Inhibition of Hypoxanthine-Guanine Phosphoribosyltransferase by

Jun 15, 2009 - The School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072 QLD, Australia. § Institute of ... Coo...
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J. Med. Chem. 2009, 52, 4391–4399 4391 DOI: 10.1021/jm900267n

Inhibition of Hypoxanthine-Guanine Phosphoribosyltransferase by Acyclic Nucleoside Phosphonates: A New Class of Antimalarial Therapeutics† )

Dianne T. Keough,‡ Dana Hockov a,§ Antonı´ n Hol y,§ Lieve M. J. Naesens, Tina S. Skinner-Adams,^ John de Jersey,‡ and Luke ,‡ W. Guddat* The School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072 QLD, Australia, §Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and ^ Malaria Biology Laboratory, The Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Brisbane, 4029 QLD, Australia

)

‡

Received March 3, 2009

The purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. Ki values for 19 ANPs were determined for Pf HGXPRT and the corresponding human enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free enzyme moves to partly cover the active site. For three ANPs, the IC50 values for Pf grown in cell culture were 1, 14, and 46 μM, while the cytotoxic concentration for the first compound was 489 μM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.

*To whom correspondence should be addressed. Phone: 61 + 7 3365 3549. Fax: 61 + 7 3365 4699. E-mail: [email protected]. † Coordinates and structure factors have been deposited in the Protein Data Bank with access codes 3GGJ, 3GGC, and 3GEP for PEEG, PEEHx, and (S)-HPMPG, respectively. a Abbreviations: PRib-PP, 5-phospho-R-D-ribosyl-1-pyrophosphate; PRTase, phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosltransferase; HGXPRT, hypoxanthineguanine-xanthine phosphoribosyltransferase; ANP, acyclic nucleoside phosphonate; immGP, (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4imino-D-ribitol 5-phosphate; immHP, (1S)-1-(9-deazahypoxanthin-9yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate; PEEG, 9-[2-(2-phosphonoethoxy)ethyl]guanine; PEEHx, 9-[2-(2-phosphonoethoxy)ethyl] hypoxanthine (S)-HPMPG, (S)-9-[3-hydroxy-2-(phosphonomethoxy) propyl]guanine.

inhibitor binding would also be likely to decrease substrate binding and, hence, the activity of the enzyme. Thus, development of resistance may prove difficult. Human patients with inherited partial deficiencies of the activity of the corresponding purine salvage enzyme HGPRT lead normal lives even if the activity of HGPRT is reduced to 3% of normal.4 The only result is urate overproduction, which is readily amenable to treatment by allopurinol. Thus, partial inhibition of human HGPRT by an antimalarial drug inhibiting HGXPRT is unlikely to have any significant consequences. The aim of this research is to produce potent inhibitors of Pf HGXPRT, which have little or no effect on the human host. 6-Oxopurine phosphoribosyltransferases (PRTases) synthesize the purine nucleoside monophosphates required for nucleic acid production. The reaction they catalyze is shown in Figure 1. The human enzyme can only use hypoxanthine or guanine as its naturally occurring purine substrates while, for the enzymes from other organisms such as Escherichia coli5,6 and Plasmodium falciparum,7,8 xanthine is also a substrate. For catalysis to occur, a divalent metal ion must be present. This is usually Mg2+ in vivo although other divalent metal ions such as Mn2+ are effective substitutes. The mechanism of action for human HGPRT is ordered: PRib-PP.Mg2+ binds first followed by the purine base.9 After the covalent reaction, pyrophosphate then dissociates from the complex and the nucleoside monophosphate is released in the rate-limiting step.9 Pf HGXPRT is thought to follow the same mechanism although this has not been fully established. The only known tight-binding inhibitors of P. falciparum and human 6-oxopurine PRTases are the transition state

r 2009 American Chemical Society

Published on Web 06/15/2009

Introduction Malaria remains a serious infectious disease in the world today, resulting in 1-2 million fatalities each year. Approximately 48% of the world’s population live in areas of risk.1,2 There are four Plasmodium species responsible for human malaria: falciparum, vivax, ovale, and malariae. Of these, P. falciparum (Pf ) is the most lethal. Emerging resistance of the malarial parasite to existing chemotherapeutic agents has made the development of new drugs and vaccines an urgent priority. Hypoxanthine-guanine-xanthine PRTase (HGXPRTa) is considered a drug target because Plasmodium falciparum lacks not only the de novo pathway for purine nucleotide synthesis but also other purine salvage enzymes that provide alternative pathways to purine nucleotides.3 The catalytic activity of HGXPRT is therefore essential for the survival of P. falciparum and inhibition of Pf HGXPRT should arrest growth and division of the parasite. Active site mutations that prevent

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PRTases. Possible explanations for the differences in Ki values for this series of compounds between human HGPRT and PfHGXPRT are suggested. These studies provide a platform for the design of more potent and selective inhibitors of the parasite enzyme as antimalarial chemotherapeutics. Results and Discussion

Figure 1. Reaction catalyzed by the 6-oxopurine phosphoribosyltransferases. The naturally occurring purine bases are guanine (R is -NH2), hypoxanthine (R is -H), and xanthine (R is OH).

analogues known as immucillin 50 -phosphates. These bind with Ki* values of 1-4 nM to both enzymes.10 The structures of human HGPRT and P. falciparum HGXPRT in complex with (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immGP) and (1S)-1-(9-deazahypoxanthin9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immHP), respectively, together with Mg2+ and pyrophosphate, have been determined to 2 A˚ resolution.11,12 Both of these structures show near to identical interactions between the inhibitor, Mg2+, PPi, and the two enzymes suggesting that, based on this scaffold, the design of selective inhibitors of the parasite enzyme may be difficult. However, we have identified a number of purine base analogues that have kcat/Km values up to 340-fold in favor of PfHGXPRT compared with human HGPRT,13 showing that it is possible to find compounds that discriminate strongly between the two enzymes. When the transition state analogues bind, a large mobile loop (∼20 amino acid residues) is closed firmly over the active site. It has been hypothesized that the primary role of this loop is to sequester the active site from solvent during the covalent step of catalysis.11 Amino acid residues in this loop region interact with the immucillin 50 -phosphates helping to anchor the inhibitor firmly in position. The structures suggest that compounds which cause the loop to be completely closed when bound in the active site should have low Ki values. Because of their structural similarity to the purine nucleoside monophosphates, which are the products of the reaction catalyzed by HGXPRT, we have investigated a class of nucleotide analogues, the acyclic nucleoside phosphonates (ANPs), as potential inhibitors. ANPs are 2-(phosphonoalkoxy)alkyl derivatives of purine and pyrimidine bases containing an isopolar phosphonomethyl ether moiety instead of the nucleotide’s phosphate ester group. The chemical and pharmacological properties of ANPs have been the subject of intensive study because of their development as antiviral agents.14 A previous report showed that one of the ANPs, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine, had an IC50 value of 4 μM for Pf grown in cell culture although its mode of action was unknown at the time.15 Herein, we report the comparative inhibition by a series of ANPs of PfHGXPRT and human HGPRT. The IC50 values of three of these compounds for P. falciparum grown in erythrocyte cell culture have been measured and their cytotoxicity to mammalian cells determined. The crystal structures of three of these compounds, 9-[2-(2-phosphonoethoxy)ethyl]guanine (PEEG), 9-[2-(2-phosphonoethoxy)ethyl]hypoxanthine (PEEHx), and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (S-HPMPG), in complex with human HGPRT are reported. The structures show how these compounds bind in the active site of the 6-oxopurine

Selection of ANPs. Figure 2 compares the structures of the mononucleotide product of the reaction (see Figure 1) with the ANPs investigated in this study. For the ANPs, the similarity with the product mononucleotides, GMP and IMP, is the presence of a purine base and the phosphonate group mimicking the 50 -phosphate group attached to the ribose ring. Eight different phosphonate moieties were attached to the 9-position of the purine base to form various 2-(phosphonoalkoxy)alkyl purines. Nine different purine bases were used. There are three major differences in chemical properties between the ANPs and mononucleotides. Firstly, for the phosphonates, the ester oxygen present in the phosphate group is replaced by a carbon atom. Thus, enzymatic dephosphorylation or chemical hydrolysis of these analogues is excluded. Secondly, the position of the oxygen atom in the acyclic chain is shifted compared with the position it would normally occupy in the ribose moiety. Thirdly, absence of a glycosidic bond in the structure of ANPs further increases their resistance to chemical and biological degradation. Another structural advantage is the flexibility of the acyclic chain, which enables the compounds to adopt a conformation suitable for interaction with the active site of the enzyme. The different purine bases should also contribute to differences in affinities toward the two enzymes as indicated by our previous work on base analogues.13 Inhibition of PfHGXPRT and Human HGPRT. In a preliminary set of experiments, Ki values were measured for seven ANPs at pH 8.5 and these are listed in Table 1. When guanine was the purine base, the Ki values for PfHGXPRT ranged between 1.4 and 37.7 μM as five different phosphonate moieties were attached to form the ANPs. The increase in Ki followed the same pattern for the human enzyme. The influence of the purine base was shown by the fact that, when PME was the phosphonate moiety, the compound with guanine as the base bound more tightly than the hypoxanthine derivative. All of the ANPs tested under these conditions had a greater affinity for PfHGXPRT compared with human HGPRT. These data illustrate that the structures of both the purine base and the phosphonate moiety play significant roles in the affinity for the active site and in selectivity between the two enzymes. The best inhibitor within this group of ANPs (guanine base; cyclic-(R)-HPMP moiety) had a Ki of 1.4 μM and a selectivity ratio of 8 in favor of the Pf enzyme. On the basis of the results of Table 1, a second series of compounds was examined to discover additional features that are important in potency and selectivity. Table 2 shows Ki values for 12 ANPs, 2 ANP monoesters, and 2 cyclic intramolecular esters, measured at pH 7.4. The results obtained are now analyzed in terms of the contribution of the phosphonate moiety and the purine base. Contribution of the Phosphonate Tail Moiety. Ki values measured for the best ANP inhibitors of both enzymes are much lower than the values for the naturally occurring nucleotides, GMP and IMP, which are the products of the catalytic reaction (Table 2). For example, replacement of the

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Figure 2. Structures of the phosphonate moieties attached to the 9-position of the purine base in comparison with that of D-ribose 50 phosphate. Abbreviations: 2-(phosphonoethoxy)ethyl (PEE), 2-(phosphonomethoxy)ethyl (PME), 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP), 2-(phosphonomethoxy)propyl (PMP), 3-fluoro-2-(phosphonomethoxy)propyl (FPMP), cyclic-3-hydroxy-2-(phosphonomethoxy)propyl (cyclic-HPMP), 2-(phosphonomethoxy)ethyl isopropyl ester (PMEiPr), and 2-(phosphonomethoxy)ethyl isooctyl ester (PMEiOc). Tested compounds were synthesized as either the (R)- or (S)-isomer and, in some cases, a mixture of isomers as indicated in the text. Table 1. Ki Values for Seven Phosphonate Derivatives at pH 8.5 Ki (μM) base

phosphonate tail

Pf HGXPRT

human HGPRT

guanine guanine guanine guanine guanine hypoxanthine hypoxanthine

cyclic-(R)-HPMP (RS)-FPMP PME (S)-HPMP cyclic-(S)-HPMP PME (R)-PMP

1.4 ( 0.4 3.6 ( 1 18.9 ( 1.5 28.4 ( 2.2 37.7 ( 5 64.8 ( 7 437.3 ( 20

12.3 ( 3 22.7 ( 4 55.9 ( 3 176.8 ( 10 115.1 ( 6 182.3 ( 9 no inhibition

ribose 50 -phosphate by the PEE moiety reduced the Ki value for Pf HGXPRT by 100-fold, indicating that it is the phosphonate moiety that is responsible for the change in affinity. For human HGPRT, the decrease in Ki was only 6-fold. When guanine was the purine base (compounds 1, 3, 11, 15, and 16), the phosphonate analogues bound to PfHGXPRT with Ki values ranging from 0.1 to 1.6 μM. The Ki value increased in the order, PEE40

7 7-deaza-8-azahypoxanthine 8 6-thioguanine 9 guanine 10 guanine 11 guanined

PME PME PMEiPr PMEiOc (RS)-HPMP

7.2 ( 1 >100 >200 >500 0.6 ( 0.2

4.3 ( 0.6 >1000e 140 ( 20 >1000e 5.9 ( 0.4

0.6 NRf NRf NRf 10

12 8-azaguanine 13 8-bromoguanine 14 8-bromoguanine

(S)-PMP (S)-PMP (R)-PMP

>45 11 ( 4 20 ( 5

15 ( 3 >300e 41 ( 8

NR >30c 2

15 guanine 16 guanineq

cyclic-(S)-HPMP cyclic-(R)-HPMP

8(1 1 ( 0.2

90 ( 10 19 ( 5

11 19

a Taken from refs 35 and 36. b Referred to in the text as PEEG. c Referred to in the text as PEEHx. d Referred to in the text as (RS)-HPMPG. e The maximum Ki value able to be determined is governed by the maximum concentration of inhibitor able to be used in the spectrophotometric assay. The number is calculated on the basis that 5% inhibition can be measured and, at the concentration of inhibitor used, no inhibition of activity was observed. f NR=no ratio could be calculated as these compounds did not inhibit Pf HGXPRT at the concentration used.

the phosphonate moiety to the binding affinity and selectivity of the ANPs. To date, the phosphonate moiety that confers the tightest binding is PEE, while the base that offers highest selectivity is 8-azaguanine. Structural Analysis of Human HGPRT-ANP Complexes. To understand how these analogues bind in the active site as a prerequisite to the design of the next generation of inhibitors, the crystal structures of the human enzyme in complex with three ANPs (compounds 1, 2, and 11) were determined. The structures of human HGPRT in complex with PEEG (compound 1) and HPMPG (compound 11) have been determined to 2.6 A˚ resolution and to 2.78 A˚ with PEEHx (compound 2). Data collection and refinement statistics are presented in Table 3. In all three complexes, the asymmetric unit is a dimer. However, the commonly observed tetrameric structure of the enzyme16 is present when adjoining asymmetric units are considered. The electron density for the inhibitor and the amino acid residues in contact with the inhibitor is unequivocal in all subunits (Figure 3a). Overall, the structure of the polypeptide backbone and side chains distal to the active site is identical to the structure of free HGPRT16 and its complexes with immGP, PPi and Mg2+, or GMP.11,17 However, the conformations of the large mobile loop (residues 100-117), a smaller loop around residues 137-141 and a flap region that covers the purine binding site all change when the ANPs bind, as discussed below. To date, we have not been able to crystallize Pf HGXPRT in the presence of the ANPs. Structural studies showing how the inhibitors bind to the human enzyme provide us with an excellent model for how they may bind to PfHGXPRT (given the 48% sequence identity between the two enzymes) and provide hypotheses as to why these inhibitors bind to the Pf enzyme with greater affinity. The

importance of the structures also lies in the fact that they can help to explain why particular phosphonates bind better than others to the human enzyme and thus provide insights into the design of inhibitors, which are selectively potent to the malarial enzyme. The structures are now analyzed in terms of three key enzyme regions: the 50 phosphate binding site, the large mobile loop, and the purine binding site. The 50 -Phosphate Binding Site (Residues 132-141). The amino acid residues in this loop are identical in human HGPRT and Pf HGXPRT where their role is the binding of the 50 -phosphate group attached to the ribose ring (Figure 3b,e,f ). When the mononucleotide product of the catalytic reaction or the transition state analogue binds in the active site, this loop radically changes its structure.17,11 This change also occurs when the phosphonates bind in this region. The phosphonate groups of (S)-HPMPG, PEEG, and PEEHx form the same constellation of hydrogen bonds between their oxygen atoms and human HGPRT as those for the 50 -phosphate group in the immGP complex (Figure 3f ). The phosphate/phosphonate group is held in position via hydrogen bonds to each of its three oxygen atoms. One of the oxygen atoms forms hydrogen bonds to the main chain amides of G139 and D137 and a water molecule, the second oxygen atom bonds to the main chain amide of T141, the hydroxyl atom of T141, and a water molecule, while the third bonds to the side chain oxygen and main chain amide atom of T138. In the GMP complex, these interactions are not as numerous with only the side chain hydroxyl group and the main chain amide nitrogen of T141 being within hydrogen bond distance (2.9 A˚).17 Although the side chain of T138 and the amide main chain nitrogens of T138, D137, and D139 are in the same vicinity, the distances are of the order of 3.6 A˚ and too far to form hydrogen bonds. Thus, the location of

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Table 3. Data Collection and Refinement Statistics PEEG

(S)-HPMPG

PEEHx

111.12 72.59 51.28 P21212 0.4  0.2  0.05

111.28 72.78 50.99 P21212 0.3  0.2  0.05

100 29.63-2.60 0.84 59526 13343 99.5 (99.4) 0.079 (0.179) 9.3 (4.5)

100 37.44-2.78 1.64 68800 10911 99.8 (99.8) 0.134 (0.254) 27.5 (15.6)

A. crystal data unit cell length (A˚) a b c space group crystal dimensions (mm)

111.19 72.75 51.39 P21212 0.4  0.2  0.05

temperature (K) resolution range (A˚) mosaicity (deg) observations unique reflections completeness (%) Rsymb ÆI/σ(I)æ

100 27.78-2.60 0.53 52649 13236 98.7 (99.2)a 0.122 (0.300) 6.8 (3.3)

resolution limits (A˚) Rworkc Rfreed monomers/asymmetric unit atoms/asymmetric unit protein non-H water molecules rmsd from ideale bond lengths (A˚) bond angles (deg) Ramachandran plot most favored (%) additionally allowed (%) generously allowed (%) disallowed (%)

23.32-2.60 24.14 29.49 2

29.63-2.60 24.73 29.83 2

37.44-2.78 21.69 28.44 2

3298 110

3287 168

3297 84

0.009 1.301

0.009 1.269

0.013 1.562

84.9 12.1 1.1 1.9

88.7 9.1 0.5 1.6

B. diffraction data

C. refinement

85.0 11.5 1.1 2.5 a ˚ for PEEG and (S)-HPMPG and 2.85-2.78 A˚ for PEEHx. b Rsym = Ph Pi| Ih,i Values in parentheses are for the outer resolution shell, 2.69-2.60 A P P P ||Fobs| ÆIhæ|/ P h i Ih,i where Ih,i is the intensity of the ith measurement of reflection h and ÆIhæ is the average value over multiple measurements. R = c d |Fcalc||/ |Fobs|. Rwork is calculated based on the reflections used in the refinement (95% of the total data) Rfree is calculated using the remaining 5% of the data. e rmsd = root-mean-square deviation.

the phosphonate group in the ANPs in relation to the purine base is essential for tight binding but it may not contribute to selectivity. The stronger hydrogen bonds may explain, at least in part, the much tighter binding of PEEG than the product of the reaction, GMP (Table 2). In the immGP complex with human HGPRT, there is an interaction between an oxygen atom of the phosphate group and the hydroxyl group of Y104, which is located in the large flexible loop11 (Figure 3f ). However, in the phosphonate complex, this area of the large flexible loop has not moved close enough to the inhibitor for such an interaction to occur. Thus, one explanation for the tighter affinity of the phosphonate inhibitor for Pf HGXPRT, cf. the human HGPRT complex, could be that the loop has closed and, in the Pf HGXPRT complex, the conserved tyrosine residue is now close enough to form a hydrogen bond. The side chain atom (OD1) of D137 is thought to abstract a proton from the N7 atom in the purine moiety as an aid to catalysis.9,11 In the immGP-human HGPRT complex, this side chain is closer to the N7 atom in the purine ring (2.8 A˚) compared to the distances in the phosphonate complex (3.6 A˚) and in the GMP complexed structure (3.7 A˚).17 This difference is as expected given the fact that immGP complex is a mimic of the transition state in the catalytic reaction. Mobile Loop Structure (Residues 100-117). In the free structure of human HGPRT16 and in the structure of the

enzyme complexed with GMP,17 this loop is disordered and flexible. In the presence of the transition state analogue, immGP, Mg2+ and PPi, it becomes fully ordered and closed over the active site (Figure 4). This conformational change results in the tip of the loop moving by up to 25 A˚. When the ANPs bind, this loop also becomes almost completely ordered, adopting a position intermediate between the two extreme cases (Figure 4). In the immGP complex, the structure of this region has two antiparallel β-strands.11 In the presence of the ANPs, the structure is mainly random coil (Figure 4). There is 64% amino acid sequence identity between human HGPRT and Pf HGXPRT in the mobile loop. The totally conserved residues are between 102 and 111. When (S)-HPMPG binds, this loop moves, resulting in the carboxylate group of D107 forming a hydrogen bond via water to one of the oxygen atoms of the phosphonate group, which also interacts with the main chain amide atom of T138. In the PEEG and PEEHx complexes, D107 has also moved closer to the phosphonate group but not close enough for a hydrogen bond network to occur. In the immGP complex, the loop is in a slightly different position and the structure is such that the OD1 side chain atom of D107 is distal from the closest phosphoryl oxygen, being 11 A˚ away.11 The movement of this loop and the interactions so formed between loop amino acid residues and the inhibitor may contribute to the differences in the Ki values for the

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Figure 3. (a) The 2Fo - Fc electron density for (S)-HPMPG in complex with human HGPRT (subunit A) contoured at 1.5 σ. (b) The specific interactions of (S)-HPMPG with human HGPRT active site residues and the large mobile loop. (c,d) Two views of the Connolly surface of human HGPRT showing the location of (S)-HPMPG (drawn as solid spheres). (e) Comparison of the binding modes of (S)-HPMPG, PEEG, and PEEHx to human HGPRT. (f) Structural comparison of (S)-HPMPG and ImmGP bound in the active site of human HGPRT showing the relative position of the phosphonate moiety with that of ribose 50 -phosphate group.

Figure 4. Comparison of the structures of (S)-HPMPG and immGP bound in the active site with that of the structure of the free enzyme. This depicts the position of the large mobile loop, which is disordered in the free enzyme (blue), partially closed when the ANPs bind (green), and the fully closed in the presence of the transition state analogue (magenta).

compounds bound in the active site. Thus, GMP binds more weakly (loop open) than PEEG (loop partly closed), which, in turn, bind more weakly than the immGP (loop fully closed). Purine Binding Site. There are three features of the purine binding site that confer selectivity and affinity. These include

the flap region around residue F186, the 6-oxo pocket (K165), and the surface that accepts the substitution in the 2-position of the purine. When guanine or hypoxanthine is attached to either ribose-50 -phosphate or the ANP phosphonate moiety and binds in the active site, the hydrogen bond between the NZ atoms of K165 and K185 is broken. K165 moves to form a hydrogen bond to the 6-exocyclic oxo atom in the purine ring. It is this bond that confers specificity for the 6-oxopurine bases over adenine. Thus, adenine with an exocyclic amino group will not bind in the active site.13 Purine bases with a 6-exocyclic chloro group bind selectively to PfHGXPRT. For example, kcat/Km(Pf )/kcat/Km(H) is g54 for 6-chloroguanine as a substrate compared with 0.2 with guanine.13 Thus, substitution in the 6-position should confer selectivity. It is proposed that, in this region of the molecule, PfHGXPRT has greater flexibility and thus can better accommodate such substitutions. A similar argument can be advanced for the contribution to the inhibition of the ANPs when the purine base has modifications in the 8-position in the imidazole ring. The purine base bound in the active site engages in a Π stacking interaction with F186 in the immGP and GMP

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complexed structures.11,17 This is also the case in the ANPhuman HGPRT complexes (Figure 3b,e,f ). The Linker Attaching the Phosphonate Group to the Purine Base: Interactions with Human HGPRT. In the free structure, the side chain of K68 occupies the space where PPi binds in the immGP complex. When PPi enters the active site, the side chain of K68 is forced to rotate 180 and, consequently, the peptide bond between L67 and K68 changes from trans to cis.11,16 However, when (S)-HPMPG binds to the free enzyme, the NZ atom of K68 is already positioned to form a hydrogen bond to the hydroxyl group in the linker (Figure 3b,e,f ) while the trans configuration between L67 and K68 remains in place. This extra bond contributes to the increased affinity of the inhibitor compared to that when the hydroxymethyl group is replaced by a hydrogen atom (29 to 6 μM cf. compounds 3 and 11). In PEEG and PEEHx, the linker is one carbon atom longer than in (S)-HPMPG and, when these two compounds bind, rotation around the CE atom occurs so that the NZ atom of K68 now forms a hydrogen bond to the OD1 atom of D134 (3 A˚) (Figure 3e). (S)-HPMPG is also anchored in position by an additional bond, via water to the OE1 atom of E133 (Figure 3b,e,f ). In the immGP complex, this amino acid side chain forms a direct link to the 30 -hydroxyl group of the iminorbital ring, which may contribute to the tighter binding of the transition state analogue. The structure of the HPMPG complex shows that only the S-enantiomer bound when crystallization occurred in the presence of the racemic mixture of HPMPG (Figure 3a,b). The carbon atom to which the -CH2OH group is attached is now only 3.9 A˚ from the OD1 atom of D107 as the long flexible loop (residues 100-117) moves to wrap around the phosphonate inhibitor (Figure 4). In this location, there may not be sufficient room for the -CH2OH group in the R-isomer to be accommodated. The hydroxymethyl group attached to the PME tail changes the location of the K68 side chain, which helps to anchor (S)-HPMPG in the active site. In the absence of this hydroxymethyl group, the shorter PME moiety is not able to reach as far into the ribose 50 -phosphate binding pocket. This is also the case when GMP binds. The weaker interactions in this region may explain why compound 3, with the PME moiety attached to guanine, has a lower affinity for human HGPRT compared to compound 1, where the longer PEE tail is attached to the guanine base. The higher Ki for GMP as compared to the phosphonates and transition state analogues could be due to the active site being open in the GMP bound structure, as GMP is preparing to leave the active site. In contrast, when the phosphonates are bound, the active site is partially closed over by the mobile loop, and for the transition state analogue, this loop completely covers the active site. Effects of ANPs on Pf in Cell Culture and on Human Cells. A previous study showed that the (S)-isomer of compound 11 inhibited the growth of Pf in culture with an IC50 value of 4 μM.14 PMEG (3), cyclic-(S)-HPMPG (15), and cyclic-(R)HPMPG (16) were examined for their ability to arrest the growth of Pf in cell culture. PMEG was selected because it had a low Ki value for PfHGPRT (1.6 μM) but relatively high Ki value for human HGPRT (29 μM). Figure 5 demonstrates that PMEG was toxic to the parasite with an IC50 of 14 μM. Cyclic-(S)-HPMPG and cyclic-(R)-HPMPG exhibited IC50 values of 1 and 46 μM, respectively. Cyclic-HPMP derivatives contain internal ester bonds and are considered to be prodrugs of HPMP compounds. These intramolecular

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Figure 5. Inhibitory effect of the PMEG (triangles) on in vitro cultivated P. falciparum clone Dd2 in comparison to parasites grown in the absence of inhibitor (circles). Data are presented as mean ( SD of two independent experiments. Parasites grown in the presence of PMEG (46-174 μM) for 48 h exhibit cellular damage and stunted development compared to control parasites grown in the absence of drug (inset).

esters have a reduced negative charge, and therefore their transport into the cell is expected to be increased. The sixmembered ring should be easily hydrolyzed inside the cell to form free phosphonate. The much greater effect of the Sisomer is consistent with the observation that only the (S)isomer of HPMPG binds in the active site of human HGPRT and, presumably, to PfHGXPRT. This result is fully consistent with the hypothesis that inhibition of HGXPRT is responsible for toxicity to the parasite but further evidence is required to fully establish this mechanism. It is recognized that other factors (transport across membranes, metabolism, efflux) will be important in vivo. The IC50 values for proliferation of human A549 cells were 489 μM [cyclic-(S)HPMPG], >1000 μM [cyclic-(R)-HPMPG], and 17 μM [PMEG]. Thus, the ratio between the antiplasmodial and cytotoxic concentration was 489 and >20 for cyclic-(S)HPMPG and cyclic-(R)-HPMPG, respectively. The parasite-selective toxicity of (S)-HPMPG is also consistent with the proposed mechanism and encouraging for further work. Conclusion The ANPs are the first compounds that have been shown to selectively inhibit Pf HGXPRT compared to human HGPRT. Three of these compounds arrest the growth of P. falciparum in erythrocyte culture, thus validating our approach in targeting this enzyme for the development of antimalarial chemotherapeutics. Analysis of the crystal structure of human HGPRT in complex with three of these inhibitors has contributed to the understanding of how these molecules bind in the active site, laying the foundation for the design and discovery of improved drug leads. Experimental Section Synthesis of ANPs and Enzyme Preparation. Acyclic nucleoside phosphonates were prepared as described by Hol y and his colleagues.18-24 All ANPs were characterized by 1H NMR, 13C NMR, melting point, and mass spectrometry. Their purity was determined by combustion elemental analysis (C, H, N) and volumetric analysis methods (P, S, Br). The purity of all the ANPs was >95%. Recombinant Plasmodium HGXPRT and human HGPRT were expressed and purified as previously described.8 The recombinant human HGPRT has three of the four cysteine residues (C22, C105, C205) replaced by alanine to stabilize the enzyme. The kinetic and structural properties of this enzyme are identical to wild-type human HGPRT.8,16

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Determination of Ki Values. The Ki values were determined using a spectrophotometric assay at 25 C.8 Assays were performed in two different buffer systems: 0.1 M Tris-HCl, 10 mM MgCl2, pH 7.4 (reflecting physiological conditions) and 0.1 M Tris-HCl, 110 mM MgCl2, pH 8.5 (where both enzymes are maximally active).8 For human HGPRT at pH 8.5, the specific activities for guanine and hypoxanthine were 46 and 27 μmol min-1 mg-1 of protein, respectively.8 At pH 7.4, these values were 34 and 19.8 Pf HGXPRT had significantly lower kcat values compared with human HGPRT under these two conditions, with specific activities for guanine and hypoxanthine at pH 8.5 being 5.4 and 1.4 μmol min-1 mg-1 of protein, respectively, while at pH 7.4, these values were 1.5 and 0.77 μmol min-1 mg-1 of protein.8 The Ki values are Ki(app), as they were measured at a single concentration of the second substrate. They approximate Ki values because the concentration of the second substrate (guanine) was saturating: 60 μM, approximately 30-fold higher than the measured Km. The concentration of PRib-PP was in the range 16-1204 μM, depending on the value of Km(app) at that concentration of inhibitor. The concentration of inhibitor ranged from 1 μM (for the best inhibitors) to 220 μM (for the weakest). Ki values were determined by Hanes’ plots and calculated using Prism4 (GraphPad Software, Inc., La Jolla, CA). The inhibitors were found to be competitive inhibitors of PRib-PP. In some instances, Ki(app) was calculated using the equations v=Vmax[S]o/[S]o +Km(app) and Km(app) =Km(1+ [I]/ Ki(app)). Crystallization. Crystals were obtained using the hanging drop vapor diffusion method. The well solution consisted of 0.1 M citrate pH 5.5, 10% isopropyl alcohol and 29% PEG 4000. The concentration of protein was 17 mg mL-1, and the concentration of inhibitor was 3.3 mM for PEEG, 3.9 mM for (RS)-HPMPG, and 3.0 mM for PEEHx. The drop consisted of an equal volume of well solution and protein inhibitor complex. For cryocooling, the crystals were transferred to a solution that contained well solution, 15-20% glycerol and 1.5-2 mM inhibitor. All X-ray data for the PEEG and (RS)-HPMPG complexes were collected using an FR-E X-ray generator and CCD detector at the University of Queensland, while data for PEEHx were collected at the Australian Synchotron in Melbourne, Australia.25 Data for PEEG and (RS)-HPMPG were merged and scaled using Crystalclear.26 Data for PEEHx were merged and scaled using HKL2000.27 The structures were solved by molecular replacement in AMoRe28 using the free human structure (1Z7G)16 as the search model. Refinement, model building and incorporation of the ligands was undertaken using WinCoot29 and Refmac5.30,31 In Vitro Inhibition of P. falciparum Growth in Tissue Culture. Laboratory-adapted Plasmodium falciparum isolate Dd2 was maintained in erythrocyte culture for 48 h according to the method of Hamzah et al.32 The in vitro efficacy of each ANP against Dd2 was determined by assessing parasitaemia after a 48 h period. Blood smears were prepared from each culture and stained with Giemsa, and smears were examined using light microscopy and the level of parasitemia determined.33 Cytotoxicity Studies. Serial dilutions of the compounds were added to semiconfluent cultures of human lung carcinoma A549 cells. The cells were incubated at 37 C and cell proliferation quantified by a protein staining assay using sulforhodamine B.34 The IC50 (compound concentration causing 50% inhibition of cell proliferation) was calculated by extrapolation.34

Acknowledgment. We acknowledge the support of the Australian Synchrotron, Victoria, Australia, and the University of Queensland Remote-Operation Crystallization and X-ray Diffraction Facility (UQROCX) for data collection. The views expressed herein are those of the authors and are not necessarily those of the owner or operator of the

Keough et al.

Australian Synchrotron. The synthesis of ANPs was performed as a part of research project OZ4055905 of the Institute of Organic Chemistry and Biochemistry and was supported by the Programme of Targeted Projects of the Academy of Sciences of the Czech Republic (no. 1QS400550501) and Centre for New Antivirals and Antineoplastics (1M0508). This study was supported by funds from the National Health and Medical Research Council, Australia, grant no. 569703. References (1) Hay, S. I.; Guerra, C. A.; Tatem, A. J.; Noor, A. M.; Snow, R. W. The global distribution and population at risk of malaria: past, present, and future. Lancet Infect. Dis. 2004, 4, 327–336. (2) Talisuna, A. O.; Bloland, P.; D0 Alessandro, U. History, dynamics, and public health importance of malaria parasite resistance. Clin. Microbiol. Rev. 2004, 17, 235–254. (3) Ting, L.-M.; Shi, W.; Lewandowiez, A.; Singh, V.; Mwakingwe, A.; Birck, M. R.; Ringia, E. A. T.; Bench, G.; Madrid, D. C.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Schramm, V. L.; Kim, K. Targeting a novel Plasmodium recycling pathway with specific immucillins. J. Biol. Chem. 2005, 280, 9547–9554. (4) Dawson, P. A.; Gordon, R. B.; Keough, D. T.; Emmerson, B. T. Normal HPRT coding region in a male with gout due to HPRT deficiency. Mol. Genet. Metab. 2005, 85, 78–80. (5) Deo, S. S.; Tseng, W. C.; Saini, R; Coles, R. S.; Athwal, R. S. Purification and characterization of Escherichia coli xanthineguanine phosphoribosyltransferase produced by plasmid pSV2gpt. Biochim. Biophys. Acta 1985, 839, 233–239. (6) Vos, S.; de Jersey, J.; Martin, J. L. Crystal structure of Escherichia coli xanthine-phosphoribosyltransferase. Biochemistry 1997, 36, 4125–4134. (7) Queen, S. A.; van der Jagt, D.; Reyes, P. Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 1982, 30, 123–134. (8) Keough, D. T.; Ng, A. L.; Winzor, D. J.; Emmerson, B. T.; de Jersey, J. Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme. Mol. Biochem. Parasitol. 1999, 98, 29–41. (9) Xu, Y.; Eads, J.; Sacchettini, J. C.; Grubmeyer, C. Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase. Biochemistry 1997, 36, 3700–3712. (10) Li, C. M.; Tyler, P. C.; Furneaux, R. H.; Kicska, G.; Xu, Y.; Grubmeyer, C.; Girvin, M. E.; Schramm, V. L. Transition state analogs as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases. Nat. Struct. Biol. 1999, 6, 582– 587. (11) Shi, W.; Li, C. M.; Tyler, P. C.; Furneaux, R. H.; Grubmeyer, C; Schramm, V. L.; Almo, S. C. The 2.0 A˚ structure of human hypoxanthine-guanine phosphoribosyltransferase with a transition-state analog inhibitor. Nat. Struct. Biol. 1999, 6, 588– 593. (12) Shi, W.; Li, C. M.; Tyler, P. C.; Furneaux, R. H.; Cahill, S. M.; Girvin, M. E.; Grubmeyer, C.; Schramm, V. L.; Almo, S. C. The 2.0 A˚ structure of malarial purine phosphoribosyltransferase in complex with transition-state analogue inhibitor. Biochemistry 1999, 38, 9872–98780. (13) Keough, D. T.; Skinner-Adams, T.; Jones, M. K.; Ng, A. L.; Brereton, I. M.; Guddat, L. W.; de Jersey, J. Lead compounds for antimalarial chemotherapy: purine base analogs discriminate between human and P. falciparum 6-oxopurine phosphoribosyltransferases. J. Med. Chem. 2006, 49, 7479–7486. (14) De Clercq, E.; Holy, A. Acyclic nucleoside phosphonates: A key class of antiviral drugs. Nat. Rev. Drug Discovery 2005, 4, 928– 940. (15) Smeijsters, L. J. J. W.; Franssen, F. F. J.; Naesens, L.; de Vries, E.; Holy, A.; Balzarini, J.; de Clercq, E.; Overdulve, J. P. Inhibition of the in vitro growth of Plasmodium falciparum by acyclic nucleoside phosphonates. Int. J. Antimicrob. Agents 1999, 12, 53–61. (16) Keough, D. T.; Brereton, I. M.; de Jersey, J.; Guddat, L. W. The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle. J. Mol. Biol. 2005, 351, 170–181. (17) Eads, J.; Scapin, G.; Xu, Y.; Grubmeyer, C.; Sacchettini, J. C. The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP. Cell 1994, 78, 325–334.

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