Genetic resistance to purine nucleoside phosphorylaseinhibition in Plasmodium falciparumRodrigo 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 EinsteinCollege 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 humanmalaria and is a global health concern. The parasite responds toantimalarial therapies by developing drug resistance. The contin-uous development of new antimalarials with novel mechanisms ofaction is a priority for drug combination therapies. The use oftransition-state analog inhibitors to block essential steps in purinesalvage has been proposed as a new antimalarial approach. Mu-tations that reduce transition-state analog binding are also expected toreduce the essential catalytic function of the target.We have previouslyreported that inhibition of host and P. falciparum purine nucleosidephosphorylase (PfPNP) by DADMe-Immucillin-G (DADMe-ImmG) causespurine starvation and parasite death in vitro and in primate infectionmodels. P. falciparum cultured under incremental DADMe-ImmG drugpressure initially exhibited increased PfPNP gene copy number and pro-tein expression. At increased drug pressure, additional PfPNP genecopies appearedwith point mutations at catalytic site residues involvedin drug binding. Mutant PfPNPs from resistant clones demonstratedreduced affinity for DADMe-ImmG, but also reduced catalytic efficiency.The catalytic defects were partially overcome by gene amplification inthe region expressing PfPNP. Crystal structures of native and mutatedPfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG.Both point mutations and gene amplification are required to overcomepurine starvation induced by DADMe-ImmG. Resistance developedslowly, over 136 generations (2136 clonal selection). Transition-stateanalog inhibitors against PfPNP are slow to induce resistance andmay have promise in malaria therapy.

malaria | drug resistance | genetic resistance mechanisms |gene amplification | gene mutation

Malaria remains a common cause of death in children underthe age of five years and pregnant women in endemic re-

gions (1, 2). Approximately 400,000 people died from malariaand 212 million cases were reported in 2015 (3). The most severeform of malaria is caused by Plasmodium falciparum, a protozoanparasite. The first-line treatment recommended by the WorldHealth Organization is artemisinin-based combination therapy(ACT) (4), which includes a quinoline or antifolate drug incombination with an artemisinin derivative. Despite the effec-tiveness of ACT in reducing mortality rates, parasite resistancehas emerged in the greater Mekong subregion of Cambodia,Myanmar, Thailand, and Vietnam (5, 6). Measures to containresistance include monitoring and surveillance of drug-resistantparasites, followed by strategies to prevent widespread dissemi-nation (7). The emergence of ACT resistance increases the ur-gency for new treatment strategies. Agents with novel mechanismsof action will permit new drug combinations for treatment andprevention of P. falciparum infections.DADMe-Immucillin-G (DADMe-ImmG) is a transition-state

analog inhibitor of P. falciparum purine nucleoside phosphory-lase (PfPNP), an essential enzyme in the purine salvage pathway(Fig. 1) (8–10). PfPNP catalyzes the reversible phosphorolysisof the N-glycosidic bond of 6-oxopurine (deoxy)ribonucleosidesto generate (deoxy)ribose 1-phosphate and the corresponding

6-oxopurine bases (11, 12). Transcripts for PfPNP are found in allP. falciparum life cycle stages (13). Targeting hypoxanthine pro-duction is strategic as P. falciparum are purine auxotrophs, andrely on purine base salvage for purine nucleotide and oligo-nucleotide synthesis (14). DADMe-ImmG inhibits the PNPsfrom both human and malaria parasites with picomolar disso-ciation constants and acts as an oral antimalarial drug for P.falciparum infections in Aotus primates (8). Transition-stateanalogs target the catalytic function of their enzyme targets suchthat binding affinity is proportional to catalytic efficiency (15).Mutations that decrease the affinity of transition-state analogs areexpected to decrease the catalytic function of the target. Thesubnanomolar affinity of transition-state analogs for their targetproteins also limits the effectiveness of drug-export mechanisms.Drug resistance in P. falciparum often comes from point mutationsin the target proteins or altered drug-export activity (16), mech-anisms that may be slower to develop with transition-state analogs.We sought to characterize the genomic response of P. falciparum

toward drug selection pressure using DADMe-ImmG. Theanalysis of genomic amplification in resistant clones charac-terizes the responses to drug pressure and identifies the mo-lecular markers of resistance (17–22). This approach hasallowed the identification of antimalarial drug resistance

Significance

Hypoxanthine salvage is essential for nucleic acid synthesis inPlasmodium falciparum but not in humans. Hypoxanthineproduction in P. falciparum can be blocked by DADMe-Immucillin-G (DADMe-ImmG), a transition-state analog in-hibitor of purine nucleoside phosphorylase (PfPNP). Parasiteshave been previously reported to die in response to DADMe-ImmG both in vitro and in experimental primate infections.Drug pressure with DADMe-ImmG selects drug-resistant P. falci-parum with increased PfPNP gene copy number, increasedprotein production, and mutations to reduce drug binding.DADMe-ImmG is slow to produce resistance in P. falciparum,supporting its potential for malaria therapy.

Author contributions: R.G.D., R.K.H., J.P.D., and V.L.S. designed research; R.G.D.,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., andJ.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. wrotethe paper.

Reviewers: T.E.W., National Institutes of Health; and E.A.W., University of California,San Diego.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: The crystallography, atomic coordinates, and structure factors have beendeposited in the Protein Data Bank, https://www.rcsb.org/ (PDB ID codes 6AQS and6AQU).1To whom correspondence may be addressed. Email: johanna.daily@einstein.yu.edu orvern.schramm@einstein.yu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1525670115/-/DCSupplemental.

Published online February 12, 2018.

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mechanisms, including copy number variants, single nucleotidevariants, and drug transport alterations (19, 22–24). For exam-ple, mefloquine resistance has been associated with increase inpfmdr1 copy number (25), and artemisinin resistance has beenattributed to mutations in the P. falciparum kelch13 gene (22).The identification of drug resistance mutations informs drugmechanism and antimalarial treatment strategies.Parasite response to modest DADMe-ImmG drug pressure

led to an increase in PfPNP gene copy number, PfPNP protein,and enzymatic function. At increased drug pressure, PNP geneamplification increased and point mutations at residues involvedin drug binding were observed. The results support PfPNP as thesole functional target for DADMe-ImmG, elucidate the mech-anisms of resistance and support the potential for targeting thepurine salvage pathway with transition-state analogs.

Results and DiscussionIn Vitro Selection of DADMe-ImmG Resistant Parasites. P. falciparumisolates resistant to DADMe-ImmG were selected by continuousin vitro drug pressure (26–28). The high (370 μM) hypoxanthineusually added to culture media was replaced with a more-physiological level of 10 μM (29). P. falciparum cells culturedin vitro are susceptible to PfPNP inhibition by DADMe-ImmGonly at growth-limiting hypoxanthine, because high hypoxanthinebypasses the physiological function of PNP (3, 10).Selection to 2 μM resistance. The initial selection for resistance toDADMe-ImmG was performed in P. falciparum 3D7 parasitesby in vitro cultivation under increasing drug pressure (100 nM to2 μM) over the course of 30 generations (Fig. 2). ParentalP. falciparum 3D7 parasites were cultured in parallel without drugas controls. Clonal parasite isolates were generated by limiting di-lution (23, 30) and cultured for DNA and protein analysis. Fiveindividual resistant clones were selected at 2 μM drug resistance.Three were characterized by IC50 analysis and expanded for geno-mic and enzymatic characterization. The remaining two clones werecultured for an additional 30 generations without drug to determineif parasites reverted to the drug-sensitive phenotype (Fig. 2). All fiveresistant clones were analyzed for IC50 values. There were no sig-nificant differences in IC50 between these groups. IC50 values in-creased 5.5- to 7.2-fold in the five resistant clones for an average6.5 ± 0.7 increase in IC50 (from 0.24 to 1.5 μM) (Fig. 3A). Becausewithdrawal of drug pressure from resistant clones did not reversethe resistance, a stable genomic resistance was established.Selection to 5 μM resistance. DADMe-ImmG drug selection wascontinued for an additional 74 generations on three clonal cul-tures resistant to 2 μM DADMe-ImmG until growth was sus-tained at 5 μM of the inhibitor (Fig. 2). IC50 values for thesethree clones increased to 65 ± 16 μM, a 276-fold increase fromthe parental strains (Fig. 3B).

Selection to 8 μM resistance. Three cultures resistant at 5 μMDADMe-ImmG were subjected to increasing drug pressure foran additional 32 generations to achieve growth at 8 μM of theinhibitor (Fig. 2). IC50 values for these three cultures growing in8 μM DADMe-ImmG varied from 65 to 230 μM (280- to 980-fold increased resistance) and, on average, 500-fold more resistantthan the native parental strain (Fig. 3B). Three independentlyconditioned cultures (Fig. 2) were subjected to genomic DNAand protein analysis. From parental strain to the fully resis-tant strains represents 136 generations under increasing drugpressure (2136 clonal expansion).

Genomic Analysis at 2 μM DADMe-ImmG Resistance. Genome se-quencing examined four independently selected resistant clonesand two independently selected clones from control cultures.Sequencing of these 2-μM-resistant isolates and controls wasat >15× sequence coverage.Four independent clones from 2-μM-resistant strains all

showed amplification of a local region in chromosome 5 thatincludes the purine nucleoside phosphorylase gene (PNP;locus_tag PFE0660c). The amplified region was ∼20 kb long intwo isolates and 60 kb in the other two. In clones with the 60-kbamplified region, the expanded genome contained the codingregion for PfPNP together with ∼18 neighboring genes (TableS1). The PfPNP locus was amplified four- to sixfold with nodetectable sequence mutations. This genetic structure and theparasite response to IC50 determination was unchanged follow-ing removal of the drug selection pressure and growth for anadditional 30 generations in the absence of DADMe-ImmG.

Point Mutations in the PfPNP Gene for Parasites Resistant to 8 μMDADMe-ImmG. Parasite growth at higher concentrations ofDADMe-ImmG in a subsequent round of selection resulted in500-fold DADMe-ImmG resistance and increased amplificationin the PNP locus (7- to 13-fold). For these resistant parasites,genomic sequencing was at >300× coverage. Three independentlydeveloped resistant isolates (Fig. 2) were genomically sequencedand had developed point mutations affecting some copies of thePfPNP protein. In two clones, a transversion of T to A at po-sition 547 caused an M-to-L change in codon 183 (M183L).This mutation was present in 65% and 50% of the sequencereads, respectively. Thus, the genome-amplified region containsa mixture of native and mutated PfPNP sequences. In the thirdclone, a T-to-A transversion at position 542 caused a V-to-Dchange at codon 181 (V181D). In this clone, 46% of the readscarry the mutation, again indicating a mixed expression of na-tive and mutant PfPNPs. Other mutations were found in thegenomes from individual clones, but were unrelated to purinemetabolism. Each of the mutations outside the PfPNP regionwas found in only a single resistant clone. These mutations were

A B

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

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attributed to background mutation rates during the 136 gener-ations of drug selection pressure (Fig. 2 and Table S2).

Cellular PNP Level in DADMe-ImmG–Resistant Parasites. PfPNPprotein levels in drug-resistant and control isolates were ana-lyzed by Western blots from cell extracts. At 2 μM DADMe-ImmG resistance, a three- to fourfold increase of PfPNP pro-tein was estimated (Fig. 3C). PfPNP catalytic activity alsoincreased in drug-resistant cell extracts (Fig. S1). Extracts from

cultures grown in the presence of DADMe-ImmG showed nocatalytic 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 ofPfPNP protein were observed in extracts that also contained theV181D and M183L mutations (Fig. 3D).

Catalytic Properties of Mutant PfPNPs. The PfPNP proteins con-taining M183L and V181D mutants were obtained by site-directed mutagenesis and expression in Escherichia coli. Nativeand mutant N-terminal His-6 PfPNPs incorporating a thrombincleavage site were expressed and purified using Ni-NTA af-finity chromatography. The enzymes were kinetically charac-terized after His-purification sequences were removed bythrombin cleavage. The His-6 purification sequence had littleeffect on the kinetic properties of the enzymes. The purity andmass of the PfPNP constructs were established by SDS/PAGEand mass spectrometry.PfPNP M183L and V181D mutants showed reduced catalytic

efficiency for hypoxanthine formation and reduced bindingaffinity for DADMe-ImmG (Table 1). Mutation V181D decreasedthe kcat and increased the Km and Ki* for DADMe-ImmG byfactors of 2, 2, and 42, respectively. Thus, the catalytic efficiency(kcat/Km) decreased fourfold, whereas the inhibitor binding affinitydecreased more, by an order of magnitude. Gene amplification ofthis mutation provides the organism with near-native catalyticcapacity but a 42-fold diminished sensitivity to DADMe-ImmG.One hypothesis for the effective action of transition-state analogson 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 inPfPNP can occur with disproportionate effects on catalysis andinhibitor binding.Mutation M183L had more profound effects on PNP catalytic

activity. The kcat decreased 30-fold, and the Km for inosine andKi* for DADMe-ImmG increased by factors of 558 and 3.9 ×104, respectively. Thus, catalytic efficiency (kcat/Km) decreased1.7 × 104-fold and the inhibitor binding affinity decreased by afactor of 3.9 × 104. In this case, loss of catalytic function andinhibitor affinity are nearly equal. Decreased catalytic func-tion by 17,000-fold in M183L PfPNP is incompatible with itsessential function to provide hypoxanthine. However, steady-state kinetic analysis of purified M183L PfPNP (as shown inTable 1) may not accurately represent the in vivo activity inresistant P. falciparum. Coexpression of M183L PfPNP withnative PfPNP may lead to the formation of hybrid multimerscontaining native and M183L PNP subunits. The M183Lmutation alters the subunit interactions observed in crystal-lographic analysis (see below). In hybrids of native and M183Lsubunits it is possible that some subunits may retain residualcatalytic activity in the presence of micromolar DADMe-ImmG. The related PNP from the human host forms a func-tional homotrimer. Binding of DADMe-ImmG exhibits strongnegative cooperativity. Binding of transition-state analog in-hibitors to the first catalytic site completely inhibits the en-zyme. In contrast, all six subunits of the PfPNP must besaturated for full inhibition. Mixed mutant and native hybridoligomers of PfPNP provide a hypothetical model for re-sistance by way of altered inhibitor binding in M183L hybridPfPNPs that retain adequate catalytic activity.

Structural Characterization of Drug-Resistant PfPNPs. PfPNP is ahomohexameric enzyme with subunit monomers containing eightα-helices and nine β-sheets (PDB ID code 1SQ6) (33). Structureshave been reported in ternary complexes with phosphate,Immucillin-H (PDB code ID code 1NW4) and DADMe-ImmG(PDB ID code 3PHC). Cocrystallization of drug-resistant PfPNP

Fig. 2. Generation of DADMe-ImmG–resistant P. falciparum. Initial clonesof resistant parasites were selected from cultures under continuous drugpressure from 100 nM to 2 μM over 30 generations. Individual resistantclones averaged 6.5-fold resistance. Clones indicated “withdrawn drugpressure” were cultured without DADMe-ImmG for an additional 30 gener-ations before analysis. Clones indicated “continued drug pressure” wereexpanded with 2 μM continued drug pressure for genomic and proteinanalysis. An additional 74 generations increased resistance to 5 μM DADMe-ImmG. Continued drug pressure for an additional 32 generations gave re-sistance to 8 μM DADMe-ImmG. One or more features of the IC50, genomicDNA, PfPNP protein, and catalytic activity were characterized at 2, 5, and8 μM drug resistance.

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mutants V181D and M183L in the presence of DADMe-ImmGproduced unliganded PfPNP crystals. Soaking preformed crystalswith DADMe-ImmG (Table S3) yielded crystals in complex withDADMe-ImmG for V181D but not the M183L protein. Dif-fraction data for the V181D PfPNP–DADMe-ImmG complexand the unliganded M183L PfPNP were obtained in the H32 andI4122 space groups, respectively (Table S4). Molecular re-placement found one protomer in the H32 space group and twoprotomers in the I4122 space group. Loop Pro209–Leu221 wasdisordered in both proteins. Electron density was also missing forGly67–Ala69 and Tyr161–Pro167 in M183L PfPNP (Fig. S2). Allother amino acids were readily fitted into the electron densitymaps. Electron density for DADMe-ImmG was well-defined inthe V181D PfPNP structure. All of the resolved amino acids arein the favored or allowed regions of the Ramachandran plotexcept for residues Ala69 and Cys208 near the disordered loopsin M183L PfPNP (Table S4). The monomer of V181D PfPNP–DADMe-ImmG is arranged into hexameric packing, similar tothose of native PfPNP (11, 33). However, the subunit structure ofM183L PfPNP differs, forming a dimer in the asymmetric unit.The subunit interaction loop (Tyr161–Pro167) is disordered, theapparent 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 ofV181D PfPNP (at 1.57 Å resolution) with native PfPNP bound toDADMe-ImmG shows a rmsd of 0.26 Å, closely related struc-tures (Fig. S2). DADMe-ImmG bound to V181D PfPNP is al-tered 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 inthe 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 tothe complex of native PfPNP with phosphate (rmsd of 0.668 Å;Fig. S2). The subunit interaction loop (Tyr161–Pro167) is dis-ordered and alters the normal hexameric form of PfPNP. Anearlier report also showed that mutation of M183A disrupts theoligomeric state of PfPNP to become a poorly active monomer(34).

Structural Basis of PfPNP Resistance Against DADMe-ImmG. NativePfPNP (PDB ID code 3PHC) binds tightly to DADMe-ImmG, inpart stabilized by hydrogen bonds to N7 and O6 of the purinering with Asp206 and Wat281, respectively. π-Stacking and vander Waals interactions with Pro209, Trp212, and Phe217 alsostabilize 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. Thismutation and tilted purine ring result in a collapsed Pro209–Leu221 loop and disrupted π-stacking. The carboxylate ofAsp206 acts to protonate N7 of the purine ring at the transitionstate. It is also moved away from N7 (Fig. 4 and Fig. S3). Thesemissing interactions to the 9-deazapurine will affect the bindingaffinity of DADMe-ImmG in V181D PfPNP.The M183L PfPNP crystallized as dimer in the asymmetric

unit, with phosphate but not DADMe-ImmG at the catalyticsites. The active site of M183L PfPNP in complex with phosphateis collapsed to prevent facile substrate binding and thereforepoor catalytic activity. Because DADMe-ImmG also binds at thecatalytic site, its binding is likewise prevented. Evidence thatthe same structural defect causes both effects is provided by thenearly equivalent loss of catalytic function and DADMe-ImmGbinding. The side chain of Tyr160 tilts into the binding site for

Fig. 3. Characteristics of parasites resistant to DADMe-ImmG. (A) DADMe-ImmG IC50 values for isolated clones at 2 μM resistance (Fig. 2). These par-asites showed 5.5- to 7.2-fold increased IC50 values (P ≤ 0.05). Drug resistanceis genetically stable as withdrawal of drug pressure had no effect on re-sistance. (B) Resistant parasite clones grown to 5 and 8 μM drug pressure(Fig. 2) showed 206- to 980-fold increased IC50 values. Error bars are the SDsof four experiments. (C) Quantitation of Western blot intensity scans fromresistant strains in A showed a three- to fourfold increase (P < 0.05) forPfPNP protein within the drug-resistant samples. (D) Quantitation of West-ern blot intensity scans from clones resistant to 8 μM DADMe-ImmG (from B)showed a 13.3 ± 1.1 increase in PfPNP protein for the M183L drug-resistantclones and 4.5 ± 0.4 for the V181D PfPNP clones. (E) Chromosome identifiersfor the P. falciparum genome are shown in the innermost circle. Data tracksshow the relative genomic content of 10-kb contiguous regions. The heightof each track is 10 units. The two inner tracks show no amplified regions intwo control isolates. The three outer tracks correspond to 2 μM DADMe-ImmG–resistant isolates (Fig. 2). Single amplified regions are seen in all re-sistant strains in chr5. In each case, this region includes the full coding regionfor the PfPNP gene. (F) Region of gene amplification in chromosome 5 for

three DADMe-ImmG–resistant clones at 8 μM DADMe-ImmG. Each clone hasdistinct boundaries for gene amplification, and each contains the full codingregion for the PfPNP gene.

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the purine ring and interferes with catalytic site filling (Fig. 4). Inthe M183L PfPNP, three loops were disordered, includingGly67–Ala69, Tyr161–Pro167, and Phe210–Pro223. Of these, theloop from Tyr161 to Pro167 is most important for subunit–sub-unit contacts and keeps the hexameric state stable, whereas theloop 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 inclinical use access a limited range of targets, and all drugs areknown to induce resistance (e.g., refs. 19 and 31). Agents actingat the cytochrome bc1 complex, such as atovaquone, induce re-sistance by point mutations in the cytochrome. Antifolates likesulfadoxine or sulfadoxine–pyrimethamine combinations inducesingle or multiple mutations, causing resistance in dihydrofolatereductase and/or dihydropteroate synthase (35). The mutationsreduce affinity for the agents while retaining sufficient catalyticactivity to permit parasite growth. Agents interfering with hemepolymerization to form hemozoin (e.g., chloroquine) induce re-sistance by point mutation in a proposed transporter gene (pfcrt).Others, including mefloquine and primaquine, induce mutationsand/or copy number variations in the pfmdr1 multiple drug re-sistance-like transporter (36). Artemisinin resistance is also as-sociated with increase in pfmdr1 copy number (25) or tomutations in the P. falciparum kelch13 gene, whose function isstill being explored (22).Transition-state analogs differ from current antimalarials by

their high affinity and high specificity for a single target, bindingat the catalytic site of the target enzyme to mimic the transition-state properties. Point mutations that decrease transition-stateanalog binding are therefore expected to reduce the biologicalfunction of the target (7). In P. falciparum, resistance toDADMe-ImmG is initially achieved by increasing the cellularcontent of the enzyme to give a modest 6.5-fold resistance. Pointmutations in the active site and additional gene copy numbersgive greater resistance. No changes in drug transport mecha-nisms were detected. The antimalarial mechanism for PfPNPdiffers from current antimalarial chemotypes. The slow devel-opment of resistance and novel mechanism suggests thatDADMe-ImmG might provide a new candidate for combinationtherapy against malaria.

Concluding RemarksP. falciparum responds to antimalarial therapy by developingdrug resistance. DADMe-ImmG is a high-affinity transition-stateanalog inhibitor of PfPNP, validated for efficacy against P. fal-ciparum at the level of Aotus primates. The mechanism ofresistance involves a combination of gene amplification anddrug-binding mutations. Resistance developed slowly, and wasaccompanied by various degrees of loss of function of the enzy-matic target. The enzymatic characterization of the resistant en-zymes demonstrates reduced affinity for DADMe-ImmG anddecreased catalytic function. In the most catalytically damagedmutant, chimeric oligomeric structure is suggested with propertiesdistinct from the parental subunits. Crystal structures demonstratehow the resistance mutations have rearranged the PfPNP bindingsites to generate drug resistance.

Materials and MethodsSupporting Information provides detailed information on the culture con-ditions for growth and development of DADMe-ImmG resistance in P. fal-ciparum. The methods used for single clone selection, IC50 evaluation,genomic DNA isolation and sequencing, mutational, and gene amplifi-cation are also detailed in Supporting Information. Other methods de-tailed in Supporting Information include protein extraction andquantitation, Western blot analysis, PfPNP kinetic assays, production ofnative and mutant PfPNPs, kinetic analysis, crystallization, X-ray datacollection, and structural analysis.

P. falciparum parasites were cultured in medium containing human bloodobtained under an approved protocol with informed consent. Human blood was

Table 1. Enzyme kinetic parameters, catalytic efficiencies, andinhibition constants for wild-type, M183L, and V181D PfPNP

Enzyme kcat, s−1 Km, μM kcat/Km, M

−1·s−1 Ki*

Wild-type 2.63 ± 0.15 7.6 ± 1.5 346 × 103 673 ± 52 pMM183L 0.08 ± 0.01 4,240 ± 925 0.02 × 103 26 ± 3 μMV181D 1.44 ± 0.05 16.8 ± 1.5 85 × 103 28 ± 1 nM

Fig. 4. Stereoview of the binding sites of native PfPNP and mutants. (A) Thecatalytic and inhibitor binding site of native PfPNP (yellow, PDB code ID3PHC) in complex with DADMe-ImmG (green) and phosphate (orange). Theamino acid residues interacting with DADMe-ImmG are indicated by dashedlines. (B) Superimposition of the binding sites of the native PfPNP complex(as in A) and the complex of V181D PfPNP (light blue; PDB ID code 6AQS),also bound with DADMe-ImmG and phosphate. The His7 (A and B) is con-tributed from the neighboring subunit. (C) Superimposition of the activesites of native PfPNP as in A and B with M183L PfPNP (green; PDB ID code6AQU) bound to phosphate (orange). The intrusion of Tyr160 into the cat-alytic site of M183L PfPNP interferes with catalysis and DADMe-ImmGbinding. Hydrogen bonding interactions are shown by dotted lines. The redspheres are water molecules interacting with DADMe-ImmG.

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collected by venipuncture every other week from a healthy volunteer underAlbert Einstein College of Medicine Institutional Review Board protocol 2000-031.

ACKNOWLEDGMENTS. We thank the laboratory of K. Kim for samples ofmouse anti-PfPNP and PfADA, and Edward Nieves from the laboratory of

R. H. Angeletti for assistance and use of the mass spectrometer. DADMe-ImmG was a generous gift from P. C. Tyler and G. B. Evans from the FerrierResearch Institute, Victoria University, Wellington, New Zealand. This workwas supported by NIH Research Grants GM041916, AI049512, and AI089683,and NIH Training Grant T32AI070117.

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