. The enantioselectivity of non-enzymatic phenomena must also be considered. For example,nucleoside transport is an important parameter in the phar-macology of drug action, which exhibits enantiomeric selec-tivity as shown in the case of adenosine (Gati et al., 1989; Guet al., 1991) and carbovir (Mahony et al., 1992).

In spite of these adverse arguments and the fact thatmuch work remains to be done, the indicative trends inenzyme enantioselectivity reported in this review may still beused as a coherent guide line to help in the search for newinteresting L-nucleoside analogues. They also show that a L-nucleoside antiviral candidate should be considered andstudied as a unique and individual entity expected to displayvery different properties (particularly biological properties)compared to the antipode, in the same way as recommendedin the evaluation of two different D-enantiomers(Sommadossi, 1993). This represents a considerable expan-sion of the field of molecules which may be selected forscreening programmes, since it is often advantageous to planthe synthesis and evaluation of both enantiomers of a newnucleoside analogue instead of only the natural stereoisomer.

Acknowledgements

The Centre National de la Recherche Scientifique (CNRS)and the Agence Nationale de Recherches sur le SIDA(ANRS) are thanked for their financial support.

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Received 6 December 1999; accepted 1 February 2000

the enantioselectivity of enzymes involved in current

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