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Drug Resistance Updates 12 (2009) 28–41

Contents lists available at ScienceDirect

Drug Resistance Updates

journa l homepage: www.e lsev ier .com/ locate /drup

utational ‘hot-spots’ in mammalian, bacterial and protozoalihydrofolate reductases associated with antifolate resistance:equence and structural comparison

ordan P. Volpatoa, Joelle N. Pelletiera,b,∗

Département de biochimie, Université de Montréal, CanadaDépartement de chimie, Université de Montréal, Canada

r t i c l e i n f o

rticle history:eceived 20 August 2008eceived in revised form 24 December 2008ccepted 4 February 2009

a b s t r a c t

Human dihydrofolate reductase (DHFR) is a primary target for antifolate drugs in cancer treatment, whileDHFRs from Plasmodium falciparum, Plasmodium vivax and various bacterial species are primary tar-gets in the treatment of malaria and bacterial infections. Mutations in each of these DHFRs can resultin resistance towards clinically relevant antifolates. We review the structural and functional impact of

eywords:ihydrofolate reductaseutations

ntifolaterug resistanceomology

active-site mutations with respect to enzyme activity and antifolate resistance of DHFRs from mammals,protozoa and bacteria. The high structural homology between DHFRs results in a number of cross-species,active-site ‘hot-spots’ for broad-based antifolate resistance. In addition, we identify mutations that conferspecies-specific resistance, or antifolate-specific resistance. This comparative review of antifolate bindingin diverse species provides new insights into the relationship between antifolate design and the devel-opment of mutational resistance. It also presents avenues for designing antifolate-resistant mammalian

e age

DHFRs as chemoprotectiv

. Introduction

Antifolates constitute a large family of structurally diverse com-ounds used in the treatment of a broad range of diseases, the most

mportant of which are various types of cancers as well as protozoalnd bacterial infections (Lynch et al., 1982; Mennes et al., 2005; Dawt al., 2006; Bell et al., 2008; Zhanel et al., 2008) (Fig. 1). The prin-ipal target of antifolates is dihydrofolate reductase (DHFR; E.C.C..5.1.3), an essential enzyme found in all living organisms. DHFRatalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate

THF), using NADPH as a coenzyme. THF is required for the de novoynthesis of purines and thymidylate. Inhibition of DHFR leads tohe arrest of cell proliferation and, eventually, to cell death.

Abbreviations: DHFR, dihydrofolate reductase; MTX, methotrexate; TMP,rimethoprim; CYC, cycloguanil; PYR, pyrimethamine; NADPH, nicotinamide ade-ine dinucleotide phosphate (reduced form); DHF, 7,8-dihydrofolate; THF, 5,6,7,8-etrahydrofolate; PMTX, pemetrexed; TMTX, trimetrexate; p-ABA, p-aminobenzoiccid; HSC, hematopoietic stem cells; RFC, reduced folate carrier; FPGS, folylolyglutamate synthase; TS, thymidylate synthase; WT, wild-type; NBMPR-P,itrobenzylmercaptopurine ribose phosphate.∗ Corresponding author at: Département de chimie, C.P. 6128, Succursale Centre-ille, Montréal, Québec H3C 3J7, Canada. Tel.: +1 514 343 2124;

ax: +1 514 343 7586.E-mail address: joelle.pelletier@umontreal.ca (J.N. Pelletier).

368-7646/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.drup.2009.02.001

nts.© 2009 Elsevier Ltd. All rights reserved.

Antifolates bind to the folate-binding site of DHFR, acting ascompetitive inhibitors. As a result of high structural homologyacross species, many antifolates exhibit cross-species inhibitionof DHFR. Nonetheless, species-specific inhibition has also beenachieved, as illustrated by trimethoprim (TMP; Fig. 1), used in thetreatment of bacterial infections. Bacterial DHFR exhibits 2500-foldtighter binding for TMP (Ki = 0.08 nM) than the human homolog(Ki = 200 nM), making TMP an effective antibiotic with few or noside effects (Margosiak et al., 1993). Similarly, pyrimethamine (PYR)and cycloguanil (CYC) (Fig. 1) are effective antifolates for the treat-ment of malaria (Ferone et al., 1969; Hitchings, 1969). The highstructural homology shared by mammalian, parasitic and bacte-rial chromosomal DHFRs suggests that species-specific inhibitionresults from slight structural differences within their active sitecavities.

Widespread clinical application of antifolates has led to resis-tance development, greatly impairing their efficacy. A number ofdifferent resistance mechanisms have been reported, includingDHFR gene amplification, decreased cellular permeability to antifo-lates and even acquisition of evolutionarily distinct, intrinsicallyantifolate-resistant DHFR variants. This review focuses on resis-

tance stemming from mutations of the DHFR gene. In protozoa andbacteria, the resulting DHFR variants no longer bind the antifolateseffectively, yet maintain sufficient catalytic activity to ensure cel-lular proliferation. Due to the slower rate of mutation in humancells relative to bacteria and protozoa, this resistance mechanism

J.P. Volpato, J.N. Pelletier / Drug Resistance Updates 12 (2009) 28–41 29

F ctivityt

itaccamlt

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ig. 1. Chemical structures of dihydrofolate and antifolates used to inhibit DHFR areatment. Pterin ring atom numbering is shown on DHF.

s not commonly found during the treatment of human prolifera-ive diseases such as cancer (Spencer et al., 1996b). Nonetheless,ntifolate-resistant variants of human DHFR offer important appli-ations, such as the potential of protecting healthy bone marrowells from the cytotoxic effects of antifolates used in cancer ther-py (Banerjee and Bertino, 2002). Hence, the identification ofutational ‘hot-spots’ associated with antifolate resistance may

ead to engineering antifolate-resistant DHFRs for myeloprotec-ion.

We attempt to map the antifolate-binding requirements athe DHFR active site utilizing cross-species structural homology,xtensive mutational data and inhibition data from a variety of

ntifolate-resistant DHFRs. Cross-species and cross-drug similar-ties and differences are highlighted. The aim is to increase ournderstanding of the balance between substrate binding and bind-

ng of specific antifolates in clinically relevant native or mutatedHFR variants.

in different species. All antifolates shown are currently used for species-specific

1.1. Antifolate resistance mechanisms in mammalian cells

Among the antifolates designed to treat proliferative diseases inhumans, no drug has been more clinically useful than methotrex-ate (MTX). First synthesized in the 1940s (Farber et al., 1948), MTXhas been used to treat various types of cancers and other prolifera-tive diseases (Chu and Allegra, 1996). MTX is a slow, tight-binding,competitive inhibitor of DHFRs from almost all species, binding sto-ichiometrically to DHFR. Due to its lack of selectivity, MTX is appliedexclusively to the treatment of human diseases (Jones et al., 2000;Mennes et al., 2005; Strojan et al., 2005; Daw et al., 2006; Hashkesand Laxer, 2006; Slamon et al., 2006).

MTX resistance has been observed in mammalian cells exposedto MTX ex vivo. At low doses, mutations in the reduced folate car-rier protein (RFC) (Rothem et al., 2004) and in folylpolyglutamatesynthetase (FPGS) decrease cellular uptake of antifolates or allowmore rapid efflux of MTX (Zhao et al., 2000) respectively. Both

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esistance mechanisms have been recently reviewed (Zhao andoldman, 2003) and will not be discussed further here. At higherTX doses, over-expression and/or amplification of the native DHFR

ene are most frequently observed (Dolnick et al., 1979; Melera etl., 1982; Chu et al., 1993; Goker et al., 1995). Mammalian DHFRctivity is regulated by binding of its cognate mRNA at the activeite (Chu et al., 1993) and binding of MTX dislodges the DHFR mRNA,endering it available for translation.

The above-mentioned MTX resistance mechanisms have alsoeen observed in tumours isolated from patients who relapseduring or following MTX-treatment (Lin et al., 1991; Trippett etl., 1992; Goker et al., 1993; Goker et al., 1995). One additionalesistance mechanism has been observed solely in ex vivo studies:utations in the mammalian DHFR gene resulting in a DHFR variantith reduced affinity for MTX.

.1.1. MTX-resistant point mutants from mammalian cell linesCharacterization of MTX-resistant mutants will increase our

nderstanding of inhibitor binding at the active site of DHFR, whichs critical to the design of novel inhibitors. In early studies, mam-

alian cells were exposed to MTX ex vivo to predict the mutationshat could result from the long-term treatment of patients. Amonghe first mutations so identified was the L22R substitution in MTX-esistant fibroblasts from murine embryos (Simonsen and Levinson,983). Leu22 is a highly conserved active site residue involved inan der Waals contacts with the pterin ring of bound MTX (Cody etl., 2005). Crystal structures of murine and human L22R mutantsuggested that introduction of an arginine residue reduces the con-act surface with MTX (Cody et al., 2005). A L22F substitution also

esulted in MTX-resistance in CHO cells (Melera et al., 1988). Char-cterization of the human L22F variant revealed that it conferredver 100-fold decrease in MTX-affinity, while retaining catalyticctivity slightly lower than that of the WT (Lewis et al., 1995)Table 1).

able 1utations providing MTX-resistance in human DHFR.

DHFR Variant Catalytic efficiency (�

ative 92osition 7 I7F 0.24osition 22 L22F 6.1

L22R 0.028L22W 10L22Y 12

osition 31 F31A 30F31G 26F31P 4.3F31R 1.5F31S 16

osition 34 F34A 0.24a

F34I 0.57a

F34S 0.017a

F34T 0.11a

F34V 0.49a

osition 115 V115A 0.2V115C 0.1

ouble mutants L22F/F31G 3.3L22F/F31S 3.6L22Y/F31G 1.4L22Y/F31S 3.8F31R/Q35E 1.9

riple mutants F31R/F34T/Q35S 0.6F31R/F34T/Q35R 0.5F31A/F34V/Q35H 0.3

a Derived from plots of rate versus [DHF] (Nakano et al.,1994) by least-squares fit to theb Ternary KMTX

Dvalues.

ance Updates 12 (2009) 28–41

The first human DHFR mutation was the F31S variant, iden-tified in MTX-resistant human colon cancer cells (Schweitzer etal., 1989). The F31S mutation increased MTX-resistance 70-foldwhile decreasing catalytic efficiency less than 6-fold relative to WThDHFR (Table 1), providing a further example of increased resis-tance with little loss of activity. Phe31 establishes van der Waalscontacts with the p-aminobenzoyl portion of bound MTX (Oefneret al., 1988). Ten years earlier, a MTX-resistant, F31W substitutionin mouse leukemia cells was identified (Goldie et al., 1980). Takentogether, the results suggested that mutations at positions 22 and31 could be ‘hot-spots’ for MTX-resistance, and that diverse aminoacid substitutions may be tolerated at these active-site positions ofmammalian DHFRs. Further work in MTX-resistant mouse leukemiacells revealed a G15W variant (Dicker et al., 1993). In vitro charac-terization demonstrated that although MTX-affinity of G15W haddecreased by over 165-fold relative to WT, the mutant was unstable.

1.1.2. MTX-resistant mammalian DHFRs from site-directedmutagenesis

While it soon became apparent that MTX-resistant DHFRmutants were not the cause of resistance in tumours isolated frompatients (Spencer et al., 1996b), MTX-resistant DHFRs became key toa promising application. Because MTX is a myeloablative drug, MTXresistant DHFRs can protect healthy cells from cytotoxicity dur-ing high-dose MTX treatment. The incorporation of MTX-resistantDHFRs in healthy hematopoietic stem cells (HSC) for engraftmentin the bone marrow has the potential to ensure myeloprotection ofpatients during treatment with MTX (Banerjee and Bertino, 2002).We highlight below structure–function studies of MTX-resistantmutants in mammalian DHFRs and present the progress made in

myeloprotection of human HSCs and in murine models.

Site-directed mutagenesis of murine and human DHFR rapidlydemonstrated the potential for decreasing MTX-affinity. Thillet etal. (1988) created point mutations in murine DHFR at active-siteresidues that form specific contacts with bound MTX, including

M−1 s−1) KMTXi

(nM) References

0.0034 Margosiak et al. (1993)24.6 Patel et al. (1997)

0.55 Lewis et al. (1995)4.6 Lewis et al. (1995)4.3 Lewis et al. (1995)

11 Lewis et al. (1995)

0.27 Chunduru et al. (1994)0.35 Chunduru et al. (1994)1.7 Volpato et al. (2007)7.2 Patel et al. (1997)0.24 Chunduru et al. (1994)

34b Nakano et al. (1994)13b Nakano et al. (1994)

210b Nakano et al. (1994)9.6b Nakano et al. (1994)

10b Nakano et al. (1994)

20 Fossati et al. (2008)20 Fossati et al. (2008)

29 Ercikan-Abali et al. (1996a)26 Ercikan-Abali et al. (1996a)

150 Ercikan-Abali et al. (1996a)42 Ercikan-Abali et al. (1996a)21 Volpato et al. (2007)

56 Volpato et al. (2007)81 Volpato et al. (2007)

180 Volpato et al. (2007)

equation for substrate inhibition.

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he potential ‘hot-spot’ positions 22 and 31. The I7S, L22R, W24R,31S, F31R, F34L, Q35R, Q35P, V115P and T136V point mutants werereated and characterized. All exhibited decreased MTX-affinityccompanied with loss of catalytic activity generally resulting fromeduced DHF affinity. This result is readily rationalized by the facthat MTX and DHF make similar contacts with the enzyme; muta-ions affecting binding of MTX are likely to affect DHF binding.he most resistant murine variant was the previously identified22R, with MTX-affinity decreased by over five orders of magnitude,hereas catalytic efficiency was decreased 1600-fold relative to WT

nzyme. Significantly, KDHFM increased only 25-fold, indicating that

HF binding was modified to a lesser extent than MTX binding. Thisesult underscores the potential for creation of ‘efficient’ mutantsor myeloprotection, exhibiting poor MTX binding but retaining sig-ificant activity. However, the tight binding of MTX (3.4 pM) relativeo DHF (100 nM) in the native human enzyme poses a challengingtart point. In another study, Morris and McIvor performed satura-ion mutagenesis at positions 22 and 31 of murine DHFR (Morrisnd McIvor, 1994). At position 22, six residues (A, F, N, W, Y and R)esulted in a higher ICMTX

50 than the WT enzyme, the highest beingutant L22R (2000-fold increase), as seen previously (Thillet et al.,

988). At position 31, eight residues yielded MTX-resistant mutantsR, S, Q, E, W, H, G and N), the most resistant mutant being F31NICMTX

50 increased 75-fold). Positions 22 and 31 thus continued torove their potential for development of resistance.

In accordance with results obtained with murine DHFR, site-irected mutagenesis of human DHFR L22 to F, R, W or Y mutantsll conferred MTX-resistance (Lewis et al., 1995) (Table 1). The L22W

nd L22Y variants were the most resistant to MTX (KMTX

iincreased

p to 3200-fold), with a 10-fold reduction in catalytic efficiency.he L22R mutant was also highly resistant, but showed a greaterecrease in catalytic efficiency. The L22F mutant was moderately

ig. 2. Superposition of hDHFR (1U72, Cody et al., 2005) with pfDHFR (A; 1J3I, Yuvaniyairawaraporn et al., 2000). For clarity only the backbone of human DHFR is shown (cartoDHFR is complexed with MTX, while the pf, pv and mt DHFRs are complexed with WR992.3 Å and 1.8 Å.

ance Updates 12 (2009) 28–41 31

resistant while maintaining catalytic efficiency in the same rangeas that of the WT enzyme.

Although human DHFR residues Phe31 and Phe34 are neigh-bours, forming similar contacts with bound MTX (Fig. 2), theirmutation to Ser had differing effects (Schweitzer et al., 1989). Thecatalytic activity of the F31S variant was comparable to that of theWT while its KMTX

D was increased 100-fold. The F34S mutation hadan even higher ternary KMTX

D (over four orders of magnitude higherthan WT), but had low catalytic activity (four orders of magnitudelower). Variants F31L, F31V and F31T were not resistant to MTX(Prendergast et al., 1989; Chunduru et al., 1994), while the F31A andF31G displayed MTX-resistance and catalytic efficiencies compara-ble to the F31S variant (Chunduru et al., 1994) (Table 1). While theseresults suggested that only small amino acids at position 31 conferMTX-resistance, the F31R mutation yielded one of the most resis-tant hDHFR point mutants reported to date, with KMTX

iincreased

by three orders of magnitude relative to WT human DHFR (Patelet al., 1997). Further mutations at residue F34 also yielded MTX-resistant variants. Like the F34S variant, mutation to A, I, T or Vall increased the ternary KMTX

D but greatly impaired catalytic effi-ciency, mainly due to an increase in KDHF

M (Nakano et al., 1994). Theresults highlighted the importance of F34 in folate and antifolatebinding in human and murine DHFRs and, consistent with its strictconservation, suggest that this feature may be a general one. Thus,mutations at position 34 in mammalian DHFRs may be less proneto development of resistance than positions 22 and 31.

Other active-site mutations yielded MTX-resistant hDHFR vari-ants. The I7F mutation generated a 7000-fold increase in KMTX

i(Patel et al., 1997) (Table 1). The I7 backbone carbonyl has beenproposed to hydrogen-bond with the 4-amino group of MTX, aninteraction that cannot be established with folate given that itspterin ring is flipped 180◦ around the C6 C9 bond relative to MTX.

ma et al., 2003), pvDHFR (B; 2BL9, Kongsaeree et al., 2005) and mtDHFR (C; 1DG5,on representation). Residues mutated in the different DHFRs are shown in sticks.

10, PYR and TMP respectively. The RMSDs for all-atom superpositions were between

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utations that disrupt this H-bond could potentially reduce affinityo MTX while maintaining native-like catalytic efficiencies. How-ver, the catalytic efficiency of the I7F mutant dropped 380-foldue to decreased DHF binding, demonstrating that this highly con-erved residue plays a role in substrate recognition and catalysisPatel et al., 1997). This is attributable to formation of van der Waalsontacts with the pterin ring of either ligand with residue I7. Fur-hermore, by analogy to 5-deazafolate (Davies et al., 1990), the I7ackbone contributes to DHF binding via carbonyl H-bonding withhe pterin ring protonated at the N8 atom, which is positioned inhe same area of the active site as the 4-amino group of MTX. R70s a highly conserved residue that forms a salt bridge with the glu-amate portion of bound ligands. Disruption of this salt bridge inhe R70K mutant yielded a binary KMTX

D that was 4 orders of magni-ude greater than the WT KMTX

i. In contrast, catalytic efficiency was

ecreased by over 100-fold, mainly due to decreased DHF affinity.Recently, we developed a two-tier screening assay that enables

apid identification and characterization of active and MTX-esistant variants of DHFR (Fossati et al., 2008). Saturationutagenesis libraries of human DHFR at residues I7, G15, W24, R70

nd V115 were selected for activity and MTX-resistance. Position15 was the most tolerant to mutations, yielding the MTX-resistantutants V115A and V115C, which displayed high KMTX

ivalues

6000-fold increase) accompanied by significant loss of catalyticctivity (Table 1). Like residue I7, the backbone carbonyl of V115-bonds with the 4-amino group of MTX, but not folate. Nonethe-

ess, the data indicate that V115 is important for binding of bothigands. Saturation mutagenesis at I7, G15, W24 and R70 failed toield MTX-resistant mutants according to our screening stringency.

.1.3. MTX-resistant multiple mutantsHighly MTX-resistant mammalian DHFRs were obtained by

ombining ‘hot-spot’ L22 and F31 point mutants that individu-lly conferred MTX-resistance (Ercikan-Abali et al., 1996a). All theL22/F31) double mutants tested had higher KMTX

ivalues than

he corresponding L22 or F31 point mutants. Furthermore, thencrease was synergistic in some cases, such that the thermody-amic change in MTX-binding (��GMTX) for the double mutantsas greater than the sum of changes for the corresponding pointutants. The increase in MTX-resistance was accompanied by

ecreased catalytic efficiency in all cases. Nonetheless, the resultsevealed the potential for increasing MTX resistance by combin-ng mutations at positions involved in MTX-binding. We recentlypplied a semi-randomized approach to simultaneously mutateesidues F31, F34 and E35 of hDHFR, selecting the most MTX-esistant variants via a bacterial complementation assay. Oneovel point mutant (F31P) and 9 combinatorial mutants were

dentified and characterized (Volpato et al., 2007) (Table 1). Asith the (L22/F31) double mutants, the (F31/F34/Q35) double and

riple mutants were all highly MTX-resistant. A trade-off betweenncreased MTX-resistance and decreased catalytic efficiency wasgain observed. Triple mutant F31A/F34V/Q35H displayed the high-st KMTX

ireported for a hDHFR variant (50000-fold increase),

ccompanied by a relatively modest 180-fold reduction in catalyticfficiency. Thus, while the L22/F31 hot-spot combination was effec-ive in providing strong MTX-resistance, various combinations of

utations can provide similar or greater effects.

.1.4. Applications of MTX-resistant mammalian DHFR mutantsMTX-resistant DHFR mutants have been tested for their poten-

ial in protecting cells from the cytotoxic effects of MTX and

ther antifolates used in cancer chemotherapy. Antifolates are par-icularly cytotoxic to healthy but rapidly dividing cells such asaematopoietic stem cells (HSC) and other bone marrow cells.he resulting myeloablation leads to immunosuppression, induc-ng secondary infections during chemotherapy (Das et al., 2006).

ance Updates 12 (2009) 28–41

Incorporation of MTX-resistant DHFRs in HSC using retroviral vec-tors has been shown to provide effective myeloprotection duringantifolate treatment in murine models. In addition, MTX-resistantDHFRs have been shown to be reliable markers for in vivo selec-tion of transduced HSCs (Zhao et al., 1997; Allay et al., 1998) orin vitro selection of transfected or transduced cells (Banerjee et al.,1994a,b; Flasshove et al., 1995; Ercikan-Abali et al., 1996b; Havengaet al., 1998; Gatlin et al., 2000; Volpato et al., 2007). To be usefulfor these applications, MTX-resistant DHFR variants should displaya high KMTX

iwhile maintaining a catalytic efficiency sufficient for

cell proliferation.Two cell types have been used most frequently to evaluate the

protection from cytotoxicity conferred by MTX-resistant hDHFRs:CHO DUKX B11 (DHFR-) cells and HSC. Human DHFR point mutantsG15W (Banerjee et al., 1994b), L22F (Ercikan-Abali et al., 1996b),L22R (Banerjee et al., 1994a,b), F31S (Banerjee et al., 1994a,b) andF34S (Banerjee et al., 1994a) all conferred moderate MTX-protectionto CHO dhfr-cells at low MTX concentrations (less than 10% survivalof cells at 10 �M MTX). The murine DHFR L22R, F31R, F31S andQ35P mutants yielded similar results (Thillet and Pictet, 1990). Werecently observed greater MTX-protection of CHO DHFR- cells withhDHFR mutant F31A/F34V/Q35H, which conferred over 70% sur-vival at 200 �M MTX (Volpato et al., 2007). Other double and triplemutants also offered efficient protection, conferring greater than50% survival at 30–56 �M MTX (Volpato et al., 2007).

While CHO DHFR- cells offer a convenient model system, theyoffer no practical applications. HSCs constitute a prime target forprotection because of their sensitivity to antifolates and their poten-tial as recipients of genetic material for engraftment. Among theMTX-resistant DHFR variants tested for myeloprotection (Hock andMiller, 1986; Banerjee et al., 1994a,b; Zhao et al., 1997; Takebe etal., 2002; Warlick et al., 2002), only two variants offered high lev-els of protection from antifolates to HSC in culture: L22Y (Spenceret al., 1996a; Allay et al., 1998; Gatlin et al., 2000; Belzile et al.,2003) and the L22F/F31S double mutant (Sauerbrey et al., 1999;Takebe et al., 2001, 2002; Capiaux et al., 2003, 2004). The humanL22F/F31S mutant transduced into human bone marrow cells con-ferred close to 100% survival on in vitro myeloid progenitor assayscontaining 100 nM MTX (Capiaux et al., 2003). In this study, thedouble mutant also contributed to dual-protection against MTX anda thymidylate synthase (TS) inhibitor, 5-fluorouracil (5-FU), wheneither covalently linked or independently co-expressed with a 5-FUresistant mutant of TS. More recently, those constructs were shownto offer myeloprotection to bone marrow cells exposed to peme-trexed (PMTX), a newer antifolate which inhibits both TS and DHFRactivities in cells (Capiaux et al., 2004). Other studies have reporteddual protection from MTX and unrelated myeloablative drugs by co-expressing the L22F/F31S double mutant with other drug resistancemutants (Sauerbrey et al., 1999; Takebe et al., 2001, 2002). Althoughthe double mutant displays good bone marrow protection from thetoxic effects of MTX in vitro, the concentration of MTX used forselection (0.1 �M) is lower than the plasma concentration of MTX(0.5–1 �M) during drug treatment with the drug (Goldman, 1974;Cheng, in press). Mutants resistant to higher MTX concentrationswould be required for myeloprotection applications.

Unlike the L22F/F31S mutant, L22Y has mainly been used as anin vivo selection marker for transduced HSC cells. Gene transferefficiencies using retroviruses or lentiviruses rarely surpass 25%,and transplantation of poorly transduced bone marrow cell pop-ulations is one of the chief limitations to the therapeutic effectsof gene therapy (Sorrentino, 2004). MTX-resistant DHFRs can be

used as in vivo selection markers against MTX, to enrich the popu-lation of transduced cells post-transplantation. Although the L22Ymutant was first characterized for its resistance to MTX (Lewiset al., 1995), trimetrexate (TMTX), a non-polyglutamated antifo-late more cytotoxic to HSC than MTX, has mostly been used as its

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elective agent in bone marrow cells. In that system, L22Y yieldedreater survival rates against TMTX than against MTX (Spencer etl., 1996a). Transplantation of L22Y-transduced bone marrow cellsn mouse recipients followed by TMTX and nucleoside transportnhibitor (NBMPR-P) administration led to over 50% enrichmentf transduced bone marrow cell populations in primary recipi-nts, and this enrichment was maintained in secondary transplantecipients (Allay et al., 1998). However, similar experiments in rhe-us macaques (Persons et al., 2004) yielded only transient in vivoelection due to important differences in transduction efficienciesnd in vivo HSC cycling in both species. It should be noted thathe use of antifolates such as MTX and TMTX as in vivo selec-ive agents is advantageous relative to selection systems such as6–methylguanine–DNA–methyltransferase (Kramer et al., 2006)r �-glutamylcysteine synthetase (Lorico et al., 2005) given thathe side effects of their administration are well characterized andell tolerated by patients.

In vitro selection of bone marrow cells offers the possibility tonrich transduced cell populations prior to transplantation, whichould increase the likelihood of successful engraftment. In vitro

election could be applied to bone marrow cells from virtually anypecies, regardless of species-specific properties of HSCs. However,his application faces a major technical hurdle: HSCs can currentlye cultured only for a limited number of days, after which theyifferentiate and cannot repopulate bone marrow populations inivo (Dolznig et al., 2005). The MTX-resistant DHFRs L22Y and22F/F31S have been tested for this application, with mixed results.

hile bone marrow cells could be selected in vitro with thoseHFR mutants (Gatlin et al., 2000; Takebe et al., 2002; Belzile etl., 2003; Capiaux et al., 2003), the HSC viability was consideredoo poor for engraftment following the period required for efficientn vitro selection. As a result, in vivo repopulation of bone marrowy selected cells was not assessed. We are currently investigating

TX-resistant DHFRs that tolerate a higher dose of MTX during

n vitro selection, permitting efficient enrichment of transducedSC on a shorter timescale that may be compatible with engraft-ent (Volpato, Mayotte, Sauvageau and Pelletier, unpublished

esults).

able 2utations providing PYR and/or CYC-resistance in Plasmodium DHFR.

nzyme Catalytic efficiency(�M−1s−1)

KPYRi

(

ative pfDHFR 6.8 1.5

oint mutants A16V 0.01 6.0N51I 0.81 0.4C59R 76 1.1S108N 3.7 13I164L 2.4 0.8

ouble mutants A16V/S108T 0.64 3.6N51I/S108N 13 37C59R/S108N 0.18 72

riple mutants N51I/C59R/S108N 0.53 120C59R/S108N/I164L 0.17 380

uadruple mutant N51I/C59R/S108N/I164L 1.1 860

ative pvDHFR 3.3 0.16

oint mutants F57L 0.0015 130S58R 1.1 1.8S117N 0.02 650I173L 3.7 1.8

ouble mutant S58R/S117N 0.25 930

ance Updates 12 (2009) 28–41 33

1.2. Antifolate resistance mechanisms in protozoa and bacteria

Of all protozoal diseases, malaria is the most common andthe most deadly, causing over one million deaths each year(http://www.who.int/mediacentre/factsheets/fs094/en/index.htmlMalaria is transmitted via the blood by mosquitoes carrying theparasite and can be caused by four distinct species of Plasmodium:P. ovale, P. malariae, P. vivax and P. falciparum, the last one causingthe most severe and prevalent form of the disease. We will focus onthe inhibition of the DHFR domain of the bifunctional P. falciparumDHFR-thymidylate synthase (DHFR-TS) by pyrimethamine, a nor-mally effective antimalarial antifolate. Pyrimethamine resistancecan result from DHFR mutations. We will also discuss resistance toantifolates in other protozoa and alternative antifolate treatmentsof Plasmodium, to provide a broad picture of parasitic resistance toantifolates.

1.2.1. Naturally occurring antifolate resistance in Plasmodiumparasites

The search for synthetic antimalarial drugs in the 1930s wasprompted by the decreased abundance of quinine. The firstsynthetic antifolates were proguanil (which metabolizes to theactive compound cycloguanil (CYC) (McGready et al., 2003)) andpyrimethamine (PYR) (Fig. 1), which efficiently inhibit P. falci-parum DHFR (Sirawaraporn et al., 1993). The effectiveness of PYR,particularly when administered with sulfadoxine (an inhibitor ofdihydropteroate synthase), led to its widespread use as a first-line treatment for malaria in several countries (Happi et al., 2005;Nzila et al., 2005). However, PYR-resistant P. falciparum strains wereidentified among clinical isolates shortly after the first PYR trials(Chongsuphajaisiddhi and Sabchareon, 1981). A S108N mutationwas responsible for the phenotype observed in one PYR-resistantstrain (Cowman et al., 1988) (Table 2; corresponding residue num-

bering for DHFRs from other species is provided in Table 3).Combination of S108N with the C59R mutation increased PYRresistance 11-fold while combination with a N51I substitutionincreased resistance 45-fold (Foote et al., 1990). The triple mutant,C59R/S108N/I164L, was five-fold more resistant than the double

nM) KCYCi

(nM) KWR99210i

(nM) References

2.6 1 Sirawaraporn et al. (1997a);Zhang and Rathod (2002)

570 Sirawaraporn et al. (1997a)4.7 Sirawaraporn et al. (1997a)4.7 Sirawaraporn et al. (1997a)

15 Sirawaraporn et al. (1997a)3 9 Sirawaraporn et al. (1997a)

2100 Sirawaraporn et al. (1997a)24 Sirawaraporn et al. (1997a)82 0.6 Hekmat-Nejad and Rathod

(1997); Sirawaraporn et al.(1997a)

100 Sirawaraporn et al. (1997a)1141 Sirawaraporn et al. (1997a)

730 1.9 Sirawaraporn et al. (1997a);Chusacultanachai et al. (2002)

0.6 6 Leartsakulpanich et al. (2002)

740 160 Leartsakulpanich et al. (2002)8.5 13 Leartsakulpanich et al. (2002)

980 51 Leartsakulpanich et al. (2002)19 21 Leartsakulpanich et al. (2002)

743 8.1 Leartsakulpanich et al. (2002)

34 J.P. Volpato, J.N. Pelletier / Drug Resist

Table 3Homologous active site residues in DHFRs from different species.

Species Corresponding residues

H. sapiens I7 L22 F31 F34 Q35 T56 V115P. falciparum I14 L46 M55 F58 C59 S108 I164P. vivax I13 L45 M54 F57 S58 S117 I173L. major V30 L45 M53 F56 K57 T83 V156ESM

msicssdmt

hmrtHafbttbD

hipeSaa(NWto

q(tK(amNtcfpofa

mC

DHFR mRNA binds to the DHFR active site to regulate expression.

. coli I5 M20 L28 F31 K32 T46 I94

. pneumoniae I8 L23 F31 F34 K35 T49 I100. tuberculosis I5 I20 Q28 F31 R32 T46 I94

utants and over 250-fold more resistant than the WT (Table 2),uggesting that the conservative I164L mutation also played a rolen PYR-resistance (Foote et al., 1990). These results, together withorroborating observations (Peterson et al., 1988; Zolg et al., 1989),uggested that an initial mutation (S108N) was necessary for acqui-ition of PYR resistance. The findings also highlight a fundamentalifference between antifolate resistance in parasites, where DHFRutations are a primary source of resistance, and antifolate resis-

ance in mammalian cells.Further studies of antifolate-resistant strains of P. falciparum

ighlighted a general trend, where most PYR-resistant DHFRutants were also resistant to CYC (Table 2), suggesting that cross-

esistance might be a barrier to the design of novel antifolates toreat PYR-resistant malaria (Foote et al., 1990; Peterson et al., 1990).owever, the same studies identified combinations of mutationsssociated with resistance to specific antifolates. Whereas the ICPYR

50or a strain containing the A16V/S108T mutations was compara-le to that of the sensitive strain, the ICCYC

50 was increased by morehan two orders of magnitude. The role of the A16V/S108T muta-ions in CYC resistance remained unclear because those tests hadeen undertaken in P. falciparum rather than in vitro and additional,HFR-independent, resistance mechanisms could not be ruled out.

In vitro characterization of the PYR-resistant P. falciparum DHFRselped elucidate the role of mutations in resistance. The activity and

nhibition values determined for the isolated domains of the WTfDHFR-TS were similar to the bifunctional enzyme (Sirawarapornt al., 1990). Using the mutated P. falciparum DHFR domain, the108N mutation was shown to provide a 10-fold increase in KPYR

i

nd a 5-fold increase in KCYCi

, with a 2-fold reduction in cat-lytic efficiency relative to the WT solely due to increased KDHF

MSirawaraporn et al., 1993). Interestingly, combination of S108N and51I not only increased KPYR

iand KCYC

i(30- and 10-fold relative to

T, respectively), but re-established catalytic efficiency (and KDHFM )

o the level of the WT (Table 2). This clearly illustrates the impactf cumulative mutations on the fitness of resistant variants.

Further naturally occurring PYR and/or CYC-resistant double touadruple mutants, all containing S108N, were also characterizedSirawaraporn et al., 1997a). KPYR

iand KCYC

iincreased synergis-

ically in all combinatorial mutants relative to S108N, such thatPYRi

was highest in the N51I/C59R/S108N/I164L quadruple mutanta 570-fold increase relative to WT). This gain in resistance wasssociated with a 6-fold loss in catalytic efficiency. The quadrupleutant was also one of the most CYC-resistant mutants in the study.onetheless, the aforementioned double mutant, A16V/S108T, had

he highest KCYCi

but remained sensitive to PYR. This observationonfirmed that the A16V/S108T mutation was solely responsibleor the CYC-resistant phenotype observed in the strain of P. falci-arum (Foote et al., 1990) and demonstrated that a specific subsetf mutations can confer selective antifolate resistance. The basisor selectivity was rationalized later, when structural data became

vailable (see below).

The impact of each mutation occurring in those multipleutants of pfDHFR was assessed (Sirawaraporn et al., 1997a). The

59R and I164L mutants did not decrease the affinity for PYR, but

ance Updates 12 (2009) 28–41

slightly decreased affinity for CYC (2 to 5-fold). Importantly, theequivalent positions in mammalian DHFRs (Gln35 and Val115, seeTable 3) have yielded MTX-resistant mutants (Thillet et al., 1988;Fossati et al., 2008), identifying these positions as cross-species hot-spots for selective antifolate resistance. The only point mutant thatconferred moderate resistance to PYR and CYC was S108N. Theseobservations support the hypothesis that the S108N mutation isessential in conferring PYR-resistance, as it remains the only pointmutation present in all naturally occurring PYR-resistant mutants.In a parallel study (Sirawaraporn et al., 1997b), kinetic characteriza-tion of the 19 possible mutations at pfDHFR position 108 confirmedthat mutant S108N exhibited the most efficient balance betweenloss of catalytic efficiency and gain of PYR-resistance, justifyingits prevalence in PYR-resistant isolates. Homology models of theP. falciparum DHFR domain (Santos-Filho et al., 2001; Delfino et al.,2002), based on the structure of avian DHFR, yielded telling resultsregarding PYR and CYC selectivity (Delfino et al., 2002): the S108Npoint mutation created greater steric clashes between bound PYRthan between bound CYC and other active-site residues. SelectiveCYC-resistance of the A16V mutant could also be rationalized bydifferential steric clashes in this model.

The first structure of a protozoal DHFR was reported in 1994, forthe Leishmania major DHFR-TS (Knighton et al., 1994). The DHFRdomain was structurally homologous to E. coli and mammalianDHFR structures (Knighton et al., 1994; Cody et al., 1997; Sawayaand Kraut, 1997). The crystal structure of P. falciparum DHFR-TS withbound PYR (Yuvaniyama et al., 2003) confirmed that the P. falci-parum DHFR model of Delfino et al. (Delfino et al., 2002) was a goodapproximation. Superimposition of WT P. falciparum, bacterial andhuman DHFR structures (Fig. 2) shows that despite additional loopsand helices found far from the active site in P. falciparum, the NADPHand folate binding sites are homologous in the three species. Thissuggests that positions involved in Plasmodium antifolate resistancemay be relevant to bacterial and mammalian antifolate resistanceand vice versa.

Second-generation antifolates are currently being tested againstclinical isolates of PYR and CYC-resistant P. falciparum strains. Themost promising compound to date is WR99210 (Fig. 1), a triazinedrug that inhibits growth of WT P. falciparum and strains containingPYR and CYC-resistant DHFR double to quadruple mutants (Hankinset al., 2001). In vitro characterization of mutant C59R/S108N in pres-ence of WR99210 demonstrated effective inhibition (Hekmat-Nejadand Rathod, 1997). Consistent with this observation, WR99210 wasreadily docked into the active site of modeled PYR-sensitive andPYR-resistant pfDHFRs with no steric hindrance (Delfino et al.,2002). Furthermore, comparison of crystal structures of WT pfDHFRand its quadruple mutant N51I/C59R/S108N/I164L complexed withWR99210 suggested that the additional flexibility conferred by the3-carbon linker to the trichlorophenyl moiety of the ligand wasa key feature in its potency towards the different DHFR variants(Yuvaniyama et al., 2003).

Because PYR, CYC and WR99210 all carry a 2,4-diaminopyr-imidine moiety, mammalian and parasitic DHFRs have compara-ble Ki values (Ma and Kovacs, 2000). For example, KWR99210

ifor

human DHFR is only 10-fold higher than for pfDHFR (Ki = 12 nMand 1 nM respectively (Zhang and Rathod, 2002)). Nonetheless,those compounds display in vivo selectivity: growth of P. falciparumwas inhibited at WR99210 concentrations 8000-fold lower thanthose required to inhibit human fibroblasts. The in vivo selectiv-ity was recently shown to be due to differences in regulation ofDHFR expression (Zhang and Rathod, 2002). In mammalian cells,

This regulation is relieved when inhibitors bind to the active siteand dislodge the bound mRNA, making it available for translation.The resulting overexpression constitutes a commonly observedantifolate-resistance mechanism in mammalian cells (Chu et al.,

Resist

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J.P. Volpato, J.N. Pelletier / Drug

993). The parasitic DHFR-TS mRNA also binds its cognate enzyme,ut not at the DHFR active site (Zhang and Rathod, 2002). Thus,ntifolate binding at the active site does not induce overexpres-ion of pfDHFR which may explain the selectivity of drugs such as

R99210 towards protozoa.

.2.2. Laboratory-generated antifolate resistance in Plasmodiumalciparum

Directed evolution of pfDHFR was performed to predict thenset of mutations conferring resistance towards WR99210,YR and its analog, m-Cl-PYR (Chusacultanachai et al., 2002).election of random mutations for PYR resistance yielded the well-haracterized S108N, C59R/S108N and N51I/C59R/S108N/I164Lutants, as well novel combinatorial mutants at previously

ncharacterized positions (Table 2). Random mutagenesis of theuadruple N51I/C59R/S108N/I164L mutant also yielded novelombinations, including the introduction of a F58L mutation,

ccompanied by a switch of mutation S108N to N108T. The resultingutant, N51I/F58L/C59R/N108T/I164L, was also identified when

he quadruple mutant library was selected against WR99210. Inact, many WR99210-selected mutants from the quadruple mutantibrary contained the F58L mutation, often accompanied by a N108S

ig. 3. Sequence alignment of DHFRs from different species. Highlights identify residues talian numbering). Alignment was generated using Espresso (3Dcoffee alignment; http:

= strictly conserved residues.

ance Updates 12 (2009) 28–41 35

reversion, consistent with earlier observations that the S108Nmutation did not confer resistance to WR99210. This suggests thatF58L is a key actor in WR99210 resistance and that mutation N108Toffers broader-based antifolate resistance than S108N.

Consistent with this hypothesis, further variations at position58 were observed. Both the F58C mutation added to the quadrupleN51I/C59R/S108N/I164L mutant and the F58L point mutant con-ferred resistance to WR99210. All Phe58 mutants exhibited lowcatalytic activities relative to the WT or quadruple mutant enzymes,but high resistance to all the inhibitors tested. F58 is homologous toF34 in mammalian DHFR (Fig. 3; Table 3), a highly conserved active-site residue involved in van der Waals interactions with boundsubstrate as well as 2,4-diaminopterin and 2,4-diaminopyrimidineinhibitors. Substituting F34 with hydrophobic residues in human ormurine DHFR conferred high resistance towards MTX, but reducedcatalytic activity (Nakano et al., 1994), (Thillet et al., 1988). Thisillustrates the similarities between the active sites of Plasmod-

ium and mammalian DHFR enzymes with respect to substrate andinhibitor binding and is consistent with the high structural homol-ogy of their active sites, despite the fact that these enzymes shareonly between 25 and 40% sequence identity (Zhang and Rathod,2002).

hat can be mutated to confer antifolate resistance in more than one species (mam-//www.igs.cnrs-mrs.fr/Tcoffee/).: = conserved residues;. = semi-conserved residues;

3 Resist

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6 J.P. Volpato, J.N. Pelletier / Drug

.2.3. Antifolate resistance in Plasmodium vivaxPlasmodium vivax, the second most frequent type of malaria, is

arely fatal. Nonetheless, important morbidity and mortality relatedo P. vivax in certain areas of the world justify the need for effec-ive treatment (Mendis et al., 2001). It was initially believed that P.ivax was intrinsically antifolate-resistant as it proved resistant toYR and CYC treatment (Young and Burgess, 1959). de Pecoulas etl. (1998) identified polymorphisms at five residues of the P. vivaxHFR-TS gene from strains originating from regions where PYR hadeen extensively used to treat P. falciparum. As the two Plasmod-

um strains often co-exist, those studies suggested that resistanceo PYR was not intrinsic, but had been acquired by P. vivax followingrug exposure during treatment of P. falciparum. The PYR-exposedtrains all contained single or combinatorial mutations at posi-ions 58, 117 and 173 (positions 59, 108 and 164, respectively, infDHFR; Table 3). In particular, the mutations constituting the P.ivax variants S58R/S117N and S58L/S117N/I173L are the homologsf the pfDHFR mutations C59R, S108N and I164L that contributeo PYR resistance (Table 2). In vitro characterization of the P. vivax58R and S117N point mutants confirmed that they were slightlyesistant to PYR and CYC, and that their combination increasedesistance to both drugs (Tahar et al., 2001) as observed for the P.alciparum C59R/S108N mutant. A recent study showed that thisouble mutant was the most frequent polymorphism identified

n antifolate-resistant clinical isolates for both malarial speciespfDHFR C59R/S108N and pvDHFR S58R/S117N)(Alam et al., 2007).

Despite the high prevalence of the S58R/S117N double mutant,57 mutations (hDHFR F34; Table 3) were also identified (deecoulas et al., 1998). The F57L variant conferred resistance toll antifolates tested, including WR99210 (Imwong et al., 2001;astings and Sibley, 2002; Leartsakulpanich et al., 2002). This againemonstrates the cross-species contribution of this strictly con-erved residue in antifolate binding, where substitution of thehenyl ring confers antifolate resistance in mammalian as wells protozoal DHFRs. A larger screen identified triple to quintu-le mutants including F57L/S58R with the further mutations I13L,117N (Imwong et al., 2001), S117T (Imwong et al., 2003) and/or61M (Imwong et al., 2003). T61 is homologous to T38 in mam-alian DHFRs, which is an important active site residue involved

n inhibitor recognition (Cody et al., 2005). I13 is homologous toesidue I7 in mammalian DHFR, which specifically interacts withhe 4-amino group of bound inhibitor (Table 3). Similarly, theuman DHFR I7L mutation can give rise to MTX-resistant mutantshen combined with additional active site mutations, includingutations at residues F34 (F57 in pvDHFR) and Q35 (S58 in pvDHFR)

Volpato, Poulin and Pelletier., unpublished results). I7, F34 and E35re thus cross-species hot-spots for antifolate resistance.

Comparison of the mutations that confer antifolate resistancen the two Plasmodium DHFRs illustrates that despite their highequence and structural homologies (Kongsaeree et al., 2005),ertain mutations or combinations of mutations provide species-pecific antifolate resistance. The homologs of the P. vivax mutants13L and T61 M have not been reported in pfDHFR (I14 and T62,espectively; Table 3) for resistance to antifolates. Mutations equiv-lent to F57 mutations (F58 of pfDHFR) were not identified inYR-resistant clinical isolates of P. falciparum. Conversely, muta-ions at residues A15 or N50 in pvDHFR (residues A16 and N51 infDHFR; Table 3) have not been reported in PYR or CYC-resistanttrains. The above studies, though not exhaustive, suggest that theseutational variations result in subtle structural differences in the

ctive site of each species (Kongsaeree et al., 2005).

.2.4. Other protozoa and bacteriaAntifolates are also used in the treatment of other human pro-

ozoa, such as Leishmania. PYR and CYC are ineffective for thereatment of Leishmania due to lack of inhibition of its DHFR

ance Updates 12 (2009) 28–41

(Gilbert, 2002). Therefore, antifolates exhibiting cross-species inhi-bition, such as MTX, are used to treat infections caused byLeishmania. Amplification of the Leishmania DHFR-TS gene has beenobserved, suggesting that, as for the mammalian enzymes, themRNA binds to its cognate active site (Coderre et al., 1983). Reduceddrug-permeability, combined with overexpression of DHFR, hasalso been reported in Leishmania (Ellenberger and Beverley, 1989).These resistance mechanisms parallel those in mammalian systemsand contrast with the point mutations, which constitute the maincause of resistance in Plasmodium species. Arrebola et al. reported astrain of Leishmania major that was resistant to MTX due to a M53Rmutation in DHFR (Arrebola et al., 1994). Met53 is analogous toPhe31 in mammalian DHFR (Table 3), where the F31R substitutionimportantly decreases the affinity for MTX (Patel et al., 1997). ThisL. major variant exhibited a 30-fold decrease in MTX-affinity, andlike the F31R variant of human DHFR, the catalytic efficiency wasmainly decreased due to loss in reactivity (kcat) relative to the WT.Conversely, some bacterial DHFRs exhibit intrinsically low affin-ity for MTX. This is the case of Mycobacterium tuberculosis DHFR,where the KMTX

iis 11 nM, which is within the range of highly resis-

tant hDHFR mutants (Table 1). The mtDHFR active-site residues arehighly homologous to mammalian DHFR, with the exception of Q28(F31 in hDHFR) and R32 (Q35 in hDHFR) (Table 3; Fig. 4). Interest-ingly, the F31Q (Morris and McIvor, 1994) and Q35R (Thillet et al.,1988) mutations confer MTX resistance in murine DHFR. Althoughwe have shown that combining human DHFR mutations at residues31 and 35 can yield high MTX-resistance (Volpato et al., 2007), thosepoint mutants (F31Q, Q35R) were not characterized.

Trimethoprim (TMP) has been the antifolate of choice for thetreatment of bacterial infections such as urinary tract infectionsand is still widely used (Hawser et al., 2006). TMP is a competi-tive inhibitor selective towards chromosomal bacterial DHFR; itsaffinity is over 2500-fold higher for E. coli DHFR than for mam-malian DHFR (Margosiak et al., 1993). Nevertheless, resistance toTMP was reported shortly after its clinical introduction. Unlikeparasitic and mammalian resistances, the main TMP resistancemechanism involves the acquisition of a secondary DHFR that isinsensitive to TMP (reviewed in (Huovinen et al., 1995)). Theseresistance enzymes are either plasmid or integron encoded andare evolutionarily unrelated to chromosomal DHFR. Nonetheless,TMP-resistant chromosomal DHFRs have been reported in differentbacterial species. Baccanari et al. (1981) reported a L28R mutationin E. coli DHFR which decreased TMP affinity 100-fold. L28 corre-sponds to F31 in mammalian DHFR and M53 in L. major (Table 3),where mutation to Arg confers resistance to MTX and other relatedantifolates (Arrebola et al., 1994; Patel et al., 1997). More recently,directed evolution of E. coli DHFR followed by in vivo selection forTMP resistance in a bacterial complementation assay (Watson et al.,2007) yielded multiple single and combinatorial mutants. The mostfrequently observed mutations occurred at residues M20 (L22 inhDHFR) and W30 (Y33 in hDHFR), two active-site residues locatedin the folate-binding site. Mutation of L22 in mammalian DHFRscan confer antifolate resistance. The E. coli M20 V point mutantconferred the highest resistance to TMP, while its combinationwith other mutations conferred lower TMP resistance (Watson etal., 2007). Surprisingly, that point mutant showed no decrease incatalytic activity, unlike mutations of hDHFR L22. W30 was alsofrequently mutated in TMP-resistant variants: mutation W30G wasidentified in a TMP-resistant clinical isolate (Flensburg and Skold,1987) and the W30R mutation was also shown to confer TMP resis-tance (Watson et al., 2007). W30 occurs on the �-helix that plays

a key role in antifolate binding. In E. coli DHFR (Sawaya and Kraut,1997), the side-chain amine of W30 is involved in a hydrogen bond-ing network with a water molecule, residue T113 and the 2-aminogroup of bound MTX. As the 2,4-diaminopyrimidine moieties ofMTX and TMP bind in similar fashions (Matthews et al., 1985),

J.P. Volpato, J.N. Pelletier / Drug Resistance Updates 12 (2009) 28–41 37

Fig. 4. Surface representation of hDHFR, ecDHFR, pcDHFR and mtDHFR complexed with MTX. A semi-transparent surface was generated for hDHFR (1U72, Cody et al., 2005),e DHFR( e cavi

n rposit

iiD3fIabcatbmi

1i

pplctpc2Me1w(teoM

cDHFR (1RH3, Sawaya and Kraut, 1997), pcDHFR (3CD2, Cody et al., 1999) and mtoxygen: red, nitrogen: blue and carbon: white). Residues surrounding the active sit

itrogen: blue, carbon: residues in yellow, MTX in green). RMSDs for complete supe

t is likely that Trp30 mutations reduce TMP binding by disrupt-ng this hydrogen bonding network. Nonetheless, no mammalianHFR mutations have been reported at the homologous position3, suggesting that this position confers antifolate specificity. Oneurther residue also conferred TMP-resistance when mutated: the94L mutation was identified in TMP-resistant E. coli (Watson etl., 2007), and its homolog in Streptococcus pneumoniae (I100L) haseen identified in clinical isolates (Pikis et al., 1998). The backbonearbonyl of Ile94 is within hydrogen-bonding distance of the 4-mino group of TMP (Matthews et al., 1985). In mammalian DHFR,he backbone carbonyl of the homologous V115 is within hydrogenonding distance of the 4-amino group of MTX (Cody et al., 2005). Asentioned previously, mutations at this ‘hot-spot’ position, includ-

ng the hDHFR V115L mutant, give rise to MTX resistance.

.3. Parallels between mutations that confer antifolate resistancen mammalian, parasitic and bacterial DHFRs

As previously discussed, mutations in a number of homologousositions confer cross-species antifolate resistance in mammalian,arasitic and bacterial DHFRs. Some positions confer broad antifo-

ate resistance, suggesting that they are essential for bindingommon features of clinically relevant antifolates. They highlighthe cross-species similarities at the DHFR active site, allowing map-ing of mutational ‘hot-spots’. Residue F34 of mammalian DHFRonstitutes such a hot-spot, reflecting its key interactions with the,4-diaminopterin ring and the p-aminobenzoyl (pABA) portion ofTX. Substitution of F34 has yielded point mutants with the low-

st reported affinity to MTX (Thillet et al., 1988; Nakano et al.,994). Low affinity to PYR, CYC and WR99210 has been associatedith mutations at the homologous residue (F57 (P. vivax) and F58

P. falciparum)) in protozoal DHFRs, suggesting a similar role forhe phenyl ring. In fact, mutations of pvDHFR F57 appear to bessential for resistance to WR99210: the trichlorophenyl portionf WR99210 is bound in the same active site area as the pABA ofTX (Fig. 2), suggesting the establishment of van der Waals and/or

(1DF7, Sirawaraporn et al., 2000). The surface representation is coloured by atomty and MTX are shown in sticks representation and coloured by atom (oxygen: red,

ions were <1.0 ´̊A.

edge-to-face aromatic contacts between the F58 phenyl of pfDHFRand the WR99210 trichlorophenyl in the WT enzyme. On this basis,we may foresee that pfDHFR mutations at residue 58 will eventu-ally be reported if WR99210 or related compounds are used to treatPYR and CYC-resistant malarias.

Val115 of mammalian DHFRs (E. coli Ile94, P. falciparum Ile164and P. vivax Ile173) is also a hot-spot for antifolate-resistance. Inall cases studied, mutation to leucine results in decreased affinityfor antifolates. As its backbone carbonyl H-bonds with the 4-aminogroup of antifolates, mutation to leucine may displace the back-bone to weaken this interaction. Conversely, the V115I mutation inhuman DHFR (which encodes the native residue found in bacterialand parasitic DHFRs) yielded an enzyme with increased affinity forMTX compared to the native enzyme (Fossati et al., 2008).

I7 of mammalian DHFR is also involved in specific H-bondingwith the 4-amino group of antifolates. Again, this feature is con-served in all species, and mutations at this residue in combinationwith other mutations can provide antifolate resistance (Patel etal., 1997; Imwong et al., 2003). It thus constitutes a hot-spot forresistance, but not as a single mutation.

Other mutation sites confer selective antifolate resistance. Drugssuch as MTX are ‘classical’ antifolates, with chemical structuresvery similar to the substrate, DHF. F31 of mammalian DHFR estab-lishes specific van der Waals contacts with the pABA portion of DHFor MTX. Mutations at this position are only observed for antifo-lates containing a pABA moiety or a functional group that occupiesthe same active site space. Therefore, mutations at this positionmay eventually arise in plasmodial DHFRs (M55 in P. falciparum,M54 in P. vivax; Table 3) following treatment with drugs such asWR99210, since the tricholorophenyl moiety binds in a similarspace and orientation as does the pABA moiety (Yuvaniyama et al.,

2003; Cody et al., 2005). A mutation at the homologous position(M53R) has been reported for the parasite L. major following treat-ment with MTX (Arrebola et al., 1994). This position may provideantifolate-specific resistance; it also confers species specificity (seebelow).

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8 J.P. Volpato, J.N. Pelletier / Drug

The S108N mutation in P. falciparum (S117N in P. vivax) DHFR pro-ides PYR and CYC resistance while remaining sensitive to WR99210Hankins et al., 2001). This results from the proximity of S108 withhe structurally constrained chlorophenyl moiety of CYC and PYR;

R99210 does not form close contacts with this residue, such thatts mutation would have little effect on binding of the drug. There isvidence only of weak resistance upon mutation of the equivalentosition in mammalian DHFRs (hDHFR Thr56) (Schweitzer et al.,990), suggesting that this position may confer species specificity.

Cross-species mapping of DHFR active-site mutations has alsorovided insights into TMP binding. TMP displays high affinitynd selectivity towards certain bacterial DHFRs, including E. coliHFR, but binds less tightly to other bacterial, protozoal and mam-alian DHFRs. The crystal structure of ecDHFR complexed with

MP (Matthews et al., 1985), for which coordinates are not avail-ble, suggests that binding of the TMP 2,4-diaminopyrimidine isimilar to the binding of the MTX 2,4-diaminopterin, involvinghe same active-site residues in hydrophobic, van der Waals andydrogen-bonding interactions. It follows that these interactionsannot account for the observed species selectivity, given thatTX binding is in homologous sites in E. coli (Sawaya and Kraut,

997) and human DHFR (Cody et al., 2005). The selectivity ofMP was attributed to binding of the trimethoxybenzyl group. Itainly interacts with helices �B and �C of ecDHFR, which har-

our sequence differences relative to human DHFR. In particular,28 of helix �B (F31 in hDHFR) was reported to form major con-acts with the TMP trimethoxybenzyl. In contrast, F31 of avianHFR produced unfavorable interactions with the TMP trimethoxy-enzyl (Matthews et al., 1985). It was thus proposed that a F31Lutation would increase the affinity of hDHFR for TMP. However,

hat mutation did not give the expected result, indicating that thisesidue was not solely responsible for the species selectivity of TMPPrendergast et al., 1989).

Watson et al. recently reported the M20 V mutation in ecDHFRhDHFR Leu22), responsible for the greatest observed decrease inMP binding in that enzyme (Watson et al., 2007). Interestingly,ost bacterial, protozoan and mammalian DHFRs encode a leucine

r isoleucine at that position (Fig. 3; Table 3), suggesting that a non-olar �-branched amino acid at this position may account for the

ntrinsic TMP-resistance observed in most DHFRs. The side-chainf M20 is positioned at the interface of the folate and NADPH bind-ng pockets in ecDHFR (Fig. 4). Baccanari et al. (Baccanari et al.,982) reported a 3000-fold increase in TMP binding to the E. colinzyme when NADPH is present, an effect not observed in mam-alian DHFRs. The difference in cooperative binding between E.

oli and hDHFR correlates with the difference in TMP affinity. Mod-

ling NADPH into ecDHFR complexed with TMP (Matthews et al.,985) suggested that this cooperativity was related to direct inter-ctions between the two ligands. However, recent NMR studies ofDHFR in complex with NADPH and TMP suggest that the coop-

able 4ross-species comparison of Ki for TMP and MTX, and amino-acid identity at positions 22

pecies 22a 31 61 KTMPi

(nM)

. coli M L G 0.080. influenzae M L G 0.082b

. aureus L L G 1b

. pneumoniae L L G 3.6b

. falciparum L M P 11

. vivax L M P 98

. major I M P 120

. carinii L I P 152. sapiens L F P 200. tuberculosis I Q P 88000

a Residue numbering according to human DHFR.b Calculated from experimental values of IC50 and KDHF

Musing the equation for competit

ance Updates 12 (2009) 28–41

erativity is mainly caused by interactions between the ligands andprotein side-chains (Kovalevskaya et al., 2005). Thus, contacts withM20 appear to promote cooperativity between ligands in ecDHFRin a manner that cannot be reproduced by �-branched amino acids.While this suggests that the cooperativity observed in ecDHFR couldbe introduced into hDHFR by the L22 M mutation, that mutant didnot yield a TMP-sensitive hDHFR (Volpato and Pelletier, unpub-lished data).

It thus appears that TMP-selectivity in ecDHFR is the con-sequence of multiple active-site features. In the absence of thecoordinates for TMP-bound E. coli DHFR, a comparison of the pro-tein surfaces of human (Cody et al., 2005), E. coli (Sawaya and Kraut,1997), M. tuberculosis (Li et al., 2000) and (recently reclassified asfungus) P. carinii (Cody et al., 1999) DHFRs bound to MTX is infor-mative (Fig. 4). This constitutes a good comparative model sinceMTX and TMP display similar binding in ecDHFR (Matthews et al.,1985)), pcDHFR (Champness et al., 1994) and mtDHFR (Li et al.,2000) (TMP-bound structures overlayed with the respective MTX-

bound structures; RMSD < 0.3 ´̊A for each overlay, results not shown).P. carinii and M. tuberculosis DHFRs are TMP-resistant (KTMP

i0.21 �M

(Ma and Kovacs, 2000) and 88 �M (Li et al., 2000), respectively),as is hDHFR (Table 4). Some features are shared among the TMP-insensitive DHFRs, but are not found in ecDHFR. Proline, located atthe C-terminus of hDHFR helix �2 (P61), is also present in pcDHFR(P66) and mtDHFRs (P51) where it creates a bulge near the pABAmoiety of MTX. The proline is replaced by glycine in ecDHFR (G51),which has also been shown to interact with the trimethoxyben-zyl ring of TMP. The presence of a proline in this helix may induceunfavorable interactions with TMP, decreasing binding.

While hDHFR L22, alone, was not sufficient to determine TMPsensitivity, there is a correlation between residues 22, 31 and 61(hDHFR numbering) and TMP binding (Table 4). Close contacts areformed between the side chains of hDHFR L22 and F31, restrict-ing the volume of the active site cavity (Fig. 4). Those interactionsalso occur with the homologous residues of pcDHFR (L25 and I33),but are not as constrictive in ecDHFR (M20 and L28), and appearto be absent in mtDHFR (I20 and Q28), which displays the low-est affinity for MTX and TMP (Table 4). A combination of theabove active-site elements may generate topographies that areideal (ecDHFR), too constrained (pc and hDHFR) or non-specific(mtDHFR) for TMP-binding. More structural information combinedwith determination of inhibitory constants from a greater numberof DHFRs is required to confirm these hypotheses.

Information about mutations conferring antifolate resistancein protozoal and bacterial enzymes may provide knowledgetowards engineering high resistance in mammalian DHFRs while

maintaining native-like catalytic efficiency. These properties aredesirable for myeloprotection applications (Nakano et al., 1994).Practically all highly MTX-resistant mammalian DHFRs displaypoor catalytic activities. Combinatorial hDHFR mutants display

, 31 and 61.

KMTXi

(nM) References

0.0010 Margosiak et al. (1993)0.415b de Groot et al. (1991)

Dale et al. (1997)Maskell et al. (2001)

0.24 Chen et al. (1987); Tarnchompoo et al. (2002)5.2 Tahar et al. (2001)0.130 Arrebola et al. (1994); Gilbert (2002)0.0081 Ma and Kovacs (2000)0.0034 Margosiak et al. (1993)

11 Li et al. (2000)

ive inhibitor binding (Segel, 1993).

Resist

temlm((ataohotWcw

bptsdarofeDtr

A

CTR

R

A

A

A

B

B

B

B

B

B

J.P. Volpato, J.N. Pelletier / Drug

he lowest affinity to MTX, but have significantly reduced catalyticfficiencies (Table 1). In contrast, combinatorial mutants of pfDHFRaintain native-like activity (Sandefur et al., 2007) while antifo-

ate resistance to PYR and CYC is increased with every additionalutation as a result of the N51I, C59R, S108N and I164L mutations

Table 2). Point mutations of Gln35 (Cys59 in pfDHFR) and Val115Ile164 in pfDHFR) of human and murine DHFRs have led tontifolate resistance (Thillet et al., 1988; Fossati et al., 2008), andhe T56N mutation (S108N in pfDHFR) slightly decreased the MTXffinity in human DHFR (Schweitzer et al., 1990). The contributionf the N51I mutation in pfDHFR is of particular interest: whileaving little effect on antifolate binding, it reestablishes the lossf catalytic efficiency of the S108N mutant to native levels. Wouldhe homologous mutant of hDHFR possess similar properties?

e are currently combining hDHFR mutations according to thisross-species information to generate highly MTX-resistant DHFRsith high catalytic efficiencies for use in myeloprotection.

Understanding the similarities and differences in antifolateinding to DHFR across species is key to understanding specificroperties essential for inhibitor binding, and could contribute tohe design of improved antifolates in the future. By comparingtructure-based sequence alignments, mutational data and kineticata for evolutionarily distant DHFRs, we have tried to identifyctive-site residues across species which provide general antifolateesistance and active-site residues which provide species specificityr antifolate specificity. Increasing throughput of structural andunctional characterizations will provide additional data to fullylucidate the requirements for antifolate binding and resistance inHFRs. We hope that this review also illustrates the utility of high-

hroughput mutational screening and structural determination ofelevant drug targets.

cknowledgments

The authors would like to thank Elena Fossati, Christopherlouthier and Brahm Yachnin for critical reading of this manuscript.his work was supported by the Canadian Institutes of Healthesearch (CIHR) grants 68851 (J.N.P.)

eferences

lam, M.T., Bora, H., Bharti, P.K., Saifi, M.A., Das, M.K., Dev, V., Kumar, A., Singh,N., Dash, A.P., Das, B., Wajihullah, Sharma, Y.D., 2007. Similar trends ofpyrimethamine resistance-associated mutations in Plasmodium vivax and P. fal-ciparum. Antimicrob. Agents Chemother. 51, 857–863.

llay, J.A., Persons, D.A., Galipeau, J., Riberdy, J.M., Ashmun, R.A., Blakley, R.L., Sor-rentino, B.P., 1998. In vivo selection of retrovirally transduced hematopoieticstem cells. Nat. Med. 4, 1136–1143.

rrebola, R., Olmo, A., Reche, P., Garvey, E.P., Santi, D.V., Ruiz-Perez, L.M., Gonzalez-Pacanowska, D., 1994. Isolation and characterization of a mutant dihydrofolatereductase-thymidylate synthase from methotrexate-resistant Leishmania cells.J. Biol. Chem. 269, 10590–10596.

accanari, D.P., Daluge, S., King, R.W., 1982. Inhibition of dihydrofolate reductase:effect of reduced nicotinamide adenine dinucleotide phosphate on the selectiv-ity and affinity of diaminobenzylpyrimidines. Biochemistry 21, 5068–5075.

accanari, D.P., Stone, D., Kuyper, L., 1981. Effect of a single amino acid substitutionon Escherichia coli dihydrofolate reductase catalysis and ligand binding. J. Biol.Chem. 256, 1738–1747.

anerjee, D., Bertino, J.R., 2002. Myeloprotection with drug-resistance genes. LancetOncol. 3, 154–158.

anerjee, D., Schweitzer, B.I., Volkenandt, M., Li, M.X., Waltham, M., Mineishi, S.,Zhao, S.C., Bertino, J.R., 1994a. Transfection with a cDNA encoding a Ser31 orSer34 mutant human dihydrofolate reductase into Chinese hamster ovary andmouse marrow progenitor cells confers methotrexate resistance. Gene 139,269–274.

anerjee, D., Zhao, S.C., Tong, Y., Steinherz, J., Gritsman, K., Bertino, J.R., 1994b.Transfection of a nonactive site mutant murine DHFR cDNA (the tryptophan 15

mutant) into Chinese hamster ovary and mouse marrow progenitor cells impartsMTX resistance in vitro. Cancer Gene Ther. 1, 181–184.

ell, D.J., Nyirongo, S.K., Mukaka, M., Zijlstra, E.E., Plowe, C.V., Molyneux, M.E., Ward,S.A., Winstanley, P.A., 2008. Sulfadoxine-pyrimethamine-based combinationsfor malaria: a randomised blinded trial to compare efficacy, safety and selectionof resistance in Malawi. PLoS ONE 3, e1578.

ance Updates 12 (2009) 28–41 39

Belzile, J.P., Karatzas, A., Shiu, H.Y., Letourneau, S., Palerme, J.S., Cournoyer, D., 2003.Increased resistance to nitrogen mustards and antifolates following in vitro selec-tion of murine fibroblasts and primary hematopoietic cells transduced with abicistronic retroviral vector expressing the rat glutathione S-transferase A3 anda mutant dihydrofolate reductase. Cancer Gene Ther. 10, 637–646.

Capiaux, G.M., Budak-Alpdogan, T., Alpdogan, O., Bornmann, W., Takebe, N., Baner-jee, D., Maley, F., Bertino, J.R., 2004. Protection of hematopoietic stem cellsfrom pemetrexed toxicity by retroviral gene transfer with a mutant dihydro-folate reductase-mutant thymidylate synthase fusion gene. Cancer Gene Ther.11, 767–773.

Capiaux, G.M., Budak-Alpdogan, T., Takebe, N., Mayer-Kuckuk, P., Banerjee, D.,Maley, F., Bertino, J.R., 2003. Retroviral transduction of a mutant dihydrofolatereductase-thymidylate synthase fusion gene into murine marrow cells con-fers resistance to both methotrexate and 5-fluorouracil. Hum. Gene Ther. 14,435–446.

Champness, J.N., Achari, A., Ballantine, S.P., Bryant, P.K., Delves, C.J., Stammers, D.K.,1994. The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 Å res-olution. Structure 2, 915–924.

Chen, G.X., Mueller, C., Wendlinger, M., Zolg, J.W., 1987. Kinetic and molecularproperties of the dihydrofolate reductase from pyrimethamine-sensitive andpyrimethamine-resistant clones of the human malaria parasite Plasmodium fal-ciparum. Mol. Pharmacol. 31, 430–437.

Cheng, K.K.-F. Association of plasma methotrexate, neutropenia, hepatic dysfunc-tion, nausea/vomiting and oral mucositis in children with cancer. Eur. J. CancerCare, in press.

Chongsuphajaisiddhi, T., Sabchareon, A., 1981. Sulfadoxine-pyrimethamine resistantfalciparum malaria in Thai children. Southeast Asian J. Trop. Med. Public Health12, 418–421.

Chu, E., Allegra, C.J., 1996. Antifolates. In: Cancer Chemotherapy and Biotherapy.Lippincott-Raven, Philadelphia, PA.

Chu, E., Takimoto, C.H., Voeller, D., Grem, J.L., Allegra, C.J., 1993. Specific binding ofhuman dihydrofolate reductase protein to dihydrofolate reductase messengerRNA in vitro. Biochemistry 32, 4756–4760.

Chunduru, S.K., Cody, V., Luft, J.R., Pangborn, W., Appleman, J.R., Blakley, R.L., 1994.Methotrexate-resistant variants of human dihydrofolate reductase. Effects ofPhe31 substitutions. J. Biol. Chem. 269, 9547–9555.

Chusacultanachai, S., Thiensathit, P., Tarnchompoo, B., Sirawaraporn, W., Yuthavong,Y., 2002. Novel antifolate resistant mutations of Plasmodium falciparum dihy-drofolate reductase selected in Escherichia coli. Mol. Biochem. Parasitol. 120,61–72.

Coderre, J.A., Beverley, S.M., Schimke, R.T., Santi, D.V., 1983. Overproduction of abifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplifi-cation in methotrexate-resistant Leishmania tropica. Proc. Natl. Acad. Sci. U.S.A.80, 2132–2136.

Cody, V., Galitsky, N., Luft, J.R., Pangborn, W., Rosowsky, A., Blakley, R.L., 1997.Comparison of two independent crystal structures of human dihydrofolatereductase ternary complexes reduced with nicotinamide adenine dinucleotidephosphate and the very tight-binding inhibitor PT523. Biochemistry 36, 13897–13903.

Cody, V., Galitsky, N., Rak, D., Luft, J.R., Pangborn, W., Queener, S.F., 1999. Ligand-induced conformational changes in the crystal structures of Pneumocystis cariniidihydrofolate reductase complexes with folate and NADP+. Biochemistry 38,4303–4312.

Cody, V., Luft, J.R., Pangborn, W., 2005. Understanding the role of Leu22 variants inmethotrexate resistance: comparison of wild-type and Leu22Arg variant mouseand human dihydrofolate reductase ternary crystal complexes with methotrex-ate and NADPH. Acta Crystallogr. D Biol. Crystallogr. 61, 147–155.

Cowman, A.F., Morry, M.J., Biggs, B.A., Cross, G.A., Foote, S.J., 1988. Amino acidchanges linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.A.85, 9109–9113.

Dale, G.E., Broger, C., D’Arcy, A., Hartman, P.G., DeHoogt, R., Jolidon, S., Kompis, I.,Labhardt, A.M., Langen, H., Locher, H., Page, M.G., Stuber, D., Then, R.L., Wipf,B., Oefner, C., 1997. A single amino acid substitution in Staphylococcus aureusdihydrofolate reductase determines trimethoprim resistance. J. Mol. Biol. 266,23–30.

Das, J.R., Fryar, E.B., Epie, N.N., Southerland, W.M., Bowen, D., 2006. Raloxifeneattenuation of methotrexate cytotoxicity in human bone marrow by sequence-dependent administration of raloxifene, 5-FU/methotrexate. Anticancer Res. 26,1877–1883.

Davies 2nd, J.F., Delcamp, T.J., Prendergast, N.J., Ashford, V.A., Freisheim, J.H., Kraut,J., 1990. Crystal structures of recombinant human dihydrofolate reductase com-plexed with folate and 5-deazafolate. Biochemistry 29, 9467–9479.

Daw, N.C., Billups, C.A., Rodriguez-Galindo, C., McCarville, M.B., Rao, B.N., Cain, A.M.,Jenkins, J.J., Neel, M.D., Meyer, W.H., 2006. Metastatic osteosarcoma. Cancer 106,403–412.

de Groot, R., Chaffin, D.O., Kuehn, M., Smith, A.L., 1991. Trimethoprim resistance inHaemophilus influenzae is due to altered dihydrofolate reductase(s). Biochem.J. 274 (Pt 3), 657–662.

de Pecoulas, P.E., Tahar, R., Ouatas, T., Mazabraud, A., Basco, L.K., 1998. Sequence

variations in the Plasmodium vivax dihydrofolate reductase-thymidylate syn-thase gene and their relationship with pyrimethamine resistance. Mol. Biochem.Parasitol. 92, 265–273.

Delfino, R.T., Santos-Filho, O.A., Figueroa-Villar, J.D., 2002. Molecular modeling ofwild-type and antifolate resistant mutant Plasmodium falciparum DHFR. Biophys.Chem. 98, 287–300.

4 Resist

D

D

D

E

E

E

F

F

F

F

F

F

G

G

G

G

G

G

H

H

H

H

H

H

H

H

H

H

0 J.P. Volpato, J.N. Pelletier / Drug

icker, A.P., Waltham, M.C., Volkenandt, M., Schweitzer, B.I., Otter, G.M., Schmid, F.A.,Sirotnak, F.M., Bertino, J.R., 1993. Methotrexate resistance in an in vivo mousetumor due to a non-active-site dihydrofolate reductase mutation. Proc. Natl.Acad. Sci. U.S.A. 90, 11797–11801.

olnick, B.J., Berenson, R.J., Bertino, J.R., Kaufman, R.J., Nunberg, J.H., Schimke, R.T.,1979. Correlation of dihydrofolate reductase elevation with gene amplificationin a homogeneously staining chromosomal region in L5178Y cells. J. Cell Biol. 83,394–402.

olznig, H., Kolbus, A., Leberbauer, C., Schmidt, U., Deiner, E.M., Mullner, E.W.,Beug, H., 2005. Expansion and differentiation of immature mouse and humanhematopoietic progenitors. Methods Mol. Med. 105, 323–344.

llenberger, T.E., Beverley, S.M., 1989. Multiple drug resistance and conserva-tive amplification of the H region in Leishmania major. J. Biol. Chem. 264,15094–15103.

rcikan-Abali, E.A., Mineishi, S., Tong, Y., Nakahara, S., Waltham, M.C., Banerjee, D.,Chen, W., Sadelain, M., Bertino, J.R., 1996a. Active site-directed double mutantsof dihydrofolate reductase. Cancer Res. 56, 4142–4145.

rcikan-Abali, E.A., Waltham, M.C., Dicker, A.P., Schweitzer, B.I., Gritsman, H., Baner-jee, D., Bertino, J.R., 1996b. Variants of human dihydrofolate reductase withsubstitutions at leucine-22: effect on catalytic and inhibitor binding properties.Mol. Pharmacol. 49, 430–437.

arber, S., Diamond, L.K., Mercer, R.D., Sylvester, R.F., Wolff, V.A., 1948. N. Engl. J. Med.238, 787–793.

erone, R., Burchall, J.J., Hitchings, G.H., 1969. Plasmodium berghei dihydrofolatereductase. Isolation, properties, and inhibition by antifolates. Mol. Pharmacol.5, 49–59.

lasshove, M., Banerjee, D., Mineishi, S., Li, M.X., Bertino, J.R., Moore, M.A., 1995. Exvivo expansion and selection of human CD34+ peripheral blood progenitor cellsafter introduction of a mutated dihydrofolate reductase cDNA via retroviral genetransfer. Blood 85, 566–574.

lensburg, J., Skold, O., 1987. Massive overproduction of dihydrofolate reductase inbacteria as a response to the use of trimethoprim. Eur. J. Biochem. 162, 473–476.

oote, S.J., Galatis, D., Cowman, A.F., 1990. Amino acids in the dihydrofolatereductase-thymidylate synthase gene of Plasmodium falciparum involved incycloguanil resistance differ from those involved in pyrimethamine resistance.Proc. Natl. Acad. Sci. U.S.A. 87, 3014–3017.

ossati, E., Volpato, J.P., Poulin, L., Guerrero, V., Dugas, D.A., Pelletier, J.N., 2008. 2-tierbacterial and in vitro selection of active and methotrexate-resistant variants ofhuman dihydrofolate reductase. J. Biomol. Screen. 13, 504–514.

atlin, J., Douglas, J., Evans, J.T., Collins, R.H., Wendel, G.D., Garcia, J.V., 2000. In vitroselection of lentivirus vector-transduced human CD34+ cells. Hum. Gene Ther.11, 1949–1957.

ilbert, I.H., 2002. Inhibitors of dihydrofolate reductase in Leishmania and try-panosomes. Biochim. Biophys. Acta 1587, 249–257.

oker, E., Lin, J.T., Trippett, T., Elisseyeff, Y., Tong, W.P., Niedzwiecki, D., Tan, C.,Steinherz, P., Schweitzer, B.I., Bertino, J.R., 1993. Decreased polyglutamylationof methotrexate in acute lymphoblastic leukemia blasts in adults compared tochildren with this disease. Leukemia 7, 1000–1004.

oker, E., Waltham, M., Kheradpour, A., Trippett, T., Mazumdar, M., Elisseyeff, Y.,Schnieders, B., Steinherz, P., Tan, C., Berman, E., et al., 1995. Amplification ofthe dihydrofolate reductase gene is a mechanism of acquired resistance tomethotrexate in patients with acute lymphoblastic leukemia and is correlatedwith p53 gene mutations. Blood 86, 677–684.

oldie, J.H., Krystal, G., Hartley, D., Gudauskas, G., Dedhar, S., 1980. A methotrexateinsensitive variant of folate reductase present in two lines of methotrexate-resistant L5178Y cells. Eur. J. Cancer 16, 1539–1546.

oldman, I.D., 1974. The mechanism of action of methotrexate. I. Interaction with alow-affinity intracellular site required for maximum inhibition of deoxyribonu-cleic acid synthesis in L-cell mouse fibroblasts. Mol. Pharmacol. 10, 257–274.

ankins, E.G., Warhurst, D.C., Sibley, C.H., 2001. Novel alleles of the Plasmodium fal-ciparum dhfr highly resistant to pyrimethamine and chlorcycloguanil, but notWR99210. Mol. Biochem. Parasitol. 117, 91–102.

appi, C.T., Gbotosho, G.O., Folarin, O.A., Akinboye, D.O., Yusuf, B.O., Ebong, O.O.,Sowunmi, A., Kyle, D.E., Milhous, W., Wirth, D.F., Oduola, A.M., 2005. Polymor-phisms in Plasmodium falciparum dhfr and dhps genes and age related in vivosulfadoxine-pyrimethamine resistance in malaria-infected patients from Nige-ria. Acta Trop. 95, 183–193.

ashkes, P.J., Laxer, R.M., 2006. Update on the medical treatment of juvenile idio-pathic arthritis. Curr. Rheumatol. Rep. 8, 450–458.

astings, M.D., Sibley, C.H., 2002. Pyrimethamine and WR99210 exert opposingselection on dihydrofolate reductase from Plasmodium vivax. Proc. Natl. Acad.Sci. U.S.A. 99, 13137–13141.

avenga, M.J., Werner, A.B., Valerio, D., van Es, H.H., 1998. Methotrexate selectableretroviral vectors for Gaucher disease. Gene Ther. 5, 1379–1388.

awser, S., Lociuro, S., Islam, K., 2006. Dihydrofolate reductase inhibitors as antibac-terial agents. Biochem. Pharmacol. 71, 941–948.

ekmat-Nejad, M., Rathod, P.K., 1997. Plasmodium falciparum: kinetic interactionsof WR99210 with pyrimethamine-sensitive and pyrimethamine-resistant dihy-drofolate reductase. Exp. Parasitol. 87, 222–228.

itchings, G.H., 1969. Species differences among dihydrofolate reductases as a basis

for chemotherapy. Postgrad. Med. J. 45 (Suppl.), 7–10.

ock, R.A., Miller, A.D., 1986. Retrovirus-mediated transfer and expression ofdrug resistance genes in human haematopoietic progenitor cells. Nature 320,275–277.

uovinen, P., Sundstrom, L., Swedberg, G., Skold, O., 1995. Trimethoprim and sulfon-amide resistance. Antimicrob. Agents Chemother. 39, 279–289.

ance Updates 12 (2009) 28–41

Imwong, M., Pukrittakayamee, S., Looareesuwan, S., Pasvol, G., Poirreiz, J., White,N.J., Snounou, G., 2001. Association of genetic mutations in Plasmodium vivaxdhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinicalcorrelates. Antimicrob. Agents Chemother. 45, 3122–3127.

Imwong, M., Pukrittayakamee, S., Renia, L., Letourneur, F., Charlieu, J.P., Leart-sakulpanich, U., Looareesuwan, S., White, N.J., Snounou, G., 2003. Novel pointmutations in the dihydrofolate reductase gene of Plasmodium vivax: evidencefor sequential selection by drug pressure. Antimicrob. Agents Chemother. 47,1514–1521.

Jones, G., Crotty, M., Brooks, P., 2000. Interventions for psoriatic arthritis. CochraneDatabase Syst. Rev. (CD000212).

Knighton, D.R., Kan, C.C., Howland, E., Janson, C.A., Hostomska, Z., Welsh, K.M.,Matthews, D.A., 1994. Structure of and kinetic channelling in bifunctional dihy-drofolate reductase-thymidylate synthase. Nat. Struct. Biol. 1, 186–194.

Kongsaeree, P., Khongsuk, P., Leartsakulpanich, U., Chitnumsub, P., Tarnchompoo,B., Walkinshaw, M.D., Yuthavong, Y., 2005. Crystal structure of dihydrofo-late reductase from Plasmodium vivax: pyrimethamine displacement linkedwith mutation-induced resistance. Proc. Natl. Acad. Sci. U.S.A. 102, 13046–13051.

Kovalevskaya, N.V., Smurnyy, Y.D., Polshakov, V.I., Birdsall, B., Bradbury, A.F., Frenkiel,T., Feeney, J., 2005. Solution structure of human dihydrofolate reductase in itscomplex with trimethoprim and NADPH. J. Biomol. NMR 33, 69–72.

Kramer, B.A., Lemckert, F.A., Alexander, I.E., Gunning, P.W., McCowage, G.B., 2006.Characterisation of a P140K mutant O6-methylguanine–DNA–methyltransferase(MGMT)-expressing transgenic mouse line with drug-selectable bone marrow.J. Gene Med. 8, 1071–1085.

Leartsakulpanich, U., Imwong, M., Pukrittayakamee, S., White, N.J., Snounou, G., Sir-awaraporn, W., Yuthavong, Y., 2002. Molecular characterization of dihydrofolatereductase in relation to antifolate resistance in Plasmodium vivax. Mol. Biochem.Parasitol. 119, 63–73.

Lewis, W.S., Cody, V., Galitsky, N., Luft, J.R., Pangborn, W., Chunduru, S.K., Spencer,H.T., Appleman, J.R., Blakley, R.L., 1995. Methotrexate-resistant variants of humandihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallogra-phy, and potential as selectable markers. J. Biol. Chem. 270, 5057–5064.

Li, R., Sirawaraporn, R., Chitnumsub, P., Sirawaraporn, W., Wooden, J., Athappilly, F.,Turley, S., Hol, W.G., 2000. Three-dimensional structure of M. tuberculosis dihy-drofolate reductase reveals opportunities for the design of novel tuberculosisdrugs. J. Mol. Biol. 295, 307–323.

Lin, J.T., Tong, W.P., Trippett, T.M., Niedzwiecki, D., Tao, Y., Tan, C., Steinherz, P.,Schweitzer, B.I., Bertino, J.R., 1991. Basis for natural resistance to methotrexatein human acute non-lymphocytic leukemia. Leuk. Res. 15, 1191–1196.

Lorico, A., Bratbak, D., Meyer, J., Kunke, D., Krauss, S., Plott, W.E., Solodushko, V.,Baum, C., Fodstad, O., Rappa, G., 2005. Gamma-glutamylcysteine synthetase andL-buthionine-(S, R)-sulfoximine: a new selection strategy for gene-transducedneural and hematopoietic stem/progenitor cells. Hum. Gene Ther. 16, 711–724.

Lynch, G., Magill, G.B., Sordillo, P., Golbey, R.B., 1982. Combination chemotherapy ofadvanced sarcomas in adults with “CYOMAD” (S7). Cancer 50, 1724–1727.

Ma, L., Kovacs, J.A., 2000. Expression and characterization of recombinant human-derived Pneumocystis carinii dihydrofolate reductase. Antimicrob. AgentsChemother. 44, 3092–3096.

Margosiak, S.A., Appleman, J.R., Santi, D.V., Blakley, R.L., 1993. Dihydrofolate reduc-tase from the pathogenic fungus Pneumocystis carinii: catalytic properties andinteraction with antifolates. Arch. Biochem. Biophys. 305, 499–508.

Maskell, J.P., Sefton, A.M., Hall, L.M., 2001. Multiple mutations modulate the functionof dihydrofolate reductase in trimethoprim-resistant Streptococcus pneumoniae.Antimicrob. Agents Chemother. 45, 1104–1108.

Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B.T.,Beddell, C.R., Champness, J.N., Stammers, D.K., Kraut, J., 1985. Refined crystalstructures of Escherichia coli and chicken liver dihydrofolate reductase containingbound trimethoprim. J. Biol. Chem. 260, 381–391.

McGready, R., Stepniewska, K., Seaton, E., Cho, T., Cho, D., Ginsberg, A., Edstein, M.D.,Ashley, E., Looareesuwan, S., White, N.J., Nosten, F., 2003. Pregnancy and use oforal contraceptives reduces the biotransformation of proguanil to cycloguanil.Eur. J. Clin. Pharmacol. 59, 553–557.

Melera, P.W., Davide, J.P., Oen, H., 1988. Antifolate-resistant Chinese hamster cells.Molecular basis for the biochemical and structural heterogeneity among dihy-drofolate reductases produced by drug-sensitive and drug-resistant cell lines. J.Biol. Chem. 263, 1978–1990.

Melera, P.W., Hession, C.A., Davide, J.P., Scotto, K.W., Biedler, J.L., Meyers, M.B.,Shanske, S., 1982. Antifolate-resistant Chinese Hamster Cells. mRNA directedoverproduction of multiple dihydrofolate reductases from a series of indepen-dently derived sublines containing amplified dihydrofolate reductase genes. J.Biol. Chem. 257, 12939–12949.

Mendis, K., Sina, B.J., Marchesini, P., Carter, R., 2001. The neglected burden of Plas-modium vivax malaria. Am. J. Trop. Med. Hyg. 64, 97–106.

Mennes, M., Stiers, P., Vandenbussche, E., Vercruysse, G., Uyttebroeck, A., De Meyer,G., Van Cool, S.W., 2005. Attention and information processing in survivors ofchildhood acute lymphoblastic leukemia treated with chemotherapy only. Pedi-atr. Blood Cancer 44, 478–486.

Morris, J.A., McIvor, R.S., 1994. Saturation mutagenesis at dihydrofolate reductasecodons 22 and 31. A variety of amino acid substitutions conferring methotrexateresistance. Biochem. Pharmacol. 47, 1207–1220.

Nakano, T., Spencer, H.T., Appleman, J.R., Blakley, R.L., 1994. Critical role of pheny-lalanine 34 of human dihydrofolate reductase in substrate and inhibitor bindingand in catalysis. Biochemistry 33, 9945–9952.

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S

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J.P. Volpato, J.N. Pelletier / Drug

zila, A., Ochong, E., Nduati, E., Gilbert, K., Winstanley, P., Ward, S., Marsh, K., 2005.Why has the dihydrofolate reductase 164 mutation not consistently been foundin Africa yet? Trans. R. Soc. Trop. Med. Hyg. 99, 341–346.

efner, C., D’Arcy, A., Winkler, F.K., 1988. Crystal structure of human dihydrofolatereductase complexed with folate. Eur. J. Biochem. 174, 377–385.

atel, M., Sleep, S.E., Lewis, W.S., Spencer, H.T., Mareya, S.M., Sorrentino, B.P., Blakley,R.L., 1997. Comparison of the protection of cells from antifolates by transducedhuman dihydrofolate reductase mutants. Hum. Gene Ther. 8, 2069–2077.

ersons, D.A., Allay, J.A., Bonifacino, A., Lu, T., Agricola, B., Metzger, M.E., Donahue,R.E., Dunbar, C.E., Sorrentino, B.P., 2004. Transient in vivo selection of transducedperipheral blood cells using antifolate drug selection in rhesus macaques thatreceived transplants with hematopoietic stem cells expressing dihydrofolatereductase vectors. Blood 103, 796–803.

eterson, D.S., Milhous, W.K., Wellems, T.E., 1990. Molecular basis of differentialresistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria.Proc. Natl. Acad. Sci. U.S.A 87, 3018–3022.

eterson, D.S., Walliker, D., Wellems, T.E., 1988. Evidence that a point muta-tion in dihydrofolate reductase-thymidylate synthase confers resistance topyrimethamine in falciparum malaria. Proc. Natl. Acad. Sci. U.S.A. 85, 9114–9118.

ikis, A., Donkersloot, J.A., Rodriguez, W.J., Keith, J.M., 1998. A conservative aminoacid mutation in the chromosome-encoded dihydrofolate reductase conferstrimethoprim resistance in Streptococcus pneumoniae. J. Infect. Dis. 178, 700–706.

rendergast, N.J., Appleman, J.R., Delcamp, T.J., Blakley, R.L., Freisheim, J.H., 1989.Effects of conversion of phenylalanine-31 to leucine on the function of humandihydrofolate reductase. Biochemistry 28, 4645–4650.

othem, L., Stark, M., Kaufman, Y., Mayo, L., Assaraf, Y.G., 2004. Reduced folate car-rier gene silencing in multiple antifolate-resistant tumor cell lines is due to asimultaneous loss of function of multiple transcription factors but not promotermethylation. J. Biol. Chem. 279, 374–384.

andefur, C.I., Wooden, J.M., Quaye, I.K., Sirawaraporn, W., Sibley, C.H., 2007.Pyrimethamine-resistant dihydrofolate reductase enzymes of Plasmodium fal-ciparum are not enzymatically compromised in vitro. Mol. Biochem. Parasitol.154, 1–5.

antos-Filho, O.A., de Alencastro, R.B., Figueroa-Villar, J.D., 2001. Homology mod-eling of wild type and pyrimethamine/cycloguanil-cross resistant mutanttype Plasmodium falciparum dihydrofolate reductase. A model for antimalarialchemotherapy resistance. Biophys. Chem. 91, 305–317.

auerbrey, A., McPherson, J.P., Zhao, S.C., Banerjee, D., Bertino, J.R., 1999. Expressionof a novel double-mutant dihydrofolate reductase-cytidine deaminase fusiongene confers resistance to both methotrexate and cytosine arabinoside. Hum.Gene Ther. 10, 2495–2504.

awaya, M.R., Kraut, J., 1997. Loop and subdomain movements in the mechanism ofEscherichia coli dihydrofolate reductase: crystallographic evidence. Biochemistry36, 586–603.

chweitzer, B.I., Dicker, A.P., Bertino, J.R., 1990. Dihydrofolate reductase as a thera-peutic target. FASEB J. 4, 2441–2452.

chweitzer, B.I., Srimatkandada, S., Gritsman, H., Sheridan, R., Venkataraghavan, R.,Bertino, J.R., 1989. Probing the role of two hydrophobic active site residues inthe human dihydrofolate reductase by site-directed mutagenesis. J. Biol. Chem.264, 20786–20795.

egel, I.H., 1993. In Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium andSteady-State Enzyme Systems. John Wiley and sons.

imonsen, C.C., Levinson, A.D., 1983. Isolation and expression of an altered mousedihydrofolate reductase cDNA. Proc. Natl. Acad. Sci. U.S.A. 80, 2495–2499.

irawaraporn, L.R., Chitnumsub, P., Sirawaraporn, W., Wooden, J., Athapilly, F., Turley,S., Hol, H.W., 2000. Three-dimensional structure of M. tuberculosis dihydrofolatereductase reveals opportunities for the design of novel tuberculosis drugs. J. Mol.Biol. 295, 307–323.

irawaraporn, W., Prapunwattana, P., Sirawaraporn, R., Yuthavong, Y., Santi, D.V.,1993. The dihydrofolate reductase domain of Plasmodium falciparum thymidylatesynthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants. J. Biol. Chem. 268, 21637–21644.

irawaraporn, W., Sathitkul, T., Sirawaraporn, R., Yuthavong, Y., Santi, D.V., 1997a.Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase.Proc. Natl. Acad. Sci. U.S.A. 94, 1124–1129.

irawaraporn, W., Sirawaraporn, R., Cowman, A.F., Yuthavong, Y., Santi, D.V., 1990.Heterologous expression of active thymidylate synthase-dihydrofolate reduc-tase from Plasmodium falciparum. Biochemistry 29, 10779–10785.

irawaraporn, W., Yongkiettrakul, S., Sirawaraporn, R., Yuthavong, Y., Santi, D.V.,1997b. Plasmodium falciparum: asparagine mutant at residue 108 of dihydro-folate reductase is an optimal antifolate-resistant single mutant. Exp. Parasitol.87, 245–252.

lamon, D.J., Romond, E.H., Perez, E.A., 2006. Advances in adjuvant therapy for breastcancer. Clin. Adv. Hematol. Oncol. 4, 4–9.

ance Updates 12 (2009) 28–41 41

Sorrentino, B.P., 2004. Clinical strategies for expansion of haematopoietic stem cells.Nat. Rev. Immunol. 4, 878–888.

Spencer, H.T., Sleep, S.E., Rehg, J.E., Blakley, R.L., Sorrentino, B.P., 1996a. A gene trans-fer strategy for making bone marrow cells resistant to trimetrexate. Blood 87,2579–2587.

Spencer, H.T., Sorrentino, B.P., Pui, C.H., Chunduru, S.K., Sleep, S.E., Blakley, R.L., 1996b.Mutations in the gene for human dihydrofolate reductase: an unlikely cause ofclinical relapse in pediatric leukemia after therapy with methotrexate. Leukemia10, 439–446.

Strojan, P., Soba, E., Budihna, M., Auersperg, M., 2005. Radiochemotherapy with Vin-blastine, Methotrexate, and Bleomycin in the treatment of verrucous carcinomaof the head and neck. J. Surg. Oncol. 92, 278–283.

Tahar, R., de Pecoulas, P.E., Basco, L.K., Chiadmi, M., Mazabraud, A., 2001. Kineticproperties of dihydrofolate reductase from wild-type and mutant Plasmodiumvivax expressed in Escherichia coli. Mol. Biochem. Parasitol. 113, 241–249.

Takebe, N., Xu, L.C., MacKenzie, K.L., Bertino, J.R., Moore, M.A., 2002. Methotrexateselection of long-term culture initiating cells following transduction of CD34(+)cells with a retrovirus containing a mutated human dihydrofolate reductasegene. Cancer Gene Ther. 9, 308–320.

Takebe, N., Zhao, S.C., Adhikari, D., Mineishi, S., Sadelain, M., Hilton, J., Colvin,M., Banerjee, D., Bertino, J.R., 2001. Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of thehuman aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reduc-tase gene. Mol. Ther. 3, 88–96.

Tarnchompoo, B., Sirichaiwat, C., Phupong, W., Intaraudom, C., Sirawaraporn, W.,Kamchonwongpaisan, S., Vanichtanankul, J., Thebtaranonth, Y., Yuthavong, Y.,2002. Development of 2,4-diaminopyrimidines as antimalarials based on inhi-bition of the S108N and C59R+S108N mutants of dihydrofolate reductasefrom pyrimethamine-resistant Plasmodium falciparum. J. Med. Chem. 45, 1244–1252.

Thillet, J., Absil, J., Stone, S.R., Pictet, R., 1988. Site-directed mutagenesis of mousedihydrofolate reductase. Mutants with increased resistance to methotrexate andtrimethoprim. J. Biol. Chem. 263, 12500–12508.

Thillet, J., Pictet, R., 1990. Transfection of DHFR− and DHFR+ mammalian cells usingmethotrexate-resistant mutants of mouse dihydrofolate reductase. FEBS Lett.269, 450–453.

Trippett, T., Schlemmer, S., Elisseyeff, Y., Goker, E., Wachter, M., Steinherz, P., Tan, C.,Berman, E., Wright, J.E., Rosowsky, A., et al., 1992. Defective transport as a mech-anism of acquired resistance to methotrexate in patients with acute lymphocyticleukemia. Blood 80, 1158–1162.

Volpato, J.P., Fossati, E., Pelletier, J.N., 2007. Increasing methotrexate resistance bycombination of active-site mutations in human dihydrofolate reductase. J. Mol.Biol. 373, 599–611.

Warlick, C.A., Diers, M.D., Wagner, J.E., McIvor, R.S., 2002. In vivo selection ofantifolate-resistant transgenic hematopoietic stem cells in a murine bone mar-row transplant model. J. Pharmacol. Exp. Ther. 300, 50–56.

Watson, M., Liu, J.W., Ollis, D., 2007. Directed evolution of trimethoprim resistancein Escherichia coli. FEBS J. 274, 2661–2671.

Young, M.D., Burgess, R.W., 1959. Pyrimethamine resistance in Plasmodium vivaxmalaria. Bull. World Health Organ. 20, 27–36.

Yuvaniyama, J., Chitnumsub, P., Kamchonwongpaisan, S., Vanichtanankul, J., Sir-awaraporn, W., Taylor, P., Walkinshaw, M.D., Yuthavong, Y., 2003. Insights intoantifolate resistance from malarial DHFR-TS structures. Nat. Struct. Biol. 10,357–365.

Zhanel, G.G., DeCorby, M., Laing, N., Weshnoweski, B., Vashisht, R., Tailor, F., Nichol,K.A., Wierzbowski, A., Baudry, P.J., Karlowsky, J.A., Lagace-Wiens, P., Walkty,A., McCracken, M., Mulvey, M.R., Johnson, J., Hoban, D.J., 2008. Antimicrobial-resistant pathogens in intensive care units in Canada: results of the CanadianNational Intensive Care Unit (CAN-ICU) study, 2005–2006. Antimicrob. AgentsChemother. 52, 1430–1437.

Zhang, K., Rathod, P.K., 2002. Divergent regulation of dihydrofolate reductasebetween malaria parasite and human host. Science 296, 545–547.

Zhao, R., Goldman, I.D., 2003. Resistance to antifolates. Oncogene 22, 7431–7457.Zhao, R., Titus, S., Gao, F., Moran, R.G., Goldman, I.D., 2000. Molecular analysis of

murine leukemia cell lines resistant to 5, 10-dideazatetrahydrofolate identifiesseveral amino acids critical to the function of folylpolyglutamate synthetase. J.Biol. Chem. 275, 26599–26606.

Zhao, S.C., Banerjee, D., Mineishi, S., Bertino, J.R., 1997. Post-transplant methotrex-

ate administration leads to improved curability of mice bearing a mammarytumor transplanted with marrow transduced with a mutant human dihydrofo-late reductase cDNA. Hum. Gene Ther. 8, 903–909.

Zolg, J.W., Plitt, J.R., Chen, G.X., Palmer, S., 1989. Point mutations in the dihydrofolatereductase-thymidylate synthase gene as the molecular basis for pyrimethamineresistance in Plasmodium falciparum. Mol. Biochem. Parasitol. 36, 253–262.