Design of Plasmodium vivax Hypoxanthine-Guanine

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The Design of Plasmodium vivax Hypoxanthine-Guanine Phosphoribosyltransferase Inhibitors as Potential Antimalarial Therapeutics Dianne T. Keough, Dominik Rejman, Radek Pohl, Eva Zborníková, Dana Hockova, Tristan Croll, Michael D. Edstein, Geoff W. Birrell, Marina Chavchich, Lieve M. J. Naesens, Gregory K. Pierens, Ian M. Brereton, and Luke W. Guddat ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00916 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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ACS Chemical Biology

The Design of Plasmodium vivax Hypoxanthine-Guanine Phosphoribosyltransferase Inhibitors as Potential Antimalarial Therapeutics

Dianne T. Keough,† Dominik Rejman,*‡ Radek Pohl,‡ Eva Zborníková,‡ Dana Hocková,

‡

Tristan Croll,§ Michael D. Edstein,∥ Geoff W. Birrell,∥ Marina

Chavchich,∥ Lieve M. J. Naesens,# Gregory K. Pierens,⊥ Ian M. Brereton⊥ and Luke W. Guddat*†

†

School of Chemistry and Molecular Biosciences, The University of Queensland,

4072, Australia ‡

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,

CZ-166 10 Prague 6, Czech Republic §

Institute of Health and Biomedical Innovation, Queensland University of

Technology, 2 George St, Brisbane, 4000, Australia ∥Department #

of Drug Evaluation, Australian Army Malaria Institute, 4051, Australia

Rega Institute for Medical Research, Katholique University, Minderbroedersstraat

10, B-3000, Leuven, Belgium ⊥Centre

for Advanced Imaging, The University of Queensland, 4072, Australia

ACS Paragon Plus Environment


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ABSTRACT Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of

hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase,

HG[X]PRT,

is

reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT which has Ki values of 8 nM and 7 nM, respectively for these two enzymes. The crystal structure of PvHGPRT in complex with this compound has been determined to 2.85 Å resolution. The corresponding complex with human HGPRT was also obtained to allow a direct comparison of the binding modes of this compound with the two enzymes. The tetra-(ethyl L-phenylalanine) tetraamide prodrug of this compound was synthesized and it has an IC50 of 11.7 ± 3.2 µM against Pf lines grown in culture and a CC50 in human A549 cell lines of 102 ± 11 µM, thus a ~10-fold selectivity index.

2 Environment ACS Paragon Plus

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Malaria remains one of the most serious infectious diseases worldwide despite concerted efforts to control its spread and its eradication. Current chemotherapeutics are restricted to artemisinin based combination therapy and chloroquine for treating Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) infections.1-4 However, the emergence of malaria strains that are resistant to these drugs is an increasing problem necessitating the urgent need for development of new drugs aimed at novel targets. Despite many years of research, effective vaccines still remain in the pipeline.5, 6 Pv is referred to as a “neglected disease”7 since the main focus for the development of antimalarial therapy has been directed towards Pf. Pv, however, is potentially more dangerous because of its ability to lie dormant in the liver for months to years before the patient presents with recurring symptoms of this disease. An attractive approach for the development of chemotherapeutics against these malarial parasites is to block the synthesis of their DNA/RNA which will effectively result in the loss of their ability to replicate and survive. This precedent has been established by the inhibition of DNA synthesis by use of substrates and/or inhibitors of DNA polymerase. This strategy resulted in the development of the successful antivirals, tenofovir, adefovir and cidofovir.8 Both Pf and Pv rely solely on the salvage of preformed bases transported from the host to make the 6-oxopurine nucleoside monophosphates, GMP/IMP/XMP, required for the synthesis of their DNA/RNA. Central in this pathway is the enzyme hypoxanthine-guanine-[xanthine] phosphoribosyltransferase (HG[X]PRT) whose function is to convert guanine and hypoxanthine and, in the case of Pf, xanthine, into their respective nucleoside monophosphates (Figure 1). Unlike the human host which possesses two pathways to synthesize these metabolites, de novo (from small molecules) and salvage, the parasites rely solely on salvage, a pathway that is more energetically cost effective.



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Analysis of the complete Pf and Pv genomes substantiates the hypothesis that these parasites have no alternative pathways to bypass HG[X]PRT for the production of their 6-oxo and 6-amino purine nucleotides.9 Thus, it is expected that tight binding inhibitors of PfHGXPRT and PvHGPRT should prevent the growth and proliferation of malaria parasites.10-12

Figure 1. The reaction catalyzed by HGXPRT. R = -H (hypoxanthine); -NH2 (guanine); -OH (xanthine). X-ray crystal structures of PfHGXPRT and human HGPRT have shown that these enzymes exist as tetramers. Ultracentrifugation studies of these two enzymes and PvHGPRT, have confirmed that, in solution, all three enzymes are tetrameric.13 The active site is located within a single subunit and is defined by four areas able to accommodate components of the substrates/products of the reaction (Figure 1). These are (i) the purine ring; (ii) the ribose ring of 5-phospho-α-D-ribosyl-1pyrophosphate (PRib-PP) or the nucleoside monophosphate; (iii) the 5′-phosphate group of PRib-PP or the nucleoside monophosphate; and (iv) the pyrophosphate group of PRib-PP or of PPi. There are also two sites where crystal structures of the 6oxopurine phosphoribosyltransferases (PRTases) have been shown to accommodate one or two divalent metal ions. Divalent metal ions position the substrates in the optimal orientation for catalysis to occur14, 15 though it has also been suggested that they could aid in maintaining the three dimensional structure of these enzymes.16 A


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common feature of the 6-oxopurine PRTases is the presence of a large mobile loop of up to 20 residues that closes over the active site after the substrates enter and then opens again to release the products. It has been proposed that this loop can fulfill two functions: The first is to exclude bulk solvent during the catalytic reaction and the second is to form hydrogen bonds with the substrates/products to assist in locking them in place in the optimal orientation.15 The loop is also closed when the tight binding inhibitor, acyclic immucillin phosphonate (AIP), is bound in the active site of PfHGXPRT together with PPi.10 Acyclic nucleoside phosphonates (ANPs) and aza-ANPs are good inhibitors of PfHGXPRT and PvHGPRT.11,

12

Pyrrolidine nucleoside phosphonates (PNPs),

where the ribose group of PRib-PP, or the nucleoside monophosphates, is replaced by a pyrrolidine ring inhibit the two Escherichia coli 6-oxopurine PRTases, XGPRT and HPRT.17 The crystal structure of E. coli XGPRT in complex with the PNP, (S)-3(guanin-9-yl)pyrrolidin-N-ylacetylphosphonic

acid,

compound

1

(Figure

2),

suggested chemical modifications to this scaffold whereby a single molecule would occupy a number of critical areas in the active site. The structures of the new molecules are based on a “hub” and “spoke” strategy whereby a central “hub” (pyrrolidine group) is covalently linked to three “spokes”. Such single entities should not only possess high affinity for the 6-oxopurine PRTases but should also be highly specific for this group of enzymes. This new class of inhibitors of PvHGPRT are the pyrrolidine nucleoside bisphosphonates (PNBPs). The chemical structure proposed for these potential inhibitors is versatile, allowing for modifications to be made so that they can be tailored to the 6-oxopurine PRTases from different organisms. Here, the first structural visualization of PvHGPRT was achieved. This in complex with one of these new inhibitors, thus providing a rationale for its tight binding.



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RESULTS AND DISCUSSION

Analysis of E. coli XGPRT.1 and E. coli XGPRT.GMP complexes. The crystal structure of (S)-3-(guanin-9-yl)pyrrolidin-N-ylacetylphosphonic acid, 1, in complex with E. coli XGPRT (Figures 3a and b; PDB accession code: 4JIT)17 provided the basis for the design of a new class of inhibitors, 2-5 (Figure 2). In the E. coli XGPRT.1 structure, the location of the “hub” (the pyrrolidine ring) mimics that of the ribose ring of GMP in the E. coli XGPRT.GMP complex (PDB accession code: 1A9712).16 Thus, these two five membered rings (i.e. ribose and pyrrolidine) are located deep into the active site (Figures 3a and b). It can also be seen that the location of the purine ring and the phosphonate/phosphate group are virtually the same for both compound 1 and GMP. The large mobile loop (residues I60-V73) is fully resolved in the E. coli XGPRT.1 structure but is in an “open” conformation (Figure 3b). The E. coli enzyme is much smaller than PvHGPRT (152 residues compared with 233) but, in lieu of the crystal structure of the parasite enzyme, the E. coli XGPRT structure was a sufficient guide for developing new PNBP inhibitors (Figure 2).

Figure 2. The chemical structures of the pyrrolidine nucleoside phosphonate (1) and the four newly designed pyrrolidine nucleoside bisphosphonates (2-5).


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(a)

T133

T133 5’-phosphate pocket

W134 E136

E136

K115 D92

purine base pocket

G38

PPi loop

R37

K115 D92

purine base pocket

Cpd 1 GMP

D140

5’-phosphate pocket

W134

T96

D140

Cpd 1 GMP

G38 R37

S36

PPi loop

T96

S36

(b) Large mobile loop GMP Cpd 1

X

borate

S62

E70

Figure 3. Superimposition of the crystal structures of the E. coli XGPRT.1 (PDB accession code: 4JIT) and E. coli XGPRT.GMP complexes (PDB accession code: 1A97). (a) Stereo view of the two ligands in the active site (b) Surface representation of E. coli XGPRT with 1 (dark grey) and GMP (coloured by element, with carbon in green) and borate (yellow). The structure of the large flexible loop (S62-E70) is shown as a cartoon with a transparent surface. Borate (present in the crystallization buffer) indicates the likely location where one of the phosphate groups of PPi would bind. “X”, therefore, indicates the position where a second phosphonate group would be expected to be located.



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This new class of inhibitor provides scope for a variety of different chemical modifications to be made to the scaffold to enhance potency. These include: (i) changing the stereochemistry of the attachment of the pyrrolidine group to the N9 atom of the purine base; (ii) changing the number and identity of the atoms in the two linkers connecting the three “spokes” to the “hub”; (iii) alteration of the stereochemistry of the attachments to the pyrrolidine ring; and (iv) attachment of different functional groups (e.g. hydroxyl groups) to each of the linkers (Figure 2). Chemical synthesis of the PNBPs.

The four pyrrolidine nucleoside

bisphosphonates (PNBPs) compounds 2-5 (Figure 2) were synthesized according to Scheme 1. The details of the synthetic procedures together with the analysis of their structures by NMR and mass spectrometry is presented in the Suplementary Information.

Boc N OEt EtO P O

(R)

iii, iv, v, vi

(S)

O

2

22%

7 OH

i, ii 59%

Boc N (R)

59%

(S)

HO 6

Boc N

vii,viii

ODMTr

O

Boc N

ix

O O P O

68%

(R)

(S)

O

ODMTr 8

(R)

(S)

O

ODMTr

OH

Boc N

x

9

O O P O

(S)

+

(R)

(S)

O

ODMTr 10-S

OTBDMS

Boc N O O P O

(R)

(R)

(S)

O

ODMTr 10-R

OTBDMS

32%

30%

H N O O P O

iv

(R) (S)

(R)

O

N

OH

NH

13 N 74%

O O P O

60%

N

O

O P

(S)

O

iii (R)

N

OTBDMS 12

N Cl

Boc N

31%

N

N

O

v

(R)

O O P O

(S)

(R)

(S)

O

OH

ii,xi,iv,v,vi

5

OTBDMS 11

xi,iv,v,vi

10%

O

8%

ii

70%

Boc N

3

O vi

N O O P O

63% (R)

O (S) OH 14

(R)

Hx

4

i. Diethyl vinylphosphonate, t-BuOH, KOH, Cs 2CO3; ii. 2%TFA/CHCl3; iii.6-chloropurine, Ph 3P, DIAD, THF; iv. 1.5M HCl/H 2O-EtOH 1:1, 75 °C; v. diisopropyl 3-phosphonopropionic acid, EDC, DMF, 80 °C; vi. Me 3SiBr, MeCN; vii. 3-bromoprop-1-ene, NaH, DMF; viii. NaIO 4, OsO 4, THF; ix.diispropyl phosphite, TEA, DCM; x. TBDMSCl, imidazole, DMF; xi. 2-amino-6-chloropurine, Ph 3P, DIAD, THF.

Scheme 1.


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PNBPs as inhibitors of PvHGPRT. Table 1 shows how the “hub” and “spoke” strategy resulted in a decrease in the Ki values for PvHGPRT by greater than 430-fold (cf 3 and 4 with 1) as well as decreases for the other two enzymes. Compounds 3 and 4 exhibit the lowest Ki values for an inhibitior of PvHGPRT yet discovered. For PfHGXPRT, compound 4 has the lowest Ki value (2 nM) which is comparable to that previously obtained for the best two inhibitors of this enzyme. These compounds are the acyclic immucillin phosphonate (AIP) and the transition state analog, (1S)-1-(9-deazahypoxantnine-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5phosphate (ImmHP), which have Ki values of 0.65 nM10 and 1 nM.14

Table 1. Ki (nM) values of the pyrrolidine containing compounds for the three 6oxopurine PRTases.

Compound

PvHGPRT

Pf HGXPRT

Human HGPRT

1

2 600 ± 500

400 ± 50

73 000 ± 5 000

2

80 ± 10

14 ± 3

20 ± 5

3

8±2

7±1

3±1

4

6±1

2 ± 0.5

1.3 ± 0.5

5

60 ± 8

10 ± 1

8±2

See Figure 2 for the chemical structures of these compounds.

The major factor that resulted in the significant decrease in the Ki values is attributed to the attachment of a second phosphonate to the pyrrolidine ring (cf. 1 with 2 in Figure 2 and Table 1). Table 1 also shows that for 3 and 4, the Ki values are independent of the nature of the purine base (i.e. hypoxanthine or guanine) (Figure 2).

This finding is in contrast to inhibitors such as the acyclic nucleoside



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phosphonates, the aza-acyclic nucleoside phosphonates and the nucleoside monophosphates where the chemical nature of the purine base can exert a large effect on the Ki or the Km values.12, 18 The attachment of the hydroxyl group to the linker connecting a carbon atom of the pyrrolidine ring to a phosphonate group (Figure 2; cf 2 and 4) decreases the Ki values for PvHGPRT by ~13-15-fold (Table 1). This demonstrates that modifications to the linker atoms can also effect the binding of these inhibitors. Changing the isomer from (S) to (R) increased the Ki value for PvHGPRT (by ~7-8-fold) (Figure 2; Table 1; cf 3 and 5). However, it had less effect on PfHGXPRT with the values being similar (7 nM vs 10 nM; Table 1). Thus, subtle variations in the structure of these inhibitors can have a profound effect on the inhibition constants. This data further shows that the PNBPs possess the inherent versatility to allow the further development of inhibitors that can specifically target PvHGPRT. The structure of PvHGPRT has not previously been visualized. Therefore, a critical step in determining why the PNBPs are potent inhibitors was to obtain the structure of the E.I complex. The structure of the human HGPRT.3 complex was also obtained to compare binding modes of compound 3 in these two enzymes. Crystal structure of the PvHGPRT.3 complex. Crystals of the PvHGPRT.3 complex were obtained that diffracted to 2.85 Å resolution. The crystal structure of PvHGPRT in the presence or absence of ligands has proven elusive, with the best value for diffraction obtained previously being 5-7 Å. However, this low- resolution data was obtained in complex with ligands that had much lower affinity for PvHGPRT than the PNBPs. It is likely that one of the difficulties in obtaining atomic resolution data of the Pv enzyme is the high level of flexibility of the mobile loops surrounding the active site. Such mobility may preclude a rigid fold of the enzyme


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essential for high resolution crystal structures. Indeed, crystal structures of the two plasmodial 6-oxopurine PRTases, Pf and now Pv, have been determined only in the presence of low nanomolar inhibitors.10, 14 Refinement of the PvHGPRT.3 structure incorporated a novel, interactive molecular dynamics flexible fitting (iMDFF) approach19 (Figure S1) coupled with traditional methods that employed the use of non-crystallographic symmetry restraints. This approach is of particular value in obtaining reliable structures when the resolution of the data is of the order of 3 Å and provides for the accurate refinement of the structure of the flexible loops. Occupancy of the active site. The final model for the PvHGPRT.3 complex gave an Rfree of 0.252 with excellent stereochemistry (Table S1). The refinement statistics for the human enzyme in complex with 3 and 5 are also provided (Table S1). The idea for the “hub” and “spoke” strategy is exemplified in Figure 4a. The basic concept has a central five membered group (i.e. the “hub”) to which three chemical “spokes” can be attached allowing these moieties to reach into separate designated areas in the active site. The presence of rotatable bonds (Figure 4a) provides flexibility to allowing optimization of interactions of the “spokes” and linker atoms with active site amino acid residues. The electron density maps (Figures 4b and 4c) of the two E.I complexes confirm that the inhibitor design concept was fulfilled, with the four components fitting into different locations in the active site.



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(a)

(b)

(c)

(d)

Figure 4. Inhibitor binding to the 6-oxopurine PRTases. (a) Concept of inhibitor design based on a central pyrrolidine hub (red) with three rotatable attachments (green, blue and yellow). (b) and (c) Fo-Fc difference electron density maps for 3 in the PvHGPRT.3 complex and 3 in the human HGPRT.3 complex, respectively. Both maps are contoured at the 3.0 σ level. (d) Comparison of the binding mode of 3 in PvHGPRT (yellow) and 1 in EcXGPRT (white).

A comparison of the binding mode of compound 3 in PvHGPRT and compound 1 in EcXGPRT (Figure 4d) shows that the pyrrolidine ring is rotated by ~90° relative to the pyrrolidine ring in the E. coli XGPRT.1 complex and also by the same amount as the ribose ring of GMP in the E. coli XGPRT.GMP complex (see also Figure 3a). In the former complex, the pyrrolidine ring appears to protrude


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slightly out of the active site whereas, in the latter complex, the ribose ring is located deeper in the active site. Comparison of PvHGPRT.3 and human HGPRT.3 complexes.

Both

enzymes crystallized as a tetramer and their polypeptides have similar overall folds and subunit arrangements (rmsd value of 1.1 Å after superimposition of all visible Cα atoms) (Figures 5a and 5b).

Figure 5. Tetrameric structure of the PvHGPRT.3 (PDB accession code: 6BO7) and human HGPRT.3 (PDB accession code: 5HIA) complexes. (a) Surface representation of the PvHGPRT.3 complex. (b) Surface representation of the human HGPRT.3 complex. From this view, 3 is partially visible as the bright red and green spheres in two of the subunits. In a different view (i.e. Figure 4b and 4c) 3 is observed almost fully exposed to the solvent. (c) Stereoview of the residues that interact diagonally across the tetramer in the PvHGPRT.3 complex.



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The two residues located on the flexible loop that bend toward each other (middle top and bottom of the image; Figure 5a) in PvHGPRT are K20 from adjacent subunits. The equivalent residue in the human HGPRT is D12 (Figure S2). These residues are located on flexible loops in both structures. R92 is located in the center of the PvHGPRT tetramer. These residues from each of the four subunits form a pairwise stacking arrangement (red dashed lines; Figure 5c). The positive charges on these residues are partially offset by the close approach of E55 (blue dashed lines) (Figure 5c). In the Pf enzyme, there is also an arginine present (R92) in this location and, therefore, the same stacking is observed to stabilize the tetramer. In the human enzyme, this residue is replaced by alanine (A83). As a result, there are no diagonally opposed interactions in the human HGPRT structure (Figure 5b). Thus, this arrangement is particular to the two Plasmodium enzymes and is absent in the human counterpart. The subunit structure of PvHGPRT.3. The crystal structure of the PvHGPRT.3 complex revealed three identical tetramers (12 subunits) in the asymmetric unit. Superimposition of the 12 subunits (Figures S3a and 3b) shows that they are all but identical to each other. The rmsd for the Cα atoms after superimposition onto chain A ranges from 0.16 Å (184 atoms) (chain B, most similar) to 0.24 Å (201 atoms) (chain J, least similar). Thus, when this inhibitor binds, the loops that surround the active site, as well as the inhibitor itself, are firmly locked into position with little variability between subunits. This high degree of rigidity provides one piece of evidence to suggest that high resolution crystal structures of PvHGPRT may only be possible in the presence of low nanomolar inhibitors that confer stability to the structure.


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Active site of the PvHGPRT.3 complex. The specific interactions between 3 and the amino acid residues that surround the active site are shown in Figures S3 and 6a with the overall fold of a subunit in Figure S4. The contribution of bound magnesium ions is highlighted in Figure 6b.

(a)

D145 D204

E144 Mg2+

Mg2+

(b) Figure 6. Active site of the PvHGPRT.3 complex. (a) Ribbon representation of the overall fold. Green and red spheres represent magnesium and water, respectively. (b) Surface representation of the enzyme. The location and ligands of the two divalent metal ions in the active site of the PvHGPRT.3 complex. Grey and red spheres represent the magnesium ions and water molecules, respectively. The magenta sphere is a water molecule present in the human HGPRT.3 complex. This water is not visible in the PvHGPRT.3 complex, possibly due to the fact it is a lower resolution structure.



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There are two magnesium ions in the active site of the PvHGPRT.3 complex (Figures 6a and 6b). In the published structures of the human or Pf enzymes, there are either none,20 one10 or two divalent metal ions12, 14, 15 depending on the nature of the ligand, so their role is variable. In this structure, the two magnesium ions lock the inhibitor into place via direct coordination to the inhibitor and active site residues and via a network of bridging waters (Figures 6a and b). They also stabilize two of the mobile loops which are between V198-D204 and E144-D145. In contrast, in the PfHGXPRT.AIP.PPi complex, only one magnesium ion is present and this is in the PPi binding site.10 Location of the pyrrolidine ring “hub”. Superimposition of the PvHGPRT.3 complex with that of the Pf enzyme in complex with ImmHP shows that pyrrolidine group of the PNBP is located in a different position from that of the iminoribitol group of the transition state analog (Figures S5a and c). However, this difference does not result in differences in the positioning of the two phosphorus atoms. Indeed, the phosphorus atom of one of the phosphonate groups of 3 in the PvHGPRT.3 complex is located in a similar position as one of the phosphorus atoms of PPi while the carbonyl group superimposes on the second phosphoryl group of PPi when it is bound in the active site of human HGPRT together with ImmGP (Figure S5c). Thus, this part of compound 3 perfectly mimics the location of PPi and contributes to the potent inhibition. There is 250 µM against D6 lines and >1000 µM against W2, demonstrating the value of the prodrug strategy. Prodrugs of the AIP, which is also nanomolar inhibitor of PfHGXPRT, exhibited IC50 values in the 2-7 µM range.10 Detailed metabolic studies demonstrated that PfHGXPRT is the target of these lead compound.10 The CC50 value for 3a in human A549 cells was also measured, and shown to be 102 ± 11 µM, ~10-fold higher than that for the Pf lines. Thus, new chemical strategies to decrease the IC50 values are needed. These investigations are in progress.


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Conclusions. A nanomolar inhibitor of PvHGPRT has been developed by rational structure based design. The foundation of this strategy incorporates the assembly of chemical building blocks that are covalently linked so that a single molecule is able to occupy four areas in the active site, enhancing its specificity for this class of enzyme. The visualization of the crystal structure of the PvHGPRT.3 complex opens the door for the design of more potent inhibitors targeting this enzyme.

METHODS Crystallization and Structure Determination. PvHGPRT at a concentration of 21 mg/mL (0.8 mM) was incubated with 6.4 mM of 3 on ice for ~5 min. For crystallization, the hanging drop method was used where 1 µL of PvHGPRT in complex with the inhibitor was added to 1 µL of well solution. The trays were stored at 18 °C. The reservoir solution for the complex was 0.3 M potassium thiocyanate, 0.1 M Bis-tris propane, pH 8.5. Crystals were cryocooled in liquid nitrogen for shipment to the Australian Synchrotron where they were robotically placed in a cryostream (100 K) on beamline MX1. X-ray data were collected remotely using BLU-ICE.25 All data sets were scaled and merged with XDS.26 The structure was solved by molecular replacement using the program PHASER27 within PHENIX 1.8.28 The protein coordinates of subunit A of human HGPRT in complex with [(2[(Guanin-9H-yl)methyl]- propane-1,3-diyl)bis(oxy)]bis(methylene)] diphosphonic acid was the starting model (PDB code 4IJQ).18 Subsequent refinement and initial model building was performed with PHENIX 1.828 and COOT 0.8,29 respectively. Further refinement and model building (particularly in regions that were relatively poorly resolved and/or varied significantly between chains) was carried out using a



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recently developed interactive molecular dynamics flexible fitting (iMDFF)19 method. Human HGPRT was stored at a concentration of 11 mg/ml (0.4 mM). The enzyme was incubated with 3 at a concentration of 4.9 mM, on ice, for ~5 minutes. For 5, the same procedure was adopted except that the concentration of inhibitor was 6 mM. The hanging drop method was used as described for the Pv enzyme. For 3, the well solution was 0.3 M sodium acetate, 17.5% PEG 3350 and, for 5, 0.2 M sodium acetate, 20% PEG 3350. Accession codes. The atomic coordinates and structure factors for the PvHGPRT.3, human HGPRT.3 and human HGPRT.5 complexes have been deposited in the Protein Data Bank with accession codes of 6BO7, 5HIA, and 6BNJ, respectively. AUTHOR INFORMATION Corresponding Authors *

E-mail: [email protected] Phone: 61 7 336 53549 or [email protected]

Phone: 42 0220 183 371

ABBREVIATIONS:

HG[X]PRT,

hypoxanthine-guanine-[xanthine]-

phosphoribosyltransferase; PRib-PP, 5-phospho-α-D-ribosyl-1-pyrophosphate; Pf, Plasmodium falciparum; Pv, Plasmodium vivax; ANP, acyclic nucleoside phosphonate; PNPs, pyrrolidine nucleoside phosphonates; PNBP, pyrrolidine nucleoside bisphosphonate; AIP, acyclic immucillin phosphonate; ImmGP, (1S)-1(9-deazaguanin-9-yl)-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


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ACKNOWLEDGMENTS Initial crystallographic conditions were determined using the University of Queensland Remote-Operation Crystallization and X-ray Diffraction facility (UQROCX). X-ray measurements were performed at the Australian Synchrotron. We thank Kerryn Rowcliffe for in vitro drug testing and the Australian Red Cross Blood Service for human blood. The views expressed here are those of the authors and not necessarily those of the Australian Synchrotron or the Australian Defence Organisation. NAMD was developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois. This work was supported by NHMRC (Grant No. 1030353), the Institute of Organic Chemistry and Biochemistry, RVO 61388963), the Czech Science Foundation (Grant No.16-06049S to D.H. and Grant No. 15-11711S to D.R.) and Gilead Sciences (Foster City, USA). Supporting Information Available: This material is available free of charge via the internet at http://pubs.acs.org. Included in this document are Figure S1-S8, Table S1, and additional experimental methods.



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REFERENCES [1] Dondorp, A. M., Fairhurst, R. M., Slutsker, L., Macarthur, J. R., Breman, J. G., Guerin, P. J., Wellems, T. E., Ringwald, P., Newman, R. D., and Plowe, C. V. (2011) The threat of artemisinin-resistant malaria, N Engl J Med 365, 10731075. [2] Price, R. N., Douglas, N. M., and Anstey, N. M. (2009) New developments in Plasmodium vivax malaria: severe disease and the rise of chloroquine resistance, Curr Opin Infect Dis 22, 430-435. [3] Severini, C., and Menegon, M. (2015) Resistance to antimalarial drugs: An endless world war against Plasmodium that we risk losing, J Glob Antimicrob Resist 3, 58-63. [4] Phillips, M. A., Burrows, J. N., Manyando, C., van Huijsduijnen, R. H., Van Voorhis, W. C., and Wells, T. N. C. (2017) Malaria, Nat Rev Dis Primers 3, 17050. [5] Tham, W. H., Beeson, J. G., and Rayner, J. C. (2017) Plasmodium vivax vaccine research - we've only just begun, Int J Parasitol 47, 111-118. [6] Matuschewski, K. (2017) Vaccines against malaria-still a long way to go, FEBS J 284, 2560-2568. [7] Price, R. N., Tjitra, E., Guerra, C. A., Yeung, S., White, N. J., and Anstey, N. M. (2007) Vivax malaria: neglected and not benign, Am J Trop Med Hyg 77, 7987. [8] De Clercq, E. (2013) The Acyclic Nucleoside Phosphonates (ANPs): Antonin Holy's Legacy, Med Res Rev, 1278-1303.


Page 26 of 32

Page 27 of 32


27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[9] Berg, M., Van der Veken, P., Goeminne, A., Haemers, A., and Augustyns, K. (2010) Inhibitors of the purine salvage pathway: a valuable approach for antiprotozoal chemotherapy?, Curr Med Chem 17, 2456-2481. [10] 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., and Schramm, V. L. (2012) Acyclic

immucillin

Plasmodium

phosphonates:

Second

falciparum

generation

inhibitors

of

hypoxanthine-guanine-xanthine

phosphoribosyltransferase, Chem Biol 19, 721-730. [11] Keough, D. T., Hocková, D., Holý, A., Naesens, L. M., Skinner-Adams, T. S., de Jersey, J., and Guddat, L. W. (2009) Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: A new class of antimalarial therapeutics, J Med Chem 52, 4391-4399. [12] Keough, D. T., Hockova, D., Janeba, Z., Wang, T. H., Naesens, L., Edstein, M. D., Chavchich, M., and Guddat, L. W. (2015) Aza-acyclic nucleoside phosphonates containing a second phosphonate group as inhibitors of the human,

Plasmodium

falciparum

and

vivax

6-oxopurine

phosphoribosyltransferases and their prodrugs as antimalarial agents, J Med Chem 58, 827-846. [13] Keough, D. T., Hocková, D., Krečmerová, M., Česnek, M., Holý, A., Naesens, L., Brereton, I. M., Winzor, D. J., de Jersey, J., and Guddat, L. W. (2010) Plasmodium vivax hypoxanthine-guanine phosphoribosyltransferase: A target for anti-malarial chemotherapy., Mol Biochem Parasitol 173, 165-169. [14] Shi, W., Li, C. M., Tyler, P. C., Furneaux, R. H., Cahill, S. M., Girvin, M. E., Grubmeyer, C., Schramm, V. L., and Almo, S. C. (1999) The 2.0 Å structure



Page 28 of 32

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of malarial purine phosphoribosyltransferase in complex with a transitionstate analogue inhibitor, Biochemistry 38, 9872-9880. [15] Shi, W., Li, C. M., Tyler, P. C., Furneaux, R. H., Grubmeyer, C., Schramm, V. L., and Almo, S. C. (1999) The 2.0 Å structure of human hypoxanthineguanine phosphoribosyltransferase in complex with a transition-state analog inhibitor, Nat Struct Biol 6, 588-593. [16] Vos, S., de Jersey, J., and Martin, J. L. (1997) Crystal structure of Escherichia coli xanthine phosphoribosyltransferase, Biochemistry 36, 4125-4134. [17] Keough, D. T., Hockova, D., Rejman, D., Spacek, P., Vrbkova, S., Krecmerova, M., Eng, W. S., Jans, H., West, N. P., Naesens, L. M., de Jersey, J., and Guddat, L. W. (2013) Inhibition of the Escherichia coli 6-oxopurine phosphoribosyltransferases by nucleoside phosphonates: potential for new antibacterial agents, J Med Chem 56, 6967-6984. [18] Keough, D. T., Spacek, P., Hockova, D., Tichy, T., Vrbkova, S., Slavetinska, L., Janeba, Z., Naesens, L., Edstein, M. D., Chavchich, M., Wang, T. H., de Jersey, J., and Guddat, L. W. (2013) Acyclic nucleoside phosphonates containing a second phosphonate group are potent inhibitors of 6-oxopurine phosphoribosyltransferases and have antimalarial activity, J Med Chem 56, 2513-2526. [19] Croll, T. I., Smith, B. J., Margetts, M. B., Whittaker, J., Weiss, M. A., Ward, C. W., and Lawrence, M. C. (2016) Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain, Structure 24, 469-476. [20] Keough, D. T., Brereton, I. M., de Jersey, J., and Guddat, L. W. (2005) The crystal

structure

of

free

human


hypoxanthine

guanine

Page 29 of 32


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phosphoribosyltransferase

reveals

extensive

conformational

plasticity

throughout the catalytic cycle, J Mol Biol 351, 170-181. [21] Clinch, K., Evans, G. B., Fleet, G. W. J., Furneaux, R. H., Johnson, S. W., Lenz, D. H., Mee, S. P. H., Rands, P. R., Schramm, V. L., Ringia, E. A. T., and Tyler, P. C. (2006) Syntheses and bio-activities of the L-enantiomers of two potent

transition

state

analogue

inhibitors

of

purine

nucleoside

phosphorylases, Organic & Biomolecular Chemistry 4, 1131-1139. [22] Cesnek, M., Hockova, D., Holy, A., Dracinsky, M., Baszczynski, O., Jersey, J., Keough, D. T., and Guddat, L. W. (2012) Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases, Bioorg Med Chem 20, 1076-1089. [23] Hecker, S. J., and Erion, M. D. (2008) Prodrugs of phosphates and phosphonates, J Med Chem 51, 2328-2345. [24] Oliveira, F. M., Barbosa, L. C. A., and Ismail, F. M. D. (2014) The diverse pharmacology and medicinal chemistry of phosphoramidates - a review, Rsc Advances 4, 18998-19012. [25] McPhillips, T. M., McPhillips, S. E., Chiu, H. J., Cohen, A. E., Deacon, A. M., Ellis, P. J., Garman, E., Gonzalez, A., Sauter, N. K., Phizackerley, R. P., Soltis, S. M., and Kuhn, P. (2002) Blu-Ice and the Distributed Control System:

software

for

data

acquisition

and

instrument

control

at

macromolecular crystallography beamlines, J Synchrotron Radiat 9, 401-406. [26] Kabsch, W. (2010) Xds, Acta Cryst D 66, 125-132.



30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[27] McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J App Cryst 40, 658-674. [28] Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Cryst D 66, 213-221. [29] Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Cryst D 66, 486-501.


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1 2 3 4 5 6 ACS Paragon Plus Environment 7 A 8 designed nucleoside phosphonate with a Ki value of 6 nM for P. 9 vivax hypoxanthine-guanine phosphoribosyltransferase.

Design of Plasmodium vivax Hypoxanthine-Guanine

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