Genetic Resistance to Purine Nucleoside Phosphorylase Inhibition in Plasmodium Falciparum

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

Rodrigo G. Ducatia, Hilda A. Namanja-Maglianoa, Rajesh K. Harijana, J. Eduardo Fajardob, Andras Fiserb, Johanna P. Dailyc,d,1, and Vern L. Schramma,1

aDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; bDepartment of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461; cDepartment of Medicine, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, NY 10461; and dDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461

Contributed by Vern L. Schramm, January 21, 2018 (sent for review December 30, 2015; reviewed by Thomas E. Wellems and Elizabeth A. Winzeler) Plasmodium falciparum causes the most lethal form of human 6-oxopurine bases (11, 12). Transcripts for PfPNP are found in all malaria and is a global health concern. The parasite responds to P. falciparum life cycle stages (13). Targeting hypoxanthine pro- antimalarial therapies by developing drug resistance. The contin- duction is strategic as P. falciparum are purine auxotrophs, and uous development of new antimalarials with novel mechanisms of rely on purine base salvage for purine nucleotide and oligo- action is a priority for drug combination therapies. The use of nucleotide synthesis (14). DADMe-ImmG inhibits the PNPs transition-state analog inhibitors to block essential steps in purine from both human and malaria parasites with picomolar disso- salvage has been proposed as a new antimalarial approach. Mu- ciation constants and acts as an oral antimalarial drug for P. tations that reduce transition-state analog binding are also expected to falciparum infections in Aotus primates (8). Transition-state reduce the essential catalytic function of the target. We have previously analogs target the catalytic function of their enzyme targets reported that inhibition of host and P. falciparum purine nucleoside such that binding affinity is proportional to catalytic efficiency Pf phosphorylase ( PNP) by DADMe-Immucillin-G (DADMe-ImmG) causes (15). Mutations that decrease the affinity of transition-state purine starvation and parasite death in vitro and in primate infection analogs are expected to decrease the catalytic function of the P. falciparum models. cultured under incremental DADMe-ImmG drug target. The subnanomolar affinity of transition-state analogs Pf pressure initially exhibited increased PNP gene copy number and pro- for their target proteins also limits the effectiveness of drug- Pf BIOCHEMISTRY tein expression. At increased drug pressure, additional PNP gene export mechanisms. Drug resistance in P. falciparum often copies appeared with point mutations at catalytic site residues involved comes from point mutations in the target proteins or altered in drug binding. Mutant PfPNPs from resistant clones demonstrated drug-export activity (16), mechanisms that may be slower to reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. develop with transition-state analogs. The catalytic defects were partially overcome by gene amplification in We sought to characterize the genomic response of P. falciparum the region expressing PfPNP. Crystal structures of native and mutated toward drug selection pressure using DADMe-ImmG. The PfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome analysis of genomic amplification in resistant clones charac- terizes the responses to drug pressure and identifies the mo- purine starvation induced by DADMe-ImmG. Resistance developed – slowly, over 136 generations (2136 clonal selection). Transition-state lecular markers of resistance (17 22). This approach has analog inhibitors against PfPNP are slow to induce resistance and allowed the identification of antimalarial drug resistance may have promise in malaria therapy. Significance malaria | drug resistance | genetic resistance mechanisms | gene amplification | gene mutation Hypoxanthine salvage is essential for nucleic acid synthesis in Plasmodium falciparum but not in humans. Hypoxanthine alaria remains a common cause of death in children under production in P. falciparum can be blocked by DADMe- the age of five years and pregnant women in endemic re- Immucillin-G (DADMe-ImmG), a transition-state analog in- M Pf gions (1, 2). Approximately 400,000 people died from malaria hibitor of purine nucleoside phosphorylase ( PNP). Parasites and 212 million cases were reported in 2015 (3). The most severe have been previously reported to die in response to DADMe- form of malaria is caused by Plasmodium falciparum, a protozoan ImmG both in vitro and in experimental primate infections. Drug pressure with DADMe-ImmG selects drug-resistant P. falci- parasite. The first-line treatment recommended by the World parum Pf Health Organization is artemisinin-based combination therapy with increased PNP gene copy number, increased (ACT) (4), which includes a quinoline or antifolate drug in protein production, and mutations to reduce drug binding. DADMe-ImmG is slow to produce resistance in P. falciparum, combination with an artemisinin derivative. Despite the effec- supporting its potential for malaria therapy. tiveness of ACT in reducing mortality rates, parasite resistance

has emerged in the greater Mekong subregion of Cambodia, Author contributions: R.G.D., R.K.H., J.P.D., and V.L.S. designed research; R.G.D., Myanmar, Thailand, and Vietnam (5, 6). Measures to contain H.A.N.-M., and R.K.H. performed research; R.G.D., H.A.N.-M., R.K.H., J.E.F., A.F., and resistance include monitoring and surveillance of drug-resistant J.P.D. analyzed data; and R.G.D., H.A.N.-M., R.K.H., J.E.F., J.P.D., and V.L.S. wrote parasites, followed by strategies to prevent widespread dissemi- the paper. nation (7). The emergence of ACT resistance increases the ur- Reviewers: T.E.W., National Institutes of Health; and E.A.W., University of California, gency for new treatment strategies. Agents with novel mechanisms San Diego. of action will permit new drug combinations for treatment and The authors declare no conflict of interest. prevention of P. falciparum infections. Published under the PNAS license. DADMe-Immucillin-G (DADMe-ImmG) is a transition-state Data deposition: The crystallography, atomic coordinates, and structure factors have been analog inhibitor of P. falciparum purine nucleoside phosphory- deposited in the Protein Data Bank, https://www.rcsb.org/ (PDB ID codes 6AQS and Pf 6AQU). lase ( PNP), an essential enzyme in the purine salvage pathway 1 To whom correspondence may be addressed. Email: [email protected] or (Fig. 1) (8–10). PfPNP catalyzes the reversible phosphorolysis [email protected]. N of the -glycosidic bond of 6-oxopurine (deoxy)ribonucleosides This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to generate (deoxy)ribose 1-phosphate and the corresponding 1073/pnas.1525670115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1525670115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 A B

Fig. 1. PfPNP reaction and transition-state analog. (A) Purine nucleoside phosphorylase catalyzes the phosphorolysis of inosine to form hypoxanthine and α-D-ribose 1-phosphate via a ribocationic transition state. Other 6-oxypurine nucleosides (e.g., guanosine) are also substrates. The transition state for PfPNP is shown in brackets. (B) DADMe-ImmG is a transition-state analog for the phosphorolysis of guanosine.

mechanisms, including copy number variants, single nucleotide Selection to 8 μM resistance. Three cultures resistant at 5 μM variants, and drug transport alterations (19, 22–24). For exam- DADMe-ImmG were subjected to increasing drug pressure for ple, mefloquine resistance has been associated with increase in an additional 32 generations to achieve growth at 8 μM of the pfmdr1 copy number (25), and artemisinin resistance has been inhibitor (Fig. 2). IC50 values for these three cultures growing in attributed to mutations in the P. falciparum kelch13 gene (22). 8 μM DADMe-ImmG varied from 65 to 230 μM (280- to 980- The identification of drug resistance mutations informs drug fold increased resistance) and, on average, 500-fold more resistant mechanism and antimalarial treatment strategies. than the native parental strain (Fig. 3B). Three independently Parasite response to modest DADMe-ImmG drug pressure conditioned cultures (Fig. 2) were subjected to genomic DNA led to an increase in PfPNP gene copy number, PfPNP protein, and protein analysis. From parental strain to the fully resis- and enzymatic function. At increased drug pressure, PNP gene tant strains represents 136 generations under increasing drug 136 amplification increased and point mutations at residues involved pressure (2 clonal expansion). Pf in drug binding were observed. The results support PNP as the μ sole functional target for DADMe-ImmG, elucidate the mech- Genomic Analysis at 2 M DADMe-ImmG Resistance. Genome se- anisms of resistance and support the potential for targeting the quencing examined four independently selected resistant clones and two independently selected clones from control cultures. purine salvage pathway with transition-state analogs. Sequencing of these 2-μM-resistant isolates and controls was Results and Discussion at >15× sequence coverage. μ In Vitro Selection of DADMe-ImmG Resistant Parasites. P. falciparum Four independent clones from 2- M-resistant strains all isolates resistant to DADMe-ImmG were selected by continuous showed amplification of a local region in chromosome 5 that includes the purine nucleoside phosphorylase gene (PNP; in vitro drug pressure (26–28). The high (370 μM) hypoxanthine locus_tag PFE0660c). The amplified region was ∼20 kb long in usually added to culture media was replaced with a more- two isolates and 60 kb in the other two. In clones with the 60-kb physiological level of 10 μM (29). P. falciparum cells cultured amplified region, the expanded genome contained the coding in vitro are susceptible to PfPNP inhibition by DADMe-ImmG region for PfPNP together with ∼18 neighboring genes (Table only at growth-limiting hypoxanthine, because high hypoxanthine S1). The PfPNP locus was amplified four- to sixfold with no bypasses the physiological function of PNP (3, 10). detectable sequence mutations. This genetic structure and the Selection to 2 μM resistance. The initial selection for resistance to parasite response to IC determination was unchanged follow- DADMe-ImmG was performed in P. falciparum 3D7 parasites 50 ing removal of the drug selection pressure and growth for an by in vitro cultivation under increasing drug pressure (100 nM to additional 30 generations in the absence of DADMe-ImmG. 2 μM) over the course of 30 generations (Fig. 2). Parental P. falciparum 3D7 parasites were cultured in parallel without drug Point Mutations in the PfPNP Gene for Parasites Resistant to 8 μM as controls. Clonal parasite isolates were generated by limiting di- DADMe-ImmG. Parasite growth at higher concentrations of lution (23, 30) and cultured for DNA and protein analysis. Five DADMe-ImmG in a subsequent round of selection resulted in individual resistant clones were selected at 2 μM drug resistance. 500-fold DADMe-ImmG resistance and increased amplification Three were characterized by IC50 analysis and expanded for geno- in the PNP locus (7- to 13-fold). For these resistant parasites, mic and enzymatic characterization. The remaining two clones were genomic sequencing was at >300× coverage. Three independently cultured for an additional 30 generations without drug to determine developed resistant isolates (Fig. 2) were genomically sequenced if parasites reverted to the drug-sensitive phenotype (Fig. 2). All five and had developed point mutations affecting some copies of the resistant clones were analyzed for IC50 values. There were no sig- PfPNP protein. In two clones, a transversion of T to A at po- nificant differences in IC50 between these groups. IC50 values in- sition 547 caused an M-to-L change in codon 183 (M183L). creased 5.5- to 7.2-fold in the five resistant clones for an average This mutation was present in 65% and 50% of the sequence 6.5 ± 0.7 increase in IC50 (from 0.24 to 1.5 μM) (Fig. 3A). Because reads, respectively. Thus, the genome-amplified region contains withdrawal of drug pressure from resistant clones did not reverse a mixture of native and mutated PfPNP sequences. In the third the resistance, a stable genomic resistance was established. clone, a T-to-A transversion at position 542 caused a V-to-D Selection to 5 μM resistance. DADMe-ImmG drug selection was change at codon 181 (V181D). In this clone, 46% of the reads continued for an additional 74 generations on three clonal cul- carry the mutation, again indicating a mixed expression of na- tures resistant to 2 μM DADMe-ImmG until growth was sus- tive and mutant PfPNPs. Other mutations were found in the tained at 5 μM of the inhibitor (Fig. 2). IC50 values for these genomes from individual clones, but were unrelated to purine three clones increased to 65 ± 16 μM, a 276-fold increase from metabolism. Each of the mutations outside the PfPNP region the parental strains (Fig. 3B). was found in only a single resistant clone. These mutations were

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1525670115 Ducati et al. Downloaded by guest on October 1, 2021 cultures grown in the presence of DADMe-ImmG showed no catalytic activity, despite overexpression of the protein. DADMe- ImmG is a tight-binding inhibitor and remains bound to PfPNP, despite high substrate concentrations in the assay mixtures (31, 32). In the highly resistant clones, 4.5- to 13-fold increases of PfPNP protein were observed in extracts that also contained the V181D and M183L mutations (Fig. 3D).

Catalytic Properties of Mutant PfPNPs. The PfPNP proteins containing M183L and V181D mutants were obtained by site- directed mutagenesis and expression in Escherichia coli. Native and mutant N-terminal His-6 PfPNPs incorporating a thrombin cleavage site were expressed and purified using Ni-NTA affinity chromatography. The enzymes were kinetically characterized after His-purification sequences were removed by thrombin cleavage. The His-6 purification sequence had little effect on the kinetic properties of the enzymes. The purity and mass of the PfPNP constructs were established by SDS/PAGE and mass spectrometry. PfPNP M183L and V181D mutants showed reduced catalytic efficiency for hypoxanthine formation and reduced binding affinity for DADMe-ImmG (Table 1). Mutation V181D decreased the kcat and increased the Km and Ki* for DADMe-ImmG by factors of 2, 2, and 42, respectively. Thus, the catalytic efficiency (kcat/Km) decreased fourfold, whereas the inhibitor binding affinity decreased more, by an order of magnitude. Gene amplification of this mutation provides the organism with near-native catalytic

capacity but a 42-fold diminished sensitivity to DADMe-ImmG. BIOCHEMISTRY One hypothesis for the effective action of transition-state analogs on their targets is that any loss of affinity toward the transition- state analog will be matched by an equal loss of catalytic efficiency. The V181D mutation clearly demonstrates that mutations in PfPNP can occur with disproportionate effects on catalysis and inhibitor binding. Mutation M183L had more profound effects on PNP catalytic activity. The kcat decreased 30-fold, and the Km for inosine and Ki* for DADMe-ImmG increased by factors of 558 and 3.9 × 4 10 , respectively. Thus, catalytic efficiency (kcat/Km) decreased 1.7 × 104-fold and the inhibitor binding affinity decreased by a factor of 3.9 × 104. In this case, loss of catalytic function and inhibitor affinity are nearly equal. Decreased catalytic function by 17,000-fold in M183L PfPNP is incompatible with its essential function to provide hypoxanthine. However, steady- state kinetic analysis of purified M183L PfPNP (as shown in Table 1) may not accurately represent the in vivo activity in resistant P. falciparum. Coexpression of M183L PfPNP with Pf Fig. 2. Generation of DADMe-ImmG–resistant P. falciparum. Initial clones native PNP may lead to the formation of hybrid multimers of resistant parasites were selected from cultures under continuous drug containing native and M183L PNP subunits. The M183L pressure from 100 nM to 2 μM over 30 generations. Individual resistant mutation alters the subunit interactions observed in crystal- clones averaged 6.5-fold resistance. Clones indicated “withdrawn drug lographic analysis (see below). In hybrids of native and M183L pressure” were cultured without DADMe-ImmG for an additional 30 gener- subunits it is possible that some subunits may retain residual ations before analysis. Clones indicated “continued drug pressure” were catalytic activity in the presence of micromolar DADMe- expanded with 2 μM continued drug pressure for genomic and protein μ ImmG. The related PNP from the human host forms a func- analysis. An additional 74 generations increased resistance to 5 M DADMe- tional homotrimer. Binding of DADMe-ImmG exhibits strong ImmG. Continued drug pressure for an additional 32 generations gave re- negative cooperativity. Binding of transition-state analog in- sistance to 8 μM DADMe-ImmG. One or more features of the IC50, genomic DNA, PfPNP protein, and catalytic activity were characterized at 2, 5, and hibitors to the first catalytic site completely inhibits the en- 8 μM drug resistance. zyme. In contrast, all six subunits of the PfPNP must be saturated for full inhibition. Mixed mutant and native hybrid oligomers of PfPNP provide a hypothetical model for re- attributed to background mutation rates during the 136 gener- sistance by way of altered inhibitor binding in M183L hybrid ations of drug selection pressure (Fig. 2 and Table S2). PfPNPs that retain adequate catalytic activity.

Cellular PNP Level in DADMe-ImmG–Resistant Parasites. PfPNP Structural Characterization of Drug-Resistant PfPNPs. PfPNP is a protein levels in drug-resistant and control isolates were ana- homohexameric enzyme with subunit monomers containing eight lyzed by Western blots from cell extracts. At 2 μM DADMe- α-helices and nine β-sheets (PDB ID code 1SQ6) (33). Structures ImmG resistance, a three- to fourfold increase of PfPNP pro- have been reported in ternary complexes with phosphate, tein was estimated (Fig. 3C). PfPNP catalytic activity also Immucillin-H (PDB code ID code 1NW4) and DADMe-ImmG increased in drug-resistant cell extracts (Fig. S1). Extracts from (PDB ID code 3PHC). Cocrystallization of drug-resistant PfPNP

Ducati et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 1, 2021 mutants V181D and M183L in the presence of DADMe-ImmG produced unliganded PfPNP crystals. Soaking preformed crystals with DADMe-ImmG (Table S3) yielded crystals in complex with DADMe-ImmG for V181D but not the M183L protein. Dif- fraction data for the V181D PfPNP–DADMe-ImmG complex and the unliganded M183L PfPNP were obtained in the H32 and I4122 space groups, respectively (Table S4). Molecular re- placement found one protomer in the H32 space group and two protomers in the I4122 space group. Loop Pro209–Leu221 was disordered in both proteins. Electron density was also missing for Gly67–Ala69 and Tyr161–Pro167 in M183L PfPNP (Fig. S2). All other amino acids were readily fitted into the electron density maps. Electron density for DADMe-ImmG was well-defined in the V181D PfPNP structure. All of the resolved amino acids are in the favored or allowed regions of the Ramachandran plot except for residues Ala69 and Cys208 near the disordered loops in M183L PfPNP (Table S4). The monomer of V181D PfPNP– DADMe-ImmG is arranged into hexameric packing, similar to those of native PfPNP (11, 33). However, the subunit structure of M183L PfPNP differs, forming a dimer in the asymmetric unit. The subunit interaction loop (Tyr161–Pro167) is disordered, the apparent cause of the altered oligomeric state (Fig. S2). DADMe-ImmG and phosphate are bound with low B-factors in V181D PfPNP (Fig. S3 and Table S4). Superimposition of V181D PfPNP (at 1.57 Å resolution) with native PfPNP bound to DADMe-ImmG shows a rmsd of 0.26 Å, closely related structures (Fig. S2). DADMe-ImmG bound to V181D PfPNP is altered by an 18° tilt of the purine ring toward Tyr160 and Trp212 (Fig. 4). The V181D mutation causes disorder in the Pro209– Leu221 loop, distorting the positions of Pro209 and Trp212 in the purine binding pocket (Fig. 4 and Fig. S2). The structure of M183L PfPNP (2.6 Å resolution), cocrystal- lized with inorganic phosphate in the catalytic site, was similar to the complex of native PfPNP with phosphate (rmsd of 0.668 Å; Fig. S2). The subunit interaction loop (Tyr161–Pro167) is disordered and alters the normal hexameric form of PfPNP. An earlier report also showed that mutation of M183A disrupts the oligomeric state of PfPNP to become a poorly active monomer (34).

Structural Basis of PfPNP Resistance Against DADMe-ImmG. Native PfPNP (PDB ID code 3PHC) binds tightly to DADMe-ImmG, in part stabilized by hydrogen bonds to N7 and O6 of the purine ring with Asp206 and Wat281, respectively. π-Stacking and van der Waals interactions with Pro209, Trp212, and Phe217 also stabilize the 9-deazaguanine of DADMe-ImmG. These interactions are missing in V181D PfPNP to permit the 9-deazaguanine ring to tilt 18° toward Tyr160 and Trp212. This mutation and tilted purine ring result in a collapsed Pro209– Leu221 loop and disrupted π-stacking. The carboxylate of Asp206 acts to protonate N7 of the purine ring at the transition Fig. 3. Characteristics of parasites resistant to DADMe-ImmG. (A) DADMe- state. It is also moved away from N7 (Fig. 4 and Fig. S3). These μ ImmG IC50 values for isolated clones at 2 M resistance (Fig. 2). These par- missing interactions to the 9-deazapurine will affect the binding ≤ asites showed 5.5- to 7.2-fold increased IC50 values (P 0.05). Drug resistance affinity of DADMe-ImmG in V181D PfPNP. is genetically stable as withdrawal of drug pressure had no effect on re- Pf sistance. (B) Resistant parasite clones grown to 5 and 8 μM drug pressure The M183L PNP crystallized as dimer in the asymmetric unit, with phosphate but not DADMe-ImmG at the catalytic (Fig. 2) showed 206- to 980-fold increased IC50 values. Error bars are the SDs of four experiments. (C) Quantitation of Western blot intensity scans from sites. The active site of M183L PfPNP in complex with phosphate resistant strains in A showed a three- to fourfold increase (P < 0.05) for is collapsed to prevent facile substrate binding and therefore PfPNP protein within the drug-resistant samples. (D) Quantitation of West- poor catalytic activity. Because DADMe-ImmG also binds at the ern blot intensity scans from clones resistant to 8 μM DADMe-ImmG (from B) catalytic site, its binding is likewise prevented. Evidence that showed a 13.3 ± 1.1 increase in PfPNP protein for the M183L drug-resistant the same structural defect causes both effects is provided by the clones and 4.5 ± 0.4 for the V181D PfPNP clones. (E) Chromosome identifiers nearly equivalent loss of catalytic function and DADMe-ImmG for the P. falciparum genome are shown in the innermost circle. Data tracks binding. The side chain of Tyr160 tilts into the binding site for show the relative genomic content of 10-kb contiguous regions. The height of each track is 10 units. The two inner tracks show no amplified regions in two control isolates. The three outer tracks correspond to 2 μM DADMe- ImmG–resistant isolates (Fig. 2). Single amplified regions are seen in all re- three DADMe-ImmG–resistant clones at 8 μM DADMe-ImmG. Each clone has sistant strains in chr5. In each case, this region includes the full coding region distinct boundaries for gene amplification, and each contains the full coding for the PfPNP gene. (F) Region of gene amplification in chromosome 5 for region for the PfPNP gene.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1525670115 Ducati et al. Downloaded by guest on October 1, 2021 Table 1. Enzyme kinetic parameters, catalytic efficiencies, and Materials and Methods Pf inhibition constants for wild-type, M183L, and V181D PNP Supporting Information provides detailed information on the culture con- −1 −1 −1 ditions for growth and development of DADMe-ImmG resistance in P. fal- Enzyme kcat,s Km, μM kcat/Km,M ·s Ki* ciparum. The methods used for single clone selection, IC50 evaluation, Wild-type 2.63 ± 0.15 7.6 ± 1.5 346 × 103 673 ± 52 pM genomic DNA isolation and sequencing, mutational, and gene amplifi- M183L 0.08 ± 0.01 4,240 ± 925 0.02 × 103 26 ± 3 μM cation are also detailed in Supporting Information. Other methods de- V181D 1.44 ± 0.05 16.8 ± 1.5 85 × 103 28 ± 1nM tailed in Supporting Information include protein extraction and quantitation, Western blot analysis, PfPNP kinetic assays, production of native and mutant PfPNPs, kinetic analysis, crystallization, X-ray data collection, and structural analysis. the purine ring and interferes with catalytic site filling (Fig. 4). In Pf P. falciparum parasites were cultured in medium containing human blood the M183L PNP, three loops were disordered, including obtained under an approved protocol with informed consent. Human blood was Gly67–Ala69, Tyr161–Pro167, and Phe210–Pro223. Of these, the loop from Tyr161 to Pro167 is most important for subunit–subunit contacts and keeps the hexameric state stable, whereas the loop from Phe210 to Pro223 is important for binding of the 9- deazapurine ring of DADMe-ImmG (Fig. S2).

Drug Resistance Mechanisms in P. falciparum. Antimalarials in clinical use access a limited range of targets, and all drugs are known to induce resistance (e.g., refs. 19 and 31). Agents acting at the cytochrome bc1 complex, such as atovaquone, induce resistance by point mutations in the cytochrome. Antifolates like sulfadoxine or sulfadoxine–pyrimethamine combinations induce single or multiple mutations, causing resistance in dihydrofolate reductase and/or dihydropteroate synthase (35). The mutations reduce affinity for the agents while retaining sufficient catalytic

activity to permit parasite growth. Agents interfering with heme BIOCHEMISTRY polymerization to form hemozoin (e.g., chloroquine) induce resistance by point mutation in a proposed transporter gene (pfcrt). Others, including mefloquine and primaquine, induce mutations and/or copy number variations in the pfmdr1 multiple drug resistance-like transporter (36). Artemisinin resistance is also associated with increase in pfmdr1 copy number (25) or to mutations in the P. falciparum kelch13 gene, whose function is still being explored (22). Transition-state analogs differ from current antimalarials by their high affinity and high specificity for a single target, binding at the catalytic site of the target enzyme to mimic the transition- state properties. Point mutations that decrease transition-state analog binding are therefore expected to reduce the biological function of the target (7). In P. falciparum,resistanceto DADMe-ImmG is initially achieved by increasing the cellular content of the enzyme to give a modest 6.5-fold resistance. Point mutations in the active site and additional gene copy numbers give greater resistance. No changes in drug transport mechanisms were detected. The antimalarial mechanism for PfPNP differs from current antimalarial chemotypes. The slow development of resistance and novel mechanism suggests that DADMe-ImmG might provide a new candidate for combination therapy against malaria. Concluding Remarks P. falciparum responds to antimalarial therapy by developing drug resistance. DADMe-ImmG is a high-affinity transition-state analog inhibitor of PfPNP, validated for efficacy against P. falciparum at the level of Aotus primates. The mechanism of Fig. 4. Stereoview of the binding sites of native PfPNP and mutants. (A) The catalytic and inhibitor binding site of native PfPNP (yellow, PDB code ID resistance involves a combination of gene amplification and 3PHC) in complex with DADMe-ImmG (green) and phosphate (orange). The drug-binding mutations. Resistance developed slowly, and was amino acid residues interacting with DADMe-ImmG are indicated by dashed accompanied by various degrees of loss of function of the enzy- lines. (B) Superimposition of the binding sites of the native PfPNP complex matic target. The enzymatic characterization of the resistant en- (as in A) and the complex of V181D PfPNP (light blue; PDB ID code 6AQS), zymes demonstrates reduced affinity for DADMe-ImmG and also bound with DADMe-ImmG and phosphate. The His7 (A and B) is con- decreased catalytic function. In the most catalytically damaged tributed from the neighboring subunit. (C) Superimposition of the active mutant, chimeric oligomeric structure is suggested with properties sites of native PfPNP as in A and B with M183L PfPNP (green; PDB ID code 6AQU) bound to phosphate (orange). The intrusion of Tyr160 into the cat- distinct from the parental subunits. Crystal structures demonstrate alytic site of M183L PfPNP interferes with catalysis and DADMe-ImmG how the resistance mutations have rearranged the PfPNP binding binding. Hydrogen bonding interactions are shown by dotted lines. The red sites to generate drug resistance. spheres are water molecules interacting with DADMe-ImmG.

Ducati et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 1, 2021 collected by venipuncture every other week from a healthy volunteer under R. H. Angeletti for assistance and use of the mass spectrometer. DADMe- Albert Einstein College of Medicine Institutional Review Board protocol 2000-031. ImmG was a generous gift from P. C. Tyler and G. B. Evans from the Ferrier Research Institute, Victoria University, Wellington, New Zealand. This work ACKNOWLEDGMENTS. We thank the laboratory of K. Kim for samples of was supported by NIH Research Grants GM041916, AI049512, and AI089683, mouse anti-PfPNP and PfADA, and Edward Nieves from the laboratory of and NIH Training Grant T32AI070117.

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