Inhibition of the Escherichia coli 6-Oxopurine

Article pubs.acs.org/jmc

Inhibition of the Escherichia coli 6‑Oxopurine Phosphoribosyltransferases by Nucleoside Phosphonates: Potential for New Antibacterial Agents Dianne T. Keough,† Dana Hocková,‡ Dominik Rejman,‡ Petr Špaček,‡ Silvie Vrbková,‡ Marcela Krečmerová,‡ Wai Soon Eng,† Harmen Jans,§ Nicholas P. West,† Lieve M. J. Naesens,§ 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 ‡

ABSTRACT: Escherichia coli (Ec) cells possess two purine salvage enzymes: xanthine-guanine phosphoribosyltransferase (XGPRT) and hypoxanthine phosphoribosyltransferase (HPRT). EcXGPRT shares a common structural feature with other members of this family, a flexible loop that closes over the active site during catalysis. The replacement of six of these amino acids by alanine has no effect on the Km for the two substrates. However, the Ki for the nucleoside monophosphate increases by 27-fold, and the kcat is reduced by ∼200-fold. Nucleoside phosphonates (NP) are good inhibitors of EcXGPRT and EcHPRT, with Ki values as low as 10 nM. In the absence of the flexible loop, these values increase by 5- to 30-fold, indicating the importance of the loop for high-affinity inhibition. Crystal structures of two NPs in complex with EcXGPRT explain the tight binding. Prodrugs of NPs with low Ki values for EcXGPRT or EcHPRT exhibit IC50 values between 5 and 23 μM against Mycobacterium tuberculosis in cell-based assays, suggesting that these compounds are therapeutic leads against pathogenic bacteria.

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INTRODUCTION Escherichia coli cells possess two 6-oxopurine phosphoribosyltransferases (PRTases): xanthine-guanine phosphoribosyltransferase (EcXGPRT) and hypoxanthine PRTase (EcHPRT). EcXGPRT and EcHPRT catalyze the Mg2+-dependent transfer of the ribosyl-5-phosphate group from 5-phospho-α-D-ribosyl1-pyrophosphate (PRib-PP) to the N9 position of guanine, hypoxanthine, or xanthine to yield the corresponding nucleoside monophosphate (GMP, IMP, or XMP, respectively) and pyrophosphate (PPi) (Figure 1). As signified by their names, EcXGPRT and EcHPRT have different specificities for the naturally occurring purine bases. EcXGPRT has a strong preference for guanine, with a kcat/Km of 6.5 μM−1 s−1, whereas for xanthine this value is 1.2 μM−1 s−1. Hypoxanthine is a weak substrate, having a kcat/Km value of 0.2 μM−1 s−1.1 In comparison to EcXGPRT, EcHPRT prefers hypoxanthine. The kcat/Km for this substrate is 4.9 μM−1 s−1, and both guanine and xanthine are weak substrates with kcat/Km values of 0.03 and 0.0003 μM−1 s−1, respectively.1 This enzyme is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both guanine and xanthine. A number of structures of EcXGPRT have been determined in the absence or presence of substrates or products. In the © 2013 American Chemical Society

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

crystal structure of the free enzyme, sulfate and magnesium ions are observed in the active site.2 In this case, the amino acid residues between 61-SSYDHDNQRELK-72 have poor electron density, suggesting that this area of the enzyme is flexible. In complex with the inactive analog of PRib-PP (1-α-pyrophosphoryl-2-α,3-α-dihydroxy-4-β-cyclopentane-methanol-5-phosphate, cPRPP)·guanine·Mg2+, or with IMP or GMP, the loop Received: May 26, 2013 Published: August 8, 2013 6967

dx.doi.org/10.1021/jm400779n | J. Med. Chem. 2013, 56, 6967−6984

Journal of Medicinal Chemistry

Article

Scheme 1

phosphate synthesis.8 Although purine metabolism is poorly understood in TB, it is known that the purine salvage pathway is used to synthesize IMP and GMP after the uptake of hypoxanthine and guanine from its environment. As a result, HGPRT has been suggested as a target for the development of new anti-TB therapies.9 This enzyme has been purified to homogeneity10 and appears to be more similar to EcHPRT in its amino acid sequence identity and in substrate specificity (i.e., xanthine is also not a substrate). In this report, we have identified a number of novel nucleoside phosphonate (NP) inhibitors of the two E. coli 6oxopurine PRTases. Using site-directed mutagenesis, we have also shown that the flexible loop is important for catalysis and for tight binding inhibition of the NPs. Crystal structures of two NPs in complex with EcXGPRT have been determined. These data provide a basis for the design of more potent inhibitors and explain the structural basis for the measured Ki values. Docking studies using these crystal structures elucidate reasons for the differences observed in the affinity for related NPs.

becomes visible, although it is not completely closed over the active site.3 XGPRT is one of the smallest of this group of enzymes (subunit consists of 152 amino acids), but the movement of this flexible loop during catalysis is a common structural feature for this class of enzyme. It has been hypothesized that the role of this loop is to help stabilize the transition state.4 A second proposition is that the closure of this loop can also occur when inhibitors bind and this could be an important element contributing to the low Ki values. Here, the function of this flexible loop has been evaluated by replacement of the six amino acids (HDNQRE, residues 65−70) located at the top of the loop by a single alanine residue. Mammalian cells possess two pathways for the synthesis of the essential 6-oxopurine nucleosides monophosphates: de novo (from small molecules) and salvage (of purine bases). However, clinically important parasites from the genera Plasmodium5 and the bacterium Helicobacter pylori6 do not possess the necessary enzymes for de novo synthesis and rely on the transport of preformed purine bases from the host cell. These differences in metabolism between the infectious agent and their host cell can be exploited in the design of drugs aimed at combating these diseases. As these organisms have only one pathway for the synthesis of the purine nucleoside monophosphates needed for DNA/RNA production, they depend on the activity of their 6-oxopurine PRTases for both reproduction and survival. This strategy is beneficial to the invading organism because the need for the energetically expensive de novo pathway is circumvented. The parasitic 6-oxopurine PRTases have been shown to be effective drug targets with inhibitors of Plasmodium falciparum HGXPRT demonstrating antimalarial activity.7 Although E. coli cells contain two 6-oxopurine PRTases, most parasitic cells and mammalian cells contain only one such enzyme. The genome of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is said to possess genes coding for the enzymes of both pathways for purine nucleoside mono-

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RESULTS

Chemical Synthesis. Pyrrolidine derivatives of guanine (compounds 1 and 3) were synthesized according to previously described procedures.11 Hydroxypyrrolidine derivatives of guanine (5 and 7) were also synthesized according to previously described procedures.12 Hydroxymethylpyrrolidine derivative 9 was prepared according to Vaněk et al.13 Pyrrolidine phosphonic acids 2, 4, 6, 8, and 10 were prepared by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)catalyzed condensation of pyrrolidine derivatives of guanine 1, 3, 5, 7, and 9 with diisopropyl 2-phosphonoacetic acid followed by removal of the isopropyl esters by Me3SiBr (Scheme 1). The synthesis and characterization of all other compounds (11−20) in this study has been previously reported (Tables 2 and 3). 6968



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Table 1. Comparison of the Km (μM), kcat (s−1), and kcat/Km (s−1 μM−1) Values for EcXGPRT, mrEcXGPRT, and EcHPRT Ga

Xb

Hxc

PRib-PPd

GMP

90.8 ± 11.3 NDe 12.5 ± 2.4

64.3 ± 3.1 61.0 ± 6.0 192 ± 7.0

Ki (μM) 4.5 ± 0.3 120 ± 10 526 ± 30

13.7 ± 1.1 NDe 59.0 ± 3.5

28 ± 2 0.14 ± 0.02 50.0 ± 2

0.2 NDe 4.9

0.20 0.002 0.26

Km (μM) 4.3 ± 0.3 1.7 ± 0.2 294 ± 13

30.5 ± 2.6 30.3 ± 0.6 25 ± 6.1

EcXGPRT mrEcXGPRT EcHPRT

28.4 ± 0.4 0.14 ± 0.02 10.2 ± 0.7


6.5 0.08 0.03

37.5 ± 7 0.11 ± 0.03 0.008 ± 0.0001 kcat/Km (s−1 μM−1) 1.2 0.004 0.0003


kcat (s−1)

a

G, guanine. bX, xanthine. cHx, hypoxanthine. dKm values were determined using guanine as the purine base. eAccurate Km data could not be obtained for mrEcXGPRT because the kcat value is too low to allow precise measurements.

Mass Spectrometry for E. coli XGPRT and the Mutant Recombinant E. coli XGPRT. The experimental mass of EcXGPRT was determined to be 16 839.4 Da, in agreement with the calculated mass of 16 839.5 Da. The experimental mass of a mutant recombinant EcXGPRT (mrEcXGPRT), where six of the amino acid residues from the flexible loop have been deleted, was determined to be 16 018 Da, compared with the calculated mass of 16 017.5 Da. Together with sequencing of the cDNA coding for the mutant enzyme, this data confirmed that the amino acids deleted in mrEcXGPRT by mutagenesis are HDNQRE (residues 65−70) and that these residues have been replaced by a single alanine residue. Comparison of the Kinetic Constants for E. coli XGPRT and the Mutant Recombinant E. coli XGPRT. The flexible loop that moves over the active site is smaller in EcXGPRT compared with that of EcHPRT and human HGPRT. In EcXGPRT, this loop is composed of only 12 amino acids, whereas in the EcHPRT and human HGPRT enzymes, it is ∼20 amino acids. The determination of the length of this flexible loop is based on amino acid sequence alignment and the absence of electron density in the published crystal structures.1,14 This difference in the length of the largest flexible loop may be correlated to the overall size of the subunits (152 amino acids in EcXGPRT; 182 in EcHPRT, and 217 in human HGPRT). The kinetic constants for the substrates and products of EcXGPRT, mrEcXGPRT, and EcHPRT are given in Table 1. The purine bases and PRib-PP bind to EcXGPRT and mrEcXGPRT with similar Km values, indicating that the six amino acid residues removed have no influence on the binding of these two substrates (Table 1). The most significant effect of the removal of these residues is the increase in the Km value for the nucleoside monophosphate product of the reaction and in the kcat value for the forward reaction (Table 1). To understand why the Km value for PRib-PP is unchanged but the Ki value for GMP is increased, the structures of EcXGPRT in complex with the stable carbocyclic analog of PRib-PP (cPRPP) and guanine· Mg2+ (PDB code 1A953) or with GMP (PDB code 1A973) were aligned (Figure 2). In the presence of cPRPP and guanine or GMP, the loop is partially open to allow the substrates to enter the active site or for GMP to exit (Figure 2).3 The flexible loop is not located close to the purine binding pocket, so it would not be expected to affect the binding of this substrate and, as found, the Km is unchanged (Table 1).

Figure 2. Binding modes of guanine·cPRPP·Mg2+ and GMP with EcXGPRT. EcXGPRT is shown as a Connolly surface, cPRPP, guanine, and GMP are drawn as stick models, and Mg2+ is drawn as a pink sphere. The invariant SY residues are in yellow, the residues in the loop that was removed by site-directed mutagenesis are in green, the tryptophan residue that forms the π-stacking arrangement with the purine base is in pink, and the amino acids in the PPi binding pocket are in light brown. The guanidinium group of R69 is in red.

There are two interactions between GMP and the amino acid residues in the flexible loop. These are hydrogen bonds between the guanidino NE2 atom of R69 and one of the phosphoryl oxygens (O3) in GMP (2.6−2.9 Å) as well as between the NE1 atom of R69 and a second phosphoryl oxygen atom (O2) (3.1−3.3 Å). These hydrogen bonds are also found in the EcXGPRT·cPRPP·guanine·Mg2+ complex (2.6−2.9 and 2.9−3.3 Å). The cPRPP, however, does have additional bonds to active site residues through the α- and βphosphate groups. These are through the O1 atom of cPRPP to Mg2+ (2.2 Å) (Figure 2). This ion is held in place by a further three bonds: to the O1β (2.5 Å) and to the ribose hydroxyl groups (2.2 and 2.9 Å). The three oxygen atoms of the βphosphate group form bonds to the amide of R37 (O1; 2.9 Å), to Mg2+ and amide of G38 (O2; 2.5 and 3.1 Å, respectively), and to the NE2 atom of R37 and the amide of R37 (O3; 3.2 and 3.1 Å, respectively) (Figure 2). In mrEcXGPRT, six residues, including R69, have been removed. The crystal structures suggest that removal of these six residues, in particular R69, should impair the ability of the enzyme to anchor the 5′-phosphate group in position (Figure 2). This reduction in affinity is observed empirically as an increase in the Ki value for GMP (4.5 cf. 120 μM; Table 1). However, when PRib-PP binds, there is little difference in the Ki 6969



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Table 2. Inhibitors of EcXGPRT and mrEcXGPRT

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Table 2. continued

a

In the presence of 33 μM of PPi. bND, not determined

Inhibitors of the E. coli XGPRT, Mutant Recombinant E. coli XGPRT, and E. coli HPRT. A number of NPs were investigated as potential inhibitors of EcXGPRT, mrEcXGPRT (Table 2), and EcHPRT (Table 3). These compounds are analogs of the nucleoside monophosphate product of the reaction. Both IMP and GMP are competitive inhibitors with PRib-PP for EcXGPRT and EcHPRT.1 However, the Ki values for the nucleotide product of the reaction are very different for these two enzymes. The Ki value for GMP for EcXGPRT is 4.5 μM, whereas the Ki value for IMP for EcHPRT is 294 μM.1

values for mrEcXGPRT and EcXGPRT (64.3 cf. 61.0 μM). This may be due to the 5′-phosphate group in PRib-PP adopting a slightly different orientation in the 5′-phosphate binding pocket compared to GMP (i.e., there is a 5° rotation and a 0.2 Å translation of the phosphoryl moiety after the superimposition of the two protein structures). This movement, therefore, appears to reduce the influence of R69 and the nearby flexible loop residues. The additional interactions between the α- and β-phosphates of PRib-PP and the amino acid residues in the active site (Figure 2) help anchor this substrate in place, resulting in an unchanged Km for this substrate. 6971



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Table 3. Inhibitors of E. coli HPRT

a

Noncompetitive with PRib-PP.

attached to the base is 2-(phosphonoethoxy)ethyl (PEE). Group 2 contains a hydroxyl attachment to the linker. The length of the linker is the same as in group 1. The difference is that the oxygen atom is closer to the phosphonate group by one carbon. The crystal structures of human HGPRT in complex with PEEG or PEEHx suggested the design of new compounds (group 3).7c This chemical modification is the addition of a second phosphonate group to the linker. It was proposed that this second group could occupy the PPi binding site, increasing affinity. Group 4 contains a five-membered ring (pyrrolidine) in

Thus, it could be expected that the NPs would bind much more weakly to EcHPRT than to EcXGPRT. The NPs in this study have been placed into four categories depending on their chemical structure. These inhibitors have two common features: a 6-oxopurine base and a phosphonate group. Their structural differences lie in the nature of the linker connecting these two groups and the moieties attached to this linker. Group 1 contains a purine base linked by a series of carbon atoms and a single oxygen atom to the phosphonate moiety. These compounds have previously been investigated as inhibitors of the human and Pf enzymes.7c The chemical moiety 6972



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Thus, the strategy of adding a second phosphonate group to increase affinity has been successful. Although compound 19 is a very good inhibitor, it does not bind as tightly as compound 17. This may be because there is one extra carbon in the phosphonate linker and/or the different chemical structure of the second phosphonate group attached to the linker. Alternatively, it may also be due to the positioning of the oxygen atom in the second phosphonate moiety. These bisphosphonate ANPs also bind much more weakly to the mutant enzyme, providing further evidence for the contribution of the loop residues to binding. If the second phosphonate group is attached to the carbon adjacent to the N9 atom of the purine ring, the Ki values for EcXGPRT become higher (compound 15, 16, and 18, Table 2) compared to compound 17. This may be because in this location the second phosphonate group is too far from the PPi binding pocket to make strong binding. Group 4. This group of NPs contain a five-membered ring to confer rigidity to the linker and possibly tighter binding. With the inclusion of the five-membered ring in the linker, these NPs more closely resemble the structure of GMP, IMP, or the transition state analogs (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (ImmGP) and (1S)-1(9-deazahypoxantnine-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5phosphate (ImmHP). Compounds 2 and 8 (Table 2) are competitive inhibitors with PRib-PP for EcXGPRT. Both compounds 2 and 8 are the (S)-isomers, but compound 8 has a hydroxyl group attached to the five-membered ring. Compound 4, the (R)-isomer of compound 2 (Table 2), binds very weakly if at all to EcXGPRT (Ki ≥ 200 μM). Apart from the different stereocenters, the only other difference is that hypoxanthine replaces guanine as the base. However, on the basis of the Ki data for the compounds in groups 1−3 (Table 2), it is unlikely that the change in the base would make such a significant contribution to the decrease in the Ki values. Thus, the abolition of affinity is likely to be due to the fact that the (R)-isomer cannot fit into the active site. Compound 6 is an alternative stereoisomer of compound 8. This compound also does not bind to EcXGPRT. Thus, again the correct stereochemistry is critical to binding. Compound 10 is strictly competitive with PRib-PP, but the affinity for EcXGPRT has not been increased compared with that of compound 2. The expectation was that the second phosphonate group could occupy the pyrophosphate binding site. In the absence of a crystal structure, docking studies were employed to try to explain this result (described later). Compound 4 is the only compound in this series that contains hypoxanthine as the base. Although not a potent inhibitor of EcHPRT (Ki = 35 μM), it does bind more tightly to EcHPRT than IMP, showing that this difference is productive in increasing affinity. Crystal Structures of EcXGPRT in Complex with Two Pyrrolidine Phosphonates. The pyrrolidine phosphonates are a new class of inhibitor of the 6-oxopurine PRTases. To understand their mode of binding, the crystal structures of compounds 2 and 8 in complex with EcXGPRT were determined. The overall fold of the enzyme when these two compounds are bound is similar. Compound 2 occupies two of the four subunits that compose the active tetrameric structure. The other two subunits are blocked by crystal contacts, preventing this compound from binding. This is the same as occurs in the EcXGPRT·GMP crystal structure. In contrast, compound 8 is bound to all of the subunits in the asymmetric

the linker. This could confer a more rigid structure, allowing tighter binding. Group 1. EcXGPRT is not highly discriminatory between these two ANPs whose only difference is the purine base, hypoxanthine, or guanine (Ki values are 1.5 and 0.7 μM, respectively; Table 2). Removal of the six residues in the flexible loop increases the Ki values of these ANPs by factors between 7- and 30-fold, suggesting a role for these amino acid residues in the effective binding of these inhibitors. Compound 11 (Table 2), containing guanine as the base, did not inhibit EcHPRT activity even at a concentration of 0.2 mM. However, when hypoxanthine replaces guanine as the base, the Ki is 17 μM, a 15-fold decrease in the Ki value compared with IMP. This demonstrates the strong preference of EcHPRT for compounds containing Hx as the base. Group 2. Compound 13 (Table 2) is a good inhibitor of EcXGPRT, but it is not a competitive inhibitor with PRib-PP (decreased Vmax). However, when 33 μM of PPi is added to the assay mixture, strict competitive inhibition was observed and the Ki value is 0.32 μM. For EcHPRT, compound 13 (Table 3) is a very weak inhibitor with a Ki value >700 μM. However, when PPi (33 μM) is added to the assay mixture together with this compound, the Ki is reduced to 35 μM. This is a low Ki given that it represents a 15-fold decrease compared with GMP (35 cf. 526 μM). This suggests that PPi assists in orientating this ANP in the active site and that the presence of PPi contributes to the reduced Ki value. It is proposed that this may occur by hydrogen bonding between phosphoryl oxygen atoms of PPi and the hydroxyl group attached to the linker. Although PPi is a competitive inhibitor for the 6-oxopurine PRTases, no inhibition occurs at the concentration used in the assay, so the inhibition cannot be attributed to PPi itself. Compound 14 (same phosphonate moiety as compound 13 but with hypoxanthine instead of guanine) is, however, completely competitive for EcXGPRT (Table 2). This is surprising because guanine is the purine base of choice for EcXGPRT. Therefore, compound 13 would be the one expected to bind with a higher affinity. The Ki value for compound 14 is, however, higher than that for compound 13 (∼50-fold), although this difference is greater than for the PEEG and PEEHx compounds (compounds 11 and 12, Table 2). This is the expected result as, for EcXGPRT, guanine is the preferred base. However, for EcHPRT, this compound is also noncompetitive, although the calculated Ki is ∼10-fold lower than when guanine is the base. This data suggests that in these NPs the chemical nature of the linker has a significant influence on how the purine-base moieties are located in the active site. For mrEcXGPRT, these NPs do not inhibit at the concentration used in the assay. This again suggests that the residues in the flexible loop play a crucial role in binding. Group 3. The effect of PPi on the binding of compound 13 to the bacterial enzymes (Tables 2 and 3) suggested the design of new ANPs containing groups that could decrease the Ki values. The hypothesis was that this could be accomplished by the covalent attachment of a second phosphonate group to partially mimic the presence of pyrophosphate. The attachment of the second phosphonate moiety was successful in increasing the affinity for EcXGPRT. Compound 17 has the lowest Ki value, 10 nM, for all of the ANPs toward EcXGPRT. Replacing guanine with hypoxanthine (compound 20, Table 3) also makes this ANP a good inhibitor of EcHPRT. The Ki value of 0.8 μM is the lowest value obtained for this enzyme. This is a reduction of ∼310-fold compared with IMP. 6973



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Favorable chemical attachments to the ring or linker could also lower the Ki values. In the EcXGPRT·compound 2 complex, there is a hydrogen bond between the carbonyl oxygen atom in the linker and the OG1 atom of T96 (2.65 Å). However, this only occurs in subunit C; in subunit B, this distance is 3.7 Å (Figure 5). In the EcXGPRT·compound 8 complex, there are hydrogen bonds between the carbonyl oxygen atom in the linker and the OG1 atom of T96 in three subunits (A, 2.96 Å; H, 2.9 Å; and I, 2.73 Å). In the other five subunits, this distance is 3.2 and 3.5 Å. Thus, this interaction does make a contribution to the affinity of compounds 2 or 8 for EcXGPRT, but it is not strong. For compounds 2 and 8, this is the only polar interaction that the linker makes with the active-site amino acid residues. The hydroxyl group attached to the fivemembered ring of compound 8 does not make any direct interactions with the enzyme and does not contribute to its affinity. Thus, for these structures, the most important function of the linker is to place the purine ring and the phosphonate group in position. Purine Ring. The purine ring in the EcXGPRT·compound 2 and EcXGPRT·compound 8 complexes forms a π-stacking arrangement with W134. However, there is a subtle difference between these two complexes in that for compound 8 the aromatic ring of W134 is slightly tilted away from its usual position with respect to the purine ring. In the EcXGPRT·compound 2 complex, the purine ring is held in position by only two hydrogen bonds. These are N1 to the carbonyl oxygen of I135 (2.9−2.7 Å) and the 6-oxo group to the amide nitrogen of I135 (2.6−2.7 Å). The exocyclic amino group in position 2 in the ring does not interact with any active-site residues and thus, for this NP, it is likely that changing the base (i.e., hypoxanthine for guanine) will have no effect on the Ki values. In contrast, the purine ring of compound 8 is held in place by a network of interactions. These are N1 to the carbonyl oxygen of I135 (2.5 Å), the exocyclic amino group to the carbonyl oxygen of I135 (2.8 Å) and to a water molecule, and the 6-oxo group to the amide of I135 (2.9 Å). This is a similar arrangement as that found for the purine base when GMP is bound. Thus, for this compound, the replacement of guanine by hypoxanthine would be expected to change the Ki in a similar way that occurs for the Km for guanine compared with hypoxanthine (4.3 cf. 64.3 μM; Table 1). The 6-oxo group of compound 8 also forms a hydrogen bond with the side chain of K115 (2.5 Å). The 6-oxo atom of the purine ring in compound 2, however, is located too far away from K115 to form such interactions (4.1 Å). Thus, the purine ring of compound 8 is bound more firmly in the active site than compound 2. 5′-Phosphate Binding Pocket. There is a network of hydrogen bonds that connect the three phosphoryl oxygen atoms to the active site. There are only three such bonds between the phosphoryl oxygen atoms and the active site residues in the EcXGPRT·compound 8 complex compared with 10 for the EcXGPRT·compound 2 complex. This is no doubt a contributing factor for explaining why the Ki value for compound 8 is higher than that for compound 2. The contact distances for this region of both complexes are highlighted in Table 5. Magnesium Ions. Magnesium ions are present in the unliganded structures of EcXGPRT and EcHPRT. Their role has been suggested to prepare the active site for catalysis.1 Magnesium ions are also present when cPRPP is bound to EcXGPRT,3 suggesting that they help to orient PRib-PP for

unit (Figure 3). The crystallographic statistics for these structures are given in Table 4.

Figure 3. Tetrameric structure of EcXGPRT showing compound 8 bound to each of the subunits.

One difference between the EcXGPRT·compound 2 complex and the EcXGPRT·compound 8 complex is found between residues S61−D80 (Figure 4). This region of the enzyme contains the residues deleted in mrEcXGPRT, H65−L70. In the complex with compound 8, the majority of these residues are disordered and not visible (Table 4). In comparison, these residues are ordered and partially closed over the active site when compound 2 is bound. This suggests that this flexible region does not play a role in anchoring compound 8 in position but is important for compound 2. For compound 2, in both subunits that are occupied by the ligand, the residues between S61 and H65 form part of a β strand that extends to V55. This is joined to a second β strand (residues Q68−K72) by a random coil (66−67). Subunit B has the same structure except that the second sheet extends to R76. The interpretation of this result is that when the residues between S61 and K72 are not involved in binding the inhibitor, the Ki values will be higher than when they are involved. This deduction is verified by the kinetic constants, with compound 8 having a Ki value of 2.2 μM, whereas the Ki value for compound 2 is 0.23 μM. The Ki value for compound 2 increases 10-fold when these loop residues are absent, as in mrEcXGPRT (0.23 cf. 3 μM; Table 2). This confirms the role of the flexible loop residues as observed in the crystal structure. Conversely, there is no change in the Ki value for compound 8 (2.2 cf. 3 μM; Table 2) in the mutant enzyme, confirming the structural data that indicates that this region is not utilized in the binding of this compound. Contribution to Binding of the Five-Membered Ring and the Linker Between the N9 Atom of the Purine Base and the Phosphonate Group. Two of the essential elements in the chemical structure of the NPs are the purine ring and the phosphonate group that occupies the 5′-phosphate binding pocket (Figure 5). For potent binding, these have to be optimally orientated in the active site. The five-membered ring in the linker of compound 2 or 8 occupies different positions in the active site in the crystal structures (Figure 5). The position of this ring is also different when GMP or cPRPP bind (Figure 2). The five-membered rings in the two pyrrolidine NPs confer rigidity to the linker. The role of these rings is to orient the purine base and the phosphonate group in the active site. 6974



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Table 4. X-ray Data Collection and Refinement Statistics for the EcXGPRT−NP Complexes compound 2

compound 8

Crystal Parameters unit cell length a, b, c (Å) 94.07, 94.07, 162.63 unit cell angle α, β, γ (deg) 90.0, 90.0, 90.0 space group P43212 crystal dimensions (mm) 0.1 × 0.05 × 0.05 Diffraction Dataa temperature (K) 100 resolution range (Å) 20.0−2.80 (2.95−2.80)a observations 254 337 (37 261) unique reflections 18 634 (2660) completeness (%) 99.7 (100.0) Rmergeb 0.103 (0.64) Rpimc 0.029 (0.28) ⟨I⟩/⟨σ(I)⟩ 17.8 (3.0) subunits per asymmetric unit 4 solvent content (%) 53.3 Refinement resolution limits (Å) 19.97−2.80 (2.95−2.80) Rwork 0.1959 (0.3261) Rfree 0.2840 (0.3770) rmsd bond lengths (Å) 0.008 rmsd angles (deg) 1.15 clashscored 33.7 Components of the Asymmetric Unit protein subunit A 3−151 subunit B 3−152 subunit C 3−152 subunit D 3−61, 70−152

no side chain visible inhibitor bound solvent Ramachandran Plot (%) favored outliers

75.69, 117.79, 156.77 90.0, 90.0, 90.0 P212121 0.1 × 0.05 × 0.05 100 20.0−2.20 (2.26−2.20)a 478 270 (66 956) 71 529 (9673) 99.5 (97.7) 0.068 (0.63) 0.028 (0.26) 19.9 (3.6) 8 51.7 19.97−2.25 (2.25−2.20) 0.1774 (0.2356) 0.2204 (0.2653) 0.009 1.26 9.15

A 3, 4, 18, 30, 63, 69, 70, 106, 150; B 3, 18, 30, 53, 72, 102, 150; C 30, 72, 82, 102, 106; D 18, 30, 60, 71, 72, 82, 106 subunits B and C only 80

subunit A 1−61, 79−152 subunit B 3−62,78−152 subunit C 3−61, 79−152 subunit D 3−62, 80−152 subunit E 3−62, 79−152 subunit H 3−62, 80−152 subunit I 3−60, 80−152 subunit J 3−63, 80−152 A 3, 106; B 3, 61, 62; C 3, 4, 30, 53, 61; D 4, 62; E 3, 4, 30, 61− 62, 106; H 3, 4, 30, 53, 62; I 3, 82; J 3, 4, 30, 62, 63, 106 all subunits 459

92.9 0.5

98.5 0.1

The values in parentheses are for the outer resolution shell. bRmerge = ∑hkl∑i |Ii(hkl) − (I(hkl))| ∑hkl∑iIi(hkl) cRpim = ∑hkl[1/[N/(hkl) − 1]1/2] ∑i |1(hkl) − (1(hkl)| ∑hkl∑iIi (hkl), where Ii(hkl) is the observed intensity and is the average intensity obtained from multiple observations of symmetry-related reflections. dClashscore is defined as the number of bad overlaps ≥0.4 Å per thousand atoms. a

poses can only be correctly analyzed if they are based on known crystal structures. The Ki values of compound 2 and 10 for EcXGPRT are 0.23 and 0.4 μM, respectively (Table 2). The difference between these two compounds is that compound 10 has an additional phosphonate group. Surprisingly, this did not lead to a decrease in the Ki value, as had been expected on the basis of the inhibition data for compounds 11 and 17 (Table 2). It had been predicted that this second group would form interactions with the pyrophosphate binding site, resulting in increased affinity. Because the potency did not improve, docking was employed to try to explain this result. In the docked structure of compound 10 in complex with EcXGPRT, the first phosphonate group was located in the 5′-phosphate binding pocket (residues 92−96), with the second phosphonate group in the pyrophosphate binding pocket (residues 37−38). However, in this docked structure, the purine ring is moved away from its position parallel to W134 (Figure 6).

catalysis to proceed. However, these divalent metal ions are not present in the EcXGPRT·GMP complex.3 Similarly, they are not found in the structures of these two inhibitors in complex with EcXGPRT and do not play a role in binding. Docking Analysis to Correlate the Ki value for Compound 10 with the Predicted Mode of Binding. Docking studies with EcXGPRT were performed in an attempt to explain the differences in the Ki values for compounds 10 and 2 (Table 2). To assess the validity of the docking studies, compound 10 was docked into the known crystal structure of the EcXGPRT·compound 2 complex using GOLD.15 Nine poses were obtained, but only four poses located the phosphonate group in the 5′-phosphate binding pocket and the purine ring in the expected base binding pocket. Of these four poses, only one positioned the carbonyl group and the pyrrolidine ring in the same place found in the X-ray crystal structure (Figure 6). Therefore, although GOLD is able to predict where the inhibitors bind in the active site, the resultant 6975



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Figure 6. Comparison of the crystal structure of the EcXGPRT· guanine·cPRPP·Mg2+ complex with compound 10 docked in the active site of EcXGPRT. Figure 4. Stereo image of the electron density map for the EcXGPRT· compound 2 complex (top panel) and the EcXGPRT·compound 8 complex (bottom panel). The top panel highlights the interaction of the guanidinium group of R69 with the phosphoryl oxygen atoms of compound 2. In the EcXGPRT·compound 8 complex, this region of the enzyme is flexible and not visible.

possibilities: either the two phosphonate groups are located in their expected places but the purine base is unable to reach into its correct binding pocket, or the purine base binds where it should, resulting in the two phosphonate groups being unable to bind in their respective pockets. Therefore, the docking studies provide a rational explanation for compound 10 not binding more tightly to EcXGPRT than compound 2. Amino Acid Sequence Comparisons between E. coli XGPRT and Helicobacter pylori XGPRT as well as E. coli HPRT and Mycobacterium tuberculosis HPRT. The amino acid sequence alignment of the 6-oxopurine PRTases from EcXGPRT and H. pylori XGPRT16 (HpXGPRT) shows that the identity and similarity between these two enzymes are 24 and 49%, respectively (Figure 7a). However, many of the critical residues involved in substrate/product binding are conserved in the two enzymes (Figure 7a). In these regions, 54% of the amino acids are identical, 21% are conserved, and 25% are different. Thus, it is likely that high-affinity inhibitors of EcXGPRT should also be strong inhibitors of the H. pylori enzyme. The sequence alignment between EcHPRT and M. tuberculosis HPRT17 (MtHPRT) shows that these enzymes share 50% identity and 71% similarity (Figure 7b), with the amino acid residues involved in the binding of the substrates/products being 65% identical and with only 17% of the substitutions being nonconserved differences. Thus, it is likely that the inhibitors of EcHPRT will also be inhibitors of MtHPRT. Activity of Prodrugs of Compounds 17, 19, and 20 against M. tuberculosis. Prodrugs of compounds 2, 17, 19, and 20 (Tables 2 and 3) have been synthesized.18 The common chemical structure of the prodrugs of compounds 17, 19, and 20 is based on the covalent attachment of hydrophobic groups to the phosphoryl oxygens via a phosphoramidate bond (Figure 8a). A prodrug of compound 2 has also been synthesized, and its structure is given in Figure 8b. The purpose of these attachments is to mask the negative charges of the phosphonate groups and thus to increase cell permeability. Once within the microbial cells, it is proposed that they are then hydolysed to their active component.19 These prodrugs arrest the growth of M. tuberculosis in cell culture, with IC50 values of between 5 and 23 μM, corresponding to MIC90 values of 20−75 μM (Figure 9). None of the parent compounds exhibited anti M. tuberculosis activity at concentrations ≥250 μM, validating the prodrug strategy. Cytotoxicity data for these four prodrugs have been determined in human lung carcinoma cells (A549). The EC50 values are >300, >300, 100, and 100 μM for the prodrugs of compounds 2, 17, 19, and 20, respectively.18 The selectivity index [cytotoxicity/MIC50] for compounds 2, 17, and 19 are all ≥17, demonstrating a potential therapeutic window. Com-

Figure 5. Stereo images of the active site for the EcXGPRT·compound 2 (top panel) and EcXGPRT·compound 8 (bottom panel) complexes.

Table 5. Comparison of the Network of Hydrogen Bonds between the Phosphonyl Oxygens and the Active-Site Amino Acid Residues in the EcXGPRT−NP Complexes distance (Å)a phosphoryl oxygen

amino acid atom

compound 2

compound 8

O1

D92N T93N T93OG1 G94N T96N T96O T93OG1 T93N T96N G95N R69NE1

2.9 2.4 2.9 3.0 3.1 3.1 2.8 4.4 3.2 3.0 2.8

2.9 3.2 3.8 2.7 2.9 3.5 3.8 3.3 4.4 3.8 NI

O2 O3

a

The distances that are too long to form hydrogen bonds are in bold. NI, no interaction because there is no electron density for this residue.

The pyrrolidine phosphonates are thought to be rigid structures and would be unable to flex to adjust to the active site. Thus, when compound 10 binds, docking suggests two 6976



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Figure 7. Amino acid sequence alignment of the 6-oxopurine PRTases from (a) Escherichia coli (EcXGPRT) (P0A9M5) and Helicobacter pylori (HpXGPRT) (WP_00559479.1) and (b) Escherichia coli HGPRT (EcHPRT) (P0A9M2) and Mycobacterium tuberculosis HGPRT (MtHGPRT) (P0A5T0).

Figure 8. (a) Chemical structure of the prodrug of compound 19 (Table 3). The attachments to the phosphoryl oxygen atoms are identical for prodrugs of compounds 17, 19, and 20. (b) Chemical structure of the prodrug of compound 2 (Table 2).

pound 20 also has a selectivity index 4-fold in favor of anti M. tuberculosis activity.

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DISCUSSION Because, in part, of the increasing use of the currently available antibiotics, resistance to these therapeutic drugs is spreading, prompting the search for new antibiotics. It has been acknowledged that existing antibiotics are losing effectiveness more rapidly than they can be replaced.20 A serious issue is that the current antibiotics have been in use for at least half a century, and mechanisms to avoid their action have had time to develop. Attention has therefore turned to the discovery of new compounds aimed at different targets from those used traditionally. One such potential target is the purine salvage enzyme, 6-oxopurine phosphoribosyltransferase. These enzymes have not previously been used as drug targets for bacterial infections, although it has been suggested that their activity is essential for the survival of organisms such as M.

Figure 9. Inhibition of growth of M. tuberculosis by three prodrugs of the ANPs. (●) represents the prodrug of compound 17 (Table 2), (▼), the prodrug of compound 19 (Table 2), and (■), the prodrug of compound 20 (Table 3). 6977



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tuberculosis and H. pylori.6,8 The idea to target the 6-oxopurine PRTases is based on a simple premise: if RNA/DNA production can be stopped, then the bacteria will be unable to proliferate. Most, but not all, bacterial cells possess two metabolic pathways to synthesize the nucleoside monophosphates that are required to produce these essential molecules: (i) de novo (synthesis of the 6-oxopurine ring) and (ii) salvage of the preformed bases obtained from the host cell by a 6-oxopurine PRTase. The de novo pathway can be blocked by well-established drugs such as azaserine, but there are no known compounds that inhibit the bacterial salvage enzymes.21 For cells that contain both pathways, combination therapies that include inhibitors of de novo and salvage should inhibit cell growth. For bacterial cells such as H. pylori, which do not contain the enzymes required for synthesis of the purine ring,6,22 only inhibition of the salvage enzymes should be necessary. Most organisms survive with only one 6-oxopurine PRTase, but E. coli cells require two, EcXGPRT and EcHPRT. The primary difference between these two enzymes lies in their substrate specificity, as suggested by the nomenclature. Both these enzymes have been well characterized and serve as models for the corresponding enzymes from M. tuberculosis and H. pylori. Amino acid sequence identity (Figures 7a,b) suggests that EcXGPRT is akin to H. pylori XGPRT, whereas EcHPRT is more similar to the M. tuberculosis enzyme. This relationship suggests that inhibitors of either or both of the E. coli 6oxopurine PRTases would also be good inhibitors of the corresponding bacterial enzymes. Such inhibitors could therefore be new drug leads against these infections. One criterion for effective therapeutics is that the target enzyme should exhibit different properties from the corresponding eukaryotic enzyme. Both EcXGPRT and EcHPRT differ significantly from human HGPRT in their 3D structure, both in the presence or absence of ligands.1,3,7c These structural differences result in differences in their kinetic constants with the bacterial enzymes having higher kcat values for their preferred substrates compared with the human enzyme. For EcXGPRT, the kcat value using guanine as the base is 28.4 s−1, and for EcHPRT, using hypoxanthine as the base, this value is 59 s−1. These values are 13.4 (guanine as substrate) and 7.4 s−1 (hypoxanthine as substrate) for human HGPRT. More importantly, these enzymes differ in their substrate preference. EcXGPRT prefers guanine over xanthine, having kcat/Km ratios of 5.5-fold in favor of guanine, although both are very good substrates for this enzyme (Table 1). However, hypoxanthine is a much weaker substrate, with a kcat/Km 33-fold less than guanine. However, xanthine is not a substrate for human HGPRT, and this enzyme does not discriminate significantly between guanine and hypoxanthine, with the kcat/Km ratio for guanine over hypoxanthine being 3.23 EcHPRT, however, has a very strong preference for hypoxanthine, with a catalytic efficiency 167-fold greater than for guanine. These differences between the human and bacterial enzymes provide some confidence that specific inhibitors for bacterial 6-oxopurine PRTases can be designed. ANPs can be inhibitors of human HGPRT, Pf HGXPRT, and PvHGPRT.7c,18,24 They can also possess antimalarial activity and therefore have potential to be developed as therapeutic drugs. A wide variety of NPs have now been investigated as inhibitors of EcXGPRT and EcHPRT. Derivatives of 2(phosphonoethoxy)ethyl (PEE) containing either guanine or hypoxanthine as the purine base (PEEG or PEEHx) are good

inhibitors of both EcXGPRT and EcHPRT (compounds 11 and 12; Tables 2 and 3). The essential elements of these compounds are the purine base, the phosphonate group, and the linker connecting these two groups. Although EcXGPRT exhibits a preference for compounds containing guanine as the base, when guanine is replaced by hypoxanthine they are still effective inhibitors (Ki of 0.7 μM (compound 11 with guanine as the base) cf. Ki of 1.5 μM (compound 12 with hypoxanthine as the base)). EcHPRT, however, binds these compounds only when hypoxanthine is the base (Ki of 17 μM; compound 12, Table 3). PEEG does not bind to EcHPRT even at concentrations of 0.2 mM. Although the Ki for PEEHx for EcHPRT is higher than PEEG is for EcXGPRT, this is a significant increase in affinity compared with the nucleoside monophosphates. For EcXGPRT, the decrease is 6-fold, but for EcHPRT, the decrease is 15-fold. Thus, this phosphonate moiety is a lead for further development. This data also shows that the nature of the purine base is an important factor contributing to tight binding. In common with all the 6-oxopurine PRTases, EcXGPRT and EcHPRT possess a large flexible loop that is proposed to move during catalysis. For human HGPRT and Pf HGXPRT in complex with tight-binding inhibitors ImmGP (human)25 or ImmHP4 or acyclic immucillin phosphonate (AIP) (Pf),7b this loop closes over the active site when these inhibitors are bound. This suggests that if an inhibitor binds resulting in the loop closing firmly over the active site, then lower Ki values will result. Removal of six of the amino acids from the apex of this flexible loop in EcXGPRT does not affect the binding of the purine base or PRib-PP, but results in an increase in the Ki value for GMP (27-fold) and a reduction in the kcat value (200fold) for guanine. Analysis of the crystal structures of EcXGPRT in complex with GMP or cPRPP·guanine·Mg2+ suggest that the increase in Km is due to the absence of hydrogen bonds between the guanidino atoms of R69 and the phosphoryl oxygen atoms. Similarly, the Ki values for the NPs are increased in the absence of these amino acids (i.e., in mrEcXGPRT) reinforcing the proposition that for tight-binding inhibitors the positioning of the phosphonate group and the amino acids in the flexible loop are critical factors. Compound 13 (Table 2), with guanine as the base, binds to EcXGPRT with reasonable affinity but is not competitive with PRib-PP. Structures of human HGPRT in complex with PEEG and PEEHx7c showed an empty cavity in the active site near the PPi binding site. It was suggested that if this cavity could be filled, then tighter binding would result. This hypothesis was first tested by the addition of PPi to the assay in the presence of these inhibitors. This resulted in compound 13 becoming competitive with PRib-PP (increased Km(app), constant Vmax). It also resulted in a decrease in Ki. This suggested the possibility that if a second phosphonate group could be covalently attached to the linker, then these new compounds should exhibit decreased Ki values. This is the case with compounds 17 and 20 having very low Ki values (10 nM for EcXGPRT and 0.8 μM for EcHPRT). This is a decrease in Ki compared with GMP (EcXGPRT) of 450-fold and a decrease in Ki compared with IMP (EcHPRT) of 300-fold. Defining the location of this second phosphonate group in the active site and the positioning of the amino acids in the flexible loop await the crystal structures of the E. coli enzymes in complex with this compound. These structures should suggest further chemical modifications to the ANP to increase affinity. 6978



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the final products was greater than 95%. Their purity was determined by LC−MS using a Waters AutoPurification System with a 2545 Quarternary Gradient Module and 3100 Single Quadrupole Mass Detector using a Luna C18 column (100 × 4.6 mm, 3 μm, Phenomenex) at a flow rate of 1 mL/min. The following mobile phase was used, where A, B, and C represent 50 mM NH4HCO3, 50 mM NH4HCO3 in 50% aq CH3CN, and CH3CN, respectively: A → B over 10 min, B → C over 10 min, and C for 5 min. Preparative RP HPLC was performed on an LC5000 liquid chromatograph (INGOSPiKRON) using a Luna C18 (2) column (250 × 21.2 mm, 5 μm) at a flow rate of 10 mL/min. A gradient elution of methanol in pH 7.5 solution of 0.1 M TEAB (A, 0.1 M TEAB; B, 0.1 M TEAB in 50% aq methanol; and C, methanol) was used. All final compounds were lyophilized. The optical rotation values were measured on an AUTOPOL IV (Rudolph Research Analytical) polarimeter for the sodium D line at 20 °C. Mass spectra were collected on an LTQ Orbitrap XL (ThermoFisher Scientific) using ESI ionization. The phosphorus content in the compounds was determined using a simultaneous energy-dispersive X-ray fluorescence spectrometer SPECTRO iQ II. NMR spectra were recorded in D2O solutions on a Bruker AVANCE 600 (1H at 600.1 MHz and 13C at 150.9 MHz) and/or Bruker AVANCE 500 (1H at 499.8 MHz, 13C at 125.7 MHz, and 31P at 202.3 MHz) NMR spectrometers. Chemical shifts (in ppm, d scale) were referenced to the 1,4-dioxane signal (3.75 ppm for 1H and 69.3 ppm for 13C) as an internal standard in D2O. 31P NMR spectra were referenced to H3PO4 (0 ppm) as an external standard. Coupling constants (J) are given in Hz. The complete assignment of 1 H and 13C signals was performed by analysis of the correlated homonuclear H,H−COSY and heteronuclear H,C−HSQC and H,C− HMBC spectra. The numbering for the signal assignment is shown below. Synthesis of the Nucleoside Phosphonates (NPs). (S)-3(Guanin-9-yl)pyrrolidin-N-ylacetylphosphonic acid (Compound 2).

Having established that there are three critical sites for tight binding, a new series of NPs were designed. These contain a pyrrolidine ring that, in part, mimics the ribose ring of the nucleoside monophosphates. Such compounds have never previously been tested as inhibitors of the 6-oxopurine PRTases. Compound 2 has a Ki value for EcXGPRT of 0.23 μM, and compound 8 has a Ki value of 2.2 μM. Crystal structures of compound 2 and 8 bound in the active site of EcXGPRT were obtained and suggest explanations for the differences in affinity. One of the contributing factors to the affinity is that in the EcXGPRT·compound 8 complex the residues in the flexible loop are not visible, whereas in the EcXGPRT·compound 2 complex they have an organized structure and form interactions with the inhibitor. To improve the potency further, a second phosphosphonate group was attached to compound 2. Surprisingly, this did not result in a decrease in the Ki values as had been found for compound 17 in comparison with compound 11 (Table 1). The docking showed that because of the rigidity of the NP only the purine base and one of the two phosphonate groups can be optimally located in the active site. Thus, if all of these groups are not in their best position to form the optimal interactions with the actives site residues, then reduced affinity will occur even though they will still be inhibitors. Analysis of these structures highlights the importance of the linker connecting the purine base with the phosphonate group. Unless this linker is perfectly designed, neither of these groups is located in its optimal position and tighter binding does not result. The proposition that ANPs have the potential to be developed as novel antibiotics was tested against M. tuberculosis in culture using prodrugs of the most potent inhibitors. The measured IC50 values of 5−20 μM provide support for this hypothesis. Compounds 17, 19, and 20 are covalently linked single-molecule analogs of the two products of the reaction (i.e., nucleoside monophosphate and pyrophosphate). Such stable and specific chemical structures should be limited in their ability to bind to enzymes in the de novo or other purine salvage pathways. Thus, the most likely target is MtHPRT, although inhibition of another enzyme cannot be completely discounted. These studies have shown that a rational approach to drug design has led to the discovery of potent inhibitors of E. coli XGPRT and E. coli HPRT. New NPs are now being synthesized to improve further the potency of these compounds, not only for these enzymes but also for the 6oxopurine PRTases from M. tuberculosis and H. pylori. It is proposed that high-affinity inhibitors of the bacterial purine salvage enzymes, perhaps given in conjunction with known inhibitors of the de novo pathway, could prove to be excellent therapeutics.

■

The mixture of pyrrolidine derivative 1 (0.22 g, 1 mmol), diisopropyl phosphonoacetic acid (0.67 g, 3 mmol), DMAP (0.73 g, 6 mmol), and EDC (1.15 g, 6 mmol) in DMF (10 mL) was stirred at 90 °C for 2 h. The reaction mixture was concentrated in vacuo, and the diisopropyl ester intermediate was obtained by chromatography on silica gel using a linear gradient of solvent mixture H1 (EtOAc/EtOH/Acetone/H2O 6:1:1:1) in ethyl acetate in the form of colorless glass. Without further characterization, the intermediate was dissolved in DMF (5 mL), and Me3SiBr (0.46 mL, 3.52 mmol) was added under argon. The reaction mixture was stirred overnight at room temperature, concentrated in vacuo, dissolved in 0.5 M TEAB (3 mL), and concentrated in vacuo. The title compound was obtained by reverse-phase preparative HPLC, converted to the sodium salt by passing through a column of Dowex 50 in Na+ form, and lyophilized from water. Overall yield of 27% (106 mg, 0.27 mmol) of a white amorphous solid.

EXPERIMENTAL SECTION

Synthesis and Analytical Chemistry. The synthesis of the compounds in Tables 2 and 3, except for the pyrrolidine compounds, was as previously described.18,26 The synthesis of the prodrugs has also been documented previously.18 Synthesis of the pyrrolidine NPs. TLC was performed on plates precoated with silica gel (silica gel/TLC-cards, UV 254, Merck). Compounds were detected using UV light (254 nm), heating (for the detection of dimethoxytrityl group; orange color), spraying with a 1% ethanolic solution of ninhydrin to visualize amines, or by spraying with a 1% solution of 4-(4-nitrobenzyl)pyridine in ethanol followed by heating and treating with gaseous ammonia (for the detection of alkylating agents such as mesyl derivatives; blue color). The purity of

H NMR (499.8 MHz, D2O, 25 °C, ref(dioxane) = 3.75 ppm): 2.35 (m, 1H, H-4′b-B), 2.43 (m, 1H, H-4′b-A), 2.49 (m, 1H, H-4′a-B), 1

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HR-ESI C11H14O5N6P [M−H]− calcd, 326.06598; found, 326.06571. [α]20 −34.1 (c 0.607, H2O). (3S,4R)-4-(Guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylacetylphosphonic acid (Compound 6). The mixture of hydroxypyrroli-

2.55 (m, 1H, H-4′a-A), 2.72−2.88 (m, 4H, CH2P-A,B), 3.58−3.70 (m, 2H, H-5′-B), 3.81 (dd, 1H, Jgem = 12.8, J2′b,3′ = 4.8, H-2′b-A), 3.91− 4.02 (m, 3H, H-5′-A, H-2′a-A), 4.10 (dd, 1H, Jgem = 11.9, J2′b,3′ = 4.9, H-2′b-B), 4.32 (dd, 1H, Jgem = 11.9, J2′b,3′ = 6.8, H-2′a-B), 5.02 (m, 1H, J3′,2′ = 6.8, 4.8, J3′,4′ = 5.6, H-3′-A), 5.06 (m, 1H, J3′,2′ = 6.8, 4.9, J3′,4′ = 5.6, H-3′-B), 7.84 (s, 1H, H-8-A), 7.91 (s, 1H, H-8-B). 13C NMR (125.7 MHz, D2O, 25 °C, ref(dioxane) = 69.30 ppm): 32.36 (CH2-4′B), 33.69 (CH2-4′-A), 40.52 (d, JC,P = 111.5, CH2P-A), 40.92 (d, JC,P = 111.5, CH2P−B), 46.94 (CH2-5′-B), 48.63 (CH2-5′-A), 53.11 (CH22′-A), 54.63 (CH2-2′-B), 55.40 (CH-3′-A), 56.56 (CH-3′-B), 118.80 (C-5-A), 118.82 (C-5-B), 140.27 (CH-8-A), 140.45 (CH-8-B), 154.13 (C-4-A), 154.14 (C-4-B), 156.37 (C-2-A), 156.38 (C-2-B), 161.68 (C6-A), 161.69 (C-6-B), 173.85, 173.92 (d, JC,P = 5.8, CO-A,B). 31P[1H] NMR (202.3 MHz, D2O, 25 °C, ref(H3PO4) = 0 ppm): 12.25 (P−B), 12.28 (P−A). HR-ESI C11H14O5N6P [M − H]− calcd, 341.07688; found, 341.07710. [α]20 +13.7 (c 0.117, H2O). (R)-3-(Hypoxanthin-9-yl)pyrrolidin-N-ylacetylphosphonic acid (Compound 4). The mixture of pyrrolidine derivative 3 (0.21 g,

dine derivative 5 (0.06 g, 0.25 mmol), diisopropyl phosphonoacetic acid (0.18 g, 0.8 mmol), DMAP (0.19 g, 1.5 mmol), and EDC (0.29 g, 1.5 mmol) in DMF (3 mL) was stirred at 90 °C for 2 h. The reaction mixture was concentrated in vacuo, and the diisopropyl ester intermediate was obtained by reverse-phase preparative HPLC in the form of colorless glass. Without further characterization, the intermediate was dissolved in DMF (2 mL), and Me3SiBr (0.13 mL, 1 mmol) was added under argon. The reaction mixture was stirred overnight at room temperature, concentrated in vacuo, dissolved in 0.5 M TEAB (3 mL), and concentrated in vacuo. The title compound was obtained by reversed-phase preparative HPLC, converted to the sodium salt by passing through a column of Dowex 50 in Na+ form, and lyophilized from water. Overall yield of 23% (22.7 mg, 0.056 mmol) of a white amorphous solid.

1.02 mmol), diisopropyl phosphonoacetic acid (0.67 g, 3 mmol), DMAP (0.73 g, 6 mmol), and EDC (1.15 g, 6 mmol) in DMF (10 mL) was stirred at 90 °C 2 h. The reaction mixture was concentrated in vacuo, and diisopropyl ester intermediate was obtained by chromatography on silica gel using linear gradient of solvent mixture H1 in ethyl acetate in the form of colorless glass. The intermediate was without further characterization dissolved in MeCN (10 mL), and Me3SiBr (0.58 mL, 4.38 mmol) was added under argon atmosphere. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, dissolved in 0.5 M TEAB (3 mL), and concentrated in vacuo. The title compound was obtained by reverse-phase preparative HPLC, converted to sodium salt by passing through a column of Dowex 50 in Na+ form, and lyophilized from water. Overall yield of 46% (150 mg, 0.46 mmol) of a white amorphous solid.

H NMR (600.1 MHz, D2O, 25 °C, ref(dioxane) = 3.75 ppm): 2.83−2.94 (m, 4H, CH2P-A,B), 3.64 (ddd, 1H, Jgem = 13.2, J2′b,3′ = 2.3, JH,P = 1.6, H-2′b-B), 3.81 (ddd, 1H, Jgem = 13.2, J2′a,3′ = 4.8, JH,P = 2.4, H-2′a-B), 3.88 (dd, 1H, Jgem = 12.0, J2′b,3′ = 2.9, H-2′b-A), 3.93 (ddd, 1H, Jgem = 12.4, J5′b,4′ = 8.4, JH,P = 2.0, H-5′b-A), 4.11 (ddd, 1H, Jgem = 12.4, J5′a,4′ = 8.0, JH,P = 1.2, H-5′a-A), 4.13 (dd, 1H, Jgem = 12.0, J2′a,3′ = 4.9, H-2′a-A), 4.23 (dd, 1H, Jgem = 10.8, J5′b,4′ = 9.3, H-5′b-B), 4.38 (dd, 1H, Jgem = 10.8, J5′a,4′ = 8.0, H-5′a-B), 4.64 (ddd, 1H, J3′,2′ = 4.8, 2.3, J3′,4′ = 4.2, H-3′-B), 4.68 (ddd, 1H, J3′,2′ = 4.9, 2.9, J3′,4′ = 4.3, H-3′A), 5.07 (ddd, 1H, J4′,5′ = 8.4, 8.0, J4′,3′ = 4.3, H-4′-A), 5.10 (ddd, 1H, J4′,5′ = 9.3, 8.0, J4′,3′ = 4.2, H-4′-B), 7.94 (s, 1H, H-8-A), 7.97 (s, 1H, H8-B). 13C NMR (150.9 MHz, D2O, 25 °C, ref(dioxane) = 69.30 ppm): 39.60, 39.94 (d, JC,P = 116.1, CH2P-A,B), 49.34 (CH2-5′-A), 50.53 (CH2-5′-B), 54.65 (CH2-2′-B), 56.01 (CH2-2′-A), 57.62 (CH-4′-A), 58.43 (CH-4′-B), 71.25 (CH-3′-B), 72.39 (CH-3′-A), 118.34, 118.37 (C-5-A,B), 141.33, 141.37 (CH-8-A,B), 154.67, 154.77 (C-4-A,B), 156.46, 156.47 (C-2-A,B), 161.70, 161.72 (C-6-A,B), 172.97, 173.01 (d, JC,P = 5.5, CO-A,B). 31P[1H] NMR (202.3 MHz, D2O, 25 °C, ref(H3PO4) = 0 ppm): 13.63 (P−A), 13.68 (P−B). HR-ESI C11H14O6N6P [M − H]− calcd, 357.0718; found, 357.0720. [α]20 +47.6 (c 0.189, H2O). (3R,4S)-4-(Guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylacetylphosphonic acid (Compound 8). The mixture of hydroxypyrroli1

H NMR (499.8 MHz, D2O, 25 °C, ref(dioxane) = 3.75 ppm): 2.44 (m, 1H, H-4′b-B), 2.52 (m, 1H, H-4′b-A), 2.61 (m, 1H, H-4′a-B), 2.70 (m, 1H, H-4′a-A), 2.94−3.07 (m, 4H, CH2P-A,B), 3.67, 3.76 (2 × m, 2 × 1H, H-5′-B), 3.94 (bdd, 1H, Jgem = 13.1, J2′b,3′ = 4.1, H-2′b-A), 3.97−4.05 (m, 3H, H-5′-A), 4.09 (ddd, 1H, Jgem = 13.1, J2′a,3′ = 6.8, JH,P = 2.2, H-2′a-A), 4.23 (dd, 1H, Jgem = 11.8, J2′b,3′ = 4.3, H-2′b-B), 4.37 (dd, 1H, Jgem = 11.8, J2′b,3′ = 6.5, H-2′a-B), 5.21 (m, 1H, H-3′-A), 5.26 (m, 1H, H-3′-B), 8.12 (s, 1H, H-8-A), 8.13 (s, 1H, H-2-A), 8.14 (s, 1H, H-2-B), 8.20 (s, 1H, H-8-B). 13C NMR (125.7 MHz, D2O, 25 °C, ref(dioxane) = 69.30 ppm): 32.50 (CH2-4′-B), 33.79 (CH2-4′-A), 39.53 (d, JC,P = 118.7, CH2P-A), 39.91 (d, JC,P = 118.5, CH2P−B), 47.08 (CH2-5′-B), 48.68 (CH2-5′-A), 53.33 (CH2-2′-A), 54.71 (CH22′-B), 56.22 (CH-3′-A), 57.47 (CH-3′-B), 125.92 (C-5-A), 125.97 (C5-B), 142.26 (CH-8-A), 142.46 (CH-8-B), 148.33 (C-2-A,B), 150.95 (C-4-A), 150.96 (C-4-B), 160.58 (C-6-A), 160.61 (C-6-B), 172.02 (d, JC,P = 6.1, CO-B), 172.15 (d, JC,P = 6.1, CO-B). 31P[1H] NMR (202.3 MHz, D2O, 25 °C, ref(H3PO4) = 0 ppm): 14.00 (P−B), 14.07 (P−A). 1

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40.26 (d, JC,P = 108.9, CH2P), 40.35 (d, JC,P = 113.1, CH2P), 40.48 (d, JC,P = 112.3, CH2P), 51.11 (CH2-5′-B), 51.89 (CH2-2′-A), 52.24 (CH2-5′-A), 53.63 (CH2-2′-B), 59.29 (CH-4′-B), 60.46 (CH-4′-B), 77.14 (CH-3′-A), 78.57 (CH-3′-B), 118.82, 118.90 (C-5-A,B), 140.55, 140.74 (CH-8-A,B), 154.44 (C-4-A,B), 156.50 (C-6-A,B), 161.83 (C2-A,B), 173.67, 173.86 (d, JC,P = 5.8, CO), 173.97, 174.02 (d, JC,P = 6.2, CO). 31P[1H] NMR (202.3 MHz, 25 °C, D2O, ref(H3PO4) = 0 ppm): 10.69, 10.71, 12.38, 12.57. HR-ESI C11H13O5N5P [M − H]− calcd, 479.04869; found, 479.04882. [α]20 +15.4 (c 1.039, H2O). Expression of EcXGPRT, mrEcXGPRT, and EcHPRT. mrEcXGPRT, where the amino acid sequence HDNQRE (residues 65−70) was replaced by a single alanine, was prepared by the method of Kunkel.27 The cDNA coding for the mutant enzyme was sequenced at the Australian Research Genomic Facility. EcXGPRT, EcHPRT, and mrEcXGPRT were expressed in E. coli SΦ606 [ara, Δpro-gpt-lac, thi, hpt, F-] cells and were purified to homogeneity as described previously.2,3,28 The enzymes were stored in 0.1 M Tris-HCl, 0.01 M MgCl2, and 1 mM DTT, pH 7.4 at −70 °C, under which conditions they are stable for ≥24 months. The activity of EcXGPRT and mrEcXGPRT were measured as described previously.2,3,28 The protein concentration was determined using an A1%1 cm value of 16.6 M−1 cm−1 for EcXGPRT, 4.4 M−1 cm−1 for EcHPRT, and 17.5 M−1 cm−1 for the loop-out mutant enzyme. Mass spectrometry was performed using a Qstar Pulsar i with electrospray interface. The concentration of the enzyme (as a tetramer) was 12 μM (EcXGPRT). For these studies, the enzyme was desalted on a C18 HPLC column with a 0−60% CH3CN gradient containing 0.1% formic acid. The mass was calculated from the amino acid sequence predicted from the cDNA sequencing results. Mass spectrometry also showed that these protein preparations were ≥99% homogeneous. The Ki values for the NPs were determined by a spectrophotometric method, as previously described.7c The concentration of the invariable substrate (guanine for EcXGPRT and hypoxanthine for EcHPRT) was 60 μM, whereas the concentration of the variable substrate (PRib-PP) was between 14 and 1000 μM, depending on the Km(app). The concentration of the inhibitor ranged from 0.1 to 140 μM, depending upon the potency of the particular compound. Crystallization and Structure Determination of E. coli XGPRT in Complex with Compounds 2 and 8. For crystallization experiments with compound 2, EcXGPRT was concentrated to 21 mg/mL (1.2 mM in terms of subunits). For experiments with compound 8, EcXGPRT was concentrated to 29.7 mg/mL (1.9 mM in terms of subunits). The specific activity of this enzyme was 80 216 μmol/min/mg using guanine as the substrate. Prior to crystallization, the enzyme was incubated with compound 2 (2 mM) or compound 8 (2.2 mM) for up to 5 min. For the crystallization trials, the hangingdrop method was used where 1 μL of reservoir solution and 1 μL of EcXGPRT in complex with the inhibitor were combined in the drops and incubated at 17 °C. The reservoir solution that yielded crystals for the EcXGPRT·compound 2 complex consisted of 8% tacsimate pH 7.0 and 20% PEG 3350; for the EcXGPRT·compound 8 complex the conditions were identical except that the pH of the tacsimate solution was 6.0. Prior to data collection, the crystals were transferred to a cryoprotectant solution that contained well solution, 2 mM inhibitor, and 20% glycerol. The crystals were then placed in a cryostream (100 K). X-ray data were collected using Beamline MX2 (wavelength 0.95369 Å) of the Australian Synchrotron. The data for the EcXGPRT· compound 2 complex were collected with a crystal-to-detector distance of 400 mm, an oscillation range of 180°, an exposure time of 2s per image, and an image width of 0.5°. For the EcXGPRT· compound 8 complex, all of the data collection parameters were the same as for the EcXGPRT·compound 2 complex except that the crystal-to-detector distance was 300 mm. Both data sets were scaled and merged using XDS.29 The unit cell and space group for the crystals of the EcXGPRT· compound 2 complex are similar to those for the EcXGPRT·GMP· borate complex (PDB code 1A97). Therefore, the structure solution for this complex was by difference Fourier. The structure of the EcXGPRT·compound 8 complex was solved by molecular replacement using the program PHASER30 within PHENIX 1.7.331 and the protein

dine derivative 7 (0.04 g, 0.17 mmol), diisopropyl phosphonoacetic acid (0.1 g, 0.45 mmol), DMAP (0.1 g, 0.9 mmol), and EDC (0.17 g, 0.9 mmol) in DMF (5 mL) was stirred at 90 °C for 2 h. The reaction mixture was concentrated in vacuo, and the diisopropyl ester intermediate was obtained by reverse-phase preparative HPLC in the form of colorless glass. Without further characterization, the intermediate was dissolved in DMF (2 mL), and Me3SiBr (0.13 mL, 1 mmol) was added under argon. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, dissolved in 0.5 M TEAB (3 mL), and concentrated in vacuo. The title compound was obtained by reverse-phase preparative HPLC, converted to the sodium salt by passing through a column of Dowex 50 in Na+ form, and lyophilized from water. Overall yield of 38% (26.1 mg, 0.065 mmol) of a white amorphous solid. The HR-ESI, 1 H NMR, 13C NMR, and 31P[1H] NMR spectra were identical to those of compound 6. [α]20 −45.3 (c 0.239, H2O). (3R,4R)-4-(Guanin-9-yl)-3-(2-phosphonoacetyl)pyrrolidin-1N-ylacetylphosphonic acid (Compound 10). The mixture of

hydroxypyrrolidine derivative 9 (0.24 g, 1 mmol), diisopropyl phosphonoacetic acid (0.95 g, 4 mmol), DMAP (0.12 g, 1 mmol), and EDC (0.96 g, 5 mmol) in DMF (10 mL) was stirred at 90 °C for 4 h. The reaction mixture was concentrated in vacuo, and the diisopropyl ester intermediate was obtained by chromatography on silica gel using a linear gradient of methanol in chloroform in the form of colorless glass. Without further characterization, the intermediate was dissolved in MeCN (10 mL), and lutidine (0.86 mL, 7.44 mmol) and Me3SiBr (0.98 mL, 7.44 mmol) were added under argon. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, dissolved in 0.5 M TEAB (3 mL), and concentrated in vacuo. The title compound was obtained by reverse-phase preparative HPLC, converted to the sodium salt by passing through a column of Dowex 50 in Na+ form, and lyophilized from water. Overall yield of 41% (232.4 mg, 0.41 mmol) of a white amorphous solid.

1 H NMR (499.8 MHz, D2O, 25 °C, ref(dioxane) = 3.75 ppm): 2.61−2.92 (m, 8H, CH2P-A,B), 3.76 (ddd, 1H, Jgem = 14.1, J2′b,3′ = 3.2, JH,P = 2.1, H-2′b-A), 3.92 (ddd, 1H, Jgem = 14.1, J2′a,3′ = 6.3, JH,P = 2.7, H-2′a-A), 4.01 (dd, 1H, Jgem = 12.5, J2′b,3′ = 3.3, H-2′b-B), 4.04 (ddd, 1H, Jgem = 13.6, J5′b,4′ = 3.9, JH,P = 1.9, H-5′b-B), 4.17 (ddd, 1H, Jgem = 13.6, J5′a,4′ = 7.6, JH,P = 2.3, H-5′a-B), 4.35 (dd, 1H, Jgem = 12.5, J2′a,3′ = 5.7, H-2′a-B), 4.10 (dd, 1H, Jgem = 12.2, J5′b,4′ = 4.5, H-5′b-A), 4.46 (dd, 1H, Jgem = 12.2, J5′a,4′ = 6.8, H-5′a-A), 5.15 (dt, 1H, J4′,5′ = 7.6, 3.9, J4′,3′ = 3.9, H-4′-B), 5.18 (ddd, 1H, J4′,5′ = 6.8, 4.5, J4′,3′ = 3.7, H-4′-A), 4.67 (ddd, 1H, J3′,2′ = 6.3, 3.2, J3′,4′ = 3.7, H-3′-A), 5.53 (ddd, 1H, J3′,2′ = 5.7, 3.3, J3′,4′ = 3.9, H-3′-B), 7.87 (s, 1H, H-8-B), 8.01 (s, 1H, H-8A). 13C NMR (125.7 MHz, D2O, 25 °C, ref(dioxane) = 69.3 ppm):

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coordinates of the tetramer of EcXGPRT from the EcXGPRT·GMP· borate complex as the starting model. Two tetramers could be fitted to the asymmetric unit, giving a translation function Z-score of 44.1 and a log likelihood-gain (LLG) of 1761 with no steric clashes. Subsequent refinement of the coordinates was with PHENIX31 and model building with COOT.32 The dictionary files for the two inhibitors were generated using the PRODGR2 server.33 The atomic coordinates and structure factors of the EcXGPRT·compound 2 and EcXGPRT· compound 8 complexes have been deposited with the Protein Data Bank as entries 4JIT and 4JLS, respectively. Inhibition of M. tuberculosis. M. tuberculosis H37Ra (ATCC 25177) was grown in Middlebrook 7H9 broth medium supplemented with OADC, 0.5% glycerol, and 0.05% Tween-80. Freshly seeded cultures were grown at 37 °C for approximately 14 days to midexponential phase (OD600 0.4−0.8) for use in the inhibition assays. The potency of the inhibitors was measured by a resazurin reduction microplate assay, as previously described,34 with some alterations. M. tuberculosis, grown to midexponential phase (OD600 0.4−0.8), was diluted to OD600 0.001 in 7H9S media (Middlebrook 7H9 with OADC, 0.5% glycerol, 0.75% tween-80, and 1% tryptone) containing 0.5% DMSO. 96-well microtiter plates were set up with 100 μL of the inhibitors, serially diluted in 7H9S media. One-hundred microliters of diluted M. tuberculosis, representing ∼2 × 104 CFU/ml, was added to each well. The plates were incubated for 5 days at 37 °C in a humidified incubator prior to the addition of 30 μL of a 0.02% resazurin solution and 12.5 μL of 20% Tween 80 to each well. Sample fluorescence was measured 30 h later on a CytoFluor multiwell plate reader (PerSeptive Biosciences) with an excitation wavelength of 530 nm and emission read at 580 nm. Changes in fluorescence relative to the positive control wells (H37Ra with no inhibitor) minus the negative control wells (no H37Ra) were plotted for the determination of the IC50. Cytotoxicity data were measured as previously described.18 Bioinformatic Analysis. The protein sequences for H. pylori XGPRT, EcXGPRT, EcHPRT, and M. tuberculosis HGPRT were obtained from UniProtKB.35 The identity/similarity of the amino acid sequences were calculated using BlastP.36 ClustalW237 was used to perform the sequence alignments of the enzymes. Docking Analysis. Coordinates for the EcXGPRT polypeptide were obtained from chain B of the complex with cPRPP·guanine·Mg2+ (PDB: 1A95) and chain B of the EcXGPRT·compound 2 complex. The program GOLD15 was used for all of the docking calculations. The active-site center was defined by a point between the ε-amino group of Lys115 and the indole of Trp134. The search radius was 15 Å. For each ligand, 20 independent docking searches were performed. Scoring was made with the default settings in the program, taking into account H-bonding energy, van der Waals energy, and ligand torsion strain.

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nos. 569703 and 1030353), the subvention for development of research organization (Institute of Organic Chemistry and Biochemistry, RVO 61388963), the Grant Agency of the Czech Republic (grant no. P207/11/0108), and Gilead Sciences (Foster City, CA, USA).

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ABBREVIATIONS USED PRib-PP, 5-phospho-α-D-ribosyl-1-pyrophosphate; PRTase, phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; HGXPRT, hypoxanthine-guaninexanthine phosphoribosyltransferase; XGPRT, xanthine-guanine phosphoribosyltransferase; Ec, Escherichia coli; mr, mutant recombinant; ANP, acyclic nucleoside phosphonate; NP, nucleoside phosphonate; ImmGP, (1S)-1-(9-deazaguanin-9yl)-1,4-dideoxy-1,4-imino-D -ribitol 5-phosphate; ImmHP, (1S)-1-(9-deazahypoxanthin-9-yl)-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; PPi, pyrophosphate; Hx, hypoxanthine; Mt, Mycobacterium tuberculosis.; MIC, minimum inhibitory concentration.

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REFERENCES

(1) Guddat, L. W.; Vos, S.; Martin, J. L.; Keough, D. T.; de Jersey, J. Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase. Protein Sci. 2002, 11, 1626− 1638. (2) Vos, S.; de Jersey, J.; Martin, J. L. Crystal structure of Escherichia coli xanthine phosphoribosyltransferase. Biochemistry 1997, 36, 4125− 4134. (3) Vos, S.; Parry, R. J.; Burns, M. R.; de Jersey, J.; Martin, J. L. Structures of free and complexed forms of Escherichia coli xanthineguanine phosphoribosyltransferase. J. Mol. Biol. 1998, 282, 875−889. (4) 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 Å structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor. Biochemistry 1999, 38, 9872− 9880. (5) Gardner, M. J.; Hall, N.; Fung, E.; White, O.; Berriman, M.; Hyman, R. W.; Carlton, J. M.; Pain, A.; Nelson, K. E.; Bowman, S.; Paulsen, I. T.; James, K.; Eisen, J. A.; Rutherford, K.; Salzberg, S. L.; Craig, A.; Kyes, S.; Chan, M.-S.; Nene, V.; Shallom, S. J.; Suh, B.; Peterson, J.; Angiuoli, S.; Pertea, M.; Allen, J.; Selengut, J.; Haft, D.; Mather, M. W.; Vaidya, A. B.; Martin, D. M. A.; Fairlamb, A. H.; Fraunholz, M. J.; Roos, D. S.; Ralph, S. A.; McFadden, G. I.; Cummings, L. M.; Subramanian, G. M.; Mungall, C.; Venter, J. C.; Carucci, D. J.; Hoffman, S. L.; Newbold, C.; Davis, R. W.; Fraser, C. M.; Barrell, B. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002, 419, 498−511. (6) Liechti, G.; Goldberg, J. B. Helicobacter pylori relies primarily on the purine salvage pathway for purine nucleotide biosynthesis. J. Bacteriol. 2012, 194, 839−854. (7) (a) deJersey, J.; Holý, A.; Hocková, D.; Naesens, L.; Keough, D. T.; Guddat, L. W. 6-Oxopurine phosphoribosyltransferase: A target for the development of antimalarial drugs. Curr. Top. Med. Chem. 2011, 11, 2085−2102. (b) Hazleton, K. Z.; Ho, M. C.; Cassera, M. B.; Clinch, K.; Crump, D. R.; Rosario, I., Jr.; Merino, E. F.; Almo, S. C.; Tyler, P. C.; Schramm, V. L. Acyclic immucillin phosphonates: Second-generation inhibitors of Plasmodium falciparum hypoxanthineguanine-xanthine phosphoribosyltransferase. Chem. Biol. 2012, 19, 721−730. (c) Keough, D. T.; Hocková, D.; Holý, A.; Naesens, L. M. J.; Skinner-Adams, T. S.; de Jersey, J.; Guddat, L. W. Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: A new class of antimalarial therapeutics. J. Med. Chem. 2009, 52, 4391−4399.

AUTHOR INFORMATION

Corresponding Author

*Phone: +61 07 33653549. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the contribution of Professor Antonin Holý to this project. Preliminary X-ray data were measured at the University of Queensland Remote-Operation Crystallization and X-ray Diffraction facility (UQROCX). The final measurements were made at the MX2 beamline, Australian Synchrotron, Clayton, Victoria with the assistance of Alan RiboldiTunnicliffe and Tom Caradoc-Davies. The views expressed herein are those of the authors and are not necessarily those of the owner or operator of the Australian Synchotron. We thank Dr. Kyra Woods for testing some of the compounds in the M. tuberculosis cell-based assay. This work was supported by funds from the National Health and Medical Research Council (grant 6982



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Inhibition of the Escherichia coli 6-Oxopurine

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